Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing

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Andrew M. Tidball

2017-09-01

Full Text Available Specifically ablating genes in human induced pluripotent stem cells (iPSCs allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels. This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.

One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system.

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Wattanapanitch, Methichit; Damkham, Nattaya; Potirat, Ponthip; Trakarnsanga, Kongtana; Janan, Montira; U-Pratya, Yaowalak; Kheolamai, Pakpoom; Klincumhom, Nuttha; Issaragrisil, Surapol

2018-02-26

Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.

Human neural progenitors derived from integration-free iPSCs for SCI therapy

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Ying Liu

2017-03-01

Full Text Available As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs provide great capability for tissue regeneration, particularly in spinal cord injury (SCI. However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.

Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications.

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Li, Yang; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2017-07-31

Remarkable achievements have been made since induced pluripotent stem cells (iPSCs) were first introduced in 2006. Compared with non-pluripotent stem cells, iPSC research faces several additional complexities, such as the choice of extracellular matrix proteins, growth and differentiation factors, as well as technical challenges related to self-renewal and directed differentiation. Overcoming these challenges requires the integration of knowledge and technologies from multiple fields including cell biology, biomaterial science, engineering, physics and medicine. Here, engineering-derived iPSC approaches are reviewed according to three aspects of iPSC studies: preparation, expansion, differentiation and applications. Engineering strategies, such as 3D systems establishment, cell-matrix mechanics and the regulation of biophysical and biochemical cues, together with engineering techniques, such as 3D scaffolds, cell microspheres and bioreactors, have been applied to iPSC studies and have generated insightful results and even mini-organs such as retinas, livers and intestines. Specific results are given to demonstrate how these approaches impact iPSC behavior, and related mechanisms are discussed. In addition, cell printing technologies are presented as an advanced engineering-derived approach since they have been applied in both iPSC studies and the construction of diverse tissues and organs. Further development and possible innovations of cell printing technologies are presented in terms of creating complex and functional iPSC-derived living tissues and organs.

Dissecting the Contributions of Cooperating Gene Mutations to Cancer Phenotypes and Drug Responses with Patient-Derived iPSCs

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Chan-Jung Chang

2018-05-01

Full Text Available Summary: Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs—with none, one, or both genetic lesions—we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases. : Papapetrou and colleagues develop a comprehensive panel of isogenic iPSC lines with SRSF2 P95L mutation and chr7q deletion. They use these cells to identify cellular phenotypes contributed by each genetic lesion and therapeutic vulnerabilities specific to each one and develop expandable hematopoietic progenitor cell lines to facilitate drug discovery. Keywords: induced pluripotent stem cells, myelodysplastic syndrome, CRISPR/Cas9, gene editing, mutational cooperation, splicing factor mutations, spliceosomal mutations, SRSF2, chr7q deletion

Efficient generation of lens progenitor cells from cataract patient-specific induced pluripotent stem cells.

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Xiaodi Qiu

Full Text Available The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs could be induced into iPSCs and if lens-specific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLEC-derived iPS (HLE-iPS colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2, and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP. In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT markers compared with human embryonic stem cells (hESCs and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract

Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

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Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A.

2016-01-01

Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co......-electroporation with episomal plasmids containing hOCT4, hSOX2, hL-MYC, hKLF4, hNANOG, hLIN28, and short hairpin RNA against TP53. The iPSCs contained the specific heterozygous mutation c.449C>T, had normal karyotype, expressed the expected pluripotency genes and displayed in vitro differentiation potential to the three germ...

Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases.

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Wiley, Luke A; Burnight, Erin R; Songstad, Allison E; Drack, Arlene V; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

2015-01-01

Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement. Copyright © 2014. Published by Elsevier Ltd.

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

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Burnight, Erin R; Gupta, Manav; Wiley, Luke A; Anfinson, Kristin R; Tran, Audrey; Triboulet, Robinson; Hoffmann, Jeremy M; Klaahsen, Darcey L; Andorf, Jeaneen L; Jiao, Chunhua; Sohn, Elliott H; Adur, Malavika K; Ross, Jason W; Mullins, Robert F; Daley, George Q; Schlaeger, Thorsten M; Stone, Edwin M; Tucker, Budd A

2017-09-06

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

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Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-07-09

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

From iPSC towards cardiac tissue-a road under construction.

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Peischard, Stefan; Piccini, Ilaria; Strutz-Seebohm, Nathalie; Greber, Boris; Seebohm, Guiscard

2017-10-01

The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.

The Combination of CRISPR/Cas9 and iPSC Technologies in the Gene Therapy of Human β-thalassemia in Mice.

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Ou, Zhanhui; Niu, Xiaohua; He, Wenyin; Chen, Yuchang; Song, Bing; Xian, Yexing; Fan, Di; Tang, Daolin; Sun, Xiaofang

2016-09-01

β-thalassemia results from point mutations or small deletions in the β-globin (HBB) gene that ultimately cause anemia. The generation of induced pluripotent stem cells (iPSCs) from the somatic cells of patients in combination with subsequent homologous recombination-based gene correction provides new approaches to cure this disease. CRISPR/Cas9 is a genome editing tool that is creating a buzz in the scientific community for treating human diseases, especially genetic disorders. Here, we reported that correction of β-thalassemia mutations in patient-specific iPSCs using the CRISPR/Cas9 tool promotes hematopoietic differentiation in vivo. CRISPR/Cas9-corrected iPSC-derived hematopoietic stem cells (HSCs) were injected into sublethally-irradiated NOD-scid-IL2Rg-/- (NSI) mice. HBB expression was observed in these HSCs after hematopoietic differentiation in the NSI mice. Importantly, no tumor was found in the livers, lungs, kidneys, or bone marrow at 10 weeks in the NSI mice after implantation with these HSCs. Collectively, our findings demonstrated that CRISPR/Cas9 successfully corrects β-thalassemia mutations in patient-specific iPSCs. These CRISPR/Cas9-corrected iPSC-derived HSCs express normal HBB in mice without tumorigenic potential, suggesting a safe strategy for personalized treatment of β-thalassemia.

Neurons derived from sporadic Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation

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Ochalek, Anna; Mihalik, Balázs; Avci, Hasan X.

2017-01-01

, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. Methods: We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer's disease (fAD), all caused...... blotting methods. Results: Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular......, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both f...

iPSC Core

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Federal Laboratory Consortium — The induced Pluripotent Stem Cells (iPSC) Core was created in 2011 to accelerate stem cell research in the NHLBI by providing investigators consultation, technical...

Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene

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Damià Romero-Moya

2017-10-01

Full Text Available We report the generation, CRISPR/Cas9-edition and characterization of induced pluripotent stem cell (iPSC lines from a patient with coenzyme Q10 deficiency harboring the heterozygous mutation c.483G > C in the COQ4 gene. iPSCs were generated using non-integrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-MYC. The iPSC lines carried the c.483G > C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4-corrected iPSCs.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

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Mandegar, Mohammad A.; Huebsch, Nathaniel; Frolov, Ekaterina B.

2016-01-01

repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range...

Variable behavior of iPSCs derived from CML patients for response to TKI and hematopoietic differentiation.

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Aurélie Bedel

Full Text Available Chronic myeloid leukemia disease (CML found effective therapy by treating patients with tyrosine kinase inhibitors (TKI, which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.

Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC

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Zeynab Nayernia

2017-10-01

Full Text Available There is emerging evidence for the involvement of reactive oxygen species (ROS in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC. High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST, and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.

Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells

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Atsushi Tanaka

2015-08-01

Full Text Available The generation of induced pluripotent stem cells (iPSCs has opened up a new scientific frontier in medicine. This technology has made it possible to obtain pluripotent stem cells from individuals with genetic disorders. Because iPSCs carry the identical genetic anomalies related to those disorders, iPSCs are an ideal platform for medical research. The pathophysiological cellular phenotypes of genetically heritable heart diseases such as arrhythmias and cardiomyopathies, have been modeled on cell culture dishes using disease-specific iPSC-derived cardiomyocytes. These model systems can potentially provide new insights into disease mechanisms and drug discoveries. This review focuses on recent progress in cardiovascular disease modeling using iPSCs, and discusses problems and future perspectives concerning their use.

iPSCs: A Minireview from Bench to Bed, including Organoids and the CRISPR System

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Andrés Javier Orqueda

2016-01-01

Full Text Available When Dolly the sheep was born, the first probe into an adult mammalian genome traveling back in time and generating a whole new animal appeared. Ten years later, the reprogramming process became a defined method of producing induced pluripotent stem cells (iPSCs through the overexpression of four transcription factors. iPSCs are capable of originating virtually all types of cells and tissues, including a whole new animal. The reprogramming strategies based on patient-derived cells should make the development of clinical applications of cell based therapy much more straightforward. Here, we analyze the current state, opportunities, and challenges of iPSCs from bench to bed, including organoids and the CRISPR system.

Introduction to thematic minireview series: Development of human therapeutics based on induced pluripotent stem cell (iPSC) technology.

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Rao, Mahendra; Gottesfeld, Joel M

2014-02-21

With the advent of human induced pluripotent stem cell (hiPSC) technology, it is now possible to derive patient-specific cell lines that are of great potential in both basic research and the development of new therapeutics for human diseases. Not only do hiPSCs offer unprecedented opportunities to study cellular differentiation and model human diseases, but the differentiated cell types obtained from iPSCs may become therapeutics themselves. These cells can also be used in the screening of therapeutics and in toxicology assays for potential liabilities of therapeutic agents. The remarkable achievement of transcription factor reprogramming to generate iPSCs was recognized by the award of the Nobel Prize in Medicine to Shinya Yamanaka in 2012, just 6 years after the first publication of reprogramming methods to generate hiPSCs (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Cell 131, 861-872). This minireview series highlights both the promises and challenges of using iPSC technology for disease modeling, drug screening, and the development of stem cell therapeutics.

Modeling the autistic cell: iPSCs recapitulate developmental principles of syndromic and nonsyndromic ASD.

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Ben-Reuven, Lihi; Reiner, Orly

2016-06-01

The opportunity to model autism spectrum disorders (ASD) through generation of patient-derived induced pluripotent stem cells (iPSCs) is currently an emerging topic. Wide-scale research of altered brain circuits in syndromic ASD, including Rett Syndrome, Fragile X Syndrome, Angelman's Syndrome and sporadic Schizophrenia, was made possible through animal models. However, possibly due to species differences, and to the possible contribution of epigenetics in the pathophysiology of these diseases, animal models fail to recapitulate many aspects of ASD. With the advent of iPSCs technology, 3D cultures of patient-derived cells are being used to study complex neuronal phenotypes, including both syndromic and nonsyndromic ASD. Here, we review recent advances in using iPSCs to study various aspects of the ASD neuropathology, with emphasis on the efforts to create in vitro model systems for syndromic and nonsyndromic ASD. We summarize the main cellular activity phenotypes and aberrant genetic interaction networks that were found in iPSC-derived neurons of syndromic and nonsyndromic autistic patients. © 2016 Japanese Society of Developmental Biologists.

Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration.

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Zhouhui Geng

Full Text Available Fidelity in pluripotent stem cell differentiation protocols is necessary for the therapeutic and commercial use of cells derived from embryonic and induced pluripotent stem cells. Recent advances in stem cell technology, especially the widespread availability of a range of chemically defined media, substrates and differentiation components, now allow the design and implementation of fully defined derivation and differentiation protocols intended for replication across multiple research and manufacturing locations. In this report we present an application of these criteria to the generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. Primary conjunctival cells from human donors aged 70-85 years were reprogrammed to derive multiple iPSC lines that were differentiated into functional RPE using a rapid and defined differentiation protocol. The combination of defined iPSC derivation and culture with a defined RPE differentiation protocol, reproducibly generated functional RPE from each donor without requiring protocol adjustments for each individual. This successful validation of a standardized, iPSC derivation and RPE differentiation process demonstrates a practical approach for applications requiring the cost-effective generation of RPE from multiple individuals such as drug testing, population studies or for therapies requiring patient-specific RPE derivations. In addition, conjunctival cells are identified as a practical source of somatic cells for deriving iPSCs from elderly individuals.

Gender Differences in Global but Not Targeted Demethylation in iPSC Reprogramming

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Inês Milagre

2017-01-01

Full Text Available Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.

Generation of an isogenic, gene-corrected iPSC line from a symptomatic 59-year-old female patient with frontotemporal dementia caused by an R406W mutation in the microtubule associated protein tau (MAPT) gene

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Nimsanor, Natakarn; Poulsen, Ulla; Rasmussen, Mikkel A.

2016-01-01

pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required cell type. Furthermore, gene-editing approaches allow generating isogenic gene-corrected controls that can be used as a very specific control. Here, we report the generation......Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the MAPT (microtubule-associated protein tau) gene can cause FTDP-17, but the underlying pathomechanisms of the disease are still unknown. Induced...... of genetically corrected iPSCs from a 59-year-old female FTD-17 patient carrying an R406W mutation in the MAPT-gene....

Generation of an isogenic, gene-corrected iPSC line from a symptomatic 57-year-old female patient with frontotemporal dementia caused by a P301L mutation in the microtubule associated protein tau (MAPT) gene

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Nimsanor, Natakarn; Kitiyanant, Narisorn; Poulsen, Ulla

2016-01-01

pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required cell type. Furthermore, gene-editing approaches allow generating isogenic gene-corrected controls that can be used as a very specific control. Here, we report the generation......Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the MAPT (microtubule-associated protein tau)-gene can cause FTDP-17, but the underlying pathomechanisms of the disease are still unknown. Induced...... of genetically corrected iPSCs from a 57-year-old female FTD-17 patient carrying an P301L mutation in the MAPT-gene....

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

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Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.

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Liu, Guang-Hui; Suzuki, Keiichiro; Li, Mo; Qu, Jing; Montserrat, Nuria; Tarantino, Carolina; Gu, Ying; Yi, Fei; Xu, Xiuling; Zhang, Weiqi; Ruiz, Sergio; Plongthongkum, Nongluk; Zhang, Kun; Masuda, Shigeo; Nivet, Emmanuel; Tsunekawa, Yuji; Soligalla, Rupa Devi; Goebl, April; Aizawa, Emi; Kim, Na Young; Kim, Jessica; Dubova, Ilir; Li, Ying; Ren, Ruotong; Benner, Chris; Del Sol, Antonio; Bueren, Juan; Trujillo, Juan Pablo; Surralles, Jordi; Cappelli, Enrico; Dufour, Carlo; Esteban, Concepcion Rodriguez; Belmonte, Juan Carlos Izpisua

2014-07-07

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.

Modeling Fanconi Anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs

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Montserrat, Nuria; Tarantino, Carolina; Gu, Ying; Yi, Fei; Xu, Xiuling; Zhang, Weiqi; Ruiz, Sergio; Plongthongkum, Nongluk; Zhang, Kun; Masuda, Shigeo; Nivet, Emmanuel; Tsunekawa, Yuji; Soligalla, Rupa Devi; Goebl, April; Aizawa, Emi; Kim, Na Young; Kim, Jessica; Dubova, Ilir; Li, Ying; Ren, Ruotong; Benner, Chris; del Sol, Antonio; Bueren, Juan; Trujillo, Juan Pablo; Surralles, Jordi; Cappelli, Enrico; Dufour, Carlo; Esteban, Concepcion Rodriguez; Belmonte, Juan Carlos Izpisua

2014-01-01

Fanconi Anemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration-free induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA-patient bone marrow cells. PMID:24999918

Development of one control and one tumor-specific induced pluripotent stem cell line from laryngeal carcinoma patient

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Yamin Zhang

2017-12-01

Full Text Available Skin fibroblasts and tumor fibroblasts were extracted from a 64-year old male patient clinically diagnosed with laryngeal carcinoma. Control and tumor specific induced pluripotent stem cells were reprogrammed with 5 reprogramming factors, Klf-4, c-Myc, Oct-4, Sox-2, and Lin-28, using the messenger RNA reprogramming system. The transgene-free iPSC lines showed pluripotency, confirmed by immunofluorescence staining. The iPSC lines also showed normal karyotype, and could form embryoid bodies in vitro and differentiate into the 3 germ layers in vivo. This in vitro cellular model can be used to study the oncogenesis and pathogenesis of laryngeal carcinoma.

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

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Sheeja Rajasingh

Full Text Available Human induced pluripotent stem cells (iPSCs derived cardiomyocytes (iCMCs would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF and human umbilical vein endothelial cells (HUVECs were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

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Rajasingh, Sheeja; Thangavel, Jayakumar; Czirok, Andras; Samanta, Saheli; Roby, Katherine F; Dawn, Buddhadeb; Rajasingh, Johnson

2015-01-01

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells.

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Zhou, Miaojin; Hu, Zhiqing; Qiu, Liyan; Zhou, Tao; Feng, Mai; Hu, Qian; Zeng, Baitao; Li, Zhuo; Sun, Qianru; Wu, Yong; Liu, Xionghao; Wu, Lingqian; Liang, Desheng

2018-05-09

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model.

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Murai, Kiyohito; Sun, Guoqiang; Ye, Peng; Tian, E; Yang, Su; Cui, Qi; Sun, Guihua; Trinh, Daniel; Sun, Olivia; Hong, Teresa; Wen, Zhexing; Kalkum, Markus; Riggs, Arthur D; Song, Hongjun; Ming, Guo-li; Shi, Yanhong

2016-03-11

Dysregulated expression of miR-219, a brain-specific microRNA, has been observed in neurodevelopmental disorders, such as schizophrenia (SCZ). However, its role in normal mammalian neural stem cells (NSCs) and in SCZ pathogenesis remains unknown. We show here that the nuclear receptor TLX, an essential regulator of NSC proliferation and self-renewal, inhibits miR-219 processing. miR-219 suppresses mouse NSC proliferation downstream of TLX. Moreover, we demonstrate upregulation of miR-219 and downregulation of TLX expression in NSCs derived from SCZ patient iPSCs and DISC1-mutant isogenic iPSCs. SCZ NSCs exhibit reduced cell proliferation. Overexpression of TLX or inhibition of miR-219 action rescues the proliferative defect in SCZ NSCs. Therefore, this study uncovers an important role for TLX and miR-219 in both normal neurodevelopment and in SCZ patient iPSC-derived NSCs. Moreover, this study reveals an unexpected role for TLX in regulating microRNA processing, independent of its well-characterized role in transcriptional regulation.

The State of Play with iPSCs and Spinal Cord Injury Models

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Stuart I. Hodgetts

2015-01-01

Full Text Available The application of induced pluripotent stem cell (iPSC technologies in cell based strategies, for the repair of the central nervous system (with particular focus on the spinal cord, is moving towards the potential use of clinical grade donor cells. The ability of iPSCs to generate donor neuronal, glial and astrocytic phenotypes for transplantation is highlighted here, and we review recent research using iPSCs in attempts to treat spinal cord injury in various animal models. Also discussed are issues relating to the production of clinical grade iPSCs, recent advances in transdifferentiation protocols for iPSC-derived donor cell populations, concerns about tumourogenicity, and whether iPSC technologies offer any advantages over previous donor cell candidates or tissues already in use as therapeutic tools in experimental spinal cord injury studies.

Nanomedicine-Based Neuroprotective Strategies in Patient Specific-iPSC and Personalized Medicine

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Shih-Fan Jang

2014-03-01

Full Text Available In recent decades, nanotechnology has attracted major interests in view of drug delivery systems and therapies against diseases, such as cancer, neurodegenerative diseases, and many others. Nanotechnology provides the opportunity for nanoscale particles or molecules (so called “Nanomedicine” to be delivered to the targeted sites, thereby, reducing toxicity (or side effects and improving drug bioavailability. Nowadays, a great deal of nano-structured particles/vehicles has been discovered, including polymeric nanoparticles, lipid-based nanoparticles, and mesoporous silica nanoparticles. Nanomedical utilizations have already been well developed in many different aspects, including disease treatment, diagnostic, medical devices designing, and visualization (i.e., cell trafficking. However, while quite a few successful progressions on chemotherapy using nanotechnology have been developed, the implementations of nanoparticles on stem cell research are still sparsely populated. Stem cell applications and therapies are being considered to offer an outstanding potential in the treatment for numbers of maladies. Human induced pluripotent stem cells (iPSCs are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state. Although the exact mechanisms underlying are still unclear, iPSCs are already being considered as useful tools for drug development/screening and modeling of diseases. Recently, personalized medicines have drawn great attentions in biological and pharmaceutical studies. Generally speaking, personalized medicine is a therapeutic model that offers a customized healthcare/cure being tailored to a specific patient based on his own genetic information. Consequently, the combination of nanomedicine and iPSCs could actually be the potent arms for remedies in transplantation medicine and personalized medicine. This review will focus on current use of nanoparticles on therapeutical applications, nanomedicine

Sirt6 Promotes DNA End Joining in iPSCs Derived from Old Mice

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Wen Chen

2017-03-01

Full Text Available Induced pluripotent stem cells (iPSCs have great potential for treating age-related diseases, but the genome integrity of iPSCs is critically important. Here, we demonstrate that non-homologous end joining (NHEJ, rather than homologous recombination (HR, is less efficient in iPSCs from old mice than young mice. We further find that Sirt6 is downregulated in iPSCs from old mice. Sirt6 directly binds to Ku80 and facilitates the Ku80/DNA-PKcs interaction, thus promoting DNA-PKcs phosphorylation at residue S2056, leading to efficient NHEJ. Rescue experiments show that introducing a combination of Sirt6 and the Yamanaka factors during reprogramming significantly promotes DNA double-strand break (DSB repair by activating NHEJ in iPSCs derived from old mice. Thus, our study suggests a strategy to improve the quality of iPSCs derived from old donors by activating NHEJ and stabilizing the genome.

Generation of a human iPSC line from a patient with congenital glaucoma caused by mutation in CYP1B1 gene

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Arantxa Bolinches-Amorós

2018-04-01

Full Text Available The human iPSC cell line, GLC-FiPS4F1 (ESi047-A, derived from dermal fibroblast from the patient with congenital glaucoma caused by the mutation of the gene CYP1B1, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors.

A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells

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Seonmi Park

2017-04-01

Full Text Available Summary: Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs from patients of different ethnicities, β-globin gene (HBB haplotypes, and fetal hemoglobin (HbF levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS mutation. : In this resource article, Mostoslavsky, Murphy, and colleagues of the NextGen consortium describe a diverse, comprehensive, and characterized library of sickle cell disease-specific induced pluripotent stem cells (iPSCs from patients of different ethnicities, β-globin gene (HBB haplotypes and fetal hemoglobin (HbF levels. This bank is readily available and accessible to all investigators. Keywords: induced pluripotent stem cells, iPSCs, sickle cell disease, disease modeling, directed differentiation, gene correction

Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

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Simon E. Vidal

2014-10-01

Full Text Available The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs. To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

Modeling neurodegenerative diseases with patient-derived induced pluripotent cells

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Poon, Anna; Zhang, Yu; Chandrasekaran, Abinaya

2017-01-01

patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide...... the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models....

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

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Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

2017-11-08

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

Translation of Human iPSCs: From Clinical Trial in a Dish to Precision Medicine

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Sayed, Nazish; Liu, Chun; Wu, Joseph C.

2016-01-01

The prospect of changing the plasticity of terminally differentiated cells toward pluripotency has completely altered the outlook of biomedical research. Human induced pluripotent stem cells (iPSCs) provide a new source of therapeutic cells free from the ethical issues or immune barriers of human embryonic stem cells. iPSCs also confer considerable advantages over conventional methods of studying human diseases. Since its advent, iPSC technology has expanded, with 3 major applications: disease modeling; regenerative therapy; and drug discovery. Here we discuss, in a comprehensive manner, the recent advances in iPSC technology in relation to basic, clinical, and population health. PMID:27151349

The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

DEFF Research Database (Denmark)

Brix, Jacob; Zhou, Yan; Luo, Yonglun

2015-01-01

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation cap...

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a KCNA5 p.D322H mutation

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Mora, Cristina; Serzanti, Marialaura; Giacomelli, Alessio

2017-01-01

. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a KCNA5 p.D322H mutation, using a commercially available non-integrating system. The generated iPSCs expressed pluripotency markers...... and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the p.D322H mutation....

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

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Mora, Cristina; Serzanti, Marialaura; Giacomelli, Alessio

2017-01-01

the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated...... toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation....

Applications of Patient-Specific Induced Pluripotent Stem Cells; Focused on Disease Modeling, Drug Screening and Therapeutic Potentials for Liver Disease

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Chun, Yong Soon; Chaudhari, Pooja; Jang, Yoon-Young

2010-01-01

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of...

Kidney-differentiated cells derived from Lowe Syndrome patient's iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex.

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Wen-Chieh Hsieh

Full Text Available Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.

Human iPSC for Therapeutic Approaches to the Nervous System: Present and Future Applications

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Maria Giuseppina Cefalo

2016-01-01

Full Text Available Many central nervous system (CNS diseases including stroke, spinal cord injury (SCI, and brain tumors are a significant cause of worldwide morbidity/mortality and yet do not have satisfying treatments. Cell-based therapy to restore lost function or to carry new therapeutic genes is a promising new therapeutic approach, particularly after human iPSCs became available. However, efficient generation of footprint-free and xeno-free human iPSC is a prerequisite for their clinical use. In this paper, we will first summarize the current methodology to obtain footprint- and xeno-free human iPSC. We will then review the current iPSC applications in therapeutic approaches for CNS regeneration and their use as vectors to carry proapoptotic genes for brain tumors and review their applications for modelling of neurological diseases and formulating new therapeutic approaches. Available results will be summarized and compared. Finally, we will discuss current limitations precluding iPSC from being used on large scale for clinical applications and provide an overview of future areas of improvement. In conclusion, significant progress has occurred in deriving iPSC suitable for clinical use in the field of neurological diseases. Current efforts to overcome technical challenges, including reducing labour and cost, will hopefully expedite the integration of this technology in the clinical setting.

Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

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Merkert, Sylvia; Martin, Ulrich

2016-06-24

The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

Generation of iPSC lines from primary human chorionic villi cells

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Björn Lichtner

2015-11-01

Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

Generation of induced pluripotent stem cells from a patient with X-linked juvenile retinoschisis

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Chi-Hsien Peng

2018-05-01

Full Text Available X-linked juvenile retinoschisis (XLRS is a hereditary retinal dystrophy manifested as splitting of anatomical layers of retina. In this report, we generated a patient-specific induced pluripotent stem cell (iPSC line, TVGH-iPSC-013-05, from the peripheral blood mononuclear cells of a male patient with XLRS by using the Sendai-virus delivery system. We believe that XLRS patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease.

Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease.

Science.gov (United States)

Chun, Yong Soon; Chaudhari, Pooja; Jang, Yoon-Young

2010-12-14

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.

Potential of Induced Pluripotent Stem Cells (iPSCs for Treating Age-Related Macular Degeneration (AMD

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Mark Fields

2016-12-01

Full Text Available The field of stem cell biology has rapidly evolved in the last few decades. In the area of regenerative medicine, clinical applications using stem cells hold the potential to be a powerful tool in the treatment of a wide variety of diseases, in particular, disorders of the eye. Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs are promising technologies that can potentially provide an unlimited source of cells for cell replacement therapy in the treatment of retinal degenerative disorders such as age-related macular degeneration (AMD, Stargardt disease, and other disorders. ESCs and iPSCs have been used to generate retinal pigment epithelium (RPE cells and their functional behavior has been tested in vitro and in vivo in animal models. Additionally, iPSC-derived RPE cells provide an autologous source of cells for therapeutic use, as well as allow for novel approaches in disease modeling and drug development platforms. Clinical trials are currently testing the safety and efficacy of these cells in patients with AMD. In this review, the current status of iPSC disease modeling of AMD is discussed, as well as the challenges and potential of this technology as a viable option for cell replacement therapy in retinal degeneration.

Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD).

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Fields, Mark; Cai, Hui; Gong, Jie; Del Priore, Lucian

2016-12-08

The field of stem cell biology has rapidly evolved in the last few decades. In the area of regenerative medicine, clinical applications using stem cells hold the potential to be a powerful tool in the treatment of a wide variety of diseases, in particular, disorders of the eye. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising technologies that can potentially provide an unlimited source of cells for cell replacement therapy in the treatment of retinal degenerative disorders such as age-related macular degeneration (AMD), Stargardt disease, and other disorders. ESCs and iPSCs have been used to generate retinal pigment epithelium (RPE) cells and their functional behavior has been tested in vitro and in vivo in animal models. Additionally, iPSC-derived RPE cells provide an autologous source of cells for therapeutic use, as well as allow for novel approaches in disease modeling and drug development platforms. Clinical trials are currently testing the safety and efficacy of these cells in patients with AMD. In this review, the current status of iPSC disease modeling of AMD is discussed, as well as the challenges and potential of this technology as a viable option for cell replacement therapy in retinal degeneration.

Generation of an induced pluripotent stem cell (iPSC line from a 40-year-old patient with the A8344G mutation of mitochondrial DNA and MERRF (myoclonic epilepsy with ragged red fibers syndrome

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Yu-Ting Wu

2018-03-01

Full Text Available Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC) verification and validation plan. version 1.

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Bartlett, Roscoe Ainsworth; Arguello, Jose Guadalupe, Jr.; Urbina, Angel; Bouchard, Julie F.; Edwards, Harold Carter; Freeze, Geoffrey A.; Knupp, Patrick Michael; Wang, Yifeng; Schultz, Peter Andrew; Howard, Robert (Oak Ridge National Laboratory, Oak Ridge, TN); McCornack, Marjorie Turner

2011-01-01

The objective of the U.S. Department of Energy Office of Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC) is to provide an integrated suite of computational modeling and simulation (M&S) capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive-waste storage facility or disposal repository. To meet this objective, NEAMS Waste IPSC M&S capabilities will be applied to challenging spatial domains, temporal domains, multiphysics couplings, and multiscale couplings. A strategic verification and validation (V&V) goal is to establish evidence-based metrics for the level of confidence in M&S codes and capabilities. Because it is economically impractical to apply the maximum V&V rigor to each and every M&S capability, M&S capabilities will be ranked for their impact on the performance assessments of various components of the repository systems. Those M&S capabilities with greater impact will require a greater level of confidence and a correspondingly greater investment in V&V. This report includes five major components: (1) a background summary of the NEAMS Waste IPSC to emphasize M&S challenges; (2) the conceptual foundation for verification, validation, and confidence assessment of NEAMS Waste IPSC M&S capabilities; (3) specifications for the planned verification, validation, and confidence-assessment practices; (4) specifications for the planned evidence information management system; and (5) a path forward for the incremental implementation of this V&V plan.

Establishment of induced pluripotent stem cell (iPSC line from a 75-year old patient with late onset Alzheimer's disease (LOAD

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Zsuzsanna Táncos

2016-07-01

Full Text Available Peripheral blood mononuclear cells (PBMCs were collected from a clinically characterised 75-year old woman with late onset Alzheimer's disease (LOAD. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

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Shamim H Rahman

2015-05-01

Full Text Available In vitro disease modeling based on induced pluripotent stem cells (iPSCs provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID, a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(DJ recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

Challenge problem and milestones for : Nuclear Energy Advanced Modeling and Simulation (NEAMS) waste Integrated Performance and Safety Codes (IPSC).

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Freeze, Geoffrey A.; Wang, Yifeng; Howard, Robert; McNeish, Jerry A.; Schultz, Peter Andrew; Arguello, Jose Guadalupe, Jr.

2010-09-01

This report describes the specification of a challenge problem and associated challenge milestones for the Waste Integrated Performance and Safety Codes (IPSC) supporting the U.S. Department of Energy (DOE) Office of Nuclear Energy Advanced Modeling and Simulation (NEAMS) Campaign. The NEAMS challenge problems are designed to demonstrate proof of concept and progress towards IPSC goals. The goal of the Waste IPSC is to develop an integrated suite of modeling and simulation capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive waste storage or disposal system. The Waste IPSC will provide this simulation capability (1) for a range of disposal concepts, waste form types, engineered repository designs, and geologic settings, (2) for a range of time scales and distances, (3) with appropriate consideration of the inherent uncertainties, and (4) in accordance with robust verification, validation, and software quality requirements. To demonstrate proof of concept and progress towards these goals and requirements, a Waste IPSC challenge problem is specified that includes coupled thermal-hydrologic-chemical-mechanical (THCM) processes that describe (1) the degradation of a borosilicate glass waste form and the corresponding mobilization of radionuclides (i.e., the processes that produce the radionuclide source term), (2) the associated near-field physical and chemical environment for waste emplacement within a salt formation, and (3) radionuclide transport in the near field (i.e., through the engineered components - waste form, waste package, and backfill - and the immediately adjacent salt). The initial details of a set of challenge milestones that collectively comprise the full challenge problem are also specified.

Challenge problem and milestones for: Nuclear Energy Advanced Modeling and Simulation (NEAMS) waste Integrated Performance and Safety Codes (IPSC)

International Nuclear Information System (INIS)

Freeze, Geoffrey A.; Wang, Yifeng; Howard, Robert; McNeish, Jerry A.; Schultz, Peter Andrew; Arguello, Jose Guadalupe Jr.

2010-01-01

This report describes the specification of a challenge problem and associated challenge milestones for the Waste Integrated Performance and Safety Codes (IPSC) supporting the U.S. Department of Energy (DOE) Office of Nuclear Energy Advanced Modeling and Simulation (NEAMS) Campaign. The NEAMS challenge problems are designed to demonstrate proof of concept and progress towards IPSC goals. The goal of the Waste IPSC is to develop an integrated suite of modeling and simulation capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive waste storage or disposal system. The Waste IPSC will provide this simulation capability (1) for a range of disposal concepts, waste form types, engineered repository designs, and geologic settings, (2) for a range of time scales and distances, (3) with appropriate consideration of the inherent uncertainties, and (4) in accordance with robust verification, validation, and software quality requirements. To demonstrate proof of concept and progress towards these goals and requirements, a Waste IPSC challenge problem is specified that includes coupled thermal-hydrologic-chemical-mechanical (THCM) processes that describe (1) the degradation of a borosilicate glass waste form and the corresponding mobilization of radionuclides (i.e., the processes that produce the radionuclide source term), (2) the associated near-field physical and chemical environment for waste emplacement within a salt formation, and (3) radionuclide transport in the near field (i.e., through the engineered components - waste form, waste package, and backfill - and the immediately adjacent salt). The initial details of a set of challenge milestones that collectively comprise the full challenge problem are also specified.

Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

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Juan Roberto Rodriguez-Madoz

Full Text Available The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272 improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

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Rodriguez-Madoz, Juan Roberto; San Jose-Eneriz, Edurne; Rabal, Obdulia; Zapata-Linares, Natalia; Miranda, Estibaliz; Rodriguez, Saray; Porciuncula, Angelo; Vilas-Zornoza, Amaia; Garate, Leire; Segura, Victor; Guruceaga, Elizabeth; Agirre, Xabier; Oyarzabal, Julen; Prosper, Felipe

2017-01-01

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis.

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Honda, Arata; Choijookhuu, Narantsog; Izu, Haruna; Kawano, Yoshihiro; Inokuchi, Mizuho; Honsho, Kimiko; Lee, Ah-Reum; Nabekura, Hiroki; Ohta, Hiroshi; Tsukiyama, Tomoyuki; Ohinata, Yasuhide; Kuroiwa, Asato; Hishikawa, Yoshitaka; Saitou, Mitinori; Jogahara, Takamichi; Koshimoto, Chihiro

2017-05-01

In mammals, the Y chromosome strictly influences the maintenance of male germ cells. Almost all mammalian species require genetic contributors to generate testes. An endangered species, Tokudaia osimensis , has a unique sex chromosome composition XO/XO, and genetic differences between males and females have not been confirmed. Although a distinctive sex-determining mechanism may exist in T. osimensis , it has been difficult to examine thoroughly in this rare animal species. To elucidate the discriminative sex-determining mechanism in T. osimensis and to find a strategy to prevent its possible extinction, we have established induced pluripotent stem cells (iPSCs) and derived interspecific chimeras using mice as the hosts and recipients. Generated iPSCs are considered to be in the so-called "true naïve" state, and T. osimensis iPSCs may contribute as interspecific chimeras to several different tissues and cells in live animals. Surprisingly, female T. osimensis iPSCs not only contributed to the female germ line in the interspecific mouse ovary but also differentiated into spermatocytes and spermatids that survived in the adult interspecific mouse testes. Thus, T. osimensis cells have high sexual plasticity through which female somatic cells can be converted to male germline cells. These findings suggest flexibility in T. osimensis cells, which can adapt their germ cell sex to the gonadal niche. The probable reduction of the extinction risk of an endangered species through the use of iPSCs is indicated by this study.

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters.

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Diana Ribeiro

Full Text Available It has been suggested that extracellular vesicles (EVs can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.

Modelling neurodegenerative diseases in vitro: Recent advances in 3D iPSC technologies

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Elodie J Siney

2018-03-01

Full Text Available The discovery of induced pluripotent stem cells (iPSC 12 years ago has fostered the development of innovative patient-derived in vitro models for better understanding of disease mechanisms. This is particularly relevant to neurodegenerative diseases, where availability of live human brain tissue for research is limited and post-mortem interval changes influence readouts from autopsy-derived human tissue. Hundreds of iPSC lines have now been prepared and banked, thanks to several large scale initiatives and cell banks. Patient- or engineered iPSC-derived neural models are now being used to recapitulate cellular and molecular aspects of a variety of neurodegenerative diseases, including early and pre-clinical disease stages. The broad relevance of these models derives from the availability of a variety of differentiation protocols to generate disease-specific cell types and the manipulation to either introduce or correct disease-relevant genetic modifications. Moreover, the use of chemical and physical three-dimensional (3D matrices improves control over the extracellular environment and cellular organization of the models. These iPSC-derived neural models can be utilised to identify target proteins and, importantly, provide high-throughput screening for drug discovery. Choosing Alzheimer’s disease (AD as an example, this review describes 3D iPSC-derived neural models and their advantages and limitations. There is now a requirement to fully characterise and validate these 3D iPSC-derived neural models as a viable research tool that is capable of complementing animal models of neurodegeneration and live human brain tissue. With further optimization of differentiation, maturation and aging protocols, as well as the 3D cellular organisation and extracellular matrix to recapitulate more closely, the molecular extracellular-environment of the human brain, 3D iPSC-derived models have the potential to deliver new knowledge, enable discovery of novel

A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells.

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Park, Seonmi; Gianotti-Sommer, Andreia; Molina-Estevez, Francisco Javier; Vanuytsel, Kim; Skvir, Nick; Leung, Amy; Rozelle, Sarah S; Shaikho, Elmutaz Mohammed; Weir, Isabelle; Jiang, Zhihua; Luo, Hong-Yuan; Chui, David H K; Figueiredo, Maria Stella; Alsultan, Abdulraham; Al-Ali, Amein; Sebastiani, Paola; Steinberg, Martin H; Mostoslavsky, Gustavo; Murphy, George J

2017-04-11

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, β-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

DEFF Research Database (Denmark)

Li, Tong; Pires, Carlota; Nielsen, Troels Tolstrup

2016-01-01

Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28...... and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may...

Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1

DEFF Research Database (Denmark)

Li, Tong; Pires, Carlota; Nielsen, Troels Tolstrup

2016-01-01

Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1), responsible for causing Alzheimer's disease (AD). Induced pluripotent stem cells (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, h......L-MYC, hLIN28 and shTP53 genes. M146I-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported M146I...

Nuclear Energy Advanced Modeling and Simulation (NEAMS) Waste Integrated Performance and Safety Codes (IPSC) : FY10 development and integration.

Energy Technology Data Exchange (ETDEWEB)

Criscenti, Louise Jacqueline; Sassani, David Carl; Arguello, Jose Guadalupe, Jr.; Dewers, Thomas A.; Bouchard, Julie F.; Edwards, Harold Carter; Freeze, Geoffrey A.; Wang, Yifeng; Schultz, Peter Andrew

2011-02-01

This report describes the progress in fiscal year 2010 in developing the Waste Integrated Performance and Safety Codes (IPSC) in support of the U.S. Department of Energy (DOE) Office of Nuclear Energy Advanced Modeling and Simulation (NEAMS) Campaign. The goal of the Waste IPSC is to develop an integrated suite of computational modeling and simulation capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive waste storage or disposal system. The Waste IPSC will provide this simulation capability (1) for a range of disposal concepts, waste form types, engineered repository designs, and geologic settings, (2) for a range of time scales and distances, (3) with appropriate consideration of the inherent uncertainties, and (4) in accordance with robust verification, validation, and software quality requirements. Waste IPSC activities in fiscal year 2010 focused on specifying a challenge problem to demonstrate proof of concept, developing a verification and validation plan, and performing an initial gap analyses to identify candidate codes and tools to support the development and integration of the Waste IPSC. The current Waste IPSC strategy is to acquire and integrate the necessary Waste IPSC capabilities wherever feasible, and develop only those capabilities that cannot be acquired or suitably integrated, verified, or validated. This year-end progress report documents the FY10 status of acquisition, development, and integration of thermal-hydrologic-chemical-mechanical (THCM) code capabilities, frameworks, and enabling tools and infrastructure.

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

Science.gov (United States)

Wong, Chui-Wei; Chen, You-Tzung; Chien, Chung-Liang; Yu, Tien-Yu; Rwei, Syang-Peng; Hsu, Shan-Hui

2018-01-01

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Global synchronization algorithms for the Intel iPSC/860

Science.gov (United States)

Seidel, Steven R.; Davis, Mark A.

1992-01-01

In a distributed memory multicomputer that has no global clock, global processor synchronization can only be achieved through software. Global synchronization algorithms are used in tridiagonal systems solvers, CFD codes, sequence comparison algorithms, and sorting algorithms. They are also useful for event simulation, debugging, and for solving mutual exclusion problems. For the Intel iPSC/860 in particular, global synchronization can be used to ensure the most effective use of the communication network for operations such as the shift, where each processor in a one-dimensional array or ring concurrently sends a message to its right (or left) neighbor. Three global synchronization algorithms are considered for the iPSC/860: the gysnc() primitive provided by Intel, the PICL primitive sync0(), and a new recursive doubling synchronization (RDS) algorithm. The performance of these algorithms is compared to the performance predicted by communication models of both the long and forced message protocols. Measurements of the cost of shift operations preceded by global synchronization show that the RDS algorithm always synchronizes the nodes more precisely and costs only slightly more than the other two algorithms.

Induced Pluripotent Stem Cell Models of Progranulin-Deficient Frontotemporal Dementia Uncover Specific Reversible Neuronal Defects

Science.gov (United States)

Almeida, Sandra; Zhang, Zhijun; Coppola, Giovanni; Mao, Wenjie; Futai, Kensuke; Karydas, Anna; Geschwind, Michael D.; Tartaglia, M. Carmela; Gao, Fuying; Gianni, Davide; Sena-Esteves, Miguel; Geschwind, Daniel H.; Miller, Bruce L.; Farese, Robert V.; Gao, Fen-Biao

2012-01-01

SUMMARY The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell (iPSC) lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel GRN mutation (PGRN S116X). In neurons and microglia differentiated from PGRN S116X iPSCs, the levels of intracellular and secreted progranulin were reduced, establishing patient-specific cellular models of progranulin haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the PI3K and MAPK pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by progranulin expression. Our findings identify cell-autonomous, reversible defects in patient neurons with progranulin deficiency and provide a new model for studying progranulin-dependent pathogenic mechanisms and testing potential therapies. PMID:23063362

Vesicular GABA Uptake Can Be Rate Limiting for Recovery of IPSCs from Synaptic Depression

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Manami Yamashita

2018-03-01

Full Text Available Summary: Synaptic efficacy plays crucial roles in neuronal circuit operation and synaptic plasticity. Presynaptic determinants of synaptic efficacy are neurotransmitter content in synaptic vesicles and the number of vesicles undergoing exocytosis at a time. Bursts of presynaptic firings depress synaptic efficacy, mainly due to depletion of releasable vesicles, whereas recovery from strong depression is initiated by endocytic vesicle retrieval followed by refilling of vesicles with neurotransmitter. We washed out presynaptic cytosolic GABA to induce a rundown of IPSCs at cerebellar inhibitory cell pairs in slices from rats and then allowed fast recovery by elevating GABA concentration using photo-uncaging. The time course of this recovery coincided with that of IPSCs from activity-dependent depression induced by a train of high-frequency stimulation. We conclude that vesicular GABA uptake can be a limiting step for the recovery of inhibitory neurotransmission from synaptic depression. : Recovery of inhibitory synaptic transmission from activity-dependent depression requires refilling of vesicles with GABA. Yamashita et al. find that vesicular uptake rate of GABA is a slow process, limiting the recovery rate of IPSCs from depression.

Aged induced pluripotent stem cell (iPSCs) as a new cellular model for studying premature aging.

Science.gov (United States)

Petrini, Stefania; Borghi, Rossella; D'Oria, Valentina; Restaldi, Fabrizia; Moreno, Sandra; Novelli, Antonio; Bertini, Enrico; Compagnucci, Claudia

2017-05-31

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

Communication overhead on the Intel iPSC-860 hypercube

Science.gov (United States)

Bokhari, Shahid H.

1990-01-01

Experiments were conducted on the Intel iPSC-860 hypercube in order to evaluate the overhead of interprocessor communication. It is demonstrated that: (1) contrary to popular belief, the distance between two communicating processors has a significant impact on communication time, (2) edge contention can increase communication time by a factor of more than 7, and (3) node contention has no measurable impact.

Efficient differentiation of steroidogenic and germ-like cells from epigenetically-related iPSCs derived from ovarian granulosa cells.

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Raymond Anchan

Full Text Available To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs, we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs was verified by demonstrating embryonic stem cell (ESC antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2 than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4 and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1 more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.

Optogenetic release of ACh induces rhythmic bursts of perisomatic IPSCs in hippocampus.

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Daniel A Nagode

Full Text Available Acetylcholine (ACh influences a vast array of phenomena in cortical systems. It alters many ionic conductances and neuronal firing behavior, often by regulating membrane potential oscillations in populations of cells. Synaptic inhibition has crucial roles in many forms of oscillation, and cholinergic mechanisms regulate both oscillations and synaptic inhibition. In vitro investigations using bath-application of cholinergic receptor agonists, or bulk tissue electrical stimulation to release endogenous ACh, have led to insights into cholinergic function, but questions remain because of the relative lack of selectivity of these forms of stimulation. To investigate the effects of selective release of ACh on interneurons and oscillations, we used an optogenetic approach in which the light-sensitive non-selective cation channel, Channelrhodopsin2 (ChR2, was virally delivered to cholinergic projection neurons in the medial septum/diagonal band of Broca (MS/DBB of adult mice expressing Cre-recombinase under the control of the choline-acetyltransferase (ChAT promoter. Acute hippocampal slices obtained from these animals weeks later revealed ChR2 expression in cholinergic axons. Brief trains of blue light pulses delivered to untreated slices initiated bursts of ACh-evoked, inhibitory post-synaptic currents (L-IPSCs in CA1 pyramidal cells that lasted for 10's of seconds after the light stimulation ceased. L-IPSC occurred more reliably in slices treated with eserine and a very low concentration of 4-AP, which were therefore used in most experiments. The rhythmic, L-IPSCs were driven primarily by muscarinic ACh receptors (mAChRs, and could be suppressed by endocannabinoid release from pyramidal cells. Finally, low-frequency oscillations (LFOs of local field potentials (LFPs were significantly cross-correlated with the L-IPSCs, and reversal of the LFPs near s. pyramidale confirmed that the LFPs were driven by perisomatic inhibition. This optogenetic approach

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

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Wei Zhang

2018-02-01

Full Text Available Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs, are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX. These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model.

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Saito, Akiko; Ooki, Akio; Nakamura, Takashi; Onodera, Shoko; Hayashi, Kamichika; Hasegawa, Daigo; Okudaira, Takahito; Watanabe, Katsuhito; Kato, Hiroshi; Onda, Takeshi; Watanabe, Akira; Kosaki, Kenjiro; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Sakamoto, Teruo; Yamaguchi, Akira; Sueishi, Kenji; Azuma, Toshifumi

2018-01-22

Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models. Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis. Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with

Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.

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Liu, Guang-Hui; Barkho, Basam Z; Ruiz, Sergio; Diep, Dinh; Qu, Jing; Yang, Sheng-Lian; Panopoulos, Athanasia D; Suzuki, Keiichiro; Kurian, Leo; Walsh, Christopher; Thompson, James; Boue, Stephanie; Fung, Ho Lim; Sancho-Martinez, Ignacio; Zhang, Kun; Yates, John; Izpisua Belmonte, Juan Carlos

2011-04-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients

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Wen-Li Deng

2018-04-01

Full Text Available Summary: Retinitis pigmentosa (RP is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence. : Jin and colleagues demonstrate that patient-specific iPSC-derived 3D retinae can recapitulate disease progress of retinitis pigmentosa through presenting defects in photoreceptor morphology, gene profile, and electrophysiology, as well as the defective ciliogenesis in iPSCs, iPSC-RPE, and 3D retinae. CRISPR/Cas9-mediated gene correction can rescue not only photoreceptor structure and electrophysiological property but also observed ciliopathy. Keywords: RPGR, photoreceptor, electrophysiology, retinitis pigmentosa, patient-derived iPSCs, retinal organoid, RPE cells, cilium, ciliopathy, disease modeling

Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

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Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

2018-01-01

Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types

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Athanasia D. Panopoulos

2017-04-01

Full Text Available Summary: Large-scale collections of induced pluripotent stem cells (iPSCs could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines. : Working as part of the NHLBI NextGen consortium, Panopoulos and colleagues report the derivation and characterization of 222 publicly available iPSCs from ethnically diverse individuals with corresponding genomic data including SNP arrays, RNA-seq, and whole-genome sequencing. This collection provides a powerful resource to investigate the function of genetic variants. Keywords: iPSCORE, iPSC, GWAS, molecular traits, physiological traits, cardiac disease, NHLBI Next Gen, LQT2, KCNH2, iPSC-derived cardiomyocytes

Origin-Dependent Neural Cell Identities in Differentiated Human iPSCs In Vitro and after Transplantation into the Mouse Brain

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Gunnar Hargus

2014-09-01

Full Text Available The differentiation capability of induced pluripotent stem cells (iPSCs toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs, dermal fibroblasts, or cord blood CD34+ hematopoietic progenitor cells. Neural progenitor cells (NPCs and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

Zfp296 is a novel, pluripotent-specific reprogramming factor.

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Gerrit Fischedick

Full Text Available Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs. However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs, indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296 led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4, and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells.

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He, Qiong; Wang, Hui-Hui; Cheng, Tao; Yuan, Wei-Ping; Ma, Yu-Po; Jiang, Yong-Ping; Ren, Zhi-Hua

2017-09-27

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6 th coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells.

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Tang, Zi-Hua; Chen, Jia-Rong; Zheng, Jing; Shi, Hao-Song; Ding, Jie; Qian, Xiao-Dan; Zhang, Cui; Chen, Jian-Ling; Wang, Cui-Cui; Li, Liang; Chen, Jun-Zhen; Yin, Shan-Kai; Huang, Tao-Sheng; Chen, Ping; Guan, Min-Xin; Wang, Jin-Fu

2016-05-01

The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A(WT/WT); C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs. ©AlphaMed Press.

NEAMS Nuclear Waste Management IPSC: evaluation and selection of tools for the quality environment

International Nuclear Information System (INIS)

Bouchard, Julie F.; Stubblefield, William Anthony; Vigil, Dena M.; Edwards, Harold Carter

2011-01-01

The objective of the U.S. Department of Energy Office of Nuclear Energy Advanced Modeling and Simulation Nuclear Waste Management Integrated Performance and Safety Codes (NEAMS Nuclear Waste Management IPSC) is to provide an integrated suite of computational modeling and simulation (M and S) capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive-waste storage facility or disposal repository. These M and S capabilities are to be managed, verified, and validated within the NEAMS Nuclear Waste Management IPSC quality environment. M and S capabilities and the supporting analysis workflow and simulation data management tools will be distributed to end-users from this same quality environment. The same analysis workflow and simulation data management tools that are to be distributed to end-users will be used for verification and validation (V and V) activities within the quality environment. This strategic decision reduces the number of tools to be supported, and increases the quality of tools distributed to end users due to rigorous use by V and V activities. This report documents an evaluation of the needs, options, and tools selected for the NEAMS Nuclear Waste Management IPSC quality environment. The objective of the U.S. Department of Energy (DOE) Office of Nuclear Energy Advanced Modeling and Simulation Nuclear Waste Management Integrated Performance and Safety Codes (NEAMS Nuclear Waste Management IPSC) program element is to provide an integrated suite of computational modeling and simulation (M and S) capabilities to assess quantitatively the long-term performance of waste forms in the engineered and geologic environments of a radioactive-waste storage facility or disposal repository. This objective will be fulfilled by acquiring and developing M and S capabilities, and establishing a defensible level of confidence in these M and S capabilities. The foundation for assessing the

Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.

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Phondeechareon, Tanapol; Wattanapanitch, Methichit; U-Pratya, Yaowalak; Damkham, Chanapa; Klincumhom, Nuttha; Lorthongpanich, Chanchao; Kheolamai, Pakpoom; Laowtammathron, Chuti; Issaragrisil, Surapol

2016-10-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients.

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

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Hallam, Dean; Collin, Joseph; Bojic, Sanja; Chichagova, Valeria; Buskin, Adriana; Xu, Yaobo; Lafage, Lucia; Otten, Elsje G; Anyfantis, George; Mellough, Carla; Przyborski, Stefan; Alharthi, Sameer; Korolchuk, Viktor; Lotery, Andrew; Saretzki, Gabriele; McKibbin, Martin; Armstrong, Lyle; Steel, David; Kavanagh, David; Lako, Majlinda

2017-11-01

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Generation of induced pluripotent stem cells (iPSCs from a Bernard–Soulier syndrome patient carrying a W71R mutation in the GPIX gene

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Lourdes Lopez-Onieva

2016-05-01

Full Text Available We generated an induced pluripotent stem cell (iPSC line from a Bernard–Soulier Syndrome (BSS patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4. Peripheral blood mononuclear cells (PBMCs were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Generation of iPSC from cardiac and tail-tip fibroblasts derived from a second heart field reporter mouse.

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Linares, Javier; Arellano-Viera, Estibaliz; Iglesias-García, Olalla; Ferreira, Carmen; Iglesias, Elena; Abizanda, Gloria; Prósper, Felipe; Carvajal-Vergara, Xonia

2016-05-01

Mef2c Anterior Heart Field (AHF) enhancer is activated during embryonic heart development and it is expressed in multipotent cardiovascular progenitors (CVP) giving rise to endothelial and myocardial components of the outflow tract, right ventricle and ventricular septum. Here we have generated iPSC from transgenic Mef2c-AHF-Cre x Ai6(RCLZsGreen) mice. These iPSC will provide a novel tool to investigate the AHF-CVP and their cell progeny. Copyright © 2016 Roslin Cells Ltd. Published by Elsevier B.V. All rights reserved.

Recent technological updates and clinical applications of induced pluripotent stem cells.

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Diecke, Sebastian; Jung, Seung Min; Lee, Jaecheol; Ju, Ji Hyeon

2014-09-01

Induced pluripotent stem cells (iPSCs) were first described in 2006 and have since emerged as a promising cell source for clinical applications. The rapid progression in iPSC technology is still ongoing and directed toward increasing the efficacy of iPSC production and reducing the immunogenic and tumorigenic potential of these cells. Enormous efforts have been made to apply iPSC-based technology in the clinic, for drug screening approaches and cell replacement therapy. Moreover, disease modeling using patient-specific iPSCs continues to expand our knowledge regarding the pathophysiology and prospective treatment of rare disorders. Furthermore, autologous stem cell therapy with patient-specific iPSCs shows great propensity for the minimization of immune reactions and the provision of a limitless supply of cells for transplantation. In this review, we discuss the recent updates in iPSC technology and the use of iPSCs in disease modeling and regenerative medicine.

Striking similarity in the gene expression levels of individual Myc module members among ESCs, EpiSCs, and partial iPSCs.

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Masataka Hirasaki

Full Text Available Predominant transcriptional subnetworks called Core, Myc, and PRC modules have been shown to participate in preservation of the pluripotency and self-renewality of embryonic stem cells (ESCs. Epiblast stem cells (EpiSCs are another cell type that possesses pluripotency and self-renewality. However, the roles of these modules in EpiSCs have not been systematically examined to date. Here, we compared the average expression levels of Core, Myc, and PRC module genes between ESCs and EpiSCs. EpiSCs showed substantially higher and lower expression levels of PRC and Core module genes, respectively, compared with those in ESCs, while Myc module members showed almost equivalent levels of average gene expression. Subsequent analyses revealed that the similarity in gene expression levels of the Myc module between these two cell types was not just overall, but striking similarities were evident even when comparing the expression of individual genes. We also observed equivalent levels of similarity in the expression of individual Myc module genes between induced pluripotent stem cells (iPSCs and partial iPSCs that are an unwanted byproduct generated during iPSC induction. Moreover, our data demonstrate that partial iPSCs depend on a high level of c-Myc expression for their self-renewal properties.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

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Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Generation of an induced pluripotent stem cell (iPSC line from a patient with maturity-onset diabetes of the young type 3 (MODY3 carrying a hepatocyte nuclear factor 1-alpha (HNF1A mutation

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Frank Griscelli

2018-05-01

Full Text Available Heterozygous non-synonymous (p.S142F mutation in HNF1A leads to maturity-onset diabetes of the young (MODY type 3, which is a subtype of dominant inherited young-onset non-autoimmune diabetes due to the defect of insulin secretion from pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs from a patient with HNF1A p.S142F mutation. Cells from this patient, which were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the HNF1A p.S142F mutation, expressed pluripotency hallmarks.

Lymphoblast-derived integration-free iPSC line AD-TREM2-1 from a 67 year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant

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Soraia Martins

2018-05-01

Full Text Available Human lymphoblast cells from a male diagnosed with Alzheimer's disease (AD expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem cells (iPSCs by over-expressing episomal-based plasmids harbouring OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. AD-TREM2–1 was defined as pluripotent based on (i expression of pluripotency-associated markers (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.947.

Generation of an isogenic, gene-corrected iPSC line from a pre-symptomatic 28-year-old woman with an R406W mutation in the microtubule associated protein tau (MAPT) gene

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Nimsanor, Natakarn; Poulsen, Ulla; Rasmussen, Mikkel A.

2016-01-01

pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required cell type. Furthermore, gene-editing approaches allow generating isogenic gene-corrected controls that can be used as a very specific control. Here, we report the generation......Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the MAPT (microtubule-associated protein tau) gene can cause FTDP-17, but the underlying pathomechanisms of the disease are still unknown. Induced...... of genetically corrected iPSCs from a pre-symptomatic carrier of the R406W mutation in the MAPT-gene....

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

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Turan, Soeren; Farruggio, Alfonso P; Srifa, Waracharee; Day, John W; Calos, Michele P

2016-04-01

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Induced pluripotent stem cells (iPSCs) derived from a patient with frontotemporal dementia caused by a R406W mutation in microtubule-associated protein tau (MAPT)

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Rasmussen, Mikkel A.; Hjermind, Lena E.; Hasholt, Lis F.

2016-01-01

Skin fibroblasts were obtained from a 59-year-old woman diagnosed with frontotemporal dementia. The disease is caused by a R406W mutation in microtubule-associated protein tau (MAPT). Induced pluripotent stem cells (iPSCs) were established by electroporation with episomal plasmids containing hOCT4...

Modeling neurodegenerative diseases with patient-derived induced pluripotent cells: Possibilities and challenges.

Science.gov (United States)

Poon, Anna; Zhang, Yu; Chandrasekaran, Abinaya; Phanthong, Phetcharat; Schmid, Benjamin; Nielsen, Troels T; Freude, Kristine K

2017-10-25

The rising prevalence of progressive neurodegenerative diseases coupled with increasing longevity poses an economic burden at individual and societal levels. There is currently no effective cure for the majority of neurodegenerative diseases and disease-affected tissues from patients have been difficult to obtain for research and drug discovery in pre-clinical settings. While the use of animal models has contributed invaluable mechanistic insights and potential therapeutic targets, the translational value of animal models could be further enhanced when combined with in vitro models derived from patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide the opportunity to model disease development, uncover novel mechanisms and test potential therapeutics. Here we review findings from iPSC-based modeling of selected neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and spinocerebellar ataxia. Furthermore, we discuss the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

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Pittermann, Erik; Lachmann, Nico; MacLean, Glenn; Emmrich, Stephan; Ackermann, Mania; Göhring, Gudrun; Schlegelberger, Brigitte; Welte, Karl; Schambach, Axel; Heckl, Dirk; Orkin, Stuart H; Cantz, Tobias; Klusmann, Jan-Henning

2017-06-13

Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 ( HAX1 ) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1 W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1 -centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1 W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

A Comparative View on Human Somatic Cell Sources for iPSC Generation

Directory of Open Access Journals (Sweden)

Stefanie Raab

2014-01-01

Full Text Available The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs. Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.

Nuclear Energy Advanced Modeling and Simulation (NEAMS) waste Integrated Performance and Safety Codes (IPSC): gap analysis for high fidelity and performance assessment code development

International Nuclear Information System (INIS)

Lee, Joon H.; Siegel, Malcolm Dean; Arguello, Jose Guadalupe Jr.; Webb, Stephen Walter; Dewers, Thomas A.; Mariner, Paul E.; Edwards, Harold Carter; Fuller, Timothy J.; Freeze, Geoffrey A.; Jove-Colon, Carlos F.; Wang, Yifeng

2011-01-01

needed for repository modeling are severely lacking. In addition, most of existing reactive transport codes were developed for non-radioactive contaminants, and they need to be adapted to account for radionuclide decay and in-growth. The accessibility to the source codes is generally limited. Because the problems of interest for the Waste IPSC are likely to result in relatively large computational models, a compact memory-usage footprint and a fast/robust solution procedure will be needed. A robust massively parallel processing (MPP) capability will also be required to provide reasonable turnaround times on the analyses that will be performed with the code. A performance assessment (PA) calculation for a waste disposal system generally requires a large number (hundreds to thousands) of model simulations to quantify the effect of model parameter uncertainties on the predicted repository performance. A set of codes for a PA calculation must be sufficiently robust and fast in terms of code execution. A PA system as a whole must be able to provide multiple alternative models for a specific set of physical/chemical processes, so that the users can choose various levels of modeling complexity based on their modeling needs. This requires PA codes, preferably, to be highly modularized. Most of the existing codes have difficulties meeting these requirements. Based on the gap analysis results, we have made the following recommendations for the code selection and code development for the NEAMS waste IPSC: (1) build fully coupled high-fidelity THCMBR codes using the existing SIERRA codes (e.g., ARIA and ADAGIO) and platform, (2) use DAKOTA to build an enhanced performance assessment system (EPAS), and build a modular code architecture and key code modules for performance assessments. The key chemical calculation modules will be built by expanding the existing CANTERA capabilities as well as by extracting useful components from other existing codes.

Nuclear Energy Advanced Modeling and Simulation (NEAMS) waste Integrated Performance and Safety Codes (IPSC) : gap analysis for high fidelity and performance assessment code development.

Energy Technology Data Exchange (ETDEWEB)

Lee, Joon H.; Siegel, Malcolm Dean; Arguello, Jose Guadalupe, Jr.; Webb, Stephen Walter; Dewers, Thomas A.; Mariner, Paul E.; Edwards, Harold Carter; Fuller, Timothy J.; Freeze, Geoffrey A.; Jove-Colon, Carlos F.; Wang, Yifeng

2011-03-01

needed for repository modeling are severely lacking. In addition, most of existing reactive transport codes were developed for non-radioactive contaminants, and they need to be adapted to account for radionuclide decay and in-growth. The accessibility to the source codes is generally limited. Because the problems of interest for the Waste IPSC are likely to result in relatively large computational models, a compact memory-usage footprint and a fast/robust solution procedure will be needed. A robust massively parallel processing (MPP) capability will also be required to provide reasonable turnaround times on the analyses that will be performed with the code. A performance assessment (PA) calculation for a waste disposal system generally requires a large number (hundreds to thousands) of model simulations to quantify the effect of model parameter uncertainties on the predicted repository performance. A set of codes for a PA calculation must be sufficiently robust and fast in terms of code execution. A PA system as a whole must be able to provide multiple alternative models for a specific set of physical/chemical processes, so that the users can choose various levels of modeling complexity based on their modeling needs. This requires PA codes, preferably, to be highly modularized. Most of the existing codes have difficulties meeting these requirements. Based on the gap analysis results, we have made the following recommendations for the code selection and code development for the NEAMS waste IPSC: (1) build fully coupled high-fidelity THCMBR codes using the existing SIERRA codes (e.g., ARIA and ADAGIO) and platform, (2) use DAKOTA to build an enhanced performance assessment system (EPAS), and build a modular code architecture and key code modules for performance assessments. The key chemical calculation modules will be built by expanding the existing CANTERA capabilities as well as by extracting useful components from other existing codes.

Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system.

Science.gov (United States)

Lyu, Cuicui; Shen, Jun; Wang, Rui; Gu, Haihui; Zhang, Jianping; Xue, Feng; Liu, Xiaofan; Liu, Wei; Fu, Rongfeng; Zhang, Liyan; Li, Huiyuan; Zhang, Xiaobing; Cheng, Tao; Yang, Renchi; Zhang, Lei

2018-04-06

Replacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 (CRISPR-Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia. A patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors. The AAVS1-Cas9-sgRNA plasmid which targets the AAVS1 locus and the AAVS1-EF1α-F9 cDNA-puromycin donor plasmid were constructed, and they were electroporated into the iPSCs. When insertion of F9 cDNA into the AAVS1 locus was confirmed, whole genome sequencing (WGS) was carried out to detect the off-target issue. The iPSCs were then differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were measured in the culture supernatant. Finally, the hepatocytes were transplanted into non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through splenic injection. The patient's iPSCs were generated from PBMNCs. Human full-length F9 cDNA was inserted into the AAVS1 locus of iPSCs of a hemophilia B patient using the CRISPR-Cas9 system. No off-target mutations were detected by WGS. The hepatocytes differentiated from the inserted iPSCs could secrete hFIX stably and had the ability to be transplanted into the NOD/SCID mice in the short term. PBMNCs are good somatic cell choices for generating iPSCs from hemophilia patients. The iPSC technique is a good tool for genetic therapy for human hereditary diseases. CRISPR-Cas9 is versatile, convenient, and safe to be used in iPSCs with low off-target effects. Our research offers new approaches for clinical gene therapy for hemophilia.

Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

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Dorothea Maetzel

2014-06-01

Full Text Available Niemann-Pick type C (NPC disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs using transcription activator-like effector nucleases (TALENs. We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

Monte Carlo method implementation on IPSC 860 for the resolution of the Boltzmann equation

International Nuclear Information System (INIS)

AloUGES, Francois

1993-01-01

This note deals with the implementation on a massively parallel machine (IPSC-860) of a Monte-Carlo method aiming at resolving the Boltzmann equation. The parallelism of the machine incites to consider a multi-domain approach and poses the problem of the automatic generation of local meshes from a non-structured 3-D global mesh [fr

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM).

Science.gov (United States)

Xiong, Kai; Zhou, Yan; Hyttel, Poul; Bolund, Lars; Freude, Kristine Karla; Luo, Yonglun

2016-11-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

OpenAIRE

Ebert, A.; Shelley, B.; Hurley, A.; Onorati, M.; Castiglioni, V.; Patitucci, T.; Svendsen, S.; Mattis, V.; Mcgivern, J.; Schwab, A.; Sareen, D.; Kim, H.; Cattaneo, E.; Svendsen, C.

2013-01-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be...

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

Science.gov (United States)

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H; Mills, Jason A; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M; Podsakoff, Gregory M; Gadue, Paul; French, Deborah L; Mason, Philip J; Bessler, Monica; Weiss, Mitchell J

2013-08-08

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Generation of six multiple sclerosis patient-derived induced pluripotent stem cell lines.

Science.gov (United States)

Miquel-Serra, L; Duarri, A; Muñoz, Y; Kuebler, B; Aran, B; Costa, C; Martí, M; Comabella, M; Malhotra, S; Montalban, X; Veiga, A; Raya, A

2017-10-01

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells.

Science.gov (United States)

Zhu, Yanxia; Liang, Yuhong; Zhu, Hongxia; Lian, Cuihong; Wang, Liang; Wang, Yiwei; Gu, Hongsheng; Zhou, Guangqian; Yu, Xiaoping

2017-06-27

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells

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Peggy Matz

2015-11-01

Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell line — H1 have a Pearson correlation of 0.899.

Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors.

Science.gov (United States)

Hwang, Yongsung; Broxmeyer, Hal E; Lee, Man Ryul

2017-07-01

Hematopoietic cell transplantation (HCT) is a successful treatment modality for patients with malignant and nonmalignant disorders, usually when no other treatment option is available. The cells supporting long-term reconstitution after HCT are the hematopoietic stem cells (HSCs), which can be limited in numbers. Moreover, finding an appropriate human leukocyte antigen-matched donor can be problematic. If HSCs can be stably produced in large numbers from autologous or allogeneic cell sources, it would benefit HCT. Induced pluripotent stem cells (iPSCs) established from patients' own somatic cells can be differentiated into hematopoietic cells in vitro. This review will highlight recent methods for regulating human (h) iPSC production of HSCs and more mature blood cells. Advancements in transcription factor-mediated regulation of the developmental stages of in-vivo hematopoietic lineage commitment have begun to provide an understanding of the molecular mechanism of hematopoiesis. Such studies involve not only directed differentiation in which transcription factors, specifically expressed in hematopoietic lineage-specific cells, are overexpressed in iPSCs, but also direct conversion in which transcription factors are introduced into patient-derived somatic cells which are dedifferentiated to hematopoietic cells. As iPSCs derived from patients suffering from genetically mutated diseases would express the same mutated genetic information, CRISPR-Cas9 gene editing has been utilized to differentiate genetically corrected iPSCs into normal hematopoietic cells. IPSCs provide a model for molecular understanding of disease, and also may function as a cell population for therapy. Efficient differentiation of patient-specific iPSCs into HSCs and progenitor cells is a potential means to overcome limitations of such cells for HCT, as well as for providing in-vitro drug screening templates as tissue-on-a-chip models.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

OpenAIRE

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors 1,2 . Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote linea...

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

Science.gov (United States)

Shinkuma, Satoru; Guo, Zongyou; Christiano, Angela M

2016-05-17

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells

Directory of Open Access Journals (Sweden)

Mostafa Kiamehr

2017-09-01

Full Text Available Hepatocyte-like cells (HLCs differentiated from human induced pluripotent stem cells (iPSCs offer an alternative model to primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a three-step protocol through the definitive endoderm (DE stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid-metabolism-related genes were performed during the entire differentiation process from iPSCs to HLCs. Additionally, fatty acid (FA composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very long FA were produced in the cells at a later stage of differentiation. This work uncovers the previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid-associated genes. Together with biochemical, functional and gene expression measurements, the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLC differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring, for example, the lipid metabolism involved in the development of fatty liver disease or atherosclerosis.

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9.

Science.gov (United States)

Yang, Yuanyuan; Zhang, Xiaobai; Yi, Li; Hou, Zhenzhen; Chen, Jiayu; Kou, Xiaochen; Zhao, Yanhong; Wang, Hong; Sun, Xiao-Fang; Jiang, Cizhong; Wang, Yixuan; Gao, Shaorong

2016-01-01

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. In the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with β-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as β-thalassemia. ©AlphaMed Press.

Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

Science.gov (United States)

Gu, Haihui; Huang, Xia; Xu, Jing; Song, Lili; Liu, Shuping; Zhang, Xiao-Bing; Yuan, Weiping; Li, Yanxin

2018-06-15

Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

Science.gov (United States)

Meraviglia, Viviana; Benzoni, Patrizia; Landi, Sara; Murano, Carmen; Langione, Marianna; Motta, Benedetta M; Baratto, Serena; Silipigni, Rosamaria; Di Segni, Marina; Pramstaller, Peter P; DiFrancesco, Dario; Gazzerro, Elisabetta; Barbuti, Andrea; Rossini, Alessandra

2018-03-01

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Patient iPSC-derived neurons for disease modeling of frontotemporal dementia with mutation in CHMP2B

DEFF Research Database (Denmark)

Zhang, Yu; Schmid, Benjamin; Nikolaisen, Nanett Kvist

2017-01-01

-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3...... in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development....

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Science.gov (United States)

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual

Generation and genetic modification of induced pluripotent stem cells.

Science.gov (United States)

Schambach, Axel; Cantz, Tobias; Baum, Christopher; Cathomen, Toni

2010-07-01

The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research. After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs. Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases. Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.

Potential of Gene Editing and Induced Pluripotent Stem Cells (iPSCs) in Treatment of Retinal Diseases.

Science.gov (United States)

Chuang, Katherine; Fields, Mark A; Del Priore, Lucian V

2017-12-01

The advent of gene editing has introduced the ability to make changes to the genome of cells, thus allowing for correction of genetic mutations in patients with monogenic diseases. Retinal diseases are particularly suitable for the application of this new technology because many retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA), are monogenic. Moreover, gene delivery techniques such as the use of adeno-associated virus (AAV) vectors have been optimized for intraocular use, and phase III trials are well underway to treat LCA, a severe form of inherited retinal degeneration, with gene therapy. This review focuses on the use of gene editing techniques and another relatively recent advent, induced pluripotent stem cells (iPSCs), and their potential for the study and treatment of retinal disease. Investment in these technologies, including overcoming challenges such as off-target mutations and low transplanted cell integration, may allow for future treatment of many debilitating inherited retinal diseases.

Transcriptomic Analysis of Induced Pluripotent Stem Cells Derived from Patients with Bipolar Disorder from an Old Order Amish Pedigree.

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Kwi Hye Kim

Full Text Available Fibroblasts from patients with Type I bipolar disorder (BPD and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs. Established iPSCs were subsequently differentiated into neuroprogenitors (NPs and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs, NPs and neurons matured in culture for either 2 weeks (termed early neurons, E or 4 weeks (termed late neurons, L. Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate, enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically, 328 genes were differentially expressed between BPD and control L neurons including GAD1, glutamate decarboxylase 1 (2.5 fold and SCN4B, the voltage gated type IV sodium channel beta subunit (-14.6 fold. Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology, GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism, protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD.

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

DEFF Research Database (Denmark)

Zhang, Yu; Schmid, Benjamin; Nikolaisen, Nanett K

2017-01-01

-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3...... in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development....

Neurosphere based differentiation of human iPSC improves astrocyte differentiation

DEFF Research Database (Denmark)

Zhou, Shuling; Szczesna, Karolina; Ochalek, Anna

2016-01-01

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development...... of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6...... expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC...

Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

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Akshay Bhinge

2017-04-01

Full Text Available Summary: Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS, it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. : In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. Keywords: ALS, SOD1, FUS, CRISPR-Cas9, p38, ERK, JNK, WNT, TP53, JUN

A parallel implementation of particle tracking with space charge effects on an INTEL iPSC/860

International Nuclear Information System (INIS)

Chang, L.; Bourianoff, G.; Cole, B.; Machida, S.

1993-05-01

Particle-tracking simulation is one of the scientific applications that is well-suited to parallel computations. At the Superconducting Super Collider, it has been theoretically and empirically demonstrated that particle tracking on a designed lattice can achieve very high parallel efficiency on a MIMD Intel iPSC/860 machine. The key to such success is the realization that the particles can be tracked independently without considering their interaction. The perfectly parallel nature of particle tracking is broken if the interaction effects between particles are included. The space charge introduces an electromagnetic force that will affect the motion of tracked particles in 3-D space. For accurate modeling of the beam dynamics with space charge effects, one needs to solve three-dimensional Maxwell field equations, usually by a particle-in-cell (PIC) algorithm. This will require each particle to communicate with its neighbor grids to compute the momentum changes at each time step. It is expected that the 3-D PIC method will degrade parallel efficiency of particle-tracking implementation on any parallel computer. In this paper, we describe an efficient scheme for implementing particle tracking with space charge effects on an INTEL iPSC/860 machine. Experimental results show that a parallel efficiency of 75% can be obtained

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS).

Science.gov (United States)

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Vascular Smooth Muscle Cells From Hypertensive Patient-Derived Induced Pluripotent Stem Cells to Advance Hypertension Pharmacogenomics.

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Biel, Nikolett M; Santostefano, Katherine E; DiVita, Bayli B; El Rouby, Nihal; Carrasquilla, Santiago D; Simmons, Chelsey; Nakanishi, Mahito; Cooper-DeHoff, Rhonda M; Johnson, Julie A; Terada, Naohiro

2015-12-01

Studies in hypertension (HTN) pharmacogenomics seek to identify genetic sources of variable antihypertensive drug response. Genetic association studies have detected single-nucleotide polymorphisms (SNPs) that link to drug responses; however, to understand mechanisms underlying how genetic traits alter drug responses, a biological interface is needed. Patient-derived induced pluripotent stem cells (iPSCs) provide a potential source for studying otherwise inaccessible tissues that may be important to antihypertensive drug response. The present study established multiple iPSC lines from an HTN pharmacogenomics cohort. We demonstrated that established HTN iPSCs can robustly and reproducibly differentiate into functional vascular smooth muscle cells (VSMCs), a cell type most relevant to vasculature tone control. Moreover, a sensitive traction force microscopy assay demonstrated that iPSC-derived VSMCs show a quantitative contractile response on physiological stimulus of endothelin-1. Furthermore, the inflammatory chemokine tumor necrosis factor α induced a typical VSMC response in iPSC-derived VSMCs. These studies pave the way for a large research initiative to decode biological significance of identified SNPs in hypertension pharmacogenomics. Treatment of hypertension remains suboptimal, and a pharmacogenomics approach seeks to identify genetic biomarkers that could be used to guide treatment decisions; however, it is important to understand the biological underpinnings of genetic associations. Mouse models do not accurately recapitulate individual patient responses based on their genetics, and hypertension-relevant cells are difficult to obtain from patients. Induced pluripotent stem cell (iPSC) technology provides a great interface to bring patient cells with their genomic data into the laboratory and to study hypertensive responses. As an initial step, the present study established an iPSC bank from patients with primary hypertension and demonstrated an effective

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

DEFF Research Database (Denmark)

Xiong, Kai; Zhou, Yan; Hyttel, Poul

2016-01-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4...... a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes....

Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

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Michael Riedel

2014-07-01

Full Text Available Advances in induced pluripotent stem cell (iPSC technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

Generation of an induced pluripotent stem cell (iPSC line from a patient with maturity-onset diabetes of the young type 13 (MODY13 with a the potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11 mutation

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Frank Griscelli

2017-08-01

Full Text Available Heterozygous activating mutation (p.Glu227Lys in KCNJ11 leads to maturity-onset diabetes of the young (MODY type 13, that is a subtype of dominant inherited young-onset non-autoimmune diabetes due to a primary defect in pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs from a patient with KCNJ11p.Glu227Lys mutation who developed MODY at 13 years old. KCNJ11p.Glu227Lys-mutated cells that were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the KCNJ11p.Glu227Lys mutation, expressed pluripotency hallmarks and had the differentiation capacity into the three germ layers.

Induced Pluripotent Stem Cells for Regenerative Cardiovascular Therapies and Biomedical Discovery

OpenAIRE

Nsair, Ali; MacLellan, W. Robb

2011-01-01

The discovery of induced pluripotent stem cells (iPSC) has, in the short time since their discovery, revolutionized the field of stem cell biology. This technology allows the generation of a virtually unlimited supply of cells with pluripotent potential similar to that of embryonic stem cells (ESC). However, in contrast to ESC, iPSC are not subject to the same ethical concerns and can be easily generated from living individuals. For the first time, patient-specific iPSC can be generated and o...

New lessons learned from disease modeling with induced Pluripotent Stem Cells

Science.gov (United States)

Onder, Tamer T.; Daley, George Q.

2012-01-01

Cellular reprogramming and generation of induced pluripotent stem cells (iPSCs) from adult cell types has enabled the creation of patient-specific stem cells for use in disease modeling. To date, many iPSC lines have been generated from a variety of disorders, which have then been differentiated into disease-relevant cell types. When a disease-specific phenotype is detectable in such differentiated cells, the reprogramming technology provides a new opportunity to identify aberrant disease-associated pathways and drugs that can block them. Here, we highlight recent progress as well as limitations in the use of iPSCs to recapitulate disease phenotypes and to screen for therapeutics in vitro. PMID:22749051

Using Patient-Specific Induced Pluripotent Stem Cells and Wild-Type Mice to Develop a Gene Augmentation-Based Strategy to Treat CLN3-Associated Retinal Degeneration.

Science.gov (United States)

Wiley, Luke A; Burnight, Erin R; Drack, Arlene V; Banach, Bailey B; Ochoa, Dalyz; Cranston, Cathryn M; Madumba, Robert A; East, Jade S; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

2016-10-01

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a childhood neurodegenerative disease with early-onset, severe central vision loss. Affected children develop seizures and CNS degeneration accompanied by severe motor and cognitive deficits. There is no cure for JNCL, and patients usually die during the second or third decade of life. In this study, independent lines of induced pluripotent stem cells (iPSCs) were generated from two patients with molecularly confirmed mutations in CLN3, the gene mutated in JNCL. Clinical-grade adeno-associated adenovirus serotype 2 (AAV2) carrying the full-length coding sequence of human CLN3 was generated in a U.S. Food and Drug Administration-registered cGMP facility. AAV2-CLN3 was efficacious in restoring full-length CLN3 transcript and protein in patient-specific fibroblasts and iPSC-derived retinal neurons. When injected into the subretinal space of wild-type mice, purified AAV2-CLN3 did not show any evidence of retinal toxicity. This study provides proof-of-principle for initiation of a clinical trial using AAV-mediated gene augmentation for the treatment of children with CLN3-associated retinal degeneration.

3D Bioprinting Human Induced Pluripotent Stem Cell Constructs for In Situ Cell Proliferation and Successive Multilineage Differentiation.

Science.gov (United States)

Gu, Qi; Tomaskovic-Crook, Eva; Wallace, Gordon G; Crook, Jeremy M

2017-09-01

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine, including individualized, patient-specific stem cell-based treatments. There are, however, few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues, ideally comprising direct-write printing of cells for encapsulation, proliferation, and differentiation. Here, such a method, employing a clinically amenable polysaccharide-based bioink, is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically, we have extrusion printed the bioink including iPSCs, alginate (Al; 5% weight/volume [w/v]), carboxymethyl-chitosan (5% w/v), and agarose (Ag; 1.5% w/v), crosslinked the bioink in calcium chloride for a stable and porous construct, proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm, ectoderm, and mesoderm, or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined, scalable, and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.

Science.gov (United States)

Barrett, Robert; Ornelas, Loren; Yeager, Nicole; Mandefro, Berhan; Sahabian, Anais; Lenaeus, Lindsay; Targan, Stephan R; Svendsen, Clive N; Sareen, Dhruv

2014-12-01

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblast-derived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications. ©AlphaMed Press.

Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses

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Kristiina Uusi-Rauva

2017-05-01

Full Text Available Neuronal ceroid lipofuscinoses (NCLs are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5 disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X, were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

Science.gov (United States)

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC).

Energy Technology Data Exchange (ETDEWEB)

Schultz, Peter Andrew

2011-12-01

The objective of the U.S. Department of Energy Office of Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC) is to provide an integrated suite of computational modeling and simulation (M&S) capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive-waste storage facility or disposal repository. Achieving the objective of modeling the performance of a disposal scenario requires describing processes involved in waste form degradation and radionuclide release at the subcontinuum scale, beginning with mechanistic descriptions of chemical reactions and chemical kinetics at the atomic scale, and upscaling into effective, validated constitutive models for input to high-fidelity continuum scale codes for coupled multiphysics simulations of release and transport. Verification and validation (V&V) is required throughout the system to establish evidence-based metrics for the level of confidence in M&S codes and capabilities, including at the subcontiunuum scale and the constitutive models they inform or generate. This Report outlines the nature of the V&V challenge at the subcontinuum scale, an approach to incorporate V&V concepts into subcontinuum scale modeling and simulation (M&S), and a plan to incrementally incorporate effective V&V into subcontinuum scale M&S destined for use in the NEAMS Waste IPSC work flow to meet requirements of quantitative confidence in the constitutive models informed by subcontinuum scale phenomena.

Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC)

International Nuclear Information System (INIS)

Schultz, Peter Andrew

2011-01-01

The objective of the U.S. Department of Energy Office of Nuclear Energy Advanced Modeling and Simulation Waste Integrated Performance and Safety Codes (NEAMS Waste IPSC) is to provide an integrated suite of computational modeling and simulation (M and S) capabilities to quantitatively assess the long-term performance of waste forms in the engineered and geologic environments of a radioactive-waste storage facility or disposal repository. Achieving the objective of modeling the performance of a disposal scenario requires describing processes involved in waste form degradation and radionuclide release at the subcontinuum scale, beginning with mechanistic descriptions of chemical reactions and chemical kinetics at the atomic scale, and upscaling into effective, validated constitutive models for input to high-fidelity continuum scale codes for coupled multiphysics simulations of release and transport. Verification and validation (V and V) is required throughout the system to establish evidence-based metrics for the level of confidence in M and S codes and capabilities, including at the subcontiunuum scale and the constitutive models they inform or generate. This Report outlines the nature of the V and V challenge at the subcontinuum scale, an approach to incorporate V and V concepts into subcontinuum scale modeling and simulation (M and S), and a plan to incrementally incorporate effective V and V into subcontinuum scale M and S destined for use in the NEAMS Waste IPSC work flow to meet requirements of quantitative confidence in the constitutive models informed by subcontinuum scale phenomena.

Telomerase Protects Werner Syndrome Lineage-Specific Stem Cells from Premature Aging

Directory of Open Access Journals (Sweden)

Hoi-Hung Cheung

2014-04-01

Full Text Available Werner syndrome (WS patients exhibit premature aging predominantly in mesenchyme-derived tissues, but not in neural lineages, a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here, we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging, we differentiated iPSCs to mesenchymal stem cells (MSCs and neural stem/progenitor cells (NPCs. We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs, but not in NPCs. We postulate this “aging” discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC, whereas inhibition of telomerase sensitized NPCs to DNA damage. Our findings unveil a role for telomerase in the protection of accelerated aging in a specific lineage of stem cells.

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

Science.gov (United States)

Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

2015-08-01

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

Modeling Kidney Disease with iPS Cells

Science.gov (United States)

Freedman, Benjamin S.

2015-01-01

Induced pluripotent stem cells (iPSCs) are somatic cells that have been transcriptionally reprogrammed to an embryonic stem cell (ESC)-like state. iPSCs are a renewable source of diverse somatic cell types and tissues matching the original patient, including nephron-like kidney organoids. iPSCs have been derived representing several kidney disorders, such as ADPKD, ARPKD, Alport syndrome, and lupus nephritis, with the goals of generating replacement tissue and ‘disease in a dish’ laboratory models. Cellular defects in iPSCs and derived kidney organoids provide functional, personalized biomarkers, which can be correlated with genetic and clinical information. In proof of principle, disease-specific phenotypes have been described in iPSCs and ESCs with mutations linked to polycystic kidney disease or focal segmental glomerulosclerosis. In addition, these cells can be used to model nephrotoxic chemical injury. Recent advances in directed differentiation and CRISPR genome editing enable more specific iPSC models and present new possibilities for diagnostics, disease modeling, therapeutic screens, and tissue regeneration using human cells. This review outlines growth opportunities and design strategies for this rapidly expanding and evolving field. PMID:26740740

Plasma turbulence calculations on the Intel iPSC/860 (rx) hypercube

International Nuclear Information System (INIS)

Lynch, V.E.; Ruiter, J.R.

1990-01-01

One approach to improving the real-time efficiency of plasma turbulence calculations is to use a parallel algorithm. A serial algorithm used for plasma turbulence calculations was modified to allocate a radial region in each node. In this way, convolutions at a fixed radius are performed in parallel, and communication is limited to boundary values for each radial region. For a semi-implicity numerical scheme (tridiagonal matrix solver), there is a factor of 3 improvement in efficiency with the Intel iPSC/860 machine using 64 processors over a single-processor Cray-II. For block-tridiagonal matrix cases (fully implicit code), a second parallelization takes place. The Fourier components are distributed in nodes. In each node, the block-tridiagonal matrix is inverted for each of allocated Fourier components. The algorithm for this second case has not yet been optimized. 10 refs., 4 figs

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

Science.gov (United States)

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

A performance study of sparse Cholesky factorization on INTEL iPSC/860

Science.gov (United States)

Zubair, M.; Ghose, M.

1992-01-01

The problem of Cholesky factorization of a sparse matrix has been very well investigated on sequential machines. A number of efficient codes exist for factorizing large unstructured sparse matrices. However, there is a lack of such efficient codes on parallel machines in general, and distributed machines in particular. Some of the issues that are critical to the implementation of sparse Cholesky factorization on a distributed memory parallel machine are ordering, partitioning and mapping, load balancing, and ordering of various tasks within a processor. Here, we focus on the effect of various partitioning schemes on the performance of sparse Cholesky factorization on the Intel iPSC/860. Also, a new partitioning heuristic for structured as well as unstructured sparse matrices is proposed, and its performance is compared with other schemes.

Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia

Science.gov (United States)

Denton, Kyle R.; Lei, Ling; Grenier, Jeremy; Rodionov, Vladimir; Blackstone, Craig; Li, Xue-Jun

2013-01-01

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. PMID:24123785

Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts

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María Questa

2016-03-01

Full Text Available Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.

Patient-Specific Computational Modeling

CERN Document Server

Peña, Estefanía

2012-01-01

This book addresses patient-specific modeling. It integrates computational modeling, experimental procedures, imagine clinical segmentation and mesh generation with the finite element method (FEM) to solve problems in computational biomedicine and bioengineering. Specific areas of interest include cardiovascular problems, ocular and muscular systems and soft tissue modeling. Patient-specific modeling has been the subject of serious research over the last seven years and interest in the area is continually growing and this area is expected to further develop in the near future.

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

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Julio Castaño

2017-05-01

Full Text Available We report the generation-characterization of a fetal liver (FL B-cell progenitor (BCP-derived human induced pluripotent stem cell (hiPSC line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the “safe locus” AAVS1 (iCas9-FL-BCP-hiPSC. Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells.

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Niu, Xiaohua; He, Wenyin; Song, Bing; Ou, Zhanhui; Fan, Di; Chen, Yuchang; Fan, Yong; Sun, Xiaofang

2016-08-05

β-Thalassemia (β-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific β-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin β (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in β-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in β-Thal iPSCs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of human allogeneic and syngeneic pluripotent stem cells and immunological implications for transplantation.

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Sackett, S D; Brown, M E; Tremmel, D M; Ellis, T; Burlingham, W J; Odorico, J S

2016-04-01

Tissues derived from induced pluripotent stem cells (iPSCs) are a promising source of cells for building various regenerative medicine therapies; from simply transplanting cells to reseeding decellularized organs to reconstructing multicellular tissues. Although reprogramming strategies for producing iPSCs have improved, the clinical use of iPSCs is limited by the presence of unique human leukocyte antigen (HLA) genes, the main immunologic barrier to transplantation. In order to overcome the immunological hurdles associated with allogeneic tissues and organs, the generation of patient-histocompatible iPSCs (autologous or HLA-matched cells) provides an attractive platform for personalized medicine. However, concerns have been raised as to the fitness, safety and immunogenicity of iPSC derivatives because of variable differentiation potential of different lines and the identification of genetic and epigenetic aberrations that can occur during the reprogramming process. In addition, significant cost and regulatory barriers may deter commercialization of patient specific therapies in the short-term. Nonetheless, recent studies provide some evidence of immunological benefit for using autologous iPSCs. Yet, more studies are needed to evaluate the immunogenicity of various autologous and allogeneic human iPSC-derived cell types as well as test various methods to abrogate rejection. Here, we present perspectives of using allogeneic vs. autologous iPSCs for transplantation therapies and the advantages and disadvantages of each related to differentiation potential, immunogenicity, genetic stability and tumorigenicity. We also review the current literature on the immunogenicity of syngeneic iPSCs and discuss evidence that questions the feasibility of HLA-matched iPSC banks. Finally, we will discuss emerging methods of abrogating or reducing host immune responses to PSC derivatives. Copyright © 2016 Elsevier Inc. All rights reserved.

Clinical Application of Induced Pluripotent Stem Cells in Cardiovascular Medicine.

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Chi, Hong-jie; Gao, Song; Yang, Xin-chun; Cai, Jun; Zhao, Wen-shu; Sun, Hao; Geng, Yong-Jian

2015-01-01

Induced pluripotent stem cells (iPSCs) are generated by reprogramming human somatic cells through the overexpression of four transcription factors: Oct4, Sox2, Klf4 and c-Myc. iPSCs are capable of indefinite self-renewal, and they can differentiate into almost any type of cell in the body. These cells therefore offer a highly valuable therapeutic strategy for tissue repair and regeneration. Recent experimental and preclinical research has revealed their potential for cardiovascular disease diagnosis, drug screening and cellular replacement therapy. Nevertheless, significant challenges remain in terms of the development and clinical application of human iPSCs. Here, we review current progress in research related to patient-specific iPSCs for ex vivo modeling of cardiovascular disorders and drug screening, and explore the potential of human iPSCs for use in the field of cardiovascular regenerative medicine. © 2015 S. Karger AG, Basel.

Development of an inducible caspase-9 safety switch for pluripotent stem cell–based therapies

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Chuanfeng Wu

2014-01-01

Full Text Available Induced pluripotent stem cell (iPSC therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-“inducible caspase-9” (iCasp9 suicide gene into mouse iPSC (miPSC and rhesus iPSC (RhiPSC via a lentivirus, driving expression from either a cytomegalovirus (CMV, elongation factor-1 α (EF1α or pluripotency-specific EOS-C(3+ promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α-iPSC, with less effective killing of EOS-C(3+-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

ALS Patient Stem Cells for Unveiling Disease Signatures of Motoneuron Susceptibility: Perspectives on the Deadly Mitochondria, ER Stress and Calcium Triad

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Kaus, Anjoscha; Sareen, Dhruv

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a largely sporadic progressive neurodegenerative disease affecting upper and lower motoneurons (MNs) whose specific etiology is incompletely understood. Mutations in superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TARDBP/TDP-43) and C9orf72, have been identified in subsets of familial and sporadic patients. Key associated molecular and neuropathological features include ubiquitinated TDP-43 inclusions, stress granules, aggregated dipeptide proteins from mutant C9orf72 transcripts, altered mitochondrial ultrastructure, dysregulated calcium homeostasis, oxidative and endoplasmic reticulum (ER) stress, and an unfolded protein response (UPR). Such impairments have been documented in ALS animal models; however, whether these mechanisms are initiating factors or later consequential events leading to MN vulnerability in ALS patients is debatable. Human induced pluripotent stem cells (iPSCs) are a valuable tool that could resolve this “chicken or egg” causality dilemma. Relevant systems for probing pathophysiologically affected cells from large numbers of ALS patients and discovering phenotypic disease signatures of early MN susceptibility are described. Performing unbiased ‘OMICS and high-throughput screening in relevant neural cells from a cohort of ALS patient iPSCs, and rescuing mitochondrial and ER stress impairments, can identify targeted therapeutics for increasing MN longevity in ALS. PMID:26635528

Reprogramming with Small Molecules instead of Exogenous Transcription Factors

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Tongxiang Lin

2015-01-01

Full Text Available Induced pluripotent stem cells (iPSCs could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

Is Human-induced Pluripotent Stem Cell the Best Optimal?

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Wang, Feng; Kong, Jie; Cui, Yi-Yao; Liu, Peng; Wen, Jian-Yan

2018-04-05

Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modeling, drug discovery, and cell therapy development. In this review, we discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, and consider the remaining challenges and the emerging opportunities in the field. Articles in this review were searched from PubMed database from January 2014 to December 2017. Original articles about iPSCs and cardiovascular diseases were included and analyzed. iPSC holds great promises for human disease modeling, drug discovery, and stem cell-based therapy, and this potential is only beginning to be realized. However, several important issues remain to be addressed. The recent availability of human cardiomyocytes derived from iPSCs opens new opportunities to build in vitro models of cardiac disease, screening for new drugs and patient-specific cardiac therapy.

Establishment of induced pluripotent stem cell line (ZZUi010-A from an Alzheimer's disease patient carrying an APP gene mutation

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Zhilei Wang

2017-12-01

Full Text Available Alzheimer's disease (AD is one of the most common neurodegenerative disorders. Previous studies have identified mutations in several genes, such as amyloid precursor protein (APP, presenilin-1 (PSEN1, and presenilin-2 (PSEN2, in patients with early-onset (<65 years familial AD. Recently, a patient with an APP gene mutation was identified; the dermal fibroblasts of the patient were obtained and a line of induced pluripotent stem cells (iPSCs was successfully generated using the Sendai-virus (SeV delivery system. The iPSC line will be useful for further study of the pathomechanism and drug screening for AD.

Translating induced pluripotent stem cells from bench to bedside: application to retinal diseases.

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Cramer, Alona O; MacLaren, Robert E

2013-04-01

Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somatic cells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the way for retinal therapies with the ongoing development of safe animal cell transplantation studies and characterization of patient- specific and disease-specific human iPSc. The retina is an optimal model for investigation of neural regeneration; amongst other advantageous attributes, it is the most accessible part of the CNS for surgery and outcome monitoring. A recent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severely degenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitro increase the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. In this review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

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Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4

DEFF Research Database (Denmark)

Di Stefano, Bruno; Collombet, Samuel; Jakobsen, Janus Schou

2016-01-01

reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell...

Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

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Naoto Ito

2016-04-01

Full Text Available X-linked dystonia-parkinsonism (XDP is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containing TAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA-type retrotransposon in intron 32 of TAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression of TAF1 and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs. Compared with control cells, XDP fibroblasts exhibited decreased expression of TAF1 transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34′, was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.

MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs.

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Dong Lu

Full Text Available miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs.We screened 52 miRNAs cloned in a piggybac (PB vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

Adolescent maturation of inhibitory inputs onto cingulate cortex neurons is cell-type specific and TrkB dependent

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Angela eVandenberg

2015-02-01

Full Text Available The maturation of inhibitory circuits during adolescence may be tied to the onset of mental health disorders such as schizophrenia. Neurotrophin signaling likely plays a critical role in supporting inhibitory circuit development and is also implicated in psychiatric disease. Within the neocortex, subcircuits may mature at different times and show differential sensitivity to neurotrophin signaling. We measured miniature inhibitory and excitatory postsynaptic currents (mIPSC and mEPSCs in Layer 5 cell-types in the mouse anterior cingulate across the periadolescent period. We differentiated cell-types mainly by Thy1 YFP transgene expression and also retrobead injection labeling in the contralateral cingulate and ipsilateral pons. We found that YFP- neurons and commissural projecting neurons had lower frequency of mIPSCs than neighboring YFP+ neurons or pons projecting neurons in juvenile mice (P21-25. YFP- neurons and to a lesser extent commissural projecting neurons also showed a significant increase in mIPSC amplitude during the periadolescent period (P21-25 vs. P40-50, which was not seen in YFP+ neurons or pons projecting neurons. Systemic disruption of tyrosine kinase receptor B (TrkB signaling during P23-50 in TrkBF616A mice blocked developmental changes in mIPSC amplitude, without affecting miniature excitatory post synaptic currents (mEPSCs. Our data suggest that the maturation of inhibitory inputs onto layer 5 pyramidal neurons is cell-type specific. These data may inform our understanding of adolescent brain development across species and aid in identifying candidate subcircuits that may show greater vulnerability in mental illness.

Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome.

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Steven D Sheridan

Full Text Available Fragile X syndrome (FXS is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1 gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP. Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid

Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems.

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Trevisan, Marta; Sinigaglia, Alessandro; Desole, Giovanna; Berto, Alessandro; Pacenti, Monia; Palù, Giorgio; Barzon, Luisa

2015-07-13

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host-pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems

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Marta Trevisan

2015-07-01

Full Text Available The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs, which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

Defining differentially methylated regions specific for the acquisition of pluripotency and maintenance in human pluripotent stem cells via microarray.

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WenYin He

Full Text Available Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina's Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.

iPSCs to the rescue in Alzheimer's research.

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Choi, Se Hoon; Tanzi, Rudolph E

2012-03-02

A crucial limitation to our understanding of Alzheimer's disease has been the inability to test hypotheses on live, patient-specific neurons. A recent study in Nature by Israel et al. (2012) reports that iPSC-derived neurons from AD patients recapitulate multiple aspects of disease pathology. Copyright © 2012 Elsevier Inc. All rights reserved.

Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs with Spanish genetic background

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Sonia Cabrera

2015-09-01

Full Text Available We have generated iPSCs from peripheral blood mononuclear cells (PBMCs of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

Patient-derived stem cells: pathways to drug discovery for brain diseases

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Alan eMackay-Sim

2013-03-01

Full Text Available The concept of drug discovery through stem cell biology is based on technological developments whose genesis is now coincident. The first is automated cell microscopy with concurrent advances in image acquisition and analysis, known as high content screening (HCS. The second is patient-derived stem cells for modelling the cell biology of brain diseases. HCS has developed from the requirements of the pharmaceutical industry for high throughput assays to screen thousands of chemical compounds in the search for new drugs. HCS combines new fluorescent probes with automated microscopy and computational power to quantify the effects of compounds on cell functions. Stem cell biology has advanced greatly since the discovery of genetic reprogramming of somatic cells into induced pluripotent stem cells (iPSCs. There is now a rush of papers describing their generation from patients with various diseases of the nervous system. Although the majority of these have been genetic diseases, iPSCs have been generated from patients with complex diseases (schizophrenia and sporadic Parkinson’s disease. Some genetic diseases are also modelled in embryonic stem cells generated from blastocysts rejected during in vitro fertilisation. Neural stem cells have been isolated from post-mortem brain of Alzheimer’s patients and neural stem cells generated from biopsies of the olfactory organ of patients is another approach. These olfactory neurosphere-derived cells demonstrate robust disease-specific phenotypes in patients with schizophrenia and Parkinson’s disease. High content screening is already in use to find small molecules for the generation and differentiation of embryonic stem cells and induced pluripotent stem cells. The challenges for using stem cells for drug discovery are to develop robust stem cell culture methods that meet the rigorous requirements for repeatable, consistent quantities of defined cell types at the industrial scale necessary for high

The Use of Stem Cells to Model Amyotrophic Lateral Sclerosis and Frontotemporal Dementia: From Basic Research to Regenerative Medicine

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Erin C. Hedges

2016-01-01

Full Text Available In recent years several genes have linked amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD as a spectrum disease; however little is known about what triggers their onset. With the ability to generate patient specific stem cell lines from somatic cells, it is possible to model disease without the need to transfect cells with exogenous DNA. These pluripotent stem cells have opened new avenues for identification of disease phenotypes and their relation to specific molecular pathways. Thus, as never before, compounds with potential applications for regenerative medicine can be specifically tailored in patient derived cultures. In this review, we discuss how patient specific induced pluripotent stem cells (iPSCs have been used to model ALS and FTD and the most recent drug screening targets for these diseases. We also discuss how an iPSC bank would improve the quality of the available cell lines and how it would increase knowledge about the ALS/FTD disease spectrum.

The Use of Stem Cells to Model Amyotrophic Lateral Sclerosis and Frontotemporal Dementia: From Basic Research to Regenerative Medicine.

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Hedges, Erin C; Mehler, Vera J; Nishimura, Agnes L

2016-01-01

In recent years several genes have linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as a spectrum disease; however little is known about what triggers their onset. With the ability to generate patient specific stem cell lines from somatic cells, it is possible to model disease without the need to transfect cells with exogenous DNA. These pluripotent stem cells have opened new avenues for identification of disease phenotypes and their relation to specific molecular pathways. Thus, as never before, compounds with potential applications for regenerative medicine can be specifically tailored in patient derived cultures. In this review, we discuss how patient specific induced pluripotent stem cells (iPSCs) have been used to model ALS and FTD and the most recent drug screening targets for these diseases. We also discuss how an iPSC bank would improve the quality of the available cell lines and how it would increase knowledge about the ALS/FTD disease spectrum.

Induced pluripotent stem cells for regenerative cardiovascular therapies and biomedical discovery.

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Nsair, Ali; MacLellan, W Robb

2011-04-30

The discovery of induced pluripotent stem cells (iPSC) has, in the short time since their discovery, revolutionized the field of stem cell biology. This technology allows the generation of a virtually unlimited supply of cells with pluripotent potential similar to that of embryonic stem cells (ESC). However, in contrast to ESC, iPSC are not subject to the same ethical concerns and can be easily generated from living individuals. For the first time, patient-specific iPSC can be generated and offer a supply of genetically identical cells that can be differentiated into all somatic cell types for potential use in regenerative therapies or drug screening and testing. As the techniques for generation of iPSC lines are constantly evolving, new uses for human iPSC are emerging from in-vitro disease modeling to high throughput drug discovery and screening. This technology promises to revolutionize the field of medicine and offers new hope for understanding and treatment of numerous diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

Science.gov (United States)

Ebert, Allison D; Shelley, Brandon C; Hurley, Amanda M; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N; Svendsen, Soshana P; Mattis, Virginia B; McGivern, Jered V; Schwab, Andrew J; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N

2013-05-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Generation of an integration-free induced pluripotent stem cell line (CSC-43 from a patient with sporadic Parkinson's disease

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Ana Marote

2018-03-01

Full Text Available An induced pluripotent stem cell (iPSC line was generated from a 36-year-old patient with sporadic Parkinson's disease (PD. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line (CSC-43 exhibits expression of common pluripotency markers, in vitro differentiation into three germ layers and normal karyotype. This iPSC line can be used to study the mechanisms underlying the development of PD.

Modeling high-temperature superconductors and metallic alloys on the Intel IPSC/860

Science.gov (United States)

Geist, G. A.; Peyton, B. W.; Shelton, W. A.; Stocks, G. M.

Oak Ridge National Laboratory has embarked on several computational Grand Challenges, which require the close cooperation of physicists, mathematicians, and computer scientists. One of these projects is the determination of the material properties of alloys from first principles and, in particular, the electronic structure of high-temperature superconductors. While the present focus of the project is on superconductivity, the approach is general enough to permit study of other properties of metallic alloys such as strength and magnetic properties. This paper describes the progress to date on this project. We include a description of a self-consistent KKR-CPA method, parallelization of the model, and the incorporation of a dynamic load balancing scheme into the algorithm. We also describe the development and performance of a consolidated KKR-CPA code capable of running on CRAYs, workstations, and several parallel computers without source code modification. Performance of this code on the Intel iPSC/860 is also compared to a CRAY 2, CRAY YMP, and several workstations. Finally, some density of state calculations of two perovskite superconductors are given.

Is Human-induced Pluripotent Stem Cell the Best Optimal?

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Feng Wang

2018-01-01

Conclusions: The recent availability of human cardiomyocytes derived from iPSCs opens new opportunities to build in vitro models of cardiac disease, screening for new drugs and patient-specific cardiac therapy.

Induced pluripotent stem cells (iPSCs) derived from af pre-symptomatic carrier of a R406W mutation in microtubule-associated protein tau (MAPT) causing frontotemporal dementia

DEFF Research Database (Denmark)

Rasmussen, Mikkel A.; Hjermind, Lena Elisabeth; Hasholt, Lis Frydenreich

2016-01-01

Skin fibroblasts were obtained from a 28-year-old pre-symptomatic woman carrying a R406W mutation in microtubule-associated protein tau (MAPT), known to cause frontotemporal dementia. Induced pluripotent stem cell (iPSCs) were established by electroporation with episomal plasmids containing hOCT4...

Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.

Science.gov (United States)

Song, Bing; Fan, Yong; He, Wenyin; Zhu, Detu; Niu, Xiaohua; Wang, Ding; Ou, Zhanhui; Luo, Min; Sun, Xiaofang

2015-05-01

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.

Generation of a human induced pluripotent stem cell line (CSC-42 from a patient with sporadic form of Parkinson's disease

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Ekaterina Savchenko

2018-03-01

Full Text Available Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD. The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs. Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.

Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.

Science.gov (United States)

Li, Hongmei Lisa; Fujimoto, Naoko; Sasakawa, Noriko; Shirai, Saya; Ohkame, Tokiko; Sakuma, Tetsushi; Tanaka, Michihiro; Amano, Naoki; Watanabe, Akira; Sakurai, Hidetoshi; Yamamoto, Takashi; Yamanaka, Shinya; Hotta, Akitsu

2015-01-13

Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

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Ji-Yon Kim

2016-01-01

Full Text Available The Charcot-Marie-Tooth disease 2F (CMT2F and distal hereditary motor neuropathy 2B (dHMN2B are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1 gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

'Shovel-Ready' applications of stem cell advances for pediatric heart disease.

Science.gov (United States)

Files, Matthew D; Boucek, Robert J

2012-10-01

The past decade has seen remarkable advances in the field of stem cell biology. Many new technologies and applications are passing the translational phase and likely will soon be relevant for the clinical pediatric cardiologist. This review will focus on two advances in basic science that are now translating into clinical trials. The first advance is the recognition, characterization, and recent therapeutic application of resident cardiac progenitor cells (CPCs). Early results of adult trials and scattered case reports in pediatric patients support expanding CPC-based trials for end-stage heart failure in pediatric patients. The relative abundance of CPCs in the neonate and young child offers greater potential benefits in heart failure treatment than has been realized to date. The second advance is the technology of induced pluripotent stem cells (iPSCs), which reprograms differentiated somatic cells to an undifferentiated embryonic-like state. When iPSCs are differentiated into cardiomyocytes, they model a patient's specific disease, test pharmaceuticals, and potentially provide an autologous source for cell-based therapy. The therapeutic recruitment and/or replacement of CPCs has potential for enhancing cardiac repair and regeneration in children with heart failure. Use of iPSCs to model heart disease holds great potential to gain new insights into diagnosis, pathophysiology, and disease-specific management for genetic-based cardiovascular diseases that are prevalent in pediatric patients.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

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Viviana Meraviglia

2018-03-01

Full Text Available Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3, encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-AL1CAV3 (EURACi002-AN1CAV3 (EURACi003-AInstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini (alessandra.rossini@eurac.eduType of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCsMethod of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hULMultiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-AHeterozygous CAV3 c.303 TGG > TGC (EURACi002-AHeterozygous CAV3 c.233 ACG > AAG (EURACi003-AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-ASeptember 2016 (EURACi002-AMay 2016 (EURACi003-ACell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the

Patient-specific models of cardiac biomechanics

Science.gov (United States)

Krishnamurthy, Adarsh; Villongco, Christopher T.; Chuang, Joyce; Frank, Lawrence R.; Nigam, Vishal; Belezzuoli, Ernest; Stark, Paul; Krummen, David E.; Narayan, Sanjiv; Omens, Jeffrey H.; McCulloch, Andrew D.; Kerckhoffs, Roy C. P.

2013-07-01

Patient-specific models of cardiac function have the potential to improve diagnosis and management of heart disease by integrating medical images with heterogeneous clinical measurements subject to constraints imposed by physical first principles and prior experimental knowledge. We describe new methods for creating three-dimensional patient-specific models of ventricular biomechanics in the failing heart. Three-dimensional bi-ventricular geometry is segmented from cardiac CT images at end-diastole from patients with heart failure. Human myofiber and sheet architecture is modeled using eigenvectors computed from diffusion tensor MR images from an isolated, fixed human organ-donor heart and transformed to the patient-specific geometric model using large deformation diffeomorphic mapping. Semi-automated methods were developed for optimizing the passive material properties while simultaneously computing the unloaded reference geometry of the ventricles for stress analysis. Material properties of active cardiac muscle contraction were optimized to match ventricular pressures measured by cardiac catheterization, and parameters of a lumped-parameter closed-loop model of the circulation were estimated with a circulatory adaptation algorithm making use of information derived from echocardiography. These components were then integrated to create a multi-scale model of the patient-specific heart. These methods were tested in five heart failure patients from the San Diego Veteran's Affairs Medical Center who gave informed consent. The simulation results showed good agreement with measured echocardiographic and global functional parameters such as ejection fraction and peak cavity pressures.

Inherent Immunogenicity or Lack Thereof of Pluripotent Stem Cells: Implications for Cell Replacement Therapy

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Arvind Chhabra

2017-08-01

Full Text Available Donor-specific induced pluripotent stem cells (iPSCs offer opportunities for personalized cell replacement therapeutic approaches due to their unlimited self-renewal potential and ability to differentiate into different somatic cells. A significant progress has been made toward generating iPSC lines that are free of integrating viral vectors, development of xeno-free culture conditions, and differentiation of pluripotent stem cells (PSCs into functional somatic cell lineages. Since donor-specific iPSC lines are genetically identical to the individual, they are expected to be immunologically matched and these iPSC lines and their cellular derivatives are not expected to be immunologically rejected. However, studies in mouse models, utilizing rejection of teratomas as a model, have claimed that syngenic iPSC lines, especially the iPSC lines derived with integrating viral vectors, could be inherently immunogenic. This manuscript reviews current understanding of inherent immunogenicity of PSC lines, especially that of the human iPSC lines and their cellular derivatives, and strategies to overcome it.

Patient-specific surgical simulation.

Science.gov (United States)

Soler, Luc; Marescaux, Jacques

2008-02-01

Technological innovations of the twentieth century have provided medicine and surgery with new tools for education and therapy definition. Thus, by combining Medical Imaging and Virtual Reality, patient-specific applications providing preoperative surgical simulation have become possible.

A Patient-Specific Airway Branching Model for Mechanically Ventilated Patients

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Nor Salwa Damanhuri

2014-01-01

Full Text Available Background. Respiratory mechanics models have the potential to guide mechanical ventilation. Airway branching models (ABMs were developed from classical fluid mechanics models but do not provide accurate models of in vivo behaviour. Hence, the ABM was improved to include patient-specific parameters and better model observed behaviour (ABMps. Methods. The airway pressure drop of the ABMps was compared with the well-accepted dynostatic algorithm (DSA in patients diagnosed with acute respiratory distress syndrome (ARDS. A scaling factor (α was used to equate the area under the pressure curve (AUC from the ABMps to the AUC of the DSA and was linked to patient state. Results. The ABMps recorded a median α value of 0.58 (IQR: 0.54–0.63; range: 0.45–0.66 for these ARDS patients. Significantly lower α values were found for individuals with chronic obstructive pulmonary disease (P<0.001. Conclusion. The ABMps model allows the estimation of airway pressure drop at each bronchial generation with patient-specific physiological measurements and can be generated from data measured at the bedside. The distribution of patient-specific α values indicates that the overall ABM can be readily improved to better match observed data and capture patient condition.

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

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Yu Zhang

2017-03-01

Full Text Available The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B is causative for frontotemporal dementia linked to chromosome 3 (FTD3. CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.

Stem Cell Therapies in Retinal Disorders

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Aakriti Garg

2017-02-01

Full Text Available Stem cell therapy has long been considered a promising mode of treatment for retinal conditions. While human embryonic stem cells (ESCs have provided the precedent for regenerative medicine, the development of induced pluripotent stem cells (iPSCs revolutionized this field. iPSCs allow for the development of many types of retinal cells, including those of the retinal pigment epithelium, photoreceptors, and ganglion cells, and can model polygenic diseases such as age-related macular degeneration. Cellular programming and reprogramming technology is especially useful in retinal diseases, as it allows for the study of living cells that have genetic variants that are specific to patients’ diseases. Since iPSCs are a self-renewing resource, scientists can experiment with an unlimited number of pluripotent cells to perfect the process of targeted differentiation, transplantation, and more, for personalized medicine. Challenges in the use of stem cells are present from the scientific, ethical, and political realms. These include transplant complications leading to anatomically incorrect placement, concern for tumorigenesis, and incomplete targeting of differentiation leading to contamination by different types of cells. Despite these limitations, human ESCs and iPSCs specific to individual patients can revolutionize the study of retinal disease and may be effective therapies for conditions currently considered incurable.

Stemcell Information: SKIP000108 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available ... -- -- ... Yes No intractable disease-specific iPSC 難病性疾患克服事業iPS細胞バンク human ES-like -- Sendai virus S... Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Stemcell Information: SKIP000128 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available ... -- -- ... Yes No intractable disease-specific iPSC 難病性疾患克服事業iPS細胞バンク human ES-like -- Sendai virus S... Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Patient-Specific Modeling in Tomorrow's Medicine

CERN Document Server

2012-01-01

This book reviews the frontier of research and clinical applications of Patient Specific Modeling, and provides a state-of-the-art update as well as perspectives on future directions in this exciting field. The book is useful for medical physicists, biomedical engineers and other engineers who are interested in the science and technology aspects of Patient Specific Modeling, as well as for radiologists and other medical specialists who wish to be updated about the state of implementation.

A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs.

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Deborah Prè

Full Text Available Many protocols have been designed to differentiate human embryonic stem cells (ESCs and human induced pluripotent stem cells (iPSCs into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48-55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of

Cellular Reprogramming – Turning the Clock Back

Indian Academy of Sciences (India)

IAS Admin

research. Interestingly enough, Gurdon's entry into biology was not as smooth as .... basically meant to the biomedical community was that, for the first time one ... therapy to correct the mutation in patient-specific iPSCs and then introduce the ...

Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy.

Science.gov (United States)

Jiao, Jiao; Tian, Weihua; Qiu, Ping; Norton, Elizabeth L; Wang, Michael M; Chen, Y Eugene; Yang, Bo

2018-03-12

The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1 -/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1 -/- and wild type cells. The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1 -/- NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1 -/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1 -/- CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1 -/- ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement.

Science.gov (United States)

Di Foggia, Valentina; Makwana, Priyanka; Ali, Robin R; Sowden, Jane C

2016-06-01

Stem cell therapies are being explored as potential treatments for retinal disease. How to replace neurons in a degenerated retina presents a continued challenge for the regenerative medicine field that, if achieved, could restore sight. The major issues are: (i) the source and availability of donor cells for transplantation; (ii) the differentiation of stem cells into the required retinal cells; and (iii) the delivery, integration, functionality, and survival of new cells in the host neural network. This review considers the use of induced pluripotent stem cells (iPSC), currently under intense investigation, as a platform for cell transplantation therapy. Moreover, patient-specific iPSC are being developed for autologous cell transplantation and as a tool for modeling specific retinal diseases, testing gene therapies, and drug screening.

Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

Science.gov (United States)

Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Gilioli, Andrea; Forghieri, Fabio; Candoni, Anna; Cesaro, Simone; Quadrelli, Chiara; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Codeluppi, Mauro; Mussini, Cristina; Colaci, Elisabetta; Messerotti, Andrea; Paolini, Ambra; Maccaferri, Monica; Fantuzzi, Valeria; Del Giovane, Cinzia; Stefani, Alessandro; Morandi, Uliano; Maffei, Rossana; Marasca, Roberto; Narni, Franco; Fanin, Renato; Comoli, Patrizia; Romani, Luigina; Beauvais, Anne; Viale, Pier Luigi; Latgè, Jean Paul; Lewis, Russell E; Luppi, Mario

2016-01-01

Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

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Leonardo Potenza

Full Text Available Invasive mucormycosis (IM is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients.By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3% of 204 patients, accounting for 32 (5.3% of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD, showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD: 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD.Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

Stemcell Information: SKIP000026 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available yndrome 176270 ... -- -- ... Yes No intractable disease-specific iPSC 難病性疾患克服事業iPS細胞バンク human ES-like -- ...kumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Patient-specific workup of adrenal incidentalomas

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Romy R. de Haan

Full Text Available Purpose: : To develop a clinical prediction model to predict a clinically relevant adrenal disorder for patients with adrenal incidentaloma. Materials and methods: : This retrospective study is approved by the institutional review board, with waiver of informed consent. Natural language processing is used for filtering of adrenal incidentaloma cases in all thoracic and abdominal CT reports from 2010 till 2012. A total of 635 patients are identified. Stepwise logistic regression is used to construct the prediction model. The model predicts if a patient is at risk for malignancy or hormonal hyperfunction of the adrenal gland at the moment of initial presentation, thus generates a predicted probability for every individual patient. The prediction model is evaluated on its usefulness in clinical practice using decision curve analysis (DCA based on different threshold probabilities. For patients whose predicted probability is lower than the predetermined threshold probability, further workup could be omitted. Results: : A prediction model is successfully developed, with an area under the curve (AUC of 0.78. Results of the DCA indicate that up to 11% of patients with an adrenal incidentaloma can be avoided from unnecessary workup, with a sensitivity of 100% and specificity of 11%. Conclusion: : A prediction model can accurately predict if an adrenal incidentaloma patient is at risk for malignancy or hormonal hyperfunction of the adrenal gland based on initial imaging features and patient demographics. However, with most adrenal incidentalomas labeled as nonfunctional adrenocortical adenomas requiring no further treatment, it is likely that more patients could be omitting from unnecessary diagnostics. Keywords: Adrenal incidentaloma, Patient-specific workup, Prediction model

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications

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Adekunle Ebenezer Omole

2018-05-01

Full Text Available The discovery of induced pluripotent stem cells (iPSCs by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling

Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications.

Science.gov (United States)

Omole, Adekunle Ebenezer; Fakoya, Adegbenro Omotuyi John

2018-01-01

The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy. However, fundamental questions have been raised regarding the molecular mechanism of iPSCs generation, a process still poorly understood by scientists. The efficiency of reprogramming of iPSCs remains low due to the effect of various barriers to reprogramming. There is also the risk of chromosomal instability and oncogenic transformation associated with the use of viral vectors, such as retrovirus and lentivirus, which deliver the reprogramming transcription factors by integration in the host cell genome. These challenges can hinder the therapeutic prospects and promise of iPSCs and their clinical applications. Consequently, extensive studies have been done to elucidate the molecular mechanism of reprogramming and novel strategies have been identified which help to improve the efficiency of reprogramming methods and overcome the safety concerns linked with iPSC generation. Distinct barriers and enhancers of reprogramming have been elucidated, and non-integrating reprogramming methods have been reported. Here, we summarize the progress and the recent advances that have been made over the last 10 years in the iPSC field, with emphasis on the molecular mechanism of reprogramming, strategies to improve the efficiency of reprogramming, characteristics and limitations of iPSCs, and the progress made in the applications of iPSCs in the field of disease modelling, drug discovery and

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11

DEFF Research Database (Denmark)

Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich

2016-01-01

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study, induced pluripotent stem cells (iPSCs) were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L...

3D-printed patient-specific applications in orthopedics

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Wong KC

2016-10-01

Full Text Available Kwok Chuen Wong Department of Orthopedics and Traumatology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong Abstract: With advances in both medical imaging and computer programming, two-dimensional axial images can be processed into other reformatted views (sagittal and coronal and three-dimensional (3D virtual models that represent a patients’ own anatomy. This processed digital information can be analyzed in detail by orthopedic surgeons to perform patient-specific orthopedic procedures. The use of 3D printing is rising and has become more prevalent in medical applications over the last decade as surgeons and researchers are increasingly utilizing the technology’s flexibility in manufacturing objects. 3D printing is a type of manufacturing process in which materials such as plastic or metal are deposited in layers to create a 3D object from a digital model. This additive manufacturing method has the advantage of fabricating objects with complex freeform geometry, which is impossible using traditional subtractive manufacturing methods. Specifically in surgical applications, the 3D printing techniques can not only generate models that give a better understanding of the complex anatomy and pathology of the patients and aid in education and surgical training, but can also produce patient-specific surgical guides or even custom implants that are tailor-made to the surgical requirements. As the clinical workflow of the 3D printing technology continues to evolve, orthopedic surgeons should embrace the latest knowledge of the technology and incorporate it into their clinical practice for patient-specific orthopedic applications. This paper is written to help orthopedic surgeons stay up-to-date on the emerging 3D technology, starting from the acquisition of clinical imaging to 3D printing for patient-specific applications in orthopedics. It 1 presents the necessary steps to prepare the medical images that are

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11

DEFF Research Database (Denmark)

Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich

2016-01-01

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation in ATXN3. We generated induced pluripotent stem cells (iPSCs) from a SCA3 patient by electroporation of dermal fibroblasts with episomal plasmids encoding L-MYC, LIN28...

Disease-specific self-efficacy in spasmodic dysphonia patients.

Science.gov (United States)

Hu, Amanda; Isetti, Derek; Hillel, Allen D; Waugh, Patricia; Comstock, Bryan; Meyer, Tanya K

2013-03-01

Self-efficacy (SE) is an optimistic self-belief that one can perform a novel task. This concept involves empowerment, self-esteem, and adaptation to a stressful situation. SE is a strong predictor of health behaviors. Our objectives were to study SE in spasmodic dysphonia (SD) and to develop a disease-specific SE-SD scale. Prospective study. Academic hospital. Disease-specific SE-SD items were developed with laryngologists, speech pathologists, and SD patients. These items, General SE Scale, Voice Handicap Index-10 (VHI-10), Consensus Auditory Perceptual Evaluation of Voice (CAPE-V), and Hospital Anxiety and Depression Scale (HADS), were administered to SD patients who presented for botulinum toxin injections. One hundred forty-five SD patients (mean age 59.5 ± 13.6 years) had a general SE score (Cronbach's α = 0.894) of 33.4 ± 5.2 out of 40. This was negatively correlated with HADS-A (r = -0.42, P < 0.001) and HADS-D (r = -0.42, P < .001), but not correlated with VHI-10 (r = -0.098, P = .243) and CAPE-V (r = -0.047, P = .57). Factor analysis selected 8 items from the general SE scale and 5 disease-specific SE-SD items to generate a 13-item disease-specific SE-SD scale (Cronbach's α = 0.907). Disease-specific SE-SD score was 42.1 ± 6.9 out of 52 and was negatively correlated with VHI-10 (r = -0.19, P = .005), HADS-A (r = -0.43, P < .001), and HADS-D (r = -0.57, P < .001), but not correlated with CAPE-V (r = -0.024, P = .60). SD patients established on botulinum toxin injections have high degrees of general and disease-specific SE. Patients with higher SE-SD demonstrate lower vocal handicap and lower levels of anxiety and depression. A 13-item disease-specific SE-SD scale has been developed.

Stemcell Information: SKIP000047 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available lateral sclerosis) 105400 ... -- -- ... Yes No intractable disease-specific iPSC, Familial ALS 難病性疾患克服事業iPS細胞...学 Kumamoto University 熊本大学 Takumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Stemcell Information: SKIP000075 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available scular dystrophy 310200 ... -- -- ... Yes No intractable disease-specific iPSC, DMD 難病性疾患克服事業iPS細胞バンク hum...ty 熊本大学 Takumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Stemcell Information: SKIP000067 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available ular dystrophy 310200 ... -- -- ... Yes No intractable disease-specific iPSC, DMD 難病性疾患克服事業iPS細胞バンク human... 熊本大学 Takumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Stemcell Information: SKIP000071 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available ular dystrophy 310200 ... -- -- ... Yes No intractable disease-specific iPSC, DMD 難病性疾患克服事業iPS細胞バンク human...sity 熊本大学 Takumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

Parallel spatial direct numerical simulations on the Intel iPSC/860 hypercube

Science.gov (United States)

Joslin, Ronald D.; Zubair, Mohammad

1993-01-01

The implementation and performance of a parallel spatial direct numerical simulation (PSDNS) approach on the Intel iPSC/860 hypercube is documented. The direct numerical simulation approach is used to compute spatially evolving disturbances associated with the laminar-to-turbulent transition in boundary-layer flows. The feasibility of using the PSDNS on the hypercube to perform transition studies is examined. The results indicate that the direct numerical simulation approach can effectively be parallelized on a distributed-memory parallel machine. By increasing the number of processors nearly ideal linear speedups are achieved with nonoptimized routines; slower than linear speedups are achieved with optimized (machine dependent library) routines. This slower than linear speedup results because the Fast Fourier Transform (FFT) routine dominates the computational cost and because the routine indicates less than ideal speedups. However with the machine-dependent routines the total computational cost decreases by a factor of 4 to 5 compared with standard FORTRAN routines. The computational cost increases linearly with spanwise wall-normal and streamwise grid refinements. The hypercube with 32 processors was estimated to require approximately twice the amount of Cray supercomputer single processor time to complete a comparable simulation; however it is estimated that a subgrid-scale model which reduces the required number of grid points and becomes a large-eddy simulation (PSLES) would reduce the computational cost and memory requirements by a factor of 10 over the PSDNS. This PSLES implementation would enable transition simulations on the hypercube at a reasonable computational cost.

Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering.

Science.gov (United States)

Jung, Moonjung; Dunbar, Cynthia E; Winkler, Thomas

2015-12-01

The combination of epigenetic reprogramming with advanced genome editing technologies opened a new avenue to study disease mechanisms, particularly of disorders with depleted target tissue. Bone marrow failure syndromes (BMFS) typically present with a marked reduction of peripheral blood cells due to a destroyed or dysfunctional bone marrow compartment. Somatic and germline mutations have been etiologically linked to many cases of BMFS. However, without the ability to study primary patient material, the exact pathogenesis for many entities remained fragmentary. Capturing the pathological genotype in induced pluripotent stem cells (iPSCs) allows studying potential developmental defects leading to a particular phenotype. The lack of hematopoietic stem and progenitor cells in these patients can also be overcome by differentiating patient-derived iPSCs into hematopoietic lineages. With fast growing genome editing techniques, such as CRISPR/Cas9, correction of disease-causing mutations in iPSCs or introduction of mutations in cells from healthy individuals enable comparative studies that may identify other genetic or epigenetic events contributing to a specific disease phenotype. In this review, we present recent progresses in disease modeling of inherited and acquired BMFS using reprogramming and genome editing techniques. We also discuss the challenges and potential shortcomings of iPSC-based models for hematological diseases.

Patient-specific hip prostheses designed by surgeons

Directory of Open Access Journals (Sweden)

Coigny Florian

2016-09-01

Full Text Available Patient-specific bone and joint replacement implants lead to better functional and aesthetic results than conventional methods [1], [2], [3]. But extracting 3D shape information from CT Data and designing individual implants is demanding and requires multiple surgeon-to-engineer interactions. For manufacturing purposes, Additive Manufacturing offers various advantages, especially for low volume manufacturing parts, such as patient specific implants. To ease these new approaches and to avoid surgeon-to-engineer interactions a new design software approach is needed which offers highly automated and user friendly planning steps.

Language-specific dysgraphia in Korean stroke patients.

Science.gov (United States)

Yoon, Ji Hye; Suh, Mee Kyung; Kim, HyangHee

2010-12-01

We investigated how changes in the writing of 14 Korean stroke patients reflect the unique features of the Korean writing system. The Korean writing system, Han-geul, has both linguistic and visuospatial/constructive characteristics. In the visuospatial construction of a syllable, the component consonant(s) and vowel(s) must be arranged from top-to-bottom and/or left-to-right within the form of a square. This syllabic organization, unique to Korean writing, may distinguish dysgraphia in Korean patients from the disorder in other languages, and reveal the effects of stroke on visuospatial/constructive abilities. We compared 2 groups of patients affected by stroke, 1 group with left hemisphere (LH) lesions and the other with right hemisphere (RH) lesions. We instructed them to write from a dictation of 90 monosyllabic stimuli, each presented with a real word cue. Patients had to repeat a target syllable and a word cue, and then to write the target syllable only. Patients with LH and RH lesions produced qualitatively different error patterns. While the LH lesion group produced primarily linguistic errors, visuospatial/constructive errors predominated in the group with RH lesions. With regard to language-specific features, these Korean patients with RH lesions produced diverse visuospatial/constructive errors not commonly observed in dysgraphia of the English language. Language-specific writing errors by Korean stroke patients reflect the unique characteristics of Korean writing, which include the arrangement of strokes and graphemes within a square syllabic form by dimensional and spatial rules. These findings support the notion that the Korean writing system possesses a language-specific nature with both linguistic and visuospatial/constructive processes. Distinctive patterns of dysgraphia in the Korean language also suggest interactivity between linguistic and visuospatial/constructive levels of processing. This study is noteworthy for its systematic description of

Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

Science.gov (United States)

Isokawa, M

1996-05-01

1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to

Muscle-Specific Mis-Splicing and Heart Disease Exemplified by RBM20.

Science.gov (United States)

Rexiati, Maimaiti; Sun, Mingming; Guo, Wei

2018-01-05

Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease.

Induced pluripotent stem cells-derived myeloid-derived suppressor cells regulate the CD8+ T cell response

Directory of Open Access Journals (Sweden)

Daniel Joyce

2018-05-01

Full Text Available Myeloid-derived suppressor cells (MDSCs are markedly increased in cancer patients and tumor-bearing mice and promote tumor growth and survival by inhibiting host innate and adaptive immunity. In this study, we generated and characterized MDSCs from murine-induced pluripotent stem cells (iPSCs. The iPSCs were co-cultured with OP9 cells, stimulated with GM-CSF, and became morphologically heterologous under co-culturing with hepatic stellate cells. Allogeneic and OVA-specific antigen stimulation demonstrated that iPS-MDSCs have a T-cell regulatory function. Furthermore, a popliteal lymph node assay and autoimmune hepatitis model showed that iPS-MDSCs also regulate immune responsiveness in vivo and have a therapeutic effect against hepatitis. Taken together, our results demonstrated a method of generating functional MDSCs from iPSCs and highlighted the potential of iPS-MDSCs as a key cell therapy resource for transplantation and autoimmune diseases. Keywords: Myeloid-derived suppressor cells, Induced pluripotent stem cells, T cell response

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

Science.gov (United States)

Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

2010-11-05

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

Directory of Open Access Journals (Sweden)

Zhiwen Ye

2017-04-01

Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy

Directory of Open Access Journals (Sweden)

Miki Ando

2015-10-01

Full Text Available The discovery of induced pluripotent stem cells (iPSCs has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9 into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

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Chunli Zhao

Full Text Available A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

Combining Patient-Reprogrammed Neural Cells and Proteomics as a Model to Study Psychiatric Disorders.

Science.gov (United States)

Zuccoli, Giuliana S; Martins-de-Souza, Daniel; Guest, Paul C; Rehen, Stevens K; Nascimento, Juliana Minardi

2017-01-01

The mechanisms underlying the pathophysiology of psychiatric disorders are still poorly known. Most of the studies about these disorders have been conducted on postmortem tissue or in limited preclinical models. The development of human induced pluripotent stem cells (iPSCs) has helped to increase the translational capacity of molecular profiling studies of psychiatric disorders through provision of human neuronal-like tissue. This approach consists of generation of pluripotent cells by genetically reprogramming somatic cells to produce the multiple neural cell types as observed within the nervous tissue. The finding that iPSCs can recapitulate the phenotype of the donor also affords the possibility of using this approach to study both the disease and control states in a given medical area. Here, we present a protocol for differentiation of human pluripotent stem cells to neural progenitor cells followed by subcellular fractionation which allows the study of specific cellular organelles and proteomic analysis.

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy.

Science.gov (United States)

Kehler, James; Greco, Marianna; Martino, Valentina; Pachiappan, Manickam; Yokoe, Hiroko; Chen, Alice; Yang, Miranda; Auerbach, Jonathan; Jessee, Joel; Gotte, Martin; Milanesi, Luciano; Albertini, Alberto; Bellipanni, Gianfranco; Zucchi, Ileana; Reinbold, Rolland A; Giordano, Antonio

2017-06-01

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Internal emitter dosimetry: are patient-specific calculations necessary?

International Nuclear Information System (INIS)

Sgouros, G.

1996-01-01

Full text: The question of whether patient-specific calculations are needed in internal emitter dosimetry arises when radionuclides are used for therapy. In diagnostic procedures the absorbed dose delivered to normal tissue is far below hazardous levels. In internal emitter therapy, the need for patient-specific dosimetry may arise if a large variability in biodistribution, normal tissue toxicity or efficacy is anticipated. Patient-specificity may be accomplished at the level of pharmacokinetics, anatomy/tumor-geometry or both. At the first level, information regarding the biodistribution of a particular radiolabeled agent is obtained and used to determine the maximum activity that may be administered for treatment. The classical example of this is radioiodine therapy for thyroid cancer. In radioiodine therapy, the therapy dose is preceded by a tracer dose of I-131-iodide which is used to measure patient kinetics by imaging and whole-body counting. Absorbed dose estimates obtained from these data are used to constrain the therapy dose to meet safety criteria established in a previously performed dose-response study. The most ambitious approach to patient-specific dosimetry, requires a three-dimensional set of images representing radionuclide distribution (SPECT or PET) and a corresponding set of registered images representing anatomy (CT or MRI). The spatial distribution of absorbed dose or dose-rate may then be obtained by convolution of a point-kernel with the radioactivity distribution or by Monte Carlo calculation. The spatial absorbed dose or dose-rate distribution may be represented as a set of images, as isodose contours, or as dose-volume histograms. The 3-D Monte Carlo approach is, in principle, the most patient-specific; it accounts for patient anatomy and tumor geometry as well as for the spatial distribution of radioactivity. It is also, however, the most logistically and technically demanding. Patients are required to undergo CT or MRI and at least one

Stemcell Information: SKIP000132 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available 301040 ... -- -- ... Yes No intractable disease-specific iPSC, ATR-X syndrome 難病性疾患克服事業iPS細胞バンク ATR-X 症候群...University 熊本大学 Takumi Era 江良　拓実 Not Available RikenBRC 理研BRC http://www.brc.riken.jp/lab/cell/english/patient_specific_ips.shtml ...

From Patient-Specific Mathematical Neuro-Oncology to Precision Medicine

Directory of Open Access Journals (Sweden)

Anne eBaldock

2013-04-01

Full Text Available Gliomas are notoriously aggressive, malignant brain tumors that have variable response to treatment. These patients often have poor prognosis, informed primarily by histopathology. Mathematical neuro-oncology (MNO is a young and burgeoning field that leverages mathematical models to predict and quantify response to therapies. These mathematical models can form the basis of modern precision medicine approaches to tailor therapy in a patient-specific manner. Patient specific models (PSMs can be used to overcome imaging limitations, improve prognostic predictions, stratify patients and assess treatment response in silico. The information gleaned from such models can aid in the construction and efficacy of clinical trials and treatment protocols, accelerating the pace of clinical research in the war on cancer. This review focuses on the growing translation of PSM to clinical neuro-oncology. It will also provide a forward-looking view on a new era of patient-specific mathematical neuro-oncology.

Representing Diversity in the Dish: Using Patient-Derived in Vitro Models to Recreate the Heterogeneity of Neurological Disease

Directory of Open Access Journals (Sweden)

Layla T. Ghaffari

2018-02-01

Full Text Available Neurological diseases, including dementias such as Alzheimer's disease (AD and fronto-temporal dementia (FTD and degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS, are responsible for an increasing fraction of worldwide fatalities. Researching these heterogeneous diseases requires models that endogenously express the full array of genetic and epigenetic factors which may influence disease development in both familial and sporadic patients. Here, we discuss the two primary methods of developing patient-derived neurons and glia to model neurodegenerative disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs, which are differentiated into neurons or glial cells, or directly converting (DC somatic cells into neurons (iNeurons or glial cells. Distinct differentiation techniques for both models result in a variety of neuronal and glial cell types, which have been successful in displaying unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease of genetic manipulation, expression of cell-specific markers, and recapitulation of disease pathogenesis are presented as determining factors in how these methods may be used separately or together to ascertain mechanisms of disease and identify therapeutics for distinct patient populations or for specific individuals in personalized medicine projects.

Patient specific ankle-foot orthoses using rapid prototyping.

Science.gov (United States)

Mavroidis, Constantinos; Ranky, Richard G; Sivak, Mark L; Patritti, Benjamin L; DiPisa, Joseph; Caddle, Alyssa; Gilhooly, Kara; Govoni, Lauren; Sivak, Seth; Lancia, Michael; Drillio, Robert; Bonato, Paolo

2011-01-12

Prefabricated orthotic devices are currently designed to fit a range of patients and therefore they do not provide individualized comfort and function. Custom-fit orthoses are superior to prefabricated orthotic devices from both of the above-mentioned standpoints. However, creating a custom-fit orthosis is a laborious and time-intensive manual process performed by skilled orthotists. Besides, adjustments made to both prefabricated and custom-fit orthoses are carried out in a qualitative manner. So both comfort and function can potentially suffer considerably. A computerized technique for fabricating patient-specific orthotic devices has the potential to provide excellent comfort and allow for changes in the standard design to meet the specific needs of each patient. In this paper, 3D laser scanning is combined with rapid prototyping to create patient-specific orthoses. A novel process was engineered to utilize patient-specific surface data of the patient anatomy as a digital input, manipulate the surface data to an optimal form using Computer Aided Design (CAD) software, and then download the digital output from the CAD software to a rapid prototyping machine for fabrication. Two AFOs were rapidly prototyped to demonstrate the proposed process. Gait analysis data of a subject wearing the AFOs indicated that the rapid prototyped AFOs performed comparably to the prefabricated polypropylene design. The rapidly prototyped orthoses fabricated in this study provided good fit of the subject's anatomy compared to a prefabricated AFO while delivering comparable function (i.e. mechanical effect on the biomechanics of gait). The rapid fabrication capability is of interest because it has potential for decreasing fabrication time and cost especially when a replacement of the orthosis is required.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

Science.gov (United States)

Lai, Frank Pui-Ling; Lau, Sin-Ting; Wong, John Kwong-Leong; Gui, Hongsheng; Wang, Reeson Xu; Zhou, Tingwen; Lai, Wing Hon; Tse, Hung-Fat; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Ngan, Elly Sau-Wai

2017-07-01

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET +/- and RET -/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can

Prospect of Induced Pluripotent Stem Cell Genetic Repair to Cure Genetic Diseases

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Jeanne Adiwinata Pawitan

2012-01-01

Full Text Available In genetic diseases, where the cells are already damaged, the damaged cells can be replaced by new normal cells, which can be differentiated from iPSC. To avoid immune rejection, iPSC from the patient’s own cell can be developed. However, iPSC from the patients’s cell harbors the same genetic aberration. Therefore, before differentiating the iPSCs into required cells, genetic repair should be done. This review discusses the various technologies to repair the genetic aberration in patient-derived iPSC, or to prevent the genetic aberration to cause further damage in the iPSC-derived cells, such as Zn finger and TALE nuclease genetic editing, RNA interference technology, exon skipping, and gene transfer method. In addition, the challenges in using the iPSC and the strategies to manage the hurdles are addressed.

Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

Science.gov (United States)

Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

2017-11-01

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

Science.gov (United States)

Chen, Jian; Lin, Mingyan; Foxe, John J; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M

2013-01-01

Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (pdisease-modeling neuropsychiatric disorder and may have some advantages over those derived from F-iPSCs.

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

Science.gov (United States)

Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

2016-03-10

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Patient specific ankle-foot orthoses using rapid prototyping

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Sivak Seth

2011-01-01

Full Text Available Abstract Background Prefabricated orthotic devices are currently designed to fit a range of patients and therefore they do not provide individualized comfort and function. Custom-fit orthoses are superior to prefabricated orthotic devices from both of the above-mentioned standpoints. However, creating a custom-fit orthosis is a laborious and time-intensive manual process performed by skilled orthotists. Besides, adjustments made to both prefabricated and custom-fit orthoses are carried out in a qualitative manner. So both comfort and function can potentially suffer considerably. A computerized technique for fabricating patient-specific orthotic devices has the potential to provide excellent comfort and allow for changes in the standard design to meet the specific needs of each patient. Methods In this paper, 3D laser scanning is combined with rapid prototyping to create patient-specific orthoses. A novel process was engineered to utilize patient-specific surface data of the patient anatomy as a digital input, manipulate the surface data to an optimal form using Computer Aided Design (CAD software, and then download the digital output from the CAD software to a rapid prototyping machine for fabrication. Results Two AFOs were rapidly prototyped to demonstrate the proposed process. Gait analysis data of a subject wearing the AFOs indicated that the rapid prototyped AFOs performed comparably to the prefabricated polypropylene design. Conclusions The rapidly prototyped orthoses fabricated in this study provided good fit of the subject's anatomy compared to a prefabricated AFO while delivering comparable function (i.e. mechanical effect on the biomechanics of gait. The rapid fabrication capability is of interest because it has potential for decreasing fabrication time and cost especially when a replacement of the orthosis is required.

Age-specific mortality among TB patients in Denmark 1998-2010

DEFF Research Database (Denmark)

Fløe, Andreas; Løkke, Anders; Ibsen, Rikke

Objective: To evaluate the age-specific mortality in a national TB cohort, and to estimate relative age-specific mortality compared with matched controls, in a retrospective case-control study. Methods: Using Danish National Patient Registry, we retrospectively identified TB-patients between 1998...... to matched controls. While the difference in survival is substantial among elderly patients, a high relative risk of dying is particularly of concern among young and middle-aged adult TB patients....

Induced pluripotent stem cells derived from a patient with autosomal dominant familial neurohypophyseal diabetes insipidus caused by a variant in the AVP gene

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Lise Bols Toustrup

2017-03-01

Full Text Available Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI is caused by variants in the arginine vasopressin (AVP gene. Here we report the generation of induced pluripotent stem cells (iPSCs from a 42-year-old man carrying an adFNDI causing variant in exon 1 of the AVP gene using lentivirus-mediated nuclear reprogramming. The iPSCs carried the expected variant in the AVP gene. Furthermore, the iPSCs expressed pluripotency markers; displayed in vitro differentiation potential to the three germ layers and had a normal karyotype consistent with the original fibroblasts. This iPSC line is useful in future studies focusing on the pathogenesis of adFNDI.

Surgeon Design Interface for Patient-Specific Concentric Tube Robots.

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Morimoto, Tania K; Greer, Joseph D; Hsieh, Michael H; Okamura, Allison M

2016-06-01

Concentric tube robots have potential for use in a wide variety of surgical procedures due to their small size, dexterity, and ability to move in highly curved paths. Unlike most existing clinical robots, the design of these robots can be developed and manufactured on a patient- and procedure-specific basis. The design of concentric tube robots typically requires significant computation and optimization, and it remains unclear how the surgeon should be involved. We propose to use a virtual reality-based design environment for surgeons to easily and intuitively visualize and design a set of concentric tube robots for a specific patient and procedure. In this paper, we describe a novel patient-specific design process in the context of the virtual reality interface. We also show a resulting concentric tube robot design, created by a pediatric urologist to access a kidney stone in a pediatric patient.

Derivation of the Duchenne muscular dystrophy patient-derived induced pluripotent stem cell line lacking DMD exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50

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Gabriella Spaltro

2017-12-01

Full Text Available Duchenne muscular dystrophy (DMD is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3 displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50.

Generation of an induced pluripotent stem cell line, IBMS-iPSC-014-05, from a female autosomal dominant polycystic kidney disease patient carrying a common mutation of R803X in PKD2

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Ming-Ching Ho

2017-12-01

Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is one of the most commonly inherited forms of polycystic kidney disease, and is characterized by the growth of numerous cysts in both kidneys. Here we generated an induced pluripotent stem cell (iPSC line from the peripheral blood mononuclear cells (PBMCs of a 63-year-old female ADPKD patient carrying an R803X mutation in the PKD2 gene using the Sendai-virus delivery system. Downstream characterization of these iPSCs showed that they possessed normal karyotyping, were free of genomic integration, retained the disease-causing PKD2 mutation, expressed pluripotency markers and could differentiate into three germ layers.

CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Giacalone, Joseph C; Sharma, Tasneem P; Burnight, Erin R; Fingert, John F; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

2018-02-28

Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Patient-specific radiation dose and cancer risk estimation in CT: Part II. Application to patients

Energy Technology Data Exchange (ETDEWEB)

Li Xiang; Samei, Ehsan; Segars, W. Paul; Sturgeon, Gregory M.; Colsher, James G.; Toncheva, Greta; Yoshizumi, Terry T.; Frush, Donald P. [Medical Physics Graduate Program, Carl E. Ravin Advanced Imaging Laboratories, Department of Radiology, Duke University Medical Center, Durham, North Carolina 27705 (United States); Carl E. Ravin Advanced Imaging Laboratories, Department of Radiology, Medical Physics Graduate Program, Department of Physics, and Department of Biomedical Engineering, Duke University Medical Center, Durham, North Carolina 27705 (United States); Carl E. Ravin Advanced Imaging Laboratories, Department of Radiology, Medical Physics Graduate Program, Duke University Medical Center, Durham, North Carolina 27705 (United States); Carl E. Ravin Advanced Imaging Laboratories, Department of Radiology, Duke University Medical Center, Durham, North Carolina 27705 and Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina 27599 (United States); Department of Radiology, Duke University Medical Center, Durham, North Carolina 27705 (United States); Duke Radiation Dosimetry Laboratory, Department of Radiology, Duke University Medical Center, Durham, North Carolina 27705 (United States); Duke Radiation Dosimetry Laboratory, Department of Radiology, Medical Physics Graduate Program, Duke University Medical Center, Durham, North Carolina 27705 (United States); Division of Pediatric Radiology, Department of Radiology, Medical Physics Graduate Program, Duke University Medical Center, Durham, North Carolina 27710 (United States)

2011-01-15

Purpose: Current methods for estimating and reporting radiation dose from CT examinations are largely patient-generic; the body size and hence dose variation from patient to patient is not reflected. Furthermore, the current protocol designs rely on dose as a surrogate for the risk of cancer incidence, neglecting the strong dependence of risk on age and gender. The purpose of this study was to develop a method for estimating patient-specific radiation dose and cancer risk from CT examinations. Methods: The study included two patients (a 5-week-old female patient and a 12-year-old male patient), who underwent 64-slice CT examinations (LightSpeed VCT, GE Healthcare) of the chest, abdomen, and pelvis at our institution in 2006. For each patient, a nonuniform rational B-spine (NURBS) based full-body computer model was created based on the patient's clinical CT data. Large organs and structures inside the image volume were individually segmented and modeled. Other organs were created by transforming an existing adult male or female full-body computer model (developed from visible human data) to match the framework defined by the segmented organs, referencing the organ volume and anthropometry data in ICRP Publication 89. A Monte Carlo program previously developed and validated for dose simulation on the LightSpeed VCT scanner was used to estimate patient-specific organ dose, from which effective dose and risks of cancer incidence were derived. Patient-specific organ dose and effective dose were compared with patient-generic CT dose quantities in current clinical use: the volume-weighted CT dose index (CTDI{sub vol}) and the effective dose derived from the dose-length product (DLP). Results: The effective dose for the CT examination of the newborn patient (5.7 mSv) was higher but comparable to that for the CT examination of the teenager patient (4.9 mSv) due to the size-based clinical CT protocols at our institution, which employ lower scan techniques for smaller

An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion.

Science.gov (United States)

Shutova, Maria V; Surdina, Anastasia V; Ischenko, Dmitry S; Naumov, Vladimir A; Bogomazova, Alexandra N; Vassina, Ekaterina M; Alekseev, Dmitry G; Lagarkova, Maria A; Kiselev, Sergey L

2016-01-01

The pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

Science.gov (United States)

Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

2014-12-01

Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

Directory of Open Access Journals (Sweden)

Xuan Guan

2014-03-01

Full Text Available The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD. Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs. USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

Overcoming reprogramming resistance of Fanconi anemia cells

Science.gov (United States)

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells

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Liang-Fang Zhu

2017-03-01

Full Text Available Objective: The present study aimed to establish an induced pluripotent stem cell (iPSC line from acute myelogenous leukemia (AML cells in vitro and identify their biological characteristics. Methods: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. Results: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. Conclusion: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

Science.gov (United States)

Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

Induced pluripotent stem cells and personalized medicine: current progress and future perspectives

OpenAIRE

Chun, Yong Soon; Byun, Kyunghee; Lee, Bonghee

2011-01-01

Generation of induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. iPSCs can self-renew and can differentiate into many cell types, offering a potentially unlimited source of cells for targeted differentiation into somatic effector cells. Hence, iPSCs are likely to be invaluable for therapeutic applications and disease-related research. In this review, we summar...

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications.

Science.gov (United States)

Yoshida, Yoshinori; Yamanaka, Shinya

2017-06-09

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that have features similar to embryonic stem cells, such as the capacity of self-renewal and differentiation into many types of cells, including cardiac myocytes. Although initially the reprogramming efficiency was low, several improvements in reprogramming methods have achieved robust and efficient generation of iPSCs without genomic insertion of transgenes. iPSCs display clonal variations in epigenetic and genomic profiles and cellular behavior in differentiation. iPSC-derived cardiac myocytes (iPSC cardiac myocytes) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models, and are useful for drug discovery and toxicology testing. In addition, iPSC cardiac myocytes can help with patient stratification in regard to drug responsiveness. Furthermore, they can be used as source cells for cardiac regeneration in animal models. Here, we review recent progress in iPSC technology and its applications to cardiac diseases. © 2017 American Heart Association, Inc.

Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.

Science.gov (United States)

Liu, Ying; Deng, Wenbin

2016-05-01

With the technology of reprogramming somatic cells by introducing defined transcription factors that enables the generation of "induced pluripotent stem cells (iPSCs)" with pluripotency comparable to that of embryonic stem cells (ESCs), it has become possible to use this technology to produce various cells and tissues that have been difficult to obtain from living bodies. This advancement is bringing forth rapid progress in iPSC-based disease modeling, drug screening, and regenerative medicine. More and more studies have demonstrated that phenotypes of adult-onset neurodegenerative disorders could be rather faithfully recapitulated in iPSC-derived neural cell cultures. Moreover, despite the adult-onset nature of the diseases, pathogenic phenotypes and cellular abnormalities often exist in early developmental stages, providing new "windows of opportunity" for understanding mechanisms underlying neurodegenerative disorders and for discovering new medicines. The cell reprogramming technology enables a reverse engineering approach for modeling the cellular degenerative phenotypes of a wide range of human disorders. An excellent example is the study of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS) using iPSCs. ALS is a progressive neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs), culminating in muscle wasting and death from respiratory failure. The iPSC approach provides innovative cell culture platforms to serve as ALS patient-derived model systems. Researchers have converted iPSCs derived from ALS patients into MNs and various types of glial cells, all of which are involved in ALS, to study the disease. The iPSC technology could be used to determine the role of specific genetic factors to track down what's wrong in the neurodegenerative disease process in the "disease-in-a-dish" model. Meanwhile, parallel experiments of targeting the same specific genes in human ESCs could also be performed to control

Dose distribution in organs: patient-specific phantoms versus reference phantoms

Energy Technology Data Exchange (ETDEWEB)

Lacerda, I.V.B., E-mail: isabelle.lacerda@ufpe.br [Universidade Federal de Pernambuco (UFPE), Recife (Brazil); Vieira, J.W. [Instituto Federal de Pernambuco (IFPE), Recife (Brazil); Oliveira, M.L.; Lima, F.R.A. [Centro Regional de Ciências Nucleares do Nordeste (CRCN-NE/CNEN-PB), Recife (Brazil)

2017-07-01

Discrepancies between ICRP phantoms and real patients lead to disparities on patient-dose estimations. This paper aims to compare distribution of dose in organs of male/female specific-phantoms and ICRP reference phantoms. The absorbed dose estimation was performed using the EGSnrc Monte Carlo code and a parallel source algorithm. In this work were used a patient-specific phantom for a man (1.73m/70.3kg) and another for a woman (1.63m/60.3kg) and the male and female ICRP reference phantoms. The comparison of the absorbed dose from each phantom gender was performed using the relative error. The results were expressed in terms of conversion coefficients to brain, lungs, liver and kidneys. The greatest absolute relative error between the organs of the patient-specific phantom and the reference phantom was 22.92% in the liver and 62.84% in the kidneys, respectively for man and woman. There are errors that cannot be disregarded. This paper shows the need for a specific study for each patient or for the population of each country, since there are different body types, which affects the distribution of the organ doses. (author)

Dose distribution in organs: patient-specific phantoms versus reference phantoms

International Nuclear Information System (INIS)

Lacerda, I.V.B.; Vieira, J.W.; Oliveira, M.L.; Lima, F.R.A.

2017-01-01

Discrepancies between ICRP phantoms and real patients lead to disparities on patient-dose estimations. This paper aims to compare distribution of dose in organs of male/female specific-phantoms and ICRP reference phantoms. The absorbed dose estimation was performed using the EGSnrc Monte Carlo code and a parallel source algorithm. In this work were used a patient-specific phantom for a man (1.73m/70.3kg) and another for a woman (1.63m/60.3kg) and the male and female ICRP reference phantoms. The comparison of the absorbed dose from each phantom gender was performed using the relative error. The results were expressed in terms of conversion coefficients to brain, lungs, liver and kidneys. The greatest absolute relative error between the organs of the patient-specific phantom and the reference phantom was 22.92% in the liver and 62.84% in the kidneys, respectively for man and woman. There are errors that cannot be disregarded. This paper shows the need for a specific study for each patient or for the population of each country, since there are different body types, which affects the distribution of the organ doses. (author)

Regeneration of cervical reserve cell-like cells from human induced pluripotent stem cells (iPSCs): A new approach to finding targets for cervical cancer stem cell treatment.

Science.gov (United States)

Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Ogishima, Juri; Eguchi, Satoko; Yamashita, Aki; Tomio, Kensuke; Wada-Hiraike, Osamu; Oda, Katsutoshi; Nagamatsu, Takeshi; Osuga, Yutaka; Fujii, Tomoyuki

2017-06-20

Cervical reserve cells are epithelial progenitor cells that are pathologically evident as the origin of cervical cancer. Thus, investigating the characteristics of cervical reserve cells could yield insight into the features of cervical cancer stem cells (CSCs). In this study, we established a method for the regeneration of cervical reserve cell-like properties from human induced pluripotent stem cells (iPSCs) and named these cells induced reserve cell-like cells (iRCs). Approximately 70% of iRCs were positive for the reserve cell markers p63, CK5 and CK8. iRCs also expressed the SC junction markers CK7, AGR2, CD63, MMP7 and GDA. While iRCs expressed neither ERα nor ERβ, they expressed CA125. These data indicated that iRCs possessed characteristics of cervical epithelial progenitor cells. iRCs secreted higher levels of several inflammatory cytokines such as macrophage migration inhibitory factor (MIF), soluble intercellular adhesion molecule 1 (sICAM-1) and C-X-C motif ligand 10 (CXCL-10) compared with normal cervical epithelial cells. iRCs also expressed human leukocyte antigen-G (HLA-G), which is an important cell-surface antigen for immune tolerance and carcinogenesis. Together with the fact that cervical CSCs can originate from reserve cells, our data suggested that iRCs were potent immune modulators that might favor cervical cancer cell survival. In conclusion, by generating reserve cell-like properties from iPSCs, we provide a new approach that may yield new insight into cervical cancer stem cells and help find new oncogenic targets.

Mucorales-Specific T Cells in Patients with Hematologic Malignancies

OpenAIRE

Potenza, L; Vallerini, D; Barozzi, P; Riva, G; Gilioli, A; Forghieri, F; Candoni, A; Cesaro, S; Quadrelli, C; Maertens, J; Rossi, G; Morselli, M; Codeluppi, M; Mussini, C; Colaci, E

2016-01-01

Background Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. Method...

ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation

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Maricela Maldonado

2016-09-01

Full Text Available Robust control of human induced pluripotent stem cell (hIPSC differentiation is essential to realize its patient-tailored therapeutic potential. Here, we demonstrate a novel application of Y-27632, a small molecule Rho-associated protein kinase (ROCK inhibitor, to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin, a cell-cell junctional protein, proportional to the increased exposure to Y-27632. Interestingly, gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36 h. Simultaneously, epithelial-to-mesenchymal (EMT transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast, an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively, these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632.

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

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Alvaro Muñoz-López

2016-10-01

Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

Patient Specific Modeling of Head-Up Tilt

DEFF Research Database (Denmark)

Williams, Nakeya; Wright, Andrew; Mehlsen, Jesper

2014-01-01

Short term cardiovascular responses to head-up tilt (HUT) experiments involve complex cardiovascular regulation in order to maintain blood pressure at homeostatic levels. This manuscript presents a patient specific compartmental model developed to predict dynamic changes in heart rate and arterial...

Applications and development of communication models for the touchstone GAMMA and DELTA prototypes

Science.gov (United States)

Seidel, Steven R.

1993-01-01

The goal of this project was to develop models of the interconnection networks of the Intel iPSC/860 and DELTA multicomputers to guide the design of efficient algorithms for interprocessor communication in problems that commonly occur in CFD codes and other applications. Interprocessor communication costs of codes for message-passing architectures such as the iPSC/860 and DELTA significantly affect the level of performance that can be obtained from those machines. This project addressed several specific problems in the achievement of efficient communication on the Intel iPSC/860 hypercube and DELTA mesh. In particular, an efficient global processor synchronization algorithm was developed for the iPSC/860 and numerous broadcast algorithms were designed for the DELTA.

New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells.

Science.gov (United States)

Garate, Zita; Davis, Brian R; Quintana-Bustamante, Oscar; Segovia, Jose C

2013-06-01

Advances in cell and gene therapy are opening up new avenues for regenerative medicine. Because of their acquired pluripotency, human induced pluripotent stem cells (hiPSCs) are a promising source of autologous cells for regenerative medicine. They show unlimited self-renewal while retaining the ability, in principle, to differentiate into any cell type of the human body. Since Yamanaka and colleagues first reported the generation of hiPSCs in 2007, significant efforts have been made to understand the reprogramming process and to generate hiPSCs with potential for clinical use. On the other hand, the development of gene-editing platforms to increase homologous recombination efficiency, namely DNA nucleases (zinc finger nucleases, TAL effector nucleases, and meganucleases), is making the application of locus-specific gene therapy in human cells an achievable goal. The generation of patient-specific hiPSC, together with gene correction by homologous recombination, will potentially allow for their clinical application in the near future. In fact, reports have shown targeted gene correction through DNA-Nucleases in patient-specific hiPSCs. Various technologies have been described to reprogram patient cells and to correct these patient hiPSCs. However, no approach has been clearly more efficient and safer than the others. In addition, there are still significant challenges for the clinical application of these technologies, such as inefficient differentiation protocols, genetic instability resulting from the reprogramming process and hiPSC culture itself, the efficacy and specificity of the engineered DNA nucleases, and the overall homologous recombination efficiency. To summarize advances in the generation of gene corrected patient-specific hiPSCs, this review focuses on the available technological platforms, including their strengths and limitations regarding future therapeutic use of gene-corrected hiPSCs.

Modeling retinal degeneration using patient-specific induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Zi-Bing Jin

Full Text Available Retinitis pigmentosa (RP is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

Science.gov (United States)

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association.

Science.gov (United States)

Musunuru, Kiran; Sheikh, Farah; Gupta, Rajat M; Houser, Steven R; Maher, Kevin O; Milan, David J; Terzic, Andre; Wu, Joseph C

2018-01-01

Induced pluripotent stem cells (iPSCs) offer an unprece-dented opportunity to study human physiology and disease at the cellular level. They also have the potential to be leveraged in the practice of precision medicine, for example, personalized drug testing. This statement comprehensively describes the provenance of iPSC lines, their use for cardiovascular disease modeling, their use for precision medicine, and strategies through which to promote their wider use for biomedical applications. Human iPSCs exhibit properties that render them uniquely qualified as model systems for studying human diseases: they are of human origin, which means they carry human genomes; they are pluripotent, which means that in principle, they can be differentiated into any of the human body's somatic cell types; and they are stem cells, which means they can be expanded from a single cell into millions or even billions of cell progeny. iPSCs offer the opportunity to study cells that are genetically matched to individual patients, and genome-editing tools allow introduction or correction of genetic variants. Initial progress has been made in using iPSCs to better understand cardiomyopathies, rhythm disorders, valvular and vascular disorders, and metabolic risk factors for ischemic heart disease. This promising work is still in its infancy. Similarly, iPSCs are only just starting to be used to identify the optimal medications to be used in patients from whom the cells were derived. This statement is intended to (1) summarize the state of the science with respect to the use of iPSCs for modeling of cardiovascular traits and disorders and for therapeutic screening; (2) identify opportunities and challenges in the use of iPSCs for disease modeling and precision medicine; and (3) outline strategies that will facilitate the use of iPSCs for biomedical applications. This statement is not intended to address the use of stem cells as regenerative therapy, such as transplantation into the body to

Morphing patient-specific musculoskeletal models

DEFF Research Database (Denmark)

Rasmussen, John; Galibarov, Pavel E.; Al-Munajjed, Amir

the resulting models do indeed represent the patients’ biomechanics. As a particularly challenging case, foot deformities based only on point sets recovered from surface scans are considered as shown in the figure. The preliminary results are promising for the cases of severe flat foot and metatarsalgia while...... other conditions may require CT or MRI data. The method and its theoretical assumptions, advantages and limitations are presented, and several examples will illustrate morphing to patient-specific models. [1] Carbes S; Tørholm S; Rasmussen, J. A Detailed Twenty-six Segments Kinematic Foot model...

Prostate Cancer–Specific Mortality After Radical Prostatectomy for Patients Treated in the Prostate-Specific Antigen Era

Science.gov (United States)

Stephenson, Andrew J.; Kattan, Michael W.; Eastham, James A.; Bianco, Fernando J.; Yossepowitch, Ofer; Vickers, Andrew J.; Klein, Eric A.; Wood, David P.; Scardino, Peter T.

2009-01-01

Purpose The long-term risk of prostate cancer–specific mortality (PCSM) after radical prostatectomy is poorly defined for patients treated in the era of widespread prostate-specific antigen (PSA) screening. Models that predict the risk of PCSM are needed for patient counseling and clinical trial design. Methods A multi-institutional cohort of 12,677 patients treated with radical prostatectomy between 1987 and 2005 was analyzed for the risk of PCSM. Patient clinical information and treatment outcome was modeled using Fine and Gray competing risk regression analysis to predict PCSM. Results Fifteen-year PCSM and all-cause mortality were 12% and 38%, respectively. The estimated PCSM ranged from 5% to 38% for patients in the lowest and highest quartiles of predicted risk of PSA-defined recurrence, based on a popular nomogram. Biopsy Gleason grade, PSA, and year of surgery were associated with PCSM. A nomogram predicting the 15-year risk of PCSM was developed, and the externally validated concordance index was 0.82. Neither preoperative PSA velocity nor body mass index improved the model's accuracy. Only 4% of contemporary patients had a predicted 15-year PCSM of greater than 5%. Conclusion Few patients will die from prostate cancer within 15 years of radical prostatectomy, despite the presence of adverse clinical features. This favorable prognosis may be related to the effectiveness of radical prostatectomy (with or without secondary therapy) or the low lethality of screen-detected cancers. Given the limited ability to identify contemporary patients at substantially elevated risk of PCSM on the basis of clinical features alone, the need for novel markers specifically associated with the biology of lethal prostate cancer is evident. PMID:19636023

CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells

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Daniel Gómez-Cabello

2017-02-01

Full Text Available Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs. All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.

Establishment of human iPSC line NCCSi003-A from CD34+cells of peripheral blood collected during apheresis of healthy donor from Indian ethnicity

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Sophia Fernandes

2018-03-01

Full Text Available We present generation of iPSCs from CD34+ cells isolated from peripheral blood, collected during apheresis of a healthy female individual. We nucleofected the CD34+cells by episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative mutation in p53. The resultant colonies showed cobble-stone appearance and stained positive for alkaline phosphatase. The colonies demonstrated presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells subsequently during passages as assessed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate to cells from all three-germ lineages in vitro.

Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency

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Adele Mucci

2016-08-01

Full Text Available Induced pluripotent stem cells (iPSCs represent an innovative source for the standardized in vitro generation of macrophages (Mφ. We here describe a robust and efficient protocol to obtain mature and functional Mφ from healthy as well as disease-specific murine iPSCs. With regard to morphology, surface phenotype, and function, our iPSC-derived Mφ (iPSC-Mφ closely resemble their counterparts generated in vitro from bone marrow cells. Moreover, when we investigated the feasibility of our differentiation system to serve as a model for rare congenital diseases associated with Mφ malfunction, we were able to faithfully recapitulate the pathognomonic defects in GM-CSF signaling and Mφ function present in hereditary pulmonary alveolar proteinosis (herPAP. Thus, our studies may help to overcome the limitations placed on research into certain rare disease entities by the lack of an adequate supply of disease-specific primary cells, and may aid the development of novel therapeutic approaches for herPAP patients.

Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein

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Marc Ehrlich

2015-07-01

Full Text Available Frontotemporal dementia (FTD is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.

Calculating patient specific doses in X-ray diagnostics and from radiopharmaceuticals

International Nuclear Information System (INIS)

Lampinen, J.

2000-01-01

The risk associated with exposure to ionising radiation is dependent on the characteristics of the exposed individual. The size and structure of the individual influences the absorbed dose distribution in the organs. Traditional methods used to calculate the patient organ doses are based on standardised calculation phantoms, which neglect the variance of the patient size or even sex. When estimating the radiation dose of an individual patient, patient specific calculation methods must be used. Methods for patient specific dosimetry in the fields of X-ray diagnostics and diagnostic and therapeutic use of radiopharmaceuticals were proposed in this thesis. A computer program, ODS-60, for calculating organ doses from diagnostic X-ray exposures was presented. The calculation is done in a patient specific phantom with depth dose and profile algorithms fitted to Monte Carlo simulation data from a previous study. Improvements to the version reported earlier were introduced, e.g. bone attenuation was implemented. The applicability of the program to determine patient doses from complex X-ray examinations (barium enema examination) was studied. The conversion equations derived for female and male patients as a function of patient weight gave the smallest deviation from the actual patient doses when compared to previous studies. Another computer program, Intdose, was presented for calculation of the dose distribution from radiopharmaceuticals. The calculation is based on convolution of an isotope specific point dose kernel with activity distribution, obtained from single photon emission computed tomography (SPECT) images. Anatomical information is taken from magnetic resonance (MR) or computed tomography (CT) images. According to a phantom study, Intdose agreed within 3 % with measurements. For volunteers administered diagnostic radiopharmaceuticals, the results given by Intdose were found to agree with traditional methods in cases of medium sized patients. For patients

Patient specific 3D printed phantom for IMRT quality assurance

International Nuclear Information System (INIS)

Ehler, Eric D; Higgins, Patrick D; Dusenbery, Kathryn E; Barney, Brett M

2014-01-01

The purpose of this study was to test the feasibility of a patient specific phantom for patient specific dosimetric verification. Using the head and neck region of an anthropomorphic phantom as a substitute for an actual patient, a soft-tissue equivalent model was constructed with the use of a 3D printer. Calculated and measured dose in the anthropomorphic phantom and the 3D printed phantom was compared for a parallel-opposed head and neck field geometry to establish tissue equivalence. A nine-field IMRT plan was constructed and dose verification measurements were performed for the 3D printed phantom as well as traditional standard phantoms. The maximum difference in calculated dose was 1.8% for the parallel-opposed configuration. Passing rates of various dosimetric parameters were compared for the IMRT plan measurements; the 3D printed phantom results showed greater disagreement at superficial depths than other methods. A custom phantom was created using a 3D printer. It was determined that the use of patient specific phantoms to perform dosimetric verification and estimate the dose in the patient is feasible. In addition, end-to-end testing on a per-patient basis was possible with the 3D printed phantom. Further refinement of the phantom construction process is needed for routine use. (paper)

Patient-specific dosimetric endpoints based treatment plan quality control in radiotherapy

International Nuclear Information System (INIS)

Song, Ting; Zhou, Linghong; Staub, David; Chen, Mingli; Lu, Weiguo; Tian, Zhen; Jia, Xun; Li, Yongbao; Jiang, Steve B; Gu, Xuejun

2015-01-01

In intensity modulated radiotherapy (IMRT), the optimal plan for each patient is specific due to unique patient anatomy. To achieve such a plan, patient-specific dosimetric goals reflecting each patient’s unique anatomy should be defined and adopted in the treatment planning procedure for plan quality control. This study is to develop such a personalized treatment plan quality control tool by predicting patient-specific dosimetric endpoints (DEs). The incorporation of patient specific DEs is realized by a multi-OAR geometry-dosimetry model, capable of predicting optimal DEs based on the individual patient’s geometry. The overall quality of a treatment plan is then judged with a numerical treatment plan quality indicator and characterized as optimal or suboptimal. Taking advantage of clinically available prostate volumetric modulated arc therapy (VMAT) treatment plans, we built and evaluated our proposed plan quality control tool. Using our developed tool, six of twenty evaluated plans were identified as sub-optimal plans. After plan re-optimization, these suboptimal plans achieved better OAR dose sparing without sacrificing the PTV coverage, and the dosimetric endpoints of the re-optimized plans agreed well with the model predicted values, which validate the predictability of the proposed tool. In conclusion, the developed tool is able to accurately predict optimally achievable DEs of multiple OARs, identify suboptimal plans, and guide plan optimization. It is a useful tool for achieving patient-specific treatment plan quality control. (paper)

Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy

Directory of Open Access Journals (Sweden)

Lara Marrone

2018-02-01

Full Text Available Summary: Perturbations in stress granule (SG dynamics may be at the core of amyotrophic lateral sclerosis (ALS. Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. : Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS. Keywords: amyotrophic lateral sclerosis, induced pluripotent stem cells, FUS, stress granules, autophagy, gene editing, CRISPR/Cas9n

Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages

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Anna-Lena Neehus

2018-01-01

Full Text Available Mendelian susceptibility to mycobacterial disease (MSMD is caused by inborn errors of interferon gamma (IFNγ immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet induced pluripotent stem cells (iPSCs from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.

Patient-specific cardiac phantom for clinical training and preprocedure surgical planning.

Science.gov (United States)

Laing, Justin; Moore, John; Vassallo, Reid; Bainbridge, Daniel; Drangova, Maria; Peters, Terry

2018-04-01

Minimally invasive mitral valve repair procedures including MitraClip ® are becoming increasingly common. For cases of complex or diseased anatomy, clinicians may benefit from using a patient-specific cardiac phantom for training, surgical planning, and the validation of devices or techniques. An imaging compatible cardiac phantom was developed to simulate a MitraClip ® procedure. The phantom contained a patient-specific cardiac model manufactured using tissue mimicking materials. To evaluate accuracy, the patient-specific model was imaged using computed tomography (CT), segmented, and the resulting point cloud dataset was compared using absolute distance to the original patient data. The result, when comparing the molded model point cloud to the original dataset, resulted in a maximum Euclidean distance error of 7.7 mm, an average error of 0.98 mm, and a standard deviation of 0.91 mm. The phantom was validated using a MitraClip ® device to ensure anatomical features and tools are identifiable under image guidance. Patient-specific cardiac phantoms may allow for surgical complications to be accounted for preoperative planning. The information gained by clinicians involved in planning and performing the procedure should lead to shorter procedural times and better outcomes for patients.

Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

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Yoshikazu Kishino

Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells

Science.gov (United States)

Kim, Kitai; Zhao, Rui; Doi, Akiko; Ng, Kitwa; Unternaehrer, Juli; Cahan, Patrick; Hongguang, Huo; Loh, Yuin-Han; Aryee, Martin J.; Lensch, M. William; Li, Hu; Collins, James J.; Feinberg, Andrew P.; Daley, George Q.

2012-01-01

We compared bona-fide human induced pluripotent stem cells (iPSC) derived from umbilical cord blood (CB) and neonatal keratinocytes (K). As a consequence of both incomplete erasure of tissue-specific methylation and aberrant de novo methylation, CB-iPSC and K-iPSC are distinct in genome-wide DNA methylation profiles and differentiation potential. Extended passage of some iPSC clones in culture didn't improve their epigenetic resemblance to ESC, implying that some human iPSC retain a residual “epigenetic memory” of their tissue of origin. PMID:22119740

Managing patients with acute and chronic non-specific neck pain

DEFF Research Database (Denmark)

Brockhusen, Simon Sidenius; Bussières, André; French, Simon David

2017-01-01

was mainly reserved for chronic patients. Danish chiropractors' compliance with guidelines for neck-pain patients was low, but is neither worse nor better than what is seen for other complaints or health disciplines. Our findings suggest a need for active knowledge translation strategies and robust......Background: Non-specific neck pain represents a quarter of all chiropractic patient visits in Denmark. Evidence informed practice can help ensure providers use best available treatment, speed up patient recovery rate and reduce healthcare utilization. It is generally believed that Danish...... chiropractors treat according to best practice, but we do not know if this is true for management of neck-pain. The objective of this study was to investigate how Danish chiropractors treat patients with acute and chronic non-specific neck pain and determine if management is compliant with recent Canadian...

Cause-specific mortality in HPV+ and HPV- oropharyngeal cancer patients

DEFF Research Database (Denmark)

Nørregaard, Cecilie; Grønhøj, Christian; Jensen, David

2018-01-01

Identifying the causes of death in head and neck cancer patients can optimize follow-up and therapeutic strategies, but studies in oropharyngeal squamous cell carcinoma (OPSCC) patients stratified by HPV status are lacking. We report cause-specific mortality in a population-based cohort of patients...... with OPSCC. Patients who had been diagnosed with OPSCC (n = 1541) between 2000 and 2014 in eastern Denmark were included in the study. Causes of death were collected through medical files and the Danish National Cause of Death registry. Deaths were grouped as (1) primary oropharyngeal cancer, (2) secondary...... malignancies, (3) cardiovascular and pulmonary disease, or (4) other/unspecified. The cumulative incidence of death and specific causes of death were determined using risk analysis. At follow-up, 723 (47.5%) patients had died. The median time to and cause of death were determined: oropharyngeal cancer (n = 432...

Emergence of a Stage-Dependent Human Liver Disease Signature with Directed Differentiation of Alpha-1 Antitrypsin-Deficient iPS Cells

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Andrew A. Wilson

2015-05-01

Full Text Available Induced pluripotent stem cells (iPSCs provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ in the gene responsible for alpha-1 antitrypsin (AAT deficiency, we find that the global transcriptomes of PiZZ iPSCs diverge from normal controls upon differentiation to hepatic cells. Expression of 135 genes distinguishes PiZZ iPSC-hepatic cells, providing potential clues to liver disease pathogenesis. The disease-specific cells display intracellular accumulation of mutant AAT protein, resulting in increased autophagic flux. Furthermore, we detect beneficial responses to the drug carbamazepine, which further augments autophagic flux, but adverse responses to known hepatotoxic drugs. Our findings support the utility of iPSCs as tools for drug development or prediction of toxicity.

Patient- and cohort-specific dose and risk estimation for abdominopelvic CT: a study based on 100 patients

Science.gov (United States)

Tian, Xiaoyu; Li, Xiang; Segars, W. Paul; Frush, Donald P.; Samei, Ehsan

2012-03-01

The purpose of this work was twofold: (a) to estimate patient- and cohort-specific radiation dose and cancer risk index for abdominopelvic computer tomography (CT) scans; (b) to evaluate the effects of patient anatomical characteristics (size, age, and gender) and CT scanner model on dose and risk conversion coefficients. The study included 100 patient models (42 pediatric models, 58 adult models) and multi-detector array CT scanners from two commercial manufacturers (LightSpeed VCT, GE Healthcare; SOMATOM Definition Flash, Siemens Healthcare). A previously-validated Monte Carlo program was used to simulate organ dose for each patient model and each scanner, from which DLP-normalized-effective dose (k factor) and DLP-normalized-risk index values (q factor) were derived. The k factor showed exponential decrease with increasing patient size. For a given gender, q factor showed exponential decrease with both increasing patient size and patient age. The discrepancies in k and q factors across scanners were on average 8% and 15%, respectively. This study demonstrates the feasibility of estimating patient-specific organ dose and cohort-specific effective dose and risk index in abdominopelvic CT requiring only the knowledge of patient size, gender, and age.

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy

Science.gov (United States)

Xu, Huaigeng

2017-01-01

In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs) and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD) has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic. PMID:28607562

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy

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Peter Gee

2017-01-01

Full Text Available In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic.

Erythroid-specific transcriptional changes in PBMCs from pulmonary hypertension patients.

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Chris Cheadle

Full Text Available Gene expression profiling of peripheral blood mononuclear cells (PBMCs is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells.The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH, 19 patients with systemic sclerosis without pulmonary hypertension (SSc, 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH, and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease.In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.

(Re-)programming of subtype specific cardiomyocytes.

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Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

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Shimamoto, Akira; Kagawa, Harunobu; Zensho, Kazumasa; Sera, Yukihiro; Kazuki, Yasuhiro; Osaki, Mitsuhiko; Oshimura, Mitsuo; Ishigaki, Yasuhito; Hamasaki, Kanya; Kodama, Yoshiaki; Yuasa, Shinsuke; Fukuda, Keiichi; Hirashima, Kyotaro; Seimiya, Hiroyuki; Koyama, Hirofumi; Shimizu, Takahiko; Takemoto, Minoru; Yokote, Koutaro; Goto, Makoto; Tahara, Hidetoshi

2014-01-01

Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

Directory of Open Access Journals (Sweden)

Akira Shimamoto

Full Text Available Werner syndrome (WS is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs. In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

ROCK inhibitor primes human induced pluripotent stem cells to selectively differentiate towards mesendodermal lineage via epithelial-mesenchymal transition-like modulation.

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Maldonado, Maricela; Luu, Rebeccah J; Ramos, Michael E P; Nam, Jin

2016-09-01

Robust control of human induced pluripotent stem cell (hIPSC) differentiation is essential to realize its patient-tailored therapeutic potential. Here, we demonstrate a novel application of Y-27632, a small molecule Rho-associated protein kinase (ROCK) inhibitor, to significantly influence the differentiation of hIPSCs in a lineage-specific manner. The application of Y-27632 to hIPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration and time-dependent manner. Such changes in cell and colony morphology were associated with decreased expression of E-cadherin, a cell-cell junctional protein, proportional to the increased exposure to Y-27632. Interestingly, gene and protein expression of pluripotency markers such as NANOG and OCT4 were not downregulated by an exposure to Y-27632 up to 36h. Simultaneously, epithelial-to-mesenchymal (EMT) transition markers were upregulated with an exposure to Y-27632. These EMT-like changes in the cells with longer exposure to Y-27632 resulted in a significant increase in the subsequent differentiation efficiency towards mesendodermal lineage. In contrast, an inhibitory effect was observed when cells were subjected to ectodermal differentiation after prolonged exposure to Y-27632. Collectively, these results present a novel method for priming hIPSCs to modulate their differentiation potential with a simple application of Y-27632. Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved.

Comparison of serum prostate specific antigen levels and bone scintigraphy in patients with prostate carcinoma

International Nuclear Information System (INIS)

Bielickaite, J.; Zadeikaite, R.; Jurkiene, N. and others

2003-01-01

The aim of this study was to analyze the levels of serum prostate specific antigen in patients with and without bone metastases detected by means of bone scintigraphy and to determine the highest prostate specific antigen level in patients without bone metastases. The 50 patients consecutively diagnosed of prostate cancer between 1999 and 2001 in our institution made up the study population. Prostate specific antigen plasmatic levels were determined and bone scintigraphy was performed (whole body study after 99mTc-methyl-diphosphonate administration) in all the patients. In patients with positive bone scans (n=23), the mean prostate specific antigen level was 71.4±35.2 ng/ml and was significantly (p<0.00005) higher than in 14 patients with negative bone scans (mean prostate specific antigen level was 10.1±10.5 ng/ml). Suspicious lesions were found in 13 patients and their mean prostate specific antigen level was 8.5±7.7 ng/ml. Regarding prostate specific antigen levels, no statistically significant differences were found between patients with suspicious lessons and normal bone scans. The highest determined prostate specific antigen level in patients without bone metastases was 18 ng/ml. The bone scintigraphy should be performed in all patients with prostate specific antigen level above 18 ng/ml, but it is of limited value in patients with prostate specific antigen level below 18 ng/ml. (author)

Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.

Science.gov (United States)

Suzuki, Naoya M; Niwa, Akira; Yabe, Miharu; Hira, Asuka; Okada, Chihiro; Amano, Naoki; Watanabe, Akira; Watanabe, Ken-Ichiro; Heike, Toshio; Takata, Minoru; Nakahata, Tatsutoshi; Saito, Megumu K

2015-04-01

Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. ©AlphaMed Press.

Parkin and PINK1 Patient iPSC-Derived Midbrain Dopamine Neurons Exhibit Mitochondrial Dysfunction and α-Synuclein Accumulation

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Sun Young Chung

2016-10-01

Full Text Available Parkinson's disease (PD is characterized by the selective loss of dopamine neurons in the substantia nigra; however, the mechanism of neurodegeneration in PD remains unclear. A subset of familial PD is linked to mutations in PARK2 and PINK1, which lead to dysfunctional mitochondria-related proteins Parkin and PINK1, suggesting that pathways implicated in these monogenic forms could play a more general role in PD. We demonstrate that the identification of disease-related phenotypes in PD-patient-specific induced pluripotent stem cell (iPSC-derived midbrain dopamine (mDA neurons depends on the type of differentiation protocol utilized. In a floor-plate-based but not a neural-rosette-based directed differentiation strategy, iPSC-derived mDA neurons recapitulate PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. We propose that these form a pathogenic loop that contributes to disease. Our study illustrates the promise of iPSC technology for examining PD pathogenesis and identifying therapeutic targets.

Parkin and PINK1 Patient iPSC-Derived Midbrain Dopamine Neurons Exhibit Mitochondrial Dysfunction and α-Synuclein Accumulation.

Science.gov (United States)

Chung, Sun Young; Kishinevsky, Sarah; Mazzulli, Joseph R; Graziotto, John; Mrejeru, Ana; Mosharov, Eugene V; Puspita, Lesly; Valiulahi, Parvin; Sulzer, David; Milner, Teresa A; Taldone, Tony; Krainc, Dimitri; Studer, Lorenz; Shim, Jae-Won

2016-10-11

Parkinson's disease (PD) is characterized by the selective loss of dopamine neurons in the substantia nigra; however, the mechanism of neurodegeneration in PD remains unclear. A subset of familial PD is linked to mutations in PARK2 and PINK1, which lead to dysfunctional mitochondria-related proteins Parkin and PINK1, suggesting that pathways implicated in these monogenic forms could play a more general role in PD. We demonstrate that the identification of disease-related phenotypes in PD-patient-specific induced pluripotent stem cell (iPSC)-derived midbrain dopamine (mDA) neurons depends on the type of differentiation protocol utilized. In a floor-plate-based but not a neural-rosette-based directed differentiation strategy, iPSC-derived mDA neurons recapitulate PD phenotypes, including pathogenic protein accumulation, cell-type-specific vulnerability, mitochondrial dysfunction, and abnormal neurotransmitter homeostasis. We propose that these form a pathogenic loop that contributes to disease. Our study illustrates the promise of iPSC technology for examining PD pathogenesis and identifying therapeutic targets. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Effect of Inlet Waveforms on Computational Hemodynamics of Patient-Specific Intracranial Aneurysms

OpenAIRE

Xiang, J.; Siddiqui, A.H.; Meng, H.

2014-01-01

Due to the lack of patient-specific inlet flow waveform measurements, most computational fluid dynamics (CFD) simulations of intracranial aneurysms usually employ waveforms that are not patient-specific as inlet boundary conditions for the computational model. The current study examined how this assumption affects the predicted hemodynamics in patient-specific aneurysm geometries. We examined wall shear stress (WSS) and oscillatory shear index (OSI), the two most widely studied hemodynamic qu...

Patient-specific prediction of functional recovery after stroke.

Science.gov (United States)

Douiri, Abdel; Grace, Justin; Sarker, Shah-Jalal; Tilling, Kate; McKevitt, Christopher; Wolfe, Charles DA; Rudd, Anthony G

2017-07-01

Background and aims Clinical predictive models for stroke recovery could offer the opportunity of targeted early intervention and more specific information for patients and carers. In this study, we developed and validated a patient-specific prognostic model for monitoring recovery after stroke and assessed its clinical utility. Methods Four hundred and ninety-five patients from the population-based South London Stroke Register were included in a substudy between 2002 and 2004. Activities of daily living were assessed using Barthel Index) at one, two, three, four, six, eight, 12, 26, and 52 weeks after stroke. Penalized linear mixed models were developed to predict patients' functional recovery trajectories. An external validation cohort included 1049 newly registered stroke patients between 2005 and 2011. Prediction errors on discrimination and calibration were assessed. The potential clinical utility was evaluated using prognostic accuracy measurements and decision curve analysis. Results Predictive recovery curves showed good accuracy, with root mean squared deviation of 3 Barthel Index points and a R 2 of 83% up to one year after stroke in the external cohort. The negative predictive values of the risk of poor recovery (Barthel Index <8) at three and 12 months were also excellent, 96% (95% CI [93.6-97.4]) and 93% [90.8-95.3], respectively, with a potential clinical utility measured by likelihood ratios (LR+:17 [10.8-26.8] at three months and LR+:11 [6.5-17.2] at 12 months). Decision curve analysis showed an increased clinical benefit, particularly at threshold probabilities of above 5% for predictive risk of poor outcomes. Conclusions A recovery curves tool seems to accurately predict progression of functional recovery in poststroke patients.

Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

NARCIS (Netherlands)

Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

2016-01-01

Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated

A Monte Carlo study of the ''minus sign problem'' in the t-J model using an intel IPSC/860 hypercube

International Nuclear Information System (INIS)

Kovarik, M.D.; Barnes, T.; Tennessee Univ., Knoxville, TN

1993-01-01

We describe a Monte Carlo simulation of the 2-dimensional t-J model on an Intel iPSC/860 hypercube. The problem studied is the determination of the dispersion relation of a dynamical hole in the t-J model of the high temperature superconductors. Since this problem involves the motion of many fermions in more than one spatial dimensions, it is representative of the class of systems that suffer from the ''minus sign problem'' of dynamical fermions which has made Monte Carlo simulation very difficult. We demonstrate that for small values of the hole hopping parameter one can extract the entire hole dispersion relation using the GRW Monte Carlo algorithm, which is a simulation of the Euclidean time Schroedinger equation, and present results on 4 x 4 and 6 x 6 lattices. We demonstrate that a qualitative picture at higher hopping parameters may be found by extrapolating weak hopping results where the minus sign problem is less severe. Generalization to physical hopping parameter values will only require use of an improved trial wavefunction for importance sampling

Generation of a human induced pluripotent stem cell line (MUSIi001-A from caesarean section scar fibroblasts using Sendai viral vectors

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Methichit Wattanapanitch

2018-03-01

Full Text Available We generated a human induced pluripotent stem cell (iPSC line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

Science.gov (United States)

Romero-Moya, Damià; Santos-Ocaña, Carlos; Castaño, Julio; Garrabou, Gloria; Rodríguez-Gómez, José A; Ruiz-Bonilla, Vanesa; Bueno, Clara; González-Rodríguez, Patricia; Giorgetti, Alessandra; Perdiguero, Eusebio; Prieto, Cristina; Moren-Nuñez, Constanza; Fernández-Ayala, Daniel J; Victoria Cascajo, Maria; Velasco, Iván; Canals, Josep Maria; Montero, Raquel; Yubero, Delia; Jou, Cristina; López-Barneo, José; Cardellach, Francesc; Muñoz-Cánoves, Pura; Artuch, Rafael; Navas, Plácido; Menendez, Pablo

2017-07-01

Coenzyme Q 10 (CoQ 10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ 10 biosynthesis (COQ genes) cause primary CoQ 10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ 10 ], CoQ 10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4 ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4 ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ 10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ 10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703. © 2017 AlphaMed Press.

Patient-Specific Dosimetry of Pretargeted Radioimmunotherapy Using CC49 Fusion Protein in Patients with Gastrointestinal Malignancies

International Nuclear Information System (INIS)

Shen, Shang; Forero, Andres; LoBuglio, Albert F.; Breitz, H.; Khazaeli, M. B.; Fisher, Darrell R.; Wang, W. Q.; Meredith, Ruby F.

2005-01-01

Patient-Specific Dosimetry of Pretargeted Radioimmunotherapy Using CC49 Fusion Protein in Patients with Gastrointestinal Malignancies. Shen S, Forero A, Lobuglio AF, Breitz H, Khazaeli MB, Fisher DR, Wang W, Meredith RF. Department of Radiation Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, and Radioisotopes Program at Pacific Northwest National Laboratory, Richland, Washington. Pretargeted radioimmunotherapy (RIT) using CC49 fusion protein, comprised of CC49-(scFv)(4) and streptavidin, in conjunction with (90)Y/(111)In-DOTA-biotin (DOTA = dodecanetetraacetic acid) provides a new opportunity to improve efficacy by increasing the tumor-to-normal tissue dose ratio. To our knowledge, the patient-specific dosimetry of pretargeted (90)Y/(111)In-DOTA-biotin after CC49 fusion protein in patients has not been reported previously. METHODS: Nine patients received 3-step pretargeted RIT: (a) 160 mg/m(2) of CC49 fusion protein, (b) synthetic clearing agent (sCA) at 48 or 72 h later, and (c) (90)Y/(111)In-DOTA-biotin 24 h after the sCA administration. Sequential whole-body (111)In images were acquired immediately and at 2-144 h after injection of (90)Y/(111)In-DOTA-biotin. Geometric-mean quantification with background and attenuation correction was used for liver and lung dosimetry. Effective point source quantification was used for spleen, kidneys, and tumors. Organ and tumor (90)Y doses were calculated based on (111)In imaging data and the MIRD formalism using patient-specific organ masses determined from CT images. Patient-specific marrow doses were determined based on radioactivity concentration in the blood. RESULTS: The (90)Y/(111)In-DOTA-biotin had a rapid plasma clearance, which was biphasic with <10% residual at 8 h. Organ masses ranged from 1,263 to 3,855 g for liver, 95 to 1,009 g for spleen, and 309 to 578 g for kidneys. The patient-specific mean (90)Y dose (cGy/37 MBq, or rad/mCi) was 0.53 (0.32-0.78) to whole body

Modeling Glaucoma: Retinal Ganglion Cells Generated from Induced Pluripotent Stem Cells of Patients with SIX6 Risk Allele Show Developmental Abnormalities.

Science.gov (United States)

Teotia, Pooja; Van Hook, Matthew J; Wichman, Christopher S; Allingham, R Rand; Hauser, Michael A; Ahmad, Iqbal

2017-11-01

Glaucoma represents a group of multifactorial diseases with a unifying pathology of progressive retinal ganglion cell (RGC) degeneration, causing irreversible vision loss. To test the hypothesis that RGCs are intrinsically vulnerable in glaucoma, we have developed an in vitro model using the SIX6 risk allele carrying glaucoma patient-specific induced pluripotent stem cells (iPSCs) for generating functional RGCs. Here, we demonstrate that the efficiency of RGC generation by SIX6 risk allele iPSCs is significantly lower than iPSCs-derived from healthy, age- and sex-matched controls. The decrease in the number of RGC generation is accompanied by repressed developmental expression of RGC regulatory genes. The SIX6 risk allele RGCs display short and simple neurites, reduced expression of guidance molecules, and immature electrophysiological signature. In addition, these cells have higher expression of glaucoma-associated genes, CDKN2A and CDKN2B, suggesting an early onset of the disease phenotype. Consistent with the developmental abnormalities, the SIX6 risk allele RGCs display global dysregulation of genes which map on developmentally relevant biological processes for RGC differentiation and signaling pathways such as mammalian target of rapamycin that integrate diverse functions for differentiation, metabolism, and survival. The results suggest that SIX6 influences different stages of RGC differentiation and their survival; therefore, alteration in SIX6 function due to the risk allele may lead to cellular and molecular abnormalities. These abnormalities, if carried into adulthood, may make RGCs vulnerable in glaucoma. Stem Cells 2017;35:2239-2252. © 2017 AlphaMed Press.

Disease modeling using human induced pluripotent stem cells: lessons from the liver.

Science.gov (United States)

Gieseck, Richard L; Colquhoun, Jennifer; Hannan, Nicholas R F

2015-01-01

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Induced pluripotent stem cells for the treatment of stroke: the potential and the pitfalls.

Science.gov (United States)

Yu, Fenggang; Li, Yingying; Morshead, Cindi M

2013-09-01

The extraordinary discovery of induced pluripotent stem cells (iPSCs) has led to the very real possibility that patient-specific cell therapy can be realized. The potential to develop cell replacement therapies outside the ethical and legal limitations, has initiated a new era of hope for regenerative strategies to treat human neurological disease including stroke. In this article, we will review and compare the current approaches to derive iPSCs from different somatic cells, and the induction into neuronal phenotypes, considering the advantages and disadvantages to the methodologies of derivation. We will highlight the work relating to the use of iPSC-based therapies in models of stroke and their potential use in clinical trials. Finally, we will consider future directions and areas of exploration which may promote the realization of iPSC-based cell replacement strategies for the treatment of stroke.

The effect of patient-specific factors on radiation-induced regional lung injury

International Nuclear Information System (INIS)

Garipagaoglu, Melahat; Munley, Michael T.; Hollis, Donna; Poulson, Jean M.; Bentel, Gunilla C.; Sibley, Gregory; Anscher, Mitchell S.; Fan Ming; Jaszczak, Ronald J.; Coleman, R. Edward; Marks, Lawrence B.

1999-01-01

Purpose: To assess the impact of patient-specific factors on radiation (RT)-induced reductions in regional lung perfusion. Methods: Fifty patients (32 lung carcinoma, 7 Hodgkin's disease, 9 breast carcinoma and 2 other thoracic tumors) had pre-RT and ≥24-week post-RT single photon emission computed tomography (SPECT) perfusion images to assess the dose dependence of RT-induced reductions in regional lung perfusion. The SPECT data were analyzed using a normalized and non-normalized approach. Furthermore, two different mathematical methods were used to assess the impact of patient-specific factors on the dose-response curve (DRC). First, DRCs for different patient subgroups were generated and compared. Second, in a more formal statistical approach, individual DRCs for regional lung injury for each patient were fit to a linear-quadratic model (reduction = coefficient 1 x dose + coefficient 2 x dose 2 ). Multiple patient-specific factors including tobacco history, pre-RT diffusion capacity to carbon monoxide (DLCO), transforming growth factor-beta (TGF-β), chemotherapy exposure, disease type, and mean lung dose were explored in a multivariate analysis to assess their impact on the coefficients. Results: None of the variables tested had a consistent impact on the radiation sensitivity of regional lung (i.e., the slope of the DRC). In the formal statistical analysis, there was a suggestion of a slight increase in radiation sensitivity in the dose range >40 Gy for nonsmokers (vs. smokers) and in those receiving chemotherapy (vs. no chemotherapy). However, this finding was very dependent on the specific statistical and normalization method used. Conclusion: Patient-specific factors do not have a dramatic effect on RT-induced reduction in regional lung perfusion. Additional studies are underway to better clarify this issue. We continue to postulate that patient-specific factors will impact on how the summation of regional injury translates into whole organ injury

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

Science.gov (United States)

Shalom-Feuerstein, Ruby; Serror, Laura; Aberdam, Edith; Müller, Franz-Josef; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing; Aberdam, Daniel; Petit, Isabelle

2013-02-05

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Patient-specific estimation of detailed cochlear shape from clinical CT images

DEFF Research Database (Denmark)

Kjer, H Martin; Fagertun, Jens; Wimmer, Wilhelm

2018-01-01

of the detailed patient-specific cochlear shape from CT images. From a collection of temporal bone [Formula: see text]CT images, we build a cochlear statistical deformation model (SDM), which is a description of how a human cochlea deforms to represent the observed anatomical variability. The model is used...... for regularization of a non-rigid image registration procedure between a patient CT scan and a [Formula: see text]CT image, allowing us to estimate the detailed patient-specific cochlear shape. We test the accuracy and precision of the predicted cochlear shape using both [Formula: see text]CT and CT images...

Patient-specific dose estimation for pediatric chest CT

Energy Technology Data Exchange (ETDEWEB)

Li Xiang; Samei, Ehsan; Segars, W. Paul; Sturgeon, Gregory M.; Colsher, James G.; Frush, Donald P. [Medical Physics Graduate Program, Duke University, Durham, North Carolina 27705 and Department of Radiology, Duke Advanced Imaging Laboratories, Duke University Medical Center, Durham, North Carolina 27705 (United States); Medical Physics Graduate Program, Duke University, Durham, North Carolina 27705 (United States); Department of Radiology, Duke Advanced Imaging Laboratories, Duke University Medical Center, Durham, North Carolina 27705 (United States); Department of Physics, Duke University, Durham, North Carolina 27710 (United States); and Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708 (United States); Medical Physics Graduate Program, Duke University, Durham, North Carolina 27705 and Department of Radiology, Duke Advanced Imaging Laboratories, Duke University Medical Center, Durham, North Carolina 27705 (United States); Department of Radiology, Duke Advanced Imaging Laboratories, Duke University Medical Center, Durham, North Carolina 27705 (United States); Medical Physics Graduate Program, Duke University, Durham, North Carolina 27705 and Global Applied Science Laboratory, GE Healthcare, Waukesha, Wisconsin 53188 (United States); Medical Physics Graduate Program, Duke University, Durham, North Carolina 27705 and Department of Radiology, Division of Pediatric Radiology, Duke University Medical Center, Durham North Carolina 27710 (United States)

2008-12-15

Current methods for organ and effective dose estimations in pediatric CT are largely patient generic. Physical phantoms and computer models have only been developed for standard/limited patient sizes at discrete ages (e.g., 0, 1, 5, 10, 15 years old) and do not reflect the variability of patient anatomy and body habitus within the same size/age group. In this investigation, full-body computer models of seven pediatric patients in the same size/protocol group (weight: 11.9-18.2 kg) were created based on the patients' actual multi-detector array CT (MDCT) data. Organs and structures in the scan coverage were individually segmented. Other organs and structures were created by morphing existing adult models (developed from visible human data) to match the framework defined by the segmented organs, referencing the organ volume and anthropometry data in ICRP Publication 89. Organ and effective dose of these patients from a chest MDCT scan protocol (64 slice LightSpeed VCT scanner, 120 kVp, 70 or 75 mA, 0.4 s gantry rotation period, pitch of 1.375, 20 mm beam collimation, and small body scan field-of-view) was calculated using a Monte Carlo program previously developed and validated to simulate radiation transport in the same CT system. The seven patients had normalized effective dose of 3.7-5.3 mSv/100 mAs (coefficient of variation: 10.8%). Normalized lung dose and heart dose were 10.4-12.6 mGy/100 mAs and 11.2-13.3 mGy/100 mAs, respectively. Organ dose variations across the patients were generally small for large organs in the scan coverage (<7%), but large for small organs in the scan coverage (9%-18%) and for partially or indirectly exposed organs (11%-77%). Normalized effective dose correlated weakly with body weight (correlation coefficient: r=-0.80). Normalized lung dose and heart dose correlated strongly with mid-chest equivalent diameter (lung: r=-0.99, heart: r=-0.93); these strong correlation relationships can be used to estimate patient-specific organ

Systematic Review of Patient-Specific Surgical Simulation: Toward Advancing Medical Education.

Science.gov (United States)

Ryu, Won Hyung A; Dharampal, Navjit; Mostafa, Ahmed E; Sharlin, Ehud; Kopp, Gail; Jacobs, William Bradley; Hurlbert, Robin John; Chan, Sonny; Sutherland, Garnette R

Simulation-based education has been shown to be an effective tool to teach foundational technical skills in various surgical specialties. However, most of the current simulations are limited to generic scenarios and do not allow continuation of the learning curve beyond basic technical skills to prepare for more advanced expertise, such as patient-specific surgical planning. The objective of this study was to evaluate the current medical literature with respect to the utilization and educational value of patient-specific simulations for surgical training. We performed a systematic review of the literature using Pubmed, Embase, and Scopus focusing on themes of simulation, patient-specific, surgical procedure, and education. The study included randomized controlled trials, cohort studies, and case-control studies published between 2005 and 2016. Two independent reviewers (W.H.R. and N.D) conducted the study appraisal, data abstraction, and quality assessment of the studies. The search identified 13 studies that met the inclusion criteria; 7 studies employed computer simulations and 6 studies used 3-dimensional (3D) synthetic models. A number of surgical specialties evaluated patient-specific simulation, including neurosurgery, vascular surgery, orthopedic surgery, and interventional radiology. However, most studies were small in size and primarily aimed at feasibility assessments and early validation. Early evidence has shown feasibility and utility of patient-specific simulation for surgical education. With further development of this technology, simulation-based education may be able to support training of higher-level competencies outside the clinical settingto aid learners in their development of surgical skills. Copyright © 2017 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.

Towards patient specific thermal modelling of the prostate

International Nuclear Information System (INIS)

Berg, Cornelis A T van den; Kamer, Jeroen B van de; Leeuw, Astrid A C ee; Jeukens, Cecile R L P N; Raaymakers, Bas W; Vulpen, Marco van; Lagendijk, Jan J W

2006-01-01

The application of thermal modelling for hyperthermia and thermal ablation is severely hampered by lack of information about perfusion and vasculature. However, recently, with the advent of sophisticated angiography and dynamic contrast enhanced (DCE) imaging techniques, it has become possible to image small vessels and blood perfusion bringing the ultimate goal of patient specific thermal modelling closer within reach. In this study dynamic contrast enhanced multi-slice CT imaging techniques are employed to investigate the feasibility of this concept for regional hyperthermia treatment of the prostate. The results are retrospectively compared with clinical thermometry data of a patient group from an earlier trial. Furthermore, the role of the prostate vasculature in the establishment of the prostate temperature distribution is studied. Quantitative 3D perfusion maps of the prostate were constructed for five patients using a distributed-parameter tracer kinetics model to analyse dynamic CT data. CT angiography was applied to construct a discrete vessel model of the pelvis. Additionally, a discrete vessel model of the prostate vasculature was constructed of a prostate taken from a human corpse. Three thermal modelling schemes with increasing inclusion of the patient specific physiological information were used to simulate the temperature distribution of the prostate during regional hyperthermia. Prostate perfusion was found to be heterogeneous and T3 prostate carcinomas are often characterized by a strongly elevated tumour perfusion (up to 70-80 ml 100 g -1 min -1 ). This elevated tumour perfusion leads to 1-2 deg. C lower tumour temperatures than thermal simulations based on a homogeneous prostate perfusion. Furthermore, the comparison has shown that the simulations with the measured perfusion maps result in consistently lower prostate temperatures than clinically achieved. The simulations with the discrete vessel model indicate that significant pre-heating takes

HPV specific testing: a requirement for oropharyngeal squamous cell carcinoma patients.

Science.gov (United States)

Robinson, Max; Schache, Andrew; Sloan, Philip; Thavaraj, Selvam

2012-07-01

Human papillomavirus (HPV) testing is now recommended as part of the work up for patients with oropharyngeal squamous cell carcinoma (OPSCC) and those patients with cervical lymph node metastasis of unknown origin. The laboratory testing strategy should accurately assess the presence or absence of oncogenic HPV infection in routinely collected tumour samples that are subject to standard fixation protocols, alcohol-fixed cytological preparations and formalin-fixed tissue samples. The HPV status should correlate with biologically relevant outcome measures such as overall, disease-specific and disease-free survival. Whilst increased expression of p16 by immunohistochemistry is considered to be a surrogate marker of oncogenic HPV infection and is a validated independent prognostic biomarker, only HPV specific tests provide definitive evidence of the aetiological agent. We provide an overview of HPV testing in OPSCC, justifying the use of HPV specific tests. We examine the analytical accuracy of HPV specific tests against the 'reference' test--high risk HPV mRNA in fresh tissue--and contrast this with the performance of p16 immunohistochemistry as a stand alone test. We highlight the added value of HPV specific tests in prognostication, clinical trial design, and population-based disease surveillance. We consider that HPV specific testing is the starting point for developing increasingly informative biomarker panels in the context of 'stratified medicine'. We briefly frame test information in the context of disclosure of HPV status to patients. We conclude that only a testing strategy that includes HPV specific tests can deliver more effective care for patients with OPSCC. The international head and neck oncology community should work together to clearly define the minimum requirements for assigning a diagnosis of HPV-related OPSCC in order to ensure consistent reporting of this emerging and increasingly prevalent disease.

Quantitative analysis of patient-specific dosimetric IMRT verification

International Nuclear Information System (INIS)

Budgell, G J; Perrin, B A; Mott, J H L; Fairfoul, J; Mackay, R I

2005-01-01

Patient-specific dosimetric verification methods for IMRT treatments are variable, time-consuming and frequently qualitative, preventing evidence-based reduction in the amount of verification performed. This paper addresses some of these issues by applying a quantitative analysis parameter to the dosimetric verification procedure. Film measurements in different planes were acquired for a series of ten IMRT prostate patients, analysed using the quantitative parameter, and compared to determine the most suitable verification plane. Film and ion chamber verification results for 61 patients were analysed to determine long-term accuracy, reproducibility and stability of the planning and delivery system. The reproducibility of the measurement and analysis system was also studied. The results show that verification results are strongly dependent on the plane chosen, with the coronal plane particularly insensitive to delivery error. Unexpectedly, no correlation could be found between the levels of error in different verification planes. Longer term verification results showed consistent patterns which suggest that the amount of patient-specific verification can be safely reduced, provided proper caution is exercised: an evidence-based model for such reduction is proposed. It is concluded that dose/distance to agreement (e.g., 3%/3 mm) should be used as a criterion of acceptability. Quantitative parameters calculated for a given criterion of acceptability should be adopted in conjunction with displays that show where discrepancies occur. Planning and delivery systems which cannot meet the required standards of accuracy, reproducibility and stability to reduce verification will not be accepted by the radiotherapy community

Patient-Specific Tailored Intervention Improves INR Time in Therapeutic Range and INR Variability in Heart Failure Patients.

Science.gov (United States)

Gotsman, Israel; Ezra, Orly; Hirsh Raccah, Bruria; Admon, Dan; Lotan, Chaim; Dekeyser Ganz, Freda

2017-08-01

Many patients with heart failure need anticoagulants, including warfarin. Good control is particularly challenging in heart failure patients, with range, thereby increasing the risk of complications. This study aimed to evaluate the effect of a patient-specific tailored intervention on anticoagulation control in patients with heart failure. Patients with heart failure taking warfarin therapy (n = 145) were randomized to either standard care or a 1-time intervention assessing potential risk factors for lability of INR, in which they received patient-specific instructions. Time in therapeutic range (TTR) using Rosendaal's linear model was assessed 3 months before and after the intervention. The patient-tailored intervention significantly increased anticoagulation control. The median TTR levels before intervention were suboptimal in the interventional and control groups (53% vs 45%, P = .14). After intervention the median TTR increased significantly in the interventional group compared with the control group (80% [interquartile range, 62%-93%] vs 44% [29%-61%], P <.0001). The intervention resulted in a significant improvement in the interventional group before versus after intervention (53% vs 80%, P <.0001) but not in the control group (45% vs 44%, P = .95). The percentage of patients with a TTR ≥60%, considered therapeutic, was substantially higher in the interventional group: 79% versus 25% (P <.0001). The INR variability (standard deviation of each patient's INR measurements) decreased significantly in the interventional group, from 0.53 to 0.32 (P <.0001) after intervention but not in the control group. Patient-specific tailored intervention significantly improves anticoagulation therapy in patients with heart failure. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of Induced Pluripotent Stem Cells and Neural Stem/Progenitor Cells from Newborns with Spina Bifida Aperta.

Science.gov (United States)

Bamba, Yohei; Nonaka, Masahiro; Sasaki, Natsu; Shofuda, Tomoko; Kanematsu, Daisuke; Suemizu, Hiroshi; Higuchi, Yuichiro; Pooh, Ritsuko K; Kanemura, Yonehiro; Okano, Hideyuki; Yamasaki, Mami

2017-12-01

We established induced pluripotent stem cells (iPSCs) and neural stem/progenitor cells (NSPCs) from three newborns with spina bifida aperta (SBa) using clinically practical methods. We aimed to develop stem cell lines derived from newborns with SBa for future therapeutic use. SBa is a common congenital spinal cord abnormality that causes defects in neurological and urological functions. Stem cell transplantation therapies are predicted to provide beneficial effects for patients with SBa. However, the availability of appropriate cell sources is inadequate for clinical use because of their limited accessibility and expandability, as well as ethical issues. Fibroblast cultures were established from small fragments of skin obtained from newborns with SBa during SBa repair surgery. The cultured cells were transfected with episomal plasmid vectors encoding reprogramming factors necessary for generating iPSCs. These cells were then differentiated into NSPCs by chemical compound treatment, and NSPCs were expanded using neurosphere technology. We successfully generated iPSC lines from the neonatal dermal fibroblasts of three newborns with SBa. We confirmed that these lines exhibited the characteristics of human pluripotent stem cells. We successfully generated NSPCs from all SBa newborn-derived iPSCs with a combination of neural induction and neurosphere technology. We successfully generated iPSCs and iPSC-NSPCs from surgical samples obtained from newborns with SBa with the goal of future clinical use in patients with SBa.

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma.

Science.gov (United States)

Bahmad, Hisham; Hadadeh, Ola; Chamaa, Farah; Cheaito, Katia; Darwish, Batoul; Makkawi, Ahmad-Kareem; Abou-Kheir, Wassim

2017-01-01

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro . The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

Disease-Specific Mortality of Differentiated Thyroid Cancer Patients in Korea: A Multicenter Cohort Study

Directory of Open Access Journals (Sweden)

Min Ji Jeon

2017-11-01

Full Text Available BackgroundLittle is known regarding disease-specific mortality of differentiated thyroid cancer (DTC patients and its risk factors in Korea.MethodsWe retrospectively reviewed a large multi-center cohort of thyroid cancer from six Korean hospitals and included 8,058 DTC patients who underwent initial surgery between 1996 and 2005.ResultsMean age of patients at diagnosis was 46.2±12.3 years; 87% were females. Most patients had papillary thyroid cancer (PTC; 97% and underwent total thyroidectomy (85%. Mean size of the primary tumor was 1.6±1.0 cm. Approximately 40% of patients had cervical lymph node (LN metastases and 1.3% had synchronous distant metastases. During 11.3 years of follow-up, 150 disease-specific mortalities (1.9% occurred; the 10-year disease-specific survival (DSS rate was 98%. According to the year of diagnosis, the number of disease-specific mortality was not different. However, the rate of disease-specific mortality decreased during the study period (from 7.7% to 0.7%. Older age (≥45 years at diagnosis, male, follicular thyroid cancer (FTC versus PTC, larger tumor size (>2 cm, presence of extrathyroidal extension (ETE, lateral cervical LN metastasis, distant metastasis and tumor node metastasis (TNM stage were independent risk factors of disease-specific mortality of DTC patients.ConclusionThe rate of disease-specific mortality of Korean DTC patients was 1.9%; the 10-year DSS rate was 98% during 1996 to 2005. Older age at diagnosis, male, FTC, larger tumor size, presence of ETE, lateral cervical LN metastasis, distant metastasis, and TNM stages were significant risk factors of disease-specific mortality of Korean DTC patients.

Patient specific 3D visualisation of human brain

African Journals Online (AJOL)

Nafiisah

development of powerful new 3D image analysis and visualization algorithms that ... The tool is aimed to provide facility to reconstruct patient-specific 3D ... In this paper we present a review of the ... medical diagnosis, procedures training, pre-operative planning, ..... Body: Handbook of Numerical Analysis, Elsevier, 2004.

Specifics of nursing care for a patient with nutritional stoma.

OpenAIRE

MUSILOVÁ, Klára

2017-01-01

Main goal of the thesis was to map out the specifics of nursing care for a patient with a nutritious stoma. Three research questions have been identified in connection to this goal. First research question was focused on mapping out the nursing care for a patient prior applying the nutritious stoma. Second research question was focusing on nursing care for a patient while the nutritious stoma is being applied, and the last third question researches the nursing care for a patient after applyin...

Psychiatric in-patients' experience of being secluded in a specific ...

African Journals Online (AJOL)

This qualitative, explorative, descriptive and contextual study was undertaken to explore and describe the experiences of psychiatric in-patients who are secluded in a specific hospital in Lesotho. Evidence about the rationale and appropriate use of seclusion as well as promotion of mental health in secluded patients has ...

Risks of all-cause and site-specific fractures among hospitalized patients with COPD

OpenAIRE

Liao, Kuang-Ming; Liang, Fu-Wen; Li, Chung-Yi

2016-01-01

Abstract Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis. The clinical sequel of osteoporosis is fracture. Patients with COPD who experience a fracture also have increased morbidity and mortality. Currently, the types of all-cause and site-specific fracture among patients with COPD are unknown. Thus, we elucidated the all-cause and site-specific fractures among patients with COPD. A retrospective, population-based, cohort study was conducted u...

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

Directory of Open Access Journals (Sweden)

Ahmed Kamel El-Sayed

2014-11-01

Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

Characterization of cat dander-specific T lymphocytes from atopic patients

NARCIS (Netherlands)

van Neerven, R. J.; van de Pol, M. M.; van Milligen, F. J.; Jansen, H. M.; Aalberse, R. C.; Kapsenberg, M. L.

1994-01-01

Fel d I, the major cat dander allergen, is recognized by serum IgE of more than 80% of all cat-allergic patients. Because IgE synthesis by B lymphocytes is under the control of T lymphocytes, we studied the specificity and lymphokine production profiles of cat dander-specific T lymphocytes.

Tolerance design of patient-specific range QA using the DMAIC framework in proton therapy.

Science.gov (United States)

Rah, Jeong-Eun; Shin, Dongho; Manger, Ryan P; Kim, Tae Hyun; Oh, Do Hoon; Kim, Dae Yong; Kim, Gwe-Ya

2018-02-01

To implement the DMAIC (Define-Measure-Analyze-Improve-Control) can be used for customizing the patient-specific QA by designing site-specific range tolerances. The DMAIC framework (process flow diagram, cause and effect, Pareto chart, control chart, and capability analysis) were utilized to determine the steps that need focus for improving the patient-specific QA. The patient-specific range QA plans were selected according to seven treatment site groups, a total of 1437 cases. The process capability index, C pm was used to guide the tolerance design of patient site-specific range. For prostate field, our results suggested that the patient range measurements were capable at the current tolerance level of ±1 mm in clinical proton plans. For other site-specific ranges, we analyzed that the tolerance tends to be overdesigned to insufficient process capability calculated by the patient-specific QA data. The customized tolerances were calculated for treatment sites. Control charts were constructed to simulate the patient QA time before and after the new tolerances were implemented. It is found that the total simulation QA time was decreased on average of approximately 20% after establishing new site-specific range tolerances. We simulated the financial impact of this project. The QA failure for whole process in proton therapy would lead up to approximately 30% increase in total cost. DMAIC framework can be used to provide an effective QA by setting customized tolerances. When tolerance design is customized, the quality is reasonably balanced with time and cost demands. © 2017 American Association of Physicists in Medicine.

Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

Directory of Open Access Journals (Sweden)

Ling Wang

2017-12-01

Full Text Available The development of human induced pluripotent stem cells (iPSCs holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4. Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

Comparing ESC and iPSC-Based Models for Human Genetic Disorders.

Science.gov (United States)

Halevy, Tomer; Urbach, Achia

2014-10-24

Traditionally, human disorders were studied using animal models or somatic cells taken from patients. Such studies enabled the analysis of the molecular mechanisms of numerous disorders, and led to the discovery of new treatments. Yet, these systems are limited or even irrelevant in modeling multiple genetic diseases. The isolation of human embryonic stem cells (ESCs) from diseased blastocysts, the derivation of induced pluripotent stem cells (iPSCs) from patients' somatic cells, and the new technologies for genome editing of pluripotent stem cells have opened a new window of opportunities in the field of disease modeling, and enabled studying diseases that couldn't be modeled in the past. Importantly, despite the high similarity between ESCs and iPSCs, there are several fundamental differences between these cells, which have important implications regarding disease modeling. In this review we compare ESC-based models to iPSC-based models, and highlight the advantages and disadvantages of each system. We further suggest a roadmap for how to choose the optimal strategy to model each specific disorder.

Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages.

Science.gov (United States)

Neehus, Anna-Lena; Lam, Jenny; Haake, Kathrin; Merkert, Sylvia; Schmidt, Nico; Mucci, Adele; Ackermann, Mania; Schubert, Madline; Happle, Christine; Kühnel, Mark Philipp; Blank, Patrick; Philipp, Friederike; Goethe, Ralph; Jonigk, Danny; Martin, Ulrich; Kalinke, Ulrich; Baumann, Ulrich; Schambach, Axel; Roesler, Joachim; Lachmann, Nico

2018-01-09

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Estimating patient-specific soft-tissue properties in a TKA knee.

Science.gov (United States)

Ewing, Joseph A; Kaufman, Michelle K; Hutter, Erin E; Granger, Jeffrey F; Beal, Matthew D; Piazza, Stephen J; Siston, Robert A

2016-03-01

Surgical technique is one factor that has been identified as critical to success of total knee arthroplasty. Researchers have shown that computer simulations can aid in determining how decisions in the operating room generally affect post-operative outcomes. However, to use simulations to make clinically relevant predictions about knee forces and motions for a specific total knee patient, patient-specific models are needed. This study introduces a methodology for estimating knee soft-tissue properties of an individual total knee patient. A custom surgical navigation system and stability device were used to measure the force-displacement relationship of the knee. Soft-tissue properties were estimated using a parameter optimization that matched simulated tibiofemoral kinematics with experimental tibiofemoral kinematics. Simulations using optimized ligament properties had an average root mean square error of 3.5° across all tests while simulations using generic ligament properties taken from literature had an average root mean square error of 8.4°. Specimens showed large variability among ligament properties regardless of similarities in prosthetic component alignment and measured knee laxity. These results demonstrate the importance of soft-tissue properties in determining knee stability, and suggest that to make clinically relevant predictions of post-operative knee motions and forces using computer simulations, patient-specific soft-tissue properties are needed. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Stem Cells as In Vitro Model of Parkinson's Disease

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Patricia L. Martínez-Morales

2012-01-01

Full Text Available Progress in understanding neurodegenerative cell biology in Parkinson's disease (PD has been hampered by a lack of predictive and relevant cellular models. In addition, the lack of an adequate in vitro human neuron cell-based model has been an obstacle for the uncover of new drugs for treating PD. The ability to generate induced pluripotent stem cells (iPSCs from PD patients and a refined capacity to differentiate these iPSCs into DA neurons, the relevant disease cell type, promises a new paradigm in drug development that positions human disease pathophysiology at the core of preclinical drug discovery. Disease models derived from iPSC that manifest cellular disease phenotypes have been established for several monogenic diseases, but iPSC can likewise be used for phenotype-based drug screens in complex diseases for which the underlying genetic mechanism is unknown. Here, we highlight recent advances as well as limitations in the use of iPSC technology for modelling PD “in a dish” and for testing compounds against human disease phenotypes in vitro. We discuss how iPSCs are being exploited to illuminate disease pathophysiology, identify novel drug targets, and enhance the probability of clinical success of new drugs.

Generation of Tumor Antigen-Specific iPSC-Derived Thymic Emigrants Using a 3D Thymic Culture System

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Raul Vizcardo

2018-03-01

Full Text Available Summary: Induced pluripotent stem cell (iPSC-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs. iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient

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Zhong Liu

2016-09-01

Full Text Available Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD. The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test.

Computational biomechanics for medicine fundamental science and patient-specific applications

CERN Document Server

Miller, Karol; Wittek, Adam; Nielsen, Poul

2014-01-01

One of the greatest challenges facing the computational engineering community is to extend the success of computational mechanics to fields outside traditional engineering, in particular to biology, the biomedical sciences, and medicine. The Computational Biomechanics for Medicine titles provide an opportunity for specialists in computational biomechanics to present their latest methodologies and advancements. This latest installment comprises nine of the latest developments in both fundamental science and patient-specific applications, from researchers in Australia, New Zealand, USA, UK, France, Ireland, and China. Some of the interesting topics discussed are: cellular mechanics; tumor growth and modeling; medical image analysis; and both patient-specific fluid dynamics and solid mechanics simulations.

Frequency and specificity of red blood cell alloimmunization in chilean transfused patients.

Science.gov (United States)

Caamaño, José; Musante, Evangelina; Contreras, Margarita; Ulloa, Hernán; Reyes, Carolina; Inaipil, Verónica; Saavedra, Nicolás; Guzmán, Neftalí

2015-01-01

Alloimmunization is an adverse effect of blood transfusions. In Chile, alloimmunization frequency is not established, and for this reason the aim of this study was to investigate the prevalence and specificity of red blood cell (RBC) alloantibodies in Chilean transfused subjects. Records from 4,716 multi-transfused patients were analyzed. In these patients, antibody screening was carried out prior to cross-matching with a commercially available two-cell panel by the microcolum gel test, and samples with a positive screen were analyzed for the specificity of the alloantibody with a 16-cell identification panel. The incidence of RBC alloimmunization in transfused patients was 1.02% (48/4,716) with a higher prevalence in women (40/48). We detected 52 antibodies, the most frequent specificities identified were anti-E (30.8%), anti-K (26.9%), anti-D (7.7%), and anti-Fy(a) (5.8%). The highest incidence of alloantibodies was observed in cancer and gastroenterology patients. The data demonstrated a low alloimmunization frequency in Chilean transfused patients, principally associated with antibodies anti-E, anti-K, anti-D, and anti-Fy(a).

Specific efficacy expectations mediate exercise compliance in patients with COPD.

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Kaplan, R M; Atkins, C J; Reinsch, S

1984-01-01

Social learning theory has generated two different approaches for the assessment of expectancies. Bandura argues that expectancies are specific and do not generalize. Therefore, he prefers measures of specific efficacy expectations. Others endorse the role of generalized expectancies measured by locus of control scales. The present study examines specific versus generalized expectancies as mediators of changes in exercise behavior among 60 older adult patients with Chronic Obstructive Pulmonary Disease. The patients were given a prescription to increase exercise and randomly assigned to experimental groups or control groups. All groups received attention but only experimental groups received training to increase their exercise. After 3 months, groups given specific training for compliance with walking significantly increased their activity in comparison to the control group receiving only attention. These changes were mediated by changes in perceived efficacy for walking, with efficacy expectations for other behaviors changing as a function of their similarity to walking. A generalized health locus of control expectancy measure was less clearly associated with behavior change. The results are interpreted as supporting Bandura's version of social theory.

A Patient-Specific Foot Model for the Estimate of Ankle Joint Forces in Patients with Juvenile Idiopathic Arthritis.

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Prinold, Joe A I; Mazzà, Claudia; Di Marco, Roberto; Hannah, Iain; Malattia, Clara; Magni-Manzoni, Silvia; Petrarca, Maurizio; Ronchetti, Anna B; Tanturri de Horatio, Laura; van Dijkhuizen, E H Pieter; Wesarg, Stefan; Viceconti, Marco

2016-01-01

Juvenile idiopathic arthritis (JIA) is the leading cause of childhood disability from a musculoskeletal disorder. It generally affects large joints such as the knee and the ankle, often causing structural damage. Different factors contribute to the damage onset, including altered joint loading and other mechanical factors, associated with pain and inflammation. The prediction of patients' joint loading can hence be a valuable tool in understanding the disease mechanisms involved in structural damage progression. A number of lower-limb musculoskeletal models have been proposed to analyse the hip and knee joints, but juvenile models of the foot are still lacking. This paper presents a modelling pipeline that allows the creation of juvenile patient-specific models starting from lower limb kinematics and foot and ankle MRI data. This pipeline has been applied to data from three children with JIA and the importance of patient-specific parameters and modelling assumptions has been tested in a sensitivity analysis focused on the variation of the joint reaction forces. This analysis highlighted the criticality of patient-specific definition of the ankle joint axes and location of the Achilles tendon insertions. Patient-specific detection of the Tibialis Anterior, Tibialis Posterior, and Peroneus Longus origins and insertions were also shown to be important.

Simulating the Euclidean time Schroedinger equations using an Intel iPSC/860 hypercube: Application to the t-J model of high-Tc superconductivity

International Nuclear Information System (INIS)

Kovarik, M.D.; Barnes, T.; Tennessee Univ., Knoxville, TN

1993-01-01

We describe a Monte Carlo simulation of a dynamical fermion problem in two spatial dimensions on an Intel iPSC/860 hypercube. The problem studied is the determination of the dispersion relation of a dynamical hole in the t-J model of the high temperature superconductors. Since this problem involves the motion of many fermions in more than one spatial dimensions, it is representative of the class of systems that suffer from the ''minus sign problem'' of dynamical fermions which has made Monte Carlo simulation very difficult. We demonstrate that for small values of the hole hopping parameter one can extract the entire hole dispersion relation using the GRW Monte Carlo algorithm, which is a simulation of the Euclidean time Schroedinger equation, and present results on 4 x 4 and 6 x 6 lattices. Generalization to physical hopping parameter values wig only require use of an improved trial wavefunction for importance sampling

Patient specific actual size 3D printed models for patient education in glioma treatment: first experiences.

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van de Belt, Tom H; Nijmeijer, Hugo; Grim, David; Engelen, Lucien Jlpg; Vreeken, Rinaldo; van Gelder, Marleen Mmj; Laan, Mark Ter

2018-06-02

Cancer patients need high quality information about the disease stage, treatment options and side effects. High quality information can also improve health literacy, shared decision-making and satisfaction. We created patient-specific 3D models of tumours including surrounding functional areas, and assessed what patients with glioma actually value (or fear) about these models when they are used to educate them about the relation between their tumour and specific brain parts, the surgical procedure, and risks. We carried out an explorative study with adult glioma patients, who underwent functional MRI and DTi as part of the pre-operative work-up. All participants received an actual size 3D model, printed based on fMRI and DTi imaging. Semi-structured interviews were held to identify facilitators and barriers for using the model, and perceived effects. A model was successfully created for all 11 participants. A total of 18 facilitators and 8 barriers were identified. The model improved patients' understanding about their situation, that it was easier to ask questions to their neurosurgeon based on their model and that it supported their decision about the preferred treatment. A perceived barrier for using the 3D model was that it could be emotionally confronting, particularly in an early phase of the disease process. Positive effects were related to psychological domains including coping, learning effects and communication. Patient-specific 3D models are promising and simple tools that could help patients with glioma to better understand their situation, treatment options and risks. They have the potential to improve shared decision-making. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Stem-Cell Based Therapies for Epidermolysis Bullosa

Science.gov (United States)

2014-10-01

immunocompromised NSG mice. This task has not been initiated. Task 4.6 Derive mesenchymal cells from genetically corrected patient-specific JEB iPSC and...determine their ability to stably engraft long-term into the BM of immunocompromised NSG mice. This task has not been initiated. Key Research...differentiation protocol below (see Note 7). 1. Prewarm complete DKSFM (with antibiotics and DKSFM supplement) in the 37 C water bath. 2. Add 5 mL of prewarmed

Skeletal Muscle Cell Induction from Pluripotent Stem Cells

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Yusaku Kodaka

2017-01-01

Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD. Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

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David J Kahler

Full Text Available Current methods to derive induced pluripotent stem cell (iPSC lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS to isolate single cells expressing the cell surface marker signature CD13(NEGSSEA4(POSTra-1-60(POS on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral or non- integrating (Sendai virus reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

Science.gov (United States)

Sun, Yishan; Paşca, Sergiu P; Portmann, Thomas; Goold, Carleton; Worringer, Kathleen A; Guan, Wendy; Chan, Karen C; Gai, Hui; Vogt, Daniel; Chen, Ying-Jiun J; Mao, Rong; Chan, Karrie; Rubenstein, John LR; Madison, Daniel V; Hallmayer, Joachim; Froehlich-Santino, Wendy M; Bernstein, Jonathan A; Dolmetsch, Ricardo E

2016-01-01

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. DOI: http://dx.doi.org/10.7554/eLife.13073.001 PMID:27458797

SPECIFIC FEATURES OF ANESTHESIA IN PATIENTS WITH MYASTHENIA GRAVIS.

Science.gov (United States)

Spasojevic, Ivana; Hajdukovic, Danica; Komarcevic, Milena; Petrovic, Stanislava; Jovanovic, Jelena; Ciric, Aleksandra

2016-09-01

Myasthenia gravis is an autoimmune disease caused by antibodies leading to the destruction of nicotinic acetylcholine receptors on the neuromuscular junction. It is characterized by muscle weakness that gets aggravated with physical activity and improves at rest. Myasthenia Gravis Foundation of America made the clinical classification of Myasthenia gravis which is still in use today. "Tensilon test" is still the gold standard for the diagnosis of Myasthenia gravis. In addition to this test repeated muscular stimulation can be used as well as the analysis of specific autoantibodies. Treatment of Myasthenia Gravis. In conservative treatment of Mysthenia gravis anticholinesterases, immunosuppressants and plasmapheresis can be used. If conservative treatment does not lead to the desired remission, surgical treatment is indicated. The most accepted indication for thymectomy is the presence of thymoma with generalized form of Myasthenia gravis in adults. How to Distinguish Myasthenic From Cholinergic Crisis.'The following is important to make a difference between these two crises: knowledge of the events that preceded the crisis, the size of pupils as well as the presence of muscarinic signs and tensilon test. Specific Features of Anesthesia in Patients with Myasthienia Gravis. Mechanism of the disease development is the reason'for the increased sensitivity or resistance of these patients to certain types of drugs used in anesthesia. Protocol of Perioperative Anesthesia in Patients with Myasthenia Gravis. Based on 35 years of experience in the surgical treatment of patients with Myasthenia gravis anesthesiologists at the Department of Thoracic Surgery, Institute for Pulmonary Diseases of Vojvodina. made the protocol of anesthesia and perioperative treatment for these patients. Anesthesiologists may have to deal with a patient with myasthenia gravis in different types of surgical interventions. The protocol for anesthesia and perioperative management of these patients

Magnetic resonance imaging of the ear for patient-specific reconstructive surgery.

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Luc Nimeskern

Full Text Available INTRODUCTION: Like a fingerprint, ear shape is a unique personal feature that should be reconstructed with a high fidelity during reconstructive surgery. Ear cartilage tissue engineering (TE advantageously offers the possibility to use novel 3D manufacturing techniques to reconstruct the ear, thus allowing for a detailed auricular shape. However it also requires detailed patient-specific images of the 3D cartilage structures of the patient's intact contralateral ear (if available. Therefore the aim of this study was to develop and evaluate an imaging strategy for acquiring patient-specific ear cartilage shape, with sufficient precision and accuracy for use in a clinical setting. METHODS AND MATERIALS: Magnetic resonance imaging (MRI was performed on 14 volunteer and six cadaveric auricles and manually segmented. Reproducibility of cartilage volume (Cg.V, surface (Cg.S and thickness (Cg.Th was assessed, to determine whether raters could repeatedly define the same volume of interest. Additionally, six cadaveric auricles were harvested, scanned and segmented using the same procedure, then dissected and scanned using high resolution micro-CT. Correlation between MR and micro-CT measurements was assessed to determine accuracy. RESULTS: Good inter- and intra-rater reproducibility was observed (precision errors 0.82, but low for Cg.Th (0.95 demonstrated high accuracy. DISCUSSION AND CONCLUSION: This study demonstrated that precision and accuracy of the proposed method was high enough to detect patient-specific variation in ear cartilage geometry. The present study provides a clinical strategy to access the necessary information required for the production of 3D ear scaffolds for TE purposes, including detailed patient-specific shape. Furthermore, the protocol is applicable in daily clinical practice with existing infrastructure.

Virus-Specific T Cells for the Immunocompromised Patient

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Amy Houghtelin

2017-10-01

Full Text Available While progress has been made in the treatment of both hematologic cancers and solid tumors, chemorefractory or relapsed disease often portends a dismal prognosis, and salvage chemotherapy or radiation expose patients to intolerable toxicities and may not be effective. Hematopoietic stem cell transplant offers the promise of cure for many patients, and while mismatched, unrelated or haploidentical donors are increasingly available, the recipients are at higher risk of severe immunosuppression and immune dysregulation due to graft versus host disease. Viral infections remain a primary cause of severe morbidity and mortality in this patient population. Again, many therapeutic options for viral disease are toxic, may be ineffective or generate resistance, or fail to convey long-term protection. Adoptive cell therapy with virus-specific T cells (VSTs is a targeted therapy that is efficacious and has minimal toxicity in immunocompromised patients with CMV and EBV infections in particular. Products have since been generated specific for multiple viral antigens (multi-VST, which are not only effective but also confer protection in 70–90% of recipients when used as prophylaxis. Notably, these products can be generated from either virus-naive or virus-experienced autologous or allogeneic sources, including partially matched HLA-matched third-party donors. Obstacles to effective VST treatment are donor availability and product generation time. Banking of third-party VST is an attractive way to overcome these constraints and provide products on an as-needed basis. Other developments include epitope discovery to broaden the number of viral antigens targets in a single product, the optimization of VST generation from naive donor sources, and the modification of VSTs to enhance persistence and efficacy in vivo.

Virus-Specific T Cells for the Immunocompromised Patient.

Science.gov (United States)

Houghtelin, Amy; Bollard, Catherine M

2017-01-01

While progress has been made in the treatment of both hematologic cancers and solid tumors, chemorefractory or relapsed disease often portends a dismal prognosis, and salvage chemotherapy or radiation expose patients to intolerable toxicities and may not be effective. Hematopoietic stem cell transplant offers the promise of cure for many patients, and while mismatched, unrelated or haploidentical donors are increasingly available, the recipients are at higher risk of severe immunosuppression and immune dysregulation due to graft versus host disease. Viral infections remain a primary cause of severe morbidity and mortality in this patient population. Again, many therapeutic options for viral disease are toxic, may be ineffective or generate resistance, or fail to convey long-term protection. Adoptive cell therapy with virus-specific T cells (VSTs) is a targeted therapy that is efficacious and has minimal toxicity in immunocompromised patients with CMV and EBV infections in particular. Products have since been generated specific for multiple viral antigens (multi-VST), which are not only effective but also confer protection in 70-90% of recipients when used as prophylaxis. Notably, these products can be generated from either virus-naive or virus-experienced autologous or allogeneic sources, including partially matched HLA-matched third-party donors. Obstacles to effective VST treatment are donor availability and product generation time. Banking of third-party VST is an attractive way to overcome these constraints and provide products on an as-needed basis. Other developments include epitope discovery to broaden the number of viral antigens targets in a single product, the optimization of VST generation from naive donor sources, and the modification of VSTs to enhance persistence and efficacy in vivo .

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

DEFF Research Database (Denmark)

Brimnes, J; Halberg, P; Jacobsen, Søren

1997-01-01

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins...

Fetal Therapy Model of Myelomeningocele with Three-Dimensional Skin Using Amniotic Fluid Cell-Derived Induced Pluripotent Stem Cells

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Kazuhiro Kajiwara

2017-06-01

Full Text Available Myelomeningocele (MMC is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth, and no effective treatment has been reported to date. Only surgical repairs have been reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs generated from a patient with Down syndrome (AF-T21-iPSCs and twin-twin transfusion syndrome (AF-TTTS-iPSCs to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

The situation-specific theory of pain experience for Asian American cancer patients.

Science.gov (United States)

Im, Eun-Ok

2008-01-01

Studies have indicated the need for theories that explain and target ethnic-specific cancer pain experiences, including those of Asian Americans. In this article, I present a situation-specific theory that explains the unique cancer pain experience of Asian Americans. Unlike other existing theories, this situation-specific theory was developed on the basis of evidence, including a systematic literature review and research findings, making it comprehensive and highly applicable to research and practice with Asian American patients with cancer. Thus, this theory would strengthen the interconnections among theory, evidence, and practice in pain management for Asian American cancer patients.

[Specific iatrogenic risks to patients with HIV infection].

Science.gov (United States)

De Tournemire, R; Yeni, P

1994-01-01

Human immunodeficiency virus-infected patients are exposed to more or less specific iatrogenic diseases. The main characteristics of the risks encountered in this field are described: drug intolerance, mostly to sulfamethoxazole-trimethoprim, is extremely frequent; nucleoside analogue antiviral toxicity is reminiscent of that of chemotherapy; nosocomial infections, in general, are more prominent than in HIV-non infected patients. Intravenous line infections are particularly frequent, but these devices are necessary for prolonged intravenous therapies such as anti-CMV treatment of parenteral nutrition. An improved understanding of different etiopathogenic mechanisms and a better approach of the toxicity/efficacy ratio for each treatment would allow to reduce the excessive morbidity due to iatrogenicity.

Differentiation of primordial germ cells from induced pluripotent stem cells of primary ovarian insufficiency.

Science.gov (United States)

Leng, Lizhi; Tan, Yueqiu; Gong, Fei; Hu, Liang; Ouyang, Qi; Zhao, Yan; Lu, Guangxiu; Lin, Ge

2015-03-01

Can the induced pluripotent stem cells (iPSCs) derived from women with primary ovarian insufficiency (POI) differentiate into germ cells for potential disease modeling in vitro? The iPSC lines derived from POI patients with 46, X, del(X)(q26) or 46, X, del(X)(q26)9qh+ could differentiate into germ cells and expressed lower levels of genes in the deletion region of the X chromosome. iPSC technology has been envisioned as an approach for generating patient-specific stem cells for disease modeling and for developing novel therapies. It has also been confirmed that iPSCs differentiate into germ cells. We compared the differentiation ability of germ cells and the gene expression level of germ cell-related genes in the X chromosome deletion region of iPSC lines derived from POI patients (n = 2) with an iPSC line derived from normal fibroblasts (n = 1). We established three iPSC lines from two patients with partial Xq deletion-induced POI and normal fibroblasts by overexpressing four factors: octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and lin-28 homolog (LIN28), using lentiviral vectors. We then generated stable-transfected fluorescent reporter cell lines under the control of the Asp-Glu-Ala-Asp box polypeptide 4 (DDX4, also called VASA) promoter, and selected clonal derived sublines. We induced subline differentiation into germ cells by adding Wnt3a (30 ng/ml) and bone morphogenetic protein 4 (100 ng/ml). After 12 days of differentiation, green fluorescent protein (GFP)-positive and GFP-negative cells were isolated via fluorescence-activated cell sorting and analyzed for endogenous VASA protein (immunostaining) and for germ cell markers and genes expressed in the deleted region of the X chromosome (quantitative RT-PCR). The POI- and normal fibroblast-derived iPSCs had typical self-renewal and pluripotency characteristics. After stable transfection with the VASA-GFP construct, the sublines POI1-iPS-V.1

Prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in active surveillance patients.

Science.gov (United States)

Iremashvili, Viacheslav; Barney, Shane L; Manoharan, Murugesan; Kava, Bruce R; Parekh, Dipen J; Punnen, Sanoj

2016-04-01

To analyze the association between prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in prostate cancer patients on active surveillance, and to study the effect of prediagnostic prostate-specific antigen values on the predictive performance of prostate-specific antigen velocity and prostate-specific antigen doubling time. The study included 137 active surveillance patients with two or more prediagnostic prostate-specific antigen levels measured over a period of at least 3 months. Two sets of analyses were carried out. First, the association between prostate-specific antigen kinetics calculated using only the prediagnostic prostate-specific antigen values and the risk of biopsy progression was studied. Second, using the same cohort of patients, the predictive value of prostate-specific antigen kinetics calculated using only post-diagnostic prostate-specific antigens and compared with that of prostate-specific antigen kinetics based on both pre- and post-diagnostic prostate-specific antigen levels was analyzed. Of 137 patients included in the analysis, 37 (27%) had biopsy progression over a median follow-up period of 3.2 years. Prediagnostic prostate-specific antigen velocity of more than 2 ng/mL/year and 3 ng/mL/year was statistically significantly associated with the risk of future biopsy progression. However, after adjustment for baseline prostate-specific antigen density, these associations were no longer significant. None of the tested prostate-specific antigen kinetics based on combined pre- and post-diagnostic prostate-specific antigen values were statistically significantly associated with the risk of biopsy progression. Historical prediagnostic prostate-specific antigens seems to be not clinically useful in patients diagnosed with low-risk prostate cancer on active surveillance. © 2016 The Japanese Urological Association.

The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

Science.gov (United States)

Liu, Ji; Nie, Huarong; Xu, Zhengliang; Niu, Xin; Guo, Shangchun; Yin, Junhui; Guo, Fei; Li, Gang; Wang, Yang; Zhang, Changqing

2014-01-01

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

Directory of Open Access Journals (Sweden)

Ji Liu

Full Text Available The discovery of induced pluripotent stem cells (iPSCs rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM, the Cell Counting Kit-8 (CCK-8, histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

Induced pluripotent stem cells reprogramming: Epigenetics and applications in the regenerative medicine

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Kátia Maria Sampaio Gomes

Full Text Available Summary Induced pluripotent stem cells (iPSCs are somatic cells reprogrammed into an embryonic-like pluripotent state by the expression of specific transcription factors. iPSC technology is expected to revolutionize regenerative medicine in the near future. Despite the fact that these cells have the capacity to self-renew, they present low efficiency of reprogramming. Recent studies have demonstrated that the previous somatic epigenetic signature is a limiting factor in iPSC performance. Indeed, the process of effective reprogramming involves a complete remodeling of the existing somatic epigenetic memory, followed by the establishment of a "new epigenetic signature" that complies with the new type of cell to be differentiated. Therefore, further investigations of epigenetic modifications associated with iPSC reprogramming are required in an attempt to improve their self-renew capacity and potency, as well as their application in regenerative medicine, with a new strategy to reduce the damage in degenerative diseases. Our review aimed to summarize the most recent findings on epigenetics and iPSC, focusing on DNA methylation, histone modifications and microRNAs, highlighting their potential in translating cell therapy into clinics.

Development and fabrication of patient-specific knee implant using additive manufacturing techniques

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Zammit, Robert; Rochman, Arif

2017-10-01

Total knee replacement is the most effective treatment to relief pain and restore normal function in a diseased knee joint. The aim of this research was to develop a patient-specific knee implant which can be fabricated using additive manufacturing techniques and has reduced wear rates using a highly wear resistant materials. The proposed design was chosen based on implant requirements, such as reduction in wear rates as well as strong fixation. The patient-specific knee implant improves on conventional knee implants by modifying the articulating surfaces and bone-implant interfaces. Moreover, tribological tests of different polymeric wear couples were carried out to determine the optimal materials to use for the articulating surfaces. Finite element analysis was utilized to evaluate the stresses sustained by the proposed design. Finally, the patient-specific knee implant was successfully built using additive manufacturing techniques.

Pathway index models for construction of patient-specific risk profiles.

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Eng, Kevin H; Wang, Sijian; Bradley, William H; Rader, Janet S; Kendziorski, Christina

2013-04-30

Statistical methods for variable selection, prediction, and classification have proven extremely useful in moving personalized genomics medicine forward, in particular, leading to a number of genomic-based assays now in clinical use for predicting cancer recurrence. Although invaluable in individual cases, the information provided by these assays is limited. Most often, a patient is classified into one of very few groups (e.g., recur or not), limiting the potential for truly personalized treatment. Furthermore, although these assays provide information on which individuals are at most risk (e.g., those for which recurrence is predicted), they provide no information on the aberrant biological pathways that give rise to the increased risk. We have developed an approach to address these limitations. The approach models a time-to-event outcome as a function of known biological pathways, identifies important genomic aberrations, and provides pathway-based patient-specific assessments of risk. As we demonstrate in a study of ovarian cancer from The Cancer Genome Atlas project, the patient-specific risk profiles are powerful and efficient characterizations useful in addressing a number of questions related to identifying informative patient subtypes and predicting survival. Copyright © 2012 John Wiley & Sons, Ltd.

Observations of pretreatment prostate-specific antigen doubling time in 107 patients referred for definitive radiotherapy

International Nuclear Information System (INIS)

Lee, W. Robert; Hanks, Gerald E.; Corn, Benjamin W.; Schultheiss, Timothy E.

1995-01-01

Purpose: To determine pretreatment prostate-specific antigen doubling times (PSADT) in patients referred for definitive radiotherapy. Methods and Materials: One hundred and seven patients with histologically proven nonmetastatic prostate cancer and an elevated prostate-specific antigen (PSA) who were referred for radiation therapy had three serum PSA values obtained prior to the start of definitive therapy. Prostate-specific antigen doubling times were calculated by linear regression. Results: Prostate-specific antigen values increased during the period of observation in 78 patients (73%). Forty-three patients (40%) had calculated PSADT of less than 2 years and of those patients with pretreatment serum PSA values of greater than 10 ng/mL more than 50% has calculated PSADT of less than 2 years. Conclusions: A significant minority of patients referred for radiotherapy have calculated PSADT of less than 2 years. The significance of this relatively fast growth rate is as yet undetermined, but suggests that patients referred for radiotherapy may have aggressive disease prior to treatment

THE USE OF SPECIFIC AND NON-SPECIFIC QUESTIONNAIRES TO ASSESS QUALITY OF LIFE IN PATIENTS WITH FUNCTIONAL DISORDERS OF INTESTINE

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A. E. Shklyaev

2016-01-01

Full Text Available Objective: comparative assessment of the quality of life of IBS patients in the treatment process with the use of specific and non-specific questionnaires.Materials and methods: a dynamic study of quality of life in 40 patients of IBS with the use of questionnaires GSRS and SF-36.Results: marked decrease in the severity of syndromes on all 6 scales of the questionnaire GSRS, and after 1 to 3 weeks of treatment dynamics was gained statistically significant, and total score improved significantly already after 1 week of therapy. Positive dynamics was obtained on the two scales of the questionnaire SF-36 (social functioning, and emotional functioning, as well as physical and psychological components of health.Conclusions: the high sensitivity of specific questionnaire GSRS and diagnostic significance of nonspecific SF-36 questionnaire in patients with IBS, the necessity to combine them.

3D-Printed Patient-Specific ACL Femoral Tunnel Guide from MRI.

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Rankin, Iain; Rehman, Haroon; Frame, Mark

2018-01-01

Traditional ACL reconstruction with non-anatomic techniques can demonstrate unsatisfactory long-term outcomes with regards instability and the degenerative knee changes observed with these results. Anatomic ACL reconstruction attempts to closely reproduce the patient's individual anatomic characteristics with the aim of restoring knee kinematics, in order to improve patient short and long-term outcomes. We designed an arthroscopic, patient-specific, ACL femoral tunnel guide to aid anatomical placement of the ACL graft within the femoral tunnel. The guide design was based on MRI scan of the subject's uninjured contralateral knee, identifying the femoral footprint and its anatomical position relative to the borders of the femoral articular cartilage. Image processing software was used to create a 3D computer aided design which was subsequently exported to a 3D-printing service. Transparent acrylic based photopolymer, PA220 plastic and 316L stainless steel patient-specific ACL femoral tunnel guides were created; the models produced were accurate with no statistical difference in size and positioning of the center of the ACL femoral footprint guide to MRI ( p =0.344, p =0.189, p =0.233 respectively). The guides aim to provide accurate marking of the starting point of the femoral tunnel in arthroscopic ACL reconstruction. This study serves as a proof of concept for the accurate creation of 3D-printed patient-specific guides for the anatomical placement of the femoral tunnel during ACL reconstruction.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

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Ramiro Olivera

Full Text Available The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647 on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I induced pluripotent stem cells (iPSCs vs. adult fibroblasts (AF fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II umbilical cord-derived mesenchymal stromal cells (UC-MSCs vs. fetal fibroblasts derived from an unborn cloned foetus (FF vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively, respect to <1h (5.2% and 2%, respectively and 1-2h (5.6% and 4.7%, respectively groups (P<0.05. However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2% or 1-2h (35.7% were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6% than using FF (8.9% or AF (9.3%, (P<0.05. Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively, viable foals (two were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Nurses' Perceptions of Implementing Fall Prevention Interventions to Mitigate Patient-Specific Fall Risk Factors.

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Wilson, Deleise S; Montie, Mary; Conlon, Paul; Reynolds, Margaret; Ripley, Robert; Titler, Marita G

2016-08-01

Evidence-based (EB) fall prevention interventions to mitigate patient-specific fall risk factors are readily available but not routinely used in practice. Few studies have examined nurses' perceptions about both the use of these EB interventions and implementation strategies designed to promote their adoption. This article reports qualitative findings of nurses' perceptions about use of EB fall prevention interventions to mitigate patient-specific fall risks, and implementation strategies to promote use of these interventions. The findings revealed five major themes: before-study fall prevention practices, use of EB fall prevention interventions tailored to patient-specific fall risk factors, beneficial implementation strategies, overall impact on approach to fall prevention, and challenges These findings are useful to guide nurses' engagement and use of EB fall prevention practices tailored to patient-specific fall risk factors. © The Author(s) 2016.

Rare myositis-specific autoantibody associations among Hungarian patients with idiopathic inflammatory myopathy.

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Bodoki, L; Nagy-Vincze, M; Griger, Z; Betteridge, Z; Szöllősi, L; Jobanputra, R; Dankó, K

2015-01-01

Idiopathic inflammatory myopathies are systemic, chronic autoimmune diseases characterized by symmetrical, proximal muscle weakness. Homogeneous groups present with similar symptoms. The response to therapy and prognosis could be facilitated by myositis-specific autoantibodies, and in this way, give rise to immunoserological classification. The myositis-specific autoantibodies are directed against specific proteins found in the cytoplasm or in the nucleus of the cells. To date, literature suggests the rarity of the co-existence of two myositis-specific autoantibodies. In this study the authors highlight rare associations of myositis-specific autoantibodies. Three hundred and thirty-seven Hungarian patients with polymyositis or dermatomyositis were studied. Their clinical findings were noted retrospectively. Specific blood tests identified six patients with the rare co-existence of myositis-specific autoantibodies, anti-Jo-1 and anti-SRP, anti-Jo-1 and anti-Mi-2, anti-Mi-2 and anti-PL-12, anti-Mi-2 and anti-SRP, and anti-SRP and anti-PL-7, respectively. This case review aims to identify the clinical importance of these rare associations and their place within the immunoserological classification.

Assessment of CT dose to the fetus and pregnant female patient using patient-specific computational models

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Xie, Tianwu; Poletti, Pierre-Alexandre; Platon, Alexandra; Becker, Christoph D. [Geneva University Hospital, Department of Medical Imaging and Information Sciences, Geneva (Switzerland); Zaidi, Habib [Geneva University Hospital, Department of Medical Imaging and Information Sciences, Geneva (Switzerland); Geneva University, Geneva Neuroscience Center, Geneva (Switzerland); University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, University of Groningen, Groningen (Netherlands); University of Southern Denmark, Department of Nuclear Medicine, Odense (Denmark); Geneva University Hospital, Division of Nuclear Medicine and Molecular Imaging, Geneva (Switzerland)

2018-03-15

This work provides detailed estimates of the foetal dose from diagnostic CT imaging of pregnant patients to enable the assessment of the diagnostic benefits considering the associated radiation risks. To produce realistic biological and physical representations of pregnant patients and the embedded foetus, we developed a methodology for construction of patient-specific voxel-based computational phantoms based on existing standardised hybrid computational pregnant female phantoms. We estimated the maternal absorbed dose and foetal organ dose for 30 pregnant patients referred to the emergency unit of Geneva University Hospital for abdominal CT scans. The effective dose to the mother varied from 1.1 mSv to 2.0 mSv with an average of 1.6 mSv, while commercial dose-tracking software reported an average effective dose of 1.9 mSv (range 1.7-2.3 mSv). The foetal dose normalised to CTDI{sub vol} varies between 0.85 and 1.63 with an average of 1.17. The methodology for construction of personalised computational models can be exploited to estimate the patient-specific radiation dose from CT imaging procedures. Likewise, the dosimetric data can be used for assessment of the radiation risks to pregnant patients and the foetus from various CT scanning protocols, thus guiding the decision-making process. (orig.)

Elevated Cancer-Specific Mortality Among HIV-Infected Patients in the United States.

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Coghill, Anna E; Shiels, Meredith S; Suneja, Gita; Engels, Eric A

2015-07-20

Despite advances in the treatment of HIV, HIV-infected people remain at increased risk for many cancers, and the number of non-AIDS-defining cancers is increasing with the aging of the HIV-infected population. No prior study has comprehensively evaluated the effect of HIV on cancer-specific mortality. We identified cases of 14 common cancers occurring from 1996 to 2010 in six US states participating in a linkage of cancer and HIV/AIDS registries. We used Cox regression to examine the association between patient HIV status and death resulting from the presenting cancer (ascertained from death certificates), adjusting for age, sex, race/ethnicity, year of cancer diagnosis, and cancer stage. We included 1,816,461 patients with cancer, 6,459 (0.36%) of whom were HIV infected. Cancer-specific mortality was significantly elevated in HIV-infected compared with HIV-uninfected patients for many cancers: colorectum (adjusted hazard ratio [HR], 1.49; 95% CI, 1.21 to 1.84), pancreas (HR, 1.71; 95% CI, 1.35 to 2.18), larynx (HR, 1.62; 95% CI, 1.06 to 2.47), lung (HR, 1.28; 95% CI, 1.17 to 1.39), melanoma (HR, 1.72; 95% CI, 1.09 to 2.70), breast (HR, 2.61; 95% CI, 2.06 to 3.31), and prostate (HR, 1.57; 95% CI, 1.02 to 2.41). HIV was not associated with increased cancer-specific mortality for anal cancer, Hodgkin lymphoma, or diffuse large B-cell lymphoma. After further adjustment for cancer treatment, HIV remained associated with elevated cancer-specific mortality for common non-AIDS-defining cancers: colorectum (HR, 1.40; 95% CI, 1.09 to 1.80), lung (HR, 1.28; 95% CI, 1.14 to 1.44), melanoma (HR, 1.93; 95% CI, 1.14 to 3.27), and breast (HR, 2.64; 95% CI, 1.86 to 3.73). HIV-infected patients with cancer experienced higher cancer-specific mortality than HIV-uninfected patients, independent of cancer stage or receipt of cancer treatment. The elevation in cancer-specific mortality among HIV-infected patients may be attributable to unmeasured stage or treatment differences as well

Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient.

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Liu, Zhong; Zhao, Rui

2016-09-01

Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD). The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Applications of patient-specific 3D printing in medicine.

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Heller, Martin; Bauer, Heide-Katharina; Goetze, Elisabeth; Gielisch, Matthias; Roth, Klaus E; Drees, Philipp; Maier, Gerrit S; Dorweiler, Bernhard; Ghazy, Ahmed; Neufurth, Meik; Müller, Werner E G; Schröder, Heinz C; Wang, Xiaohong; Vahl, Christian-Friedrich; Al-Nawas, Bilal

Already three decades ago, the potential of medical 3D printing (3DP) or rapid prototyping for improved patient treatment began to be recognized. Since then, more and more medical indications in different surgical disciplines have been improved by using this new technique. Numerous examples have demonstrated the enormous benefit of 3DP in the medical care of patients by, for example, planning complex surgical interventions preoperatively, reducing implantation steps and anesthesia times, and helping with intraoperative orientation. At the beginning of every individual 3D model, patient-specific data on the basis of computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound data is generated, which is then digitalized and processed using computer-aided design/computer-aided manufacturing (CAD/CAM) software. Finally, the resulting data sets are used to generate 3D-printed models or even implants. There are a variety of different application areas in the various medical fields, eg, drill or positioning templates, or surgical guides in maxillofacial surgery, or patient-specific implants in orthopedics. Furthermore, in vascular surgery it is possible to visualize pathologies such as aortic aneurysms so as to improve the planning of surgical treatment. Although rapid prototyping of individual models and implants is already applied very successfully in regenerative medicine, most of the materials used for 3DP are not yet suitable for implantation in the body. Therefore, it will be necessary in future to develop novel therapy approaches and design new materials in order to completely reconstruct natural tissue.

iPSC-Derived Regulatory Dendritic Cells Inhibit Allograft Rejection by Generating Alloantigen-Specific Regulatory T Cells

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Songjie Cai

2017-05-01

Full Text Available Regulatory dendritic cell (DCregs-based immunotherapy is a potential therapeutic tool for transplant rejection. We generated DCregs from murine induced pluripotent stem cells (iPSCs, which could remain in a “stable immature stage” even under strong stimulation. Harnessing this characteristic, we hypothesized that iPS-DCregs worked as a negative vaccine to generate regulatory T cells (Tregs, and induced donor-specific allograft acceptance. We immunized naive CBA (H-2Kk mice with B6 (H-2Kb iPS-DCregs and found that Tregs (CD4+CD25+FOXP3+ significantly increased in CBA splenocytes. Moreover, immunized CBA recipients permanently accepted B6 cardiac grafts in a donor-specific pattern. We demonstrated mechanistically that donor-type iPS-DCregs triggered transforming growth factor β1 secretion, under which the donor-antigen peptides directed naive CD4+ T cells to differentiate into donor-specific FOXP3+ Tregs instead of into effector T cells in vivo. These findings highlight the potential of iPS-DCregs as a key cell therapy resource in clinical transplantation.

Assessment of CT dose to the fetus and pregnant female patient using patient-specific computational models

DEFF Research Database (Denmark)

Xie, Tianwu; Poletti, Pierre-Alexandre; Platon, Alexandra

2018-01-01

of pregnant patients and the embedded foetus, we developed a methodology for construction of patient-specific voxel-based computational phantoms based on existing standardised hybrid computational pregnant female phantoms. We estimated the maternal absorbed dose and foetal organ dose for 30 pregnant patients...... for assessment of the radiation risks to pregnant patients and the foetus from various CT scanning protocols, thus guiding the decision-making process. KEY POINTS: • In CT examinations, the absorbed dose is non-uniformly distributed within foetal organs. • This work reports, for the first time, estimates...

Patient specific root-analogue dental implants – additive manufacturing and finite element analysis

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Gattinger Johannes

2016-09-01

Full Text Available Aim of this study was to prove the possibility of manufacturing patient specific root analogue two-part (implant and abutment implants by direct metal laser sintering. The two-part implant design enables covered healing of the implant. Therefore, CT-scans of three patients are used for reverse engineering of the implants, abutments and crowns. Patient specific implants are manufactured and measured concerning dimensional accuracy and surface roughness. Impacts of occlusal forces are simulated via FEA and compared to those of standard implants.

Patient-Specific Variations in Biomarkers across Gingivitis and Periodontitis

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Nagarajan, Radhakrishnan; Miller, Craig S.; Dawson, Dolph; Al-Sabbagh, Mohanad; Ebersole, Jeffrey L.

2015-01-01

This study investigates the use of saliva, as an emerging diagnostic fluid in conjunction with classification techniques to discern biological heterogeneity in clinically labelled gingivitis and periodontitis subjects (80 subjects; 40/group) A battery of classification techniques were investigated as traditional single classifier systems as well as within a novel selective voting ensemble classification approach (SVA) framework. Unlike traditional single classifiers, SVA is shown to reveal patient-specific variations within disease groups, which may be important for identifying proclivity to disease progression or disease stability. Salivary expression profiles of IL-1ß, IL-6, MMP-8, and MIP-1α from 80 patients were analyzed using four classification algorithms (LDA: Linear Discriminant Analysis [LDA], Quadratic Discriminant Analysis [QDA], Naïve Bayes Classifier [NBC] and Support Vector Machines [SVM]) as traditional single classifiers and within the SVA framework (SVA-LDA, SVA-QDA, SVA-NB and SVA-SVM). Our findings demonstrate that performance measures (sensitivity, specificity and accuracy) of traditional classification as single classifier were comparable to that of the SVA counterparts using clinical labels of the samples as ground truth. However, unlike traditional single classifier approaches, the normalized ensemble vote-counts from SVA revealed varying proclivity of the subjects for each of the disease groups. More importantly, the SVA identified a subset of gingivitis and periodontitis samples that demonstrated a biological proclivity commensurate with the other clinical group. This subset was confirmed across SVA-LDA, SVA-QDA, SVA-NB and SVA-SVM. Heatmap visualization of their ensemble sets revealed lack of consensus between these subsets and the rest of the samples within the respective disease groups indicating the unique nature of the patients in these subsets. While the source of variation is not known, the results presented clearly elucidate the

Fetal Therapy Model of Myelomeningocele with Three-Dimensional Skin Using Amniotic Fluid Cell-Derived Induced Pluripotent Stem Cells.

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Kajiwara, Kazuhiro; Tanemoto, Tomohiro; Wada, Seiji; Karibe, Jurii; Ihara, Norimasa; Ikemoto, Yu; Kawasaki, Tomoyuki; Oishi, Yoshie; Samura, Osamu; Okamura, Kohji; Takada, Shuji; Akutsu, Hidenori; Sago, Haruhiko; Okamoto, Aikou; Umezawa, Akihiro

2017-06-06

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth, and no effective treatment has been reported to date. Only surgical repairs have been reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Specificity of Health-Related Autobiographical Memories in Patients With Somatic Symptom Disorder.

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Walentynowicz, Marta; Raes, Filip; Van Diest, Ilse; Van den Bergh, Omer

2017-01-01

Patients with somatic symptom disorder (SSD) have persistent distressing somatic symptoms that are associated with excessive thoughts, feelings, and behaviors. Reduced autobiographical memory specificity (rAMS) is related to a range of emotional disorders and is considered a vulnerability factor for an unfavorable course of pathology. The present study investigated whether the specificity of health-related autobiographical memories is reduced in patients with SSD with medically unexplained dyspnea complaints, compared with healthy controls. Female patients with SSD (n = 30) and matched healthy controls (n = 24) completed a health-related Autobiographical Memory Test, the Beck Depression Inventory, the Ruminative Response Scale, and rumination scales concerning bodily reactions. Depressive symptoms and rumination were assessed because both variables previously showed associations with rAMS. Patients with SSD recalled fewer specific (F(1,52) = 13.63, p = .001) and more categoric (F(1,52) = 7.62, p = .008) autobiographical memories to health-related cue words than healthy controls. Patients also reported higher levels of depressive symptoms and rumination (all t > 3.00, p < .01). Importantly, the differences in memory specificity were independent of depressive symptoms and trait rumination. The present study extends findings on rAMS to a previously unstudied sample of patients with SSD. Importantly, the presence of rAMS could not be explained by increased levels of depressive symptoms and rumination. We submit that rAMS in this group reflects how health-related episodes and associated symptoms are encoded in memory.

Coupling of EIT with computational lung modeling for predicting patient-specific ventilatory responses.

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Roth, Christian J; Becher, Tobias; Frerichs, Inéz; Weiler, Norbert; Wall, Wolfgang A

2017-04-01

Providing optimal personalized mechanical ventilation for patients with acute or chronic respiratory failure is still a challenge within a clinical setting for each case anew. In this article, we integrate electrical impedance tomography (EIT) monitoring into a powerful patient-specific computational lung model to create an approach for personalizing protective ventilatory treatment. The underlying computational lung model is based on a single computed tomography scan and able to predict global airflow quantities, as well as local tissue aeration and strains for any ventilation maneuver. For validation, a novel "virtual EIT" module is added to our computational lung model, allowing to simulate EIT images based on the patient's thorax geometry and the results of our numerically predicted tissue aeration. Clinically measured EIT images are not used to calibrate the computational model. Thus they provide an independent method to validate the computational predictions at high temporal resolution. The performance of this coupling approach has been tested in an example patient with acute respiratory distress syndrome. The method shows good agreement between computationally predicted and clinically measured airflow data and EIT images. These results imply that the proposed framework can be used for numerical prediction of patient-specific responses to certain therapeutic measures before applying them to an actual patient. In the long run, definition of patient-specific optimal ventilation protocols might be assisted by computational modeling. NEW & NOTEWORTHY In this work, we present a patient-specific computational lung model that is able to predict global and local ventilatory quantities for a given patient and any selected ventilation protocol. For the first time, such a predictive lung model is equipped with a virtual electrical impedance tomography module allowing real-time validation of the computed results with the patient measurements. First promising results

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

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Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by

Age Is Relative—Impact of Donor Age on Induced Pluripotent Stem Cell-Derived Cell Functionality

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Elisabeth Tamara Strässler

2018-01-01

Full Text Available Induced pluripotent stem cells (iPSCs avoid many of the restrictions that hamper the application of human embryonic stem cells: limited availability of source material due to legal restrictions in some countries, immunogenic rejection and ethical concerns. Also, the donor’s clinical phenotype is often known when working with iPSCs. Therefore, iPSCs seem ideal to tackle the two biggest tasks of regenerative medicine: degenerative diseases with genetic cause (e.g., Duchenne’s muscular dystrophy and organ replacement in age-related diseases (e.g., end-stage heart or renal failure, especially in combination with recently developed gene-editing tools. In the setting of autologous transplantation in elderly patients, donor age becomes a potentially relevant factor that needs to be assessed. Here, we review and critically discuss available data pertinent to the questions: How does donor age influence the reprogramming process and iPSC functionality? Would it even be possible to reprogram senescent somatic cells? How does donor age affect iPSC differentiation into specialised cells and their functionality? We also identify research needs, which might help resolve current unknowns. Until recently, most hallmarks of ageing were attributed to an accumulation of DNA damage over time, and it was thus expected that DNA damage from a somatic cell would accumulate in iPSCs and the cells derived from them. In line with this, a decreased lifespan of cloned organisms compared with the donor was also observed in early cloning experiments. Therefore, it was questioned for a time whether iPSC derived from an old individual’s somatic cells would suffer from early senescence and, thus, may not be a viable option either for disease modelling nor future clinical applications. Instead, typical signs of cellular ageing are reverted in the process of iPSC reprogramming, and iPSCs from older donors do not show diminished differentiation potential nor do i

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

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Yumlu, Saniye; Stumm, Jürgen; Bashir, Sanum; Dreyer, Anne-Kathrin; Lisowski, Pawel; Danner, Eric; Kühn, Ralf

2017-05-15

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy.

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Dhruv Sareen

Full Text Available Spinal muscular atrophy (SMA is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC lines generated from two Type I SMA subjects-one produced with lentiviral constructs and the second using a virus-free plasmid-based approach-recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients.

Site-Specific Difference of Bone Geometry Indices in Hypoparathyroid Patients

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Hye-Sun Park

2017-02-01

Full Text Available BackgroundHypoparathyroid patients often have a higher bone mineral density (BMD than the general population. However, an increase in BMD does not necessarily correlate with a solid bone microstructure. This study aimed to evaluate the bone microstructure of hypoparathyroid patients by using hip structure analysis (HSA.MethodsNinety-five hypoparathyroid patients >20 years old were enrolled and 31 of them had eligible data for analyzing bone geometry parameters using HSA. And among the control data, we extracted sex-, age-, and body mass index-matched three control subjects to each patient. The BMD data were reviewed retrospectively and the bone geometry parameters of the patients were analyzed by HSA.ResultsThe mean Z-scores of hypoparathyroid patients at the lumbar spine, femoral neck, and total hip were above zero (0.63±1.17, 0.48±1.13, and 0.62±1.10, respectively. The differences in bone geometric parameters were site specific. At the femoral neck and intertrochanter, the cross-sectional area (CSA and cortical thickness (C.th were higher, whereas the buckling ratio (BR was lower than in controls. However, those trends were opposite at the femoral shaft; that is, the CSA and C.th were low and the BR was high.ConclusionOur study shows the site-specific effects of hypoparathyroidism on the bone. Differences in bone components, marrow composition, or modeling based bone formation may explain these findings. However, further studies are warranted to investigate the mechanism, and its relation to fracture risk.

Violence and Personality in Forensic Patients: Is There a Forensic Patient-Specific Personality Profile?

Science.gov (United States)

Stupperich, Alexandra; Ihm, Helga; Strack, Micha

2009-01-01

Concerning the discussion about the connection of personality traits, personality disorders, and mental illness, this study focused on the personality profiles of male forensic patients, prison inmates, and young men without criminal reports. The main topic centered on group-specific personality profiles and identifying personality facets…

Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific homozygous mutation in CHMP2B

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Yu Zhang

2016-07-01

Full Text Available Frontotemporal dementia (FTD is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B on chromosome 3 (FTD3, a component of the endosomal sorting complex required for transport III (ESCRT-III. We have generated an induced pluripotent stem cell (iPSC line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into both alleles, resulting in aberrant splicing. This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.

Disease specific knowledge about cystic fibrosis, patient education and counselling in Poland

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Sławomir Chomik

2014-06-01

Full Text Available introduction and objective. The presented study assesses levels of specific knowledge of the disease among cystic fibrosis (CF patients and their families, and evaluates the effectiveness of a targeted, disease specific education programme. materials and methods. A cross-sectional survey among 462 families with a CF child evaluated their knowledge of the disease. A one year follow up survey among 200 families assessed the effectiveness of an educational programme developed to correct gaps, errors and misconceptions identified in the previously administered survey. Self-administered, comprehensive, 5-domains, 45-item multiple-choice CF Disease Knowledge Questionnaire (CFDKQ was anonymously completed by 462 subjects. results. 228 respondents were male (49%, 234 female (51%. The level of disease-specific knowledge in the age groups 0–6 and 7–10 years, was significantly higher than in 11–14 and 15–18 years of age groups (p<0.005. General medical and Genetics/Reproduction knowledge was low in all patients. Significant predictors of patient and parental knowledge were age and domicile. Patients and parents rely heavily on doctors for information about CF (77%. The follow-up survey (CFDKQ emphasized that special education programmes significantly improved levels of disease specific knowledge (p<0.0001. conclusions. If left uncorrected, the misconceptions, gaps and errors in CF knowledge identified in the presented study could result in inadvertent non-adherence to treatment, and impact on the progression and outcome of the disease. Secondly, the results demonstrate the effectiveness of targeted, disease specific information in improving disease knowledge of CF patients and their families, and highlights the value and need for the development of educational programmes for chronically ill patients and their families.

Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation

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Shishi Li

2018-03-01

Full Text Available Summary: Hypertrophic cardiomyopathy (HCM is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2. The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM. : In this article, Yan Q, Liu Z, Huang W, and colleagues show that patient-specific iPSCs as well as their derived cardiomyocytes carrying the m.2336T>C mutation in MT-RNR2 were generated to understand the pathogenic mechanism of maternally inherited HCM. MT-RNR2 mutation resulted in mitochondrial dysfunctions and ultrastructure defects, which induced abnormal Ca2+ homeostasis, then HCM-specific cellular and electrophysiological characteristics in iPSC-CMs. Keywords: mitochondrion, hypertrophic cardiomyopathy, induced pluripotent stem cells, MT-RNR2, maternal inheritance

Patient specific 3D visualisation of human brain | Baichoo ...

African Journals Online (AJOL)

University of Mauritius Research Journal. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 15, No 1 (2009) >. Log in or Register to get access to full text downloads. Username, Password, Remember me, or Register. Patient specific 3D visualisation of human brain.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

Science.gov (United States)

Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

2014-12-21

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Muller cell-specific autoantibodies in a patient with progressive loss of vision

NARCIS (Netherlands)

Peek, R.; Verbraak, F.; Coevoet, H. M.; Kijlstra, A.

1998-01-01

PURPOSE. To study the specificity of circulating retinal autoantibodies in a patient with progressive loss of vision resembling cancer-associated retinopathy in the absence of systemic malignancy. METHODS. Patient's serum was tested for the presence of antiretinal antibodies by Western blot

Porcine induced pluripotent stem cells produce chimeric offspring.

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West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

2010-08-01

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

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Duan, Jubao

2015-02-01

Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.

Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature.

Science.gov (United States)

Haberman, Yael; Tickle, Timothy L; Dexheimer, Phillip J; Kim, Mi-Ok; Tang, Dora; Karns, Rebekah; Baldassano, Robert N; Noe, Joshua D; Rosh, Joel; Markowitz, James; Heyman, Melvin B; Griffiths, Anne M; Crandall, Wallace V; Mack, David R; Baker, Susan S; Huttenhower, Curtis; Keljo, David J; Hyams, Jeffrey S; Kugathasan, Subra; Walters, Thomas D; Aronow, Bruce; Xavier, Ramnik J; Gevers, Dirk; Denson, Lee A

2014-08-01

Interactions between the host and gut microbial community likely contribute to Crohn disease (CD) pathogenesis; however, direct evidence for these interactions at the onset of disease is lacking. Here, we characterized the global pattern of ileal gene expression and the ileal microbial community in 359 treatment-naive pediatric patients with CD, patients with ulcerative colitis (UC), and control individuals. We identified core gene expression profiles and microbial communities in the affected CD ilea that are preserved in the unaffected ilea of patients with colon-only CD but not present in those with UC or control individuals; therefore, this signature is specific to CD and independent of clinical inflammation. An abnormal increase of antimicrobial dual oxidase (DUOX2) expression was detected in association with an expansion of Proteobacteria in both UC and CD, while expression of lipoprotein APOA1 gene was downregulated and associated with CD-specific alterations in Firmicutes. The increased DUOX2 and decreased APOA1 gene expression signature favored oxidative stress and Th1 polarization and was maximally altered in patients with more severe mucosal injury. A regression model that included APOA1 gene expression and microbial abundance more accurately predicted month 6 steroid-free remission than a model using clinical factors alone. These CD-specific host and microbe profiles identify the ileum as the primary inductive site for all forms of CD and may direct prognostic and therapeutic approaches.

Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications.

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Chang, Chia-Yu; Ting, Hsiao-Chien; Su, Hong-Lin; Jeng, Jing-Ren

2018-01-01

In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.

Induced pluripotent stem cell-based therapy for age-related macular degeneration.

Science.gov (United States)

Bracha, Peter; Moore, Nicholas A; Ciulla, Thomas A

2017-09-01

In age-related macular degeneration (AMD), stem cells could possibly replace or regenerate disrupted pathologic retinal pigment epithelium (RPE), and produce supportive growth factors and cytokines such as brain-derived neurotrophic factor.  Induced pluripotent stem cells (iPSCs)-derived RPE was first subretinally transplanted in a neovascular AMD patient in 2014. Areas covered: Induced PSCs are derived from the introduction of transcription factors to adult cells under specific cell culture conditions, followed by differentiation into RPE cells. Induced PSC-derived RPE cells exhibit ion transport, membrane potential, polarized VEGF secretion and gene expression that is similar to native RPE. Despite having similar in vitro function, morphology, immunostaining and microscopic analysis, it remains to be seen if iPSC-derived RPE can replicate the myriad of in vivo functions, including immunomodulatory effects, of native RPE cells.  Historically, adjuvant RPE transplantation during CNV resections were technically difficult and complicated by immune rejection. Autologous iPSCs are hypothesized to reduce the risk of immune rejection, but their production is time-consuming and expensive.  Alternatively, allogenic transplantation using human leukocyte antigen (HLA)-matched iPSCs, similar to HLA-matched organ transplantation, is currently being investigated. Expert opinion: Challenges to successful transplantation with iPSCs include surgical technique, a pathologic subretinal microenvironment, possible immune rejection, and complications of immunosuppression.

Risks of all-cause and site-specific fractures among hospitalized patients with COPD

Science.gov (United States)

Liao, Kuang-Ming; Liang, Fu-Wen; Li, Chung-Yi

2016-01-01

Abstract Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis. The clinical sequel of osteoporosis is fracture. Patients with COPD who experience a fracture also have increased morbidity and mortality. Currently, the types of all-cause and site-specific fracture among patients with COPD are unknown. Thus, we elucidated the all-cause and site-specific fractures among patients with COPD. A retrospective, population-based, cohort study was conducted utilizing the Taiwan Longitudinal Health Insurance Database. Patients with COPD were defined as those who were hospitalized with an International Classification of Diseases, Ninth Revision, Clinical Modification code of 490 to 492 or 496 between 2001 and 2011. The index date was set as the date of discharge. The study patients were followed from the index date to the date when they sought care for any type of fracture, date of death, date of health insurance policy termination, or the last day of 2013. The types of fracture analyzed in this study included vertebral, rib, humeral, radial and ulnar/wrist, pelvic, femoral, and tibial and fibular fractures. The cohort consisted of 11,312 patients with COPD. Among these patients, 1944 experienced fractures. The most common site-specific fractures were vertebral, femoral, rib, and forearm fractures (radius, ulna, and wrist) at 32.4%, 31%, 12%, and 11.8%, respectively. The adjusted hazard ratios of fracture were 1.71 [95% confidence interval (95% CI) = 1.56–1.87] for female patient with COPD and 1.50 (95% CI = 1.39–1.52) for patients with osteoporosis after covariate adjustment. Vertebral and hip fractures are common among patients with COPD, especially among males with COPD. Many comorbidities contribute to the high risk of fracture among patients with COPD. PMID:27749576

Automatic selective feature retention in patient specific elastic surface registration

CSIR Research Space (South Africa)

Jansen van Rensburg, GJ

2011-01-01

Full Text Available The accuracy with which a recent elastic surface registration algorithm deforms the complex geometry of a skull is examined. This algorithm is then coupled to a line based algorithm as is frequently used in patient specific feature registration...

A 4D digital phantom for patient-specific simulation of brain CT perfusion protocols.

Science.gov (United States)

van den Boom, Rieneke; Manniesing, Rashindra; Oei, Marcel T H; van der Woude, Willem-Jan; Smit, Ewoud J; Laue, Hendrik O A; van Ginneken, Bram; Prokop, Mathias

2014-07-01

Optimizing CT brain perfusion protocols is a challenge because of the complex interaction between image acquisition, calculation of perfusion data, and patient hemodynamics. Several digital phantoms have been developed to avoid unnecessary patient exposure or suboptimum choice of parameters. The authors expand this idea by using realistic noise patterns and measured tissue attenuation curves representing patient-specific hemodynamics. The purpose of this work is to validate that this approach can realistically simulate mean perfusion values and noise on perfusion data for individual patients. The proposed 4D digital phantom consists of three major components: (1) a definition of the spatial structure of various brain tissues within the phantom, (2) measured tissue attenuation curves, and (3) measured noise patterns. Tissue attenuation curves were measured in patient data using regions of interest in gray matter and white matter. By assigning the tissue attenuation curves to the corresponding tissue curves within the phantom, patient-specific CTP acquisitions were retrospectively simulated. Noise patterns were acquired by repeatedly scanning an anthropomorphic skull phantom at various exposure settings. The authors selected 20 consecutive patients that were scanned for suspected ischemic stroke and constructed patient-specific 4D digital phantoms using the individual patients' hemodynamics. The perfusion maps of the patient data were compared with the digital phantom data. Agreement between phantom- and patient-derived data was determined for mean perfusion values and for standard deviation in de perfusion data using intraclass correlation coefficients (ICCs) and a linear fit. ICCs ranged between 0.92 and 0.99 for mean perfusion values. ICCs for the standard deviation in perfusion maps were between 0.86 and 0.93. Linear fitting yielded slope values between 0.90 and 1.06. A patient-specific 4D digital phantom allows for realistic simulation of mean values and

Are specific antiretrovirals associated with an increased risk of discontinuation due to toxicities or patient/physician choice in patients with hepatitis C virus coinfection?

DEFF Research Database (Denmark)

Mocroft, A; Rockstroh, J; Soriano, V

2005-01-01

clinical trials as patients with HCV are often excluded. AIMS: To compare incidence rates of discontinuation due to TOXPC associated with specific antiretrovial drugs in patients with or without HCV. PATIENTS/METHODS: A total of 4929 patients from EuroSIDA under follow-up from January 1999 on a specific...

3D printing of patient-specific anatomy: A tool to improve patient consent and enhance imaging interpretation by trainees.

Science.gov (United States)

Liew, Yaoren; Beveridge, Erin; Demetriades, Andreas K; Hughes, Mark A

2015-01-01

We report the use of three-dimensional or 3D printed, patient-specific anatomy as a tool to improve informed patient consent and patient understanding in a case of posterior lumbar fixation. Next, we discuss its utility as an educational tool to enhance imaging interpretation by neurosurgery trainees.

High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Ying Ao

Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

Reliability of patient specific instrumentation in total knee arthroplasty.

Science.gov (United States)

Jennart, Harold; Ngo Yamben, Marie-Ange; Kyriakidis, Theofylaktos; Zorman, David

2015-12-01

The aim of this study was to compare the precision between Patient Specific Instrumentation (PSI) and Conventional Instrumentation (CI) as determined intra-operatively by a pinless navigation system. Eighty patients were included in this prospective comparative study and they were divided into two homogeneous groups. We defined an original score from 6 to 30 points to evaluate the accuracy of the position of the cutting guides. This score is based on 6 objective criteria. The analysis indicated that PSI was not superior to conventional instrumentation in the overall score (p = 0.949). Moreover, no statistically significant difference was observed for any individual criteria of our score. Level of evidence II.

Association between PSA kinetics and cancer-specific mortality in patients with localised prostate cancer

DEFF Research Database (Denmark)

Thomsen, Frederik Birkebæk; Brasso, Klaus; Berg, Kasper Drimer

2016-01-01

BACKGROUND: The prognostic value of prostate-specific antigen (PSA) kinetics in untreated prostate cancer (PCa) patients is debatable. We investigated the association between PSA doubling time (PSAdt), PSA velocity (PSAvel) and PSAvel risk count (PSAvRC) and PCa mortality in a cohort of patients...... with localised PCa managed on watchful waiting. PATIENTS AND METHODS: Patients with clinically localised PCa managed observationally, who were randomised to and remained on placebo for minimum 18 months in the SPCG-6 study, were included. All patients survived at least 2 years and had a minimum of three PSA...... determinations available. The prognostic value of PSA kinetics was analysed and patients were stratified according to their PSA at consent: ≤10, 10.1-25, and >25 ng/ml. Cumulative incidences of PCa-specific mortality were estimated with the Aalen-Johansen method. RESULTS: Two hundred and sixty-three patients...

Using an EPID for patient-specific VMAT quality assurance

International Nuclear Information System (INIS)

Bakhtiari, M.; Kumaraswamy, L.; Bailey, D. W.; Boer, S. de; Malhotra, H. K.; Podgorsak, M. B.

2011-01-01

Purpose: A patient-specific quality assurance (QA) method was developed to verify gantry-specific individual multileaf collimator (MLC) apertures (control points) in volumetric modulated arc therapy (VMAT) plans using an electronic portal imaging device (EPID). Methods: VMAT treatment plans were generated in an Eclipse treatment planning system (TPS). DICOM images from a Varian EPID (aS1000) acquired in continuous acquisition mode were used for pretreatment QA. Each cine image file contains the grayscale image of the MLC aperture related to its specific control point and the corresponding gantry angle information. The TPS MLC file of this RapidArc plan contains the leaf positions for all 177 control points (gantry angles). In-house software was developed that interpolates the measured images based on the gantry angle and overlays them with the MLC pattern for all control points. The 38% isointensity line was used to define the edge of the MLC leaves on the portal images. The software generates graphs and tables that provide analysis for the number of mismatched leaf positions for a chosen distance to agreement at each control point and the frequency in which each particular leaf mismatches for the entire arc. Results: Seven patients plans were analyzed using this method. The leaves with the highest mismatched rate were found to be treatment plan dependent. Conclusions: This in-house software can be used to automatically verify the MLC leaf positions for all control points of VMAT plans using cine images acquired by an EPID.

Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.

Science.gov (United States)

Kotini, Andriana G; Chang, Chan-Jung; Chow, Arthur; Yuan, Han; Ho, Tzu-Chieh; Wang, Tiansu; Vora, Shailee; Solovyov, Alexander; Husser, Chrystel; Olszewska, Malgorzata; Teruya-Feldstein, Julie; Perumal, Deepak; Klimek, Virginia M; Spyridonidis, Alexandros; Rampal, Raajit K; Silverman, Lewis; Reddy, E Premkumar; Papaemmanuil, Elli; Parekh, Samir; Greenbaum, Benjamin D; Leslie, Christina S; Kharas, Michael G; Papapetrou, Eirini P

2017-03-02

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

Integrated analysis of hematopoietic differentiation outcomes and molecular characterization reveals unbiased differentiation capacity and minor transcriptional memory in HPC/HSC-iPSCs.

Science.gov (United States)

Gao, Shuai; Hou, Xinfeng; Jiang, Yonghua; Xu, Zijian; Cai, Tao; Chen, Jiajie; Chang, Gang

2017-01-23

Transcription factor-mediated reprogramming can reset the epigenetics of somatic cells into a pluripotency compatible state. Recent studies show that induced pluripotent stem cells (iPSCs) always inherit starting cell-specific characteristics, called epigenetic memory, which may be advantageous, as directed differentiation into specific cell types is still challenging; however, it also may be unpredictable when uncontrollable differentiation occurs. In consideration of biosafety in disease modeling and personalized medicine, the availability of high-quality iPSCs which lack a biased differentiation capacity and somatic memory could be indispensable. Herein, we evaluate the hematopoietic differentiation capacity and somatic memory state of hematopoietic progenitor and stem cell (HPC/HSC)-derived-iPSCs (HPC/HSC-iPSCs) using a previously established sequential reprogramming system. We found that HPC/HSCs are amenable to being reprogrammed into iPSCs with unbiased differentiation capacity to hematopoietic progenitors and mature hematopoietic cells. Genome-wide analyses revealed that no global epigenetic memory was detectable in HPC/HSC-iPSCs, but only a minor transcriptional memory of HPC/HSCs existed in a specific tetraploid complementation (4 N)-incompetent HPC/HSC-iPSC line. However, the observed minor transcriptional memory had no influence on the hematopoietic differentiation capacity, indicating the reprogramming of the HPC/HSCs was nearly complete. Further analysis revealed the correlation of minor transcriptional memory with the aberrant distribution of H3K27me3. This work provides a comprehensive framework for obtaining high-quality iPSCs from HPC/HSCs with unbiased hematopoietic differentiation capacity and minor transcriptional memory.

Are Hip-Specific Items Useful in a Quality of Life Questionnaire for Patients with Hip Fractures?

Science.gov (United States)

Yao, Kai-Ping Grace; Lee, Hsin-Yi; Tsauo, Jau-Yih

2009-01-01

Researchers measure the significance of hip fracture by the patient's impairment. The patient's quality of life (QOL) is usually also substantially affected. However, there is no specific quality of life (QOL) questionnaire for patients with hip fractures. This study was designed to determine whether adding a new set of specific questions about…

Induced pluripotent stem cells in research and therapy of diseases: review article

Directory of Open Access Journals (Sweden)

Mohammad Reza Noori Daloii

2014-10-01

Full Text Available Differentiated cells can change to embryonic stem cells by reprograming. Generation of induced pluripotent stem cells (iPSCs has revolutionized the field of regenerative and personalized medicine. iPSCs can self-renew and differentiate into many cell types. iPSC cells offer a potentially unlimited source for targeted differentiation. Through the expression of a set of transcription factors, iPSCs can be generated from different kinds of embryonic and adult cells. This technology for the first time enabled the researchers to take differentiated cells from an individual, and convert them to another cell type of interest, which is particularly to that person. When the set of master transcription factors containing OCT4, SOX2, KLF4, and MYC is expressed ectopically in somatic cells, the transcriptional network is propelled to organize itself in such a way as to maintenance a pluripotent state. Since iPSCs are similar to Embryonic Stem Cell (ESC, they can be considered as sources for modeling different diseases. iPSCs which are induced from somatic cells of patient can be useful for screening and drugs selection, and also introduce treatment via grafting the cells. Although this technology has been successful in different fields, the tumorigenesis of viral vectors during induction of reprogramming is a major challenge. Nevertheless, iPSCs are valuable for clinical applications and research. By discovery of these cells many challenges related to the safety, efficacy, and bioethics of ESCs are solved. Pluripotency is defined in two aspect of functional and molecular, by which functional regards the capacity of cell is generate three kinds of embryonic layers and germ line, and molecular aspect regards the identifying of molecules and genes that support functional features. Identification of these genes has been placed at the center of fields related to development and stem cell research. In this review, we discuss the process of generation of these

Generation of a human induced pluripotent stem cell line from urinary cells of a patient with primary congenital glaucoma using integration free Sendai technology.

Science.gov (United States)

Zhang, Jingxue; Wu, Shen; Hu, Man; Liu, Qian

2018-04-09

We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 10years old patient with primary congenital glaucoma (PCG). The cells were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system and shown to have full differentiation potential. The line is available and registered in the human pluripotent stem cell registry as BIOi001-A. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Retinal Ganglion Cell Diversity and Subtype Specification from Human Pluripotent Stem Cells

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Kirstin B. Langer

2018-04-01

Full Text Available Summary: Retinal ganglion cells (RGCs are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. : In this article, Langer and colleagues present extensive characterization of RGC subtypes derived from human pluripotent stem cells, with multiple subtypes identified by subtype-specific molecular markers. Their results present a more detailed analysis of RGC diversity in human cells and yield the use of different markers to identify RGC subtypes. Keywords: iPSC, retina, retinal ganglion cell, RGC subtype, stem cell, ipRGC, alpha RGC, direction selective RGC, RNA-seq

Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

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Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J

2017-12-01

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.

Patient-specific coronary blood supply territories for quantitative perfusion analysis

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Zakkaroff, Constantine; Biglands, John D.; Greenwood, John P.; Plein, Sven; Boyle, Roger D.; Radjenovic, Aleksandra; Magee, Derek R.

2018-01-01

Abstract Myocardial perfusion imaging, coupled with quantitative perfusion analysis, provides an important diagnostic tool for the identification of ischaemic heart disease caused by coronary stenoses. The accurate mapping between coronary anatomy and under-perfused areas of the myocardium is important for diagnosis and treatment. However, in the absence of the actual coronary anatomy during the reporting of perfusion images, areas of ischaemia are allocated to a coronary territory based on a population-derived 17-segment (American Heart Association) AHA model of coronary blood supply. This work presents a solution for the fusion of 2D Magnetic Resonance (MR) myocardial perfusion images and 3D MR angiography data with the aim to improve the detection of ischaemic heart disease. The key contribution of this work is a novel method for the mediated spatiotemporal registration of perfusion and angiography data and a novel method for the calculation of patient-specific coronary supply territories. The registration method uses 4D cardiac MR cine series spanning the complete cardiac cycle in order to overcome the under-constrained nature of non-rigid slice-to-volume perfusion-to-angiography registration. This is achieved by separating out the deformable registration problem and solving it through phase-to-phase registration of the cine series. The use of patient-specific blood supply territories in quantitative perfusion analysis (instead of the population-based model of coronary blood supply) has the potential of increasing the accuracy of perfusion analysis. Quantitative perfusion analysis diagnostic accuracy evaluation with patient-specific territories against the AHA model demonstrates the value of the mediated spatiotemporal registration in the context of ischaemic heart disease diagnosis. PMID:29392098

Patient-specific quality assurance for intracranial cases in robotic radiosurgery system.

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Koksal, Canan; Akbas, Ugur; Donmez Kesen, Nazmiye; Okutan, Murat; Bilge, Hatice; Kemikler, Gonul

2018-01-01

The purpose of this study was to perform pretreatment patient-specific quality assurance (QA) for intracranial irradiation using CyberKnife with an ion chamber. Twenty-five intracranial plans created using the ray-tracing algorithm were used for this study. Computed tomography (CT) images of the water-equivalent RW3 slab phantom with PinPoint ionization chamber were acquired with 1-mm slice thickness and transferred to the MultiPlan treatment planning system (TPS). Four gold fiducial markers embedded into two different plates were used to tracking during the irradiation. Intracranial plans were transferred to CT images of the RW3 phantom. The isodose curves and sensitive volume of ion chamber were overlapped. Point dose measurements were performed three times and the mean point doses were calculated for each plan. The mean doses measured by the PinPoint ion chamber were compared with those of the calculated by MultiPlan TPS in the sensitive volume of PinPoint. The mean percentage difference (MPD) in point dose measurements was -2.44±1.97 for 25 plans. The maximum and minimum percentage differences between the measured and calculated absolute point doses were -7.14 and 0.23, respectively. The MPD was -1.70±1.90 for 12 plans using a fixed collimator and -3.11±1.86 for 13 plans using an IRIS cone. Point dose measurement is a reliable and functional method for pre-treatment patient-specific QA in intracranial CyberKnife plans. Point dose verification should be performed to correct any possible errors prior to patient treatment. It is recommended for use in patient-specific QA process in the CyberKnife plans.

Potential and Challenges of Induced Pluripotent Stem Cells in Liver Diseases Treatment

Directory of Open Access Journals (Sweden)

Yue Yu

2014-09-01

Full Text Available Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from cell therapy involving living metabolically active cells, either by treatment of their liver disease, or by prevention of their disease phenotype. Cell therapies, including hepatocyte transplantation and bioartificial liver (BAL devices, have been proposed as therapeutic alternatives to the shortage of transplantable livers. Both BAL and hepatocyte transplantation are cellular therapies that avoid use of a whole liver. Hepatocytes are also widely used in drug screening and liver disease modelling. However, the demand for human hepatocytes, heavily outweighs their availability by conventional means. Induced pluripotent stem cells (iPSCs technology brings together the potential benefits of embryonic stem cells (ESCs (i.e., self-renewal, pluripotency and addresses the major ethical and scientific concerns of ESCs: embryo destruction and immune-incompatibility. It has been shown that hepatocyte-like cells (HLCs can be generated from iPSCs. Furthermore, human iPSCs (hiPSCs can provide an unlimited source of human hepatocytes and hold great promise for applications in regenerative medicine, drug screening and liver diseases modelling. Despite steady progress, there are still several major obstacles that need to be overcome before iPSCs will reach the bedside. This review will focus on the current state of efforts to derive hiPSCs for potential use in modelling and treatment of liver disease.

MMCTP: a radiotherapy research environment for Monte Carlo and patient-specific treatment planning

International Nuclear Information System (INIS)

Alexander, A; DeBlois, F; Stroian, G; Al-Yahya, K; Heath, E; Seuntjens, J

2007-01-01

Radiotherapy research lacks a flexible computational research environment for Monte Carlo (MC) and patient-specific treatment planning. The purpose of this study was to develop a flexible software package on low-cost hardware with the aim of integrating new patient-specific treatment planning with MC dose calculations suitable for large-scale prospective and retrospective treatment planning studies. We designed the software package 'McGill Monte Carlo treatment planning' (MMCTP) for the research development of MC and patient-specific treatment planning. The MMCTP design consists of a graphical user interface (GUI), which runs on a simple workstation connected through standard secure-shell protocol to a cluster for lengthy MC calculations. Treatment planning information (e.g., images, structures, beam geometry properties and dose distributions) is converted into a convenient MMCTP local file storage format designated, the McGill RT format. MMCTP features include (a) DICOM R T, RTOG and CADPlan CART format imports; (b) 2D and 3D visualization views for images, structure contours, and dose distributions; (c) contouring tools; (d) DVH analysis, and dose matrix comparison tools; (e) external beam editing; (f) MC transport calculation from beam source to patient geometry for photon and electron beams. The MC input files, which are prepared from the beam geometry properties and patient information (e.g., images and structure contours), are uploaded and run on a cluster using shell commands controlled from the MMCTP GUI. The visualization, dose matrix operation and DVH tools offer extensive options for plan analysis and comparison between MC plans and plans imported from commercial treatment planning systems. The MMCTP GUI provides a flexible research platform for the development of patient-specific MC treatment planning for photon and electron external beam radiation therapy. The impact of this tool lies in the fact that it allows for systematic, platform

The value of urine specific gravity in detecting diabetes insipidus in a patient with uncontrolled diabetes mellitus: urine specific gravity in differential diagnosis.

Science.gov (United States)

Akarsu, Ersin; Buyukhatipoglu, Hakan; Aktaran, Sebnem; Geyik, Ramazan

2006-11-01

When a patient with diabetes mellitus presents with worsening polyuria and polydipsia, what is a sensible, cost-effective approach? We report the unique coincidence of type 2 diabetes mellitus and diabetes insipidus. A 46-year-old woman with poorly controlled type 2 diabetes complained of polyuria with a daily output of 5 L. Although urinalysis demonstrated significant glucosuria, diabetes insipidus was suspected owing to a low urine specific gravity (1.008). The low specific gravity persisted during a water deprivation test. Ultimately, diabetes insipidus was confirmed when urine specific gravity and urine osmolality normalized following desmopressin administration. This case emphasizes the importance of accurately interpreting the urine specific gravity in patients with polyuria and diabetes mellitus to detect diabetes insipidus.

Design and manufacturing of patient-specific orthodontic appliances by computer-aided engineering techniques.

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Barone, Sandro; Neri, Paolo; Paoli, Alessandro; Razionale, Armando Viviano

2018-01-01

Orthodontic treatments are usually performed using fixed brackets or removable oral appliances, which are traditionally made from alginate impressions and wax registrations. Among removable devices, eruption guidance appliances are used for early orthodontic treatments in order to intercept and prevent malocclusion problems. Commercially available eruption guidance appliances, however, are symmetric devices produced using a few standard sizes. For this reason, they are not able to meet all the specific patient's needs since the actual dental anatomies present various geometries and asymmetric conditions. In this article, a computer-aided design-based methodology for the design and manufacturing of a patient-specific eruption guidance appliances is presented. The proposed approach is based on the digitalization of several steps of the overall process: from the digital reconstruction of patients' anatomies to the manufacturing of customized appliances. A finite element model has been developed to evaluate the temporomandibular joint disks stress level caused by using symmetric eruption guidance appliances with different teeth misalignment conditions. The developed model can then be used to guide the design of a patient-specific appliance with the aim at reducing the patient discomfort. At this purpose, two different customization levels are proposed in order to face both arches and single tooth misalignment issues. A low-cost manufacturing process, based on an additive manufacturing technique, is finally presented and discussed.

Prostate specific antigen in the diagnosis and treatment of adenocarcinoma of the prostate. III. Radiation treated patients

International Nuclear Information System (INIS)

Stamey, T.A.; Kabalin, J.N.; Ferrari, M.

1989-01-01

Serum prostate specific antigen was determined (Yang polyclonal radioimmunoassay) in 183 men after radiation therapy for adenocarcinoma of the prostate. A total of 163 men had received 7,000 rad external beam radiotherapy and 20 had been implanted with iodine-125 seeds. Only 11 per cent of these 183 patients had undetectable prostate specific antigen levels at a mean interval of 5 years since completion of radiotherapy. Prostate specific antigen levels after radiotherapy were directly related to initial clinical stage and Gleason score before treatment. Multiple prostate specific antigen determinations were performed with time in 124 of 183 patients. During year 1 after radiotherapy prostate specific antigen levels were decreasing in 82 per cent of the patients but only 8 per cent continued to decrease beyond year 1. Of 80 patients observed greater than 1 year after completion of radiotherapy 51 per cent had increasing values and 41 per cent had stable values. Increasing prostate specific antigen values after radiotherapy were correlated with progression to metastastic disease and residual cancer on prostate biopsy. Total serum acid phosphatase levels were poorly related to prostate specific antigen levels, were less effective in discriminating patients with metastatic disease and provided no additional information beyond that provided by prostate specific antigen

Progress and challenges in the development of a cell-based therapy for hemophilia A

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Fomin, Marina E.; Togarrati, Padma Priya; Muench, Marcus O.

2015-01-01

Hemophilia A results from an insufficiency of factor VIII (FVIII). Although replacement therapy with plasma-derived or recombinant FVIII is a life-saving therapy for hemophilia A patients, such therapy is a life-long treatment rather than a cure for the disease. In this review we discuss the possibilities, progress and challenges that remain in the development of a cell-based cure for hemophilia A. The success of cell therapy depends on the type and availability of donor cells, the age of the host and method of transplantation, and the levels of engraftment and production of FVIII by the graft. Early therapy, possibly even prenatal transplantation, may yield the highest levels of engraftment by avoiding immunological rejection of the graft. Potential cell sources of FVIII include a specialized subset of endothelial cells known as liver sinusoidal endothelial cells (LSECs) present in the adult and fetal liver, or patient-specific endothelial cells derived from induced pluripotent stem cells (iPSCs) that have undergone gene editing to produce FVIII. Achieving sufficient engraftment of transplanted LSECs is one of the obstacles to successful cell therapy for hemophilia A. We discuss recent results from transplants performed in animals that show production of functional and clinically relevant levels of FVIII obtained from donor LSECs. Hence, the possibility of treating hemophilia A can be envisioned through persistent production of FVIII from transplanted donor cells derived from a number of potential cell sources or through creation of donor endothelial cells from patient-specific iPSCs. PMID:25297648

COPE-ICD: Patient experience of participation in an ICD specific rehabilitation programme

DEFF Research Database (Denmark)

Berg, Selina Kikkenborg; Pedersen, Birthe Dagmar; Svendsen, Jesper Hastrup

2012-01-01

individualized care. Four themes emerged: Knowledge: patients gained much needed understanding; Physical attention: patients interpreted body signals and adjusted their exercise behaviour; Trust: patients regained trust, felt secure and dared to live again; Strategies of living: patients' coping was supported...... through reflection and professional dialogue, and they dealt with the risk of shock or death. CONCLUSION: Participating in an ICD-specific rehabilitation programme can make patients feel inspired and secure through individualized care. They discover that they have to rethink some of their strategies......PURPOSE: Evaluating rehabilitation programmes from the patient's perspective is much needed, as the patients are the most important stakeholders in the health care system. A comprehensive rehabilitation programme, COPE-ICD programme, consists of exercise training and nursing consultations during...

Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage

DEFF Research Database (Denmark)

Rasmussen, Mikkel Aabech; Holst, Bjørn; Tümer, Zeynep

2014-01-01

The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led...... to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis...... and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion...

Trans-differentiation via Epigenetics: A New Paradigm in the Bone Regeneration.

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Cho, Young-Dan; Ryoo, Hyun-Mo

2018-02-01

In regenerative medicine, growing cells or tissues in the laboratory is necessary when damaged cells can not heal by themselves. Acquisition of the required cells from the patient's own cells or tissues is an ideal option without additive side effects. In this context, cell reprogramming methods, including the use of induced pluripotent stem cells (iPSCs) and trans-differentiation, have been widely studied in regenerative research. Both approaches have advantages and disadvantages, and the possibility of de-differentiation because of the epigenetic memory of iPSCs has strengthened the need for controlling the epigenetic background for successful cell reprogramming. Therefore, interest in epigenetics has increased in the field of regenerative medicine. Herein, we outline in detail the cell trans-differentiation method using epigenetic modification for bone regeneration in comparison to the use of iPSCs.

WE-D-BRA-05: Pseudo In Vivo Patient Dosimetry Using a 3D-Printed Patient-Specific Phantom

International Nuclear Information System (INIS)

Ger, R; Craft, DF; Burgett, EA; Price, RR; Kry, SF; Howell, RM

2015-01-01

Purpose: To test the feasibility of using 3D-printed patient-specific phantoms for intensity-modulated radiation therapy (IMRT) quality assurance (QA). Methods: We created a patient-specific whole-head phantom using a 3D printer. The printer data file was created from high-resolution DICOM computed tomography (CT) images of 3-year old child treated at our institution for medulloblastoma. A custom-modified extruder system was used to create tissue-equivalent materials. For the printing process, the Hounsfield Units from the CT images were converted to proportional volumetric densities. A 5-field IMRT plan was created from the patient CT and delivered to the 3D- phantom. Dose was measured by an ion chamber placed through the eye. The ion chamber was placed at the posterior edge of the planning target volume in a high dose gradient region. CT scans of the patient and 3D-phantom were fused by using commercial treatment planning software (TPS). The patient’s plan was calculated on the phantom CT images. The ion chamber’s active volume was delineated in the TPS; dose per field and total dose were obtained. Measured and calculated doses were compared. Results: The 3D-phantom dimensions and tissue densities were in good agreement with the patient. However, because of a printing error, there was a large discrepancy in the density in the frontal cortex. The calculated and measured treatment plan doses were 1.74 Gy and 1.72 Gy, respectively. For individual fields, the absolute dose difference between measured and calculated values was on average 3.50%. Conclusion: This study demonstrated the feasibility of using 3D-printed patient-specific phantoms for IMRT QA. Such phantoms would be particularly advantageous for complex IMRT treatment plans featuring high dose gradients and/or for anatomical sites with high variation in tissue densities. Our preliminary findings are promising. We anticipate that, once the printing process is further refined, the agreement between

WE-D-BRA-05: Pseudo In Vivo Patient Dosimetry Using a 3D-Printed Patient-Specific Phantom

Energy Technology Data Exchange (ETDEWEB)

Ger, R; Craft, DF [The University of Texas Graduate School of Biomedical Sciences (United States); Burgett, EA [Idaho State University, Pocatello, idaho (United States); Price, RR [RANDJ Consulting, Frederick, MD (United States); Kry, SF; Howell, RM [The University of Texas Graduate School of Biomedical Sciences (United States); The University of Texas MD Anderson Cancer Ctr., Houston, TX (United States)

2015-06-15

Purpose: To test the feasibility of using 3D-printed patient-specific phantoms for intensity-modulated radiation therapy (IMRT) quality assurance (QA). Methods: We created a patient-specific whole-head phantom using a 3D printer. The printer data file was created from high-resolution DICOM computed tomography (CT) images of 3-year old child treated at our institution for medulloblastoma. A custom-modified extruder system was used to create tissue-equivalent materials. For the printing process, the Hounsfield Units from the CT images were converted to proportional volumetric densities. A 5-field IMRT plan was created from the patient CT and delivered to the 3D- phantom. Dose was measured by an ion chamber placed through the eye. The ion chamber was placed at the posterior edge of the planning target volume in a high dose gradient region. CT scans of the patient and 3D-phantom were fused by using commercial treatment planning software (TPS). The patient’s plan was calculated on the phantom CT images. The ion chamber’s active volume was delineated in the TPS; dose per field and total dose were obtained. Measured and calculated doses were compared. Results: The 3D-phantom dimensions and tissue densities were in good agreement with the patient. However, because of a printing error, there was a large discrepancy in the density in the frontal cortex. The calculated and measured treatment plan doses were 1.74 Gy and 1.72 Gy, respectively. For individual fields, the absolute dose difference between measured and calculated values was on average 3.50%. Conclusion: This study demonstrated the feasibility of using 3D-printed patient-specific phantoms for IMRT QA. Such phantoms would be particularly advantageous for complex IMRT treatment plans featuring high dose gradients and/or for anatomical sites with high variation in tissue densities. Our preliminary findings are promising. We anticipate that, once the printing process is further refined, the agreement between

Wildtype p53-specific Antibody and T-Cell Responses in Cancer Patients

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Stryhn, Anette; Justesen, Sune

2011-01-01

patients. Detection of antibodies against wt p53 protein has been used as a diagnostic and prognostic marker and discovery of new T-cell epitopes has enabled design of cancer vaccination protocols with promising results. Here, we identified wt p53-specific antibodies in various cancer patients......(264-272) in breast cancer patients and against HLA-A*01:01 binding peptide wt p53(226-234) and HLA-B*07:02 binding peptide wt p53(74-82) in renal cell cancer and breast cancer patients, respectively. Finally, we analyzed antibody and T-cell responses against wt p53 15-mer peptides in patients with metastatic renal...

Patient-specific 3D FLAIR for enhanced visualization of brain white matter lesions in multiple sclerosis.

Science.gov (United States)

Gabr, Refaat E; Pednekar, Amol S; Govindarajan, Koushik A; Sun, Xiaojun; Riascos, Roy F; Ramírez, María G; Hasan, Khader M; Lincoln, John A; Nelson, Flavia; Wolinsky, Jerry S; Narayana, Ponnada A

2017-08-01

To improve the conspicuity of white matter lesions (WMLs) in multiple sclerosis (MS) using patient-specific optimization of single-slab 3D fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI). Sixteen MS patients were enrolled in a prospective 3.0T MRI study. FLAIR inversion time and echo time were automatically optimized for each patient during the same scan session based on measurements of the relative proton density and relaxation times of the brain tissues. The optimization criterion was to maximize the contrast between gray matter (GM) and white matter (WM), while suppressing cerebrospinal fluid. This criterion also helps increase the contrast between WMLs and WM. The performance of the patient-specific 3D FLAIR protocol relative to the fixed-parameter protocol was assessed both qualitatively and quantitatively. Patient-specific optimization achieved a statistically significant 41% increase in the GM-WM contrast ratio (P < 0.05) and 32% increase in the WML-WM contrast ratio (P < 0.01) compared with fixed-parameter FLAIR. The increase in WML-WM contrast ratio correlated strongly with echo time (P < 10 -11 ). Two experienced neuroradiologists indicated substantially higher lesion conspicuity on the patient-specific FLAIR images over conventional FLAIR in 3-4 cases (intrarater correlation coefficient ICC = 0.72). In no case was the image quality of patient-specific FLAIR considered inferior to conventional FLAIR by any of the raters (ICC = 0.32). Changes in proton density and relaxation times render fixed-parameter FLAIR suboptimal in terms of lesion contrast. Patient-specific optimization of 3D FLAIR increases lesion conspicuity without scan time penalty, and has potential to enhance the detection of subtle and small lesions in MS. 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;46:557-564. © 2016 International Society for Magnetic Resonance in Medicine.

Residual symptoms and specific functional impairments in euthymic patients with bipolar disorder.

Science.gov (United States)

Samalin, Ludovic; de Chazeron, Ingrid; Vieta, Eduard; Bellivier, Frank; Llorca, Pierre-Michel

2016-03-01

The aims of the present study were to confirm the impact of residual symptoms on overall functioning in a large sample of euthymic patients with bipolar disorder in real-life conditions and to explore the relationship between residual symptoms and specific areas of functional impairment. This was a multicenter, cross-sectional, non-interventional study of euthymic outpatients with bipolar disorder. The Functioning Assessment Short Test was used to assess overall and specific domains of functioning (autonomy, occupational functioning, cognitive functioning, financial issues, interpersonal relationships, and leisure time). Various residual symptoms were assessed (residual mood symptoms, emotional dysregulation, sleep and sexual disorders, stigma, and perceived cognitive impairment). Logistic regression was used to determine the best model of association between functional domains and residual symptoms. Almost half of the 468 patients included (42%) had poor overall functioning. Residual depressive symptoms appeared to have an impact on overall functioning and in nearly all areas of functioning. In addition, specific residual symptoms had significantly more negative effects on some domains of functioning in euthymic patients with bipolar disorder (residual manic symptoms and occupational stigma on autonomy, emotional inhibition on occupational functioning, residual manic symptoms on financial issues, family stigma on interpersonal relationships, and sexual function and occupational stigma on leisure time). Our findings highlight the importance of evaluating overall functioning in clinical practice as well as functional domains. They also indicate that some residuals symptoms in patients with bipolar disorder should be targeted in personalized treatment plans, in order to improve functioning in the domains in which the patient is most impaired. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of Functional Correlation of Task-Specific Muscle Synergies with Motor Performance in Patients Poststroke

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Si Li

2017-07-01

Full Text Available The central nervous system produces movements by activating specifically programmed muscle synergies that are also altered with injuries in the brain, such as stroke. In this study, we hypothesize that there exists a positive correlation between task-specific muscle synergy and motor functions at joint and task levels in patients following stroke. The purpose here is to define and evaluate neurophysiological metrics based on task-specific muscle synergy for assessing motor functions in patients. A patient group of 10 subjects suffering from stroke and a control group of nine age-matched healthy subjects were recruited to participate in this study. Electromyography (EMG signals and movement kinematics were recorded in patients and control subjects while performing arm reaching tasks. Muscle synergies of individual patients were extracted off-line from EMG records of each patient, and a baseline pattern of muscle synergy was obtained from the pooled EMG data of all nine control subjects. Peak velocities and movement durations of each reaching movement were computed from measured kinematics. Similarity indices of matching components to those of the baseline synergy were defined by synergy vectors and time profiles, respectively, as well as by a combined similarity of vector and time profile. Results showed that pathological synergies of patients were altered from the characteristics of baseline synergy with missing components, or varied vector patterns and time profiles. The kinematic performance measured by peak velocities and movement durations was significantly poorer for the patient group than the control group. In patients, all three similarity indices were found to correlate significantly to the kinematics of movements for the reaching tasks. The correlation to the Fugl-Meyer score of arm was the highest with the vector index, the lowest with the time profile index, and in between with the combined index. These findings illustrate that the

Evaluation of a patient specific femoral alignment guide for hip resurfacing.

Science.gov (United States)

Olsen, Michael; Naudie, Douglas D; Edwards, Max R; Sellan, Michael E; McCalden, Richard W; Schemitsch, Emil H

2014-03-01

A novel alternative to conventional instrumentation for femoral component insertion in hip resurfacing is a patient specific, computed tomography based femoral alignment guide. A benchside study using cadaveric femora was performed comparing a custom alignment guide to conventional instrumentation and computer navigation. A clinical series of twenty-five hip resurfacings utilizing a custom alignment guide was conducted by three surgeons experienced in hip resurfacing. Using cadaveric femora, the custom guide was comparable to conventional instrumentation with computer navigation proving superior to both. Clinical femoral component alignment accuracy was 3.7° and measured within ± 5° of plan in 20 of 24 cases. Patient specific femoral alignment guides provide a satisfactory level of accuracy and may be a better alternative to conventional instrumentation for initial femoral guidewire placement in hip resurfacing. Crown Copyright © 2014. All rights reserved.

Patients with Multiple Myeloma Develop SOX2-Specific Autoantibodies after Allogeneic Stem Cell Transplantation

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Sebastian Kobold

2011-01-01

Full Text Available The occurrence of SOX2-specific autoantibodies seems to be associated with an improved prognosis in patients with monoclonal gammopathy of undetermined significance (MGUS. However, it is unclear if SOX2-specific antibodies also develop in established multiple myeloma (MM. Screening 1094 peripheral blood (PB sera from 196 MM patients and 100 PB sera from healthy donors, we detected SOX2-specific autoantibodies in 7.7% and 2.0% of patients and donors, respectively. We identified SOX2211–230 as an immunodominant antibody-epitope within the full protein sequence. SOX2 antigen was expressed in most healthy tissues and its expression did not correlate with the number of BM-resident plasma cells. Accordingly, anti-SOX2 immunity was not related to SOX2 expression levels or tumor burden in the patients’ BM. The only clinical factor predicting the development of anti-SOX2 immunity was application of allogeneic stem cell transplantation (alloSCT. Anti-SOX2 antibodies occurred more frequently in patients who had received alloSCT (n=74. Moreover, most SOX2-seropositive patients had only developed antibodies after alloSCT. This finding indicates that alloSCT is able to break tolerance towards this commonly expressed antigen. The questions whether SOX2-specific autoantibodies merely represent an epiphenomenon, are related to graft-versus-host effects or participate in the immune control of myeloma needs to be answered in prospective studies.

Generation of Transplantable Beta Cells for Patient-Specific Cell Therapy

Directory of Open Access Journals (Sweden)

Xiaojie Wang

2012-01-01

Full Text Available Islet cell transplantation offers a potential cure for type 1 diabetes, but it is challenged by insufficient donor tissue and side effects of current immunosuppressive drugs. Therefore, alternative sources of insulin-producing cells and isletfriendly immunosuppression are required to increase the efficiency and safety of this procedure. Beta cells can be transdifferentiated from precursors or another heterologous (non-beta-cell source. Recent advances in beta cell regeneration from somatic cells such as fibroblasts could circumvent the usage of immunosuppressive drugs. Therefore, generation of patient-specific beta cells provides the potential of an evolutionary treatment for patients with diabetes.

Patient-specific system for prognosis of surgical treatment outcomes of human cardiovascular system

Science.gov (United States)

Golyadkina, Anastasiya A.; Kalinin, Aleksey A.; Kirillova, Irina V.; Kossovich, Elena L.; Kossovich, Leonid Y.; Menishova, Liyana R.; Polienko, Asel V.

2015-03-01

Object of study: Improvement of life quality of patients with high stroke risk ia the main goal for development of system for patient-specific modeling of cardiovascular system. This work is dedicated at increase of safety outcomes for surgical treatment of brain blood supply alterations. The objects of study are common carotid artery, internal and external carotid arteries and bulb. Methods: We estimated mechanical properties of carotid arteries tissues and patching materials utilized at angioplasty. We studied angioarchitecture features of arteries. We developed and clinically adapted computer biomechanical models, which are characterized by geometrical, physical and mechanical similarity with carotid artery in norm and with pathology (atherosclerosis, pathological tortuosity, and their combination). Results: Collaboration of practicing cardiovascular surgeons and specialists in the area of Mathematics and Mechanics allowed to successfully conduct finite-element modeling of surgical treatment taking into account various features of operation techniques and patching materials for a specific patient. Numerical experiment allowed to reveal factors leading to brain blood supply decrease and atherosclerosis development. Modeling of carotid artery reconstruction surgery for a specific patient on the basis of the constructed biomechanical model demonstrated the possibility of its application in clinical practice at approximation of numerical experiment to the real conditions.

iPS Cells—The Triumphs and Tribulations

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