Neurosphere based differentiation of human iPSC improves astrocyte differentiation

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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...

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.

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.

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

2010-12-14

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.

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

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.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons

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Sundberg, Maria; Bogetofte, Helle; Lawson, Tristan

2013-01-01

of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations......The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and h......ESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation...

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.

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

Generation of iPSC line from desmin-related cardiomyopathy patient carrying splice site mutation of DES gene

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Aleksandr Khudiakov

2017-10-01

Full Text Available Human iPSC line was generated from patient-specific adipose tissue-derived mesenchymal multipotent stromal cells carrying desmin (DES gene heterozygous splice site mutation using non-integrative reprogramming method. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai viruses. iPSCs were characterized by sequencing, karyotype analysis, STR analysis, immunocytochemistry, RT-PCR and teratoma formation.

Generation of human iPSCs from an essential thrombocythemia patient carrying a V501L mutation in the MPL gene.

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Liu, Senquan; Ye, Zhaohui; Gao, Yongxing; He, Chaoxia; Williams, Donna W; Moliterno, Alison; Spivak, Jerry; Huang, He; Cheng, Linzhao

2017-01-01

Activating point mutations in the MPL gene encoding the thrombopoietin receptor are found in 3%-10% of essential thrombocythemia (ET) and myelofibrosis patients. Here, we report the derivation of induced pluripotent stem cells (iPSCs) from an ET patient with a heterozygous MPL V501L mutation. Peripheral blood CD34 + progenitor cells were reprogrammed by transient plasmid expression of OCT4, SOX2, KLF4, c-MYC plus BCL2L1 (BCL-xL) genes. The derived line M494 carries a MPL V501L mutation, displays typical iPSC morphology and characteristics, are pluripotent and karyotypically normal. Upon differentiation, the iPSCs are able to differentiate into cells derived from three germ layers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

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Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

2016-05-06

Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

International Nuclear Information System (INIS)

Yin, Xiaohui; Li, Yang; Li, Jingwen; Li, Peng; Liu, Yinan; Wen, Jinhua; Luan, Qingxian

2016-01-01

Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

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.

Regenerative therapy for vestibular disorders using human induced pluripotent stem cells (iPSCs): neural differentiation of human iPSC-derived neural stem cells after in vitro transplantation into mouse vestibular epithelia.

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Taura, Akiko; Nakashima, Noriyuki; Ohnishi, Hiroe; Nakagawa, Takayuki; Funabiki, Kazuo; Ito, Juichi; Omori, Koichi

2016-10-01

Vestibular ganglion cells, which convey sense of motion from vestibular hair cells to the brainstem, are known to degenerate with aging and after vestibular neuritis. Thus, regeneration of vestibular ganglion cells is important to aid in the recovery of balance for associated disorders. The present study derived hNSCs from induced pluripotent stem cells (iPSCs) and transplanted these cells into mouse utricle tissues. After a 7-day co-culture period, histological and electrophysiological examinations of transplanted hNSCs were performed. Injected hNSC-derived cells produced elongated axon-like structures within the utricle tissue that made contact with vestibular hair cells. A proportion of hNSC-derived cells showed spontaneous firing activities, similar to those observed in cultured mouse vestibular ganglion cells. However, hNSC-derived cells around the mouse utricle persisted as immature neurons or occasionally differentiated into putative astrocytes. Moreover, electrophysiological examination showed hNSC-derived cells around utricles did not exhibit any obvious spontaneous firing activities. Injected human neural stem cells (hNSCs) showed signs of morphological maturation including reconnection to denervated hair cells and partial physiological maturation, suggesting hNSC-derived cells possibly differentiated into neurons.

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.

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.

A Comparative View on Human Somatic Cell Sources for iPSC Generation

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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.

Generation of human iPSC line from a patient with laterality defects and associated congenital heart anomalies carrying a DAND5 missense alteration

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Fernando Cristo

2017-12-01

Full Text Available A human iPSC line was generated from exfoliated renal epithelial (ERE cells of a patient affected with Congenital Heart Disease (CHD and Laterality Defects carrying tshe variant p.R152H in the DAND5 gene. The transgene-free iPSCs were generated with the human OSKM transcription factor using the Sendai-virus reprogramming system. The established iPSC line had the specific heterozygous alteration, a stable karyotype, expressed pluripotency markers and generated embryoid bodies that can differentiate towards the three germ layers in vitro. This iPSC line offers a useful resource to study the molecular mechanisms of cardiomyocyte proliferation, as well as for drug testing.

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

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

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Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

2016-01-01

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases.

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Sugai, Keiko; Fukuzawa, Ryuji; Shofuda, Tomoko; Fukusumi, Hayato; Kawabata, Soya; Nishiyama, Yuichiro; Higuchi, Yuichiro; Kawai, Kenji; Isoda, Miho; Kanematsu, Daisuke; Hashimoto-Tamaoki, Tomoko; Kohyama, Jun; Iwanami, Akio; Suemizu, Hiroshi; Ikeda, Eiji; Matsumoto, Morio; Kanemura, Yonehiro; Nakamura, Masaya; Okano, Hideyuki

2016-09-19

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

Structural Analysis of Three-dimensional Human Neural Tissue derived from Induced Pluripotent Stem Cells

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Terrence Brooks, Patrick; Rasmussen, Mikkel Aabech; Hyttel, Poul

2016-01-01

Objective: The present study aimed at establishing a method for production of a three-dimensional (3D) human neural tissue derived from induced pluripotent stem cells (iPSCs) and analyzing the outcome by a combination of tissue ultrastructure and expression of neural markers. Methods: A two......-step cell culture procedure was implemented by subjecting human iPSCs to a 3D scaffoldbased neural differentiation protocol. First, neural fate-inducing small molecules were used to create a neuroepithelial monolayer. Second, the monolayer was trypsinized into single cells and seeded into a porous...... polystyrene scaffold and further cultured to produce a 3D neural tissue. The neural tissue was characterized by a combination of immunohistochemistry and transmission electron microscopy (TEM). Results: iPSCs developed into a 3D neural tissue expressing markers for neural progenitor cells, early neural...

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.

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.

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.

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.

Focal Transplantation of Human iPSC-Derived Glial-Rich Neural Progenitors Improves Lifespan of ALS Mice

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Takayuki Kondo

2014-08-01

Full Text Available Transplantation of glial-rich neural progenitors has been demonstrated to attenuate motor neuron degeneration and disease progression in rodent models of mutant superoxide dismutase 1 (SOD1-mediated amyotrophic lateral sclerosis (ALS. However, translation of these results into a clinical setting requires a renewable human cell source. Here, we derived glial-rich neural progenitors from human iPSCs and transplanted them into the lumbar spinal cord of ALS mouse models. The transplanted cells differentiated into astrocytes, and the treated mouse group showed prolonged lifespan. Our data suggest a potential therapeutic mechanism via activation of AKT signal. The results demonstrated the efficacy of cell therapy for ALS by the use of human iPSCs as cell source.

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

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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...

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

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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.

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

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.

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs.

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Fu, Lina; Xu, Xiuling; Ren, Ruotong; Wu, Jun; Zhang, Weiqi; Yang, Jiping; Ren, Xiaoqing; Wang, Si; Zhao, Yang; Sun, Liang; Yu, Yang; Wang, Zhaoxia; Yang, Ze; Yuan, Yun; Qiao, Jie; Izpisua Belmonte, Juan Carlos; Qu, Jing; Liu, Guang-Hui

2016-03-01

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patient-specific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.

Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

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Xinjian Qu

Full Text Available Induced pluripotent stem cells (iPSCs can be generated from somatic cells by ectopic expression of defined transcription factors (TFs. However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs. The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

Human induced pluripotent stem cell-derived beating cardiac tissues on paper.

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Wang, Li; Xu, Cong; Zhu, Yujuan; Yu, Yue; Sun, Ning; Zhang, Xiaoqing; Feng, Ke; Qin, Jianhua

2015-11-21

There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create "a beating heart on paper." This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40-70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

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Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

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Hiroko Shimada

Full Text Available The common marmoset (Callithrix jacchus is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs derived from mouse and human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

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

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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.

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells

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Marta Trevisan

2017-01-01

Full Text Available Induced pluripotent stem cells (iPSCs are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

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.

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

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

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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.

Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

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Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

2017-08-15

Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

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.

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.

Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics.

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Panopoulos, Athanasia D; Smith, Erin N; Arias, Angelo D; Shepard, Peter J; Hishida, Yuriko; Modesto, Veronica; Diffenderfer, Kenneth E; Conner, Clay; Biggs, William; Sandoval, Efren; D'Antonio-Chronowska, Agnieszka; Berggren, W Travis; Izpisua Belmonte, Juan Carlos; Frazer, Kelly A

2017-04-06

Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming. Published by Elsevier Inc.

Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells.

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Deepak A Lamba

2010-01-01

Full Text Available Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.In this report we have used a similar method to direct induced pluripotent stem cells (iPS from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

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.

Differentiation of blood T cells: Reprogramming human induced pluripotent stem cells into neuronal cells

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Ping-Hsing Tsai

2015-06-01

Conclusion: We have developed a safer method to generate integration-free and nonviral human iPSCs from adult somatic cells. This induction method will be useful for the derivation of human integration-free iPSCs and will also be applicable to the generation of iPSCs-derived neuronal cells for drug screening or therapeutics in the near future.

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.

Characterization of Induced Pluripotent Stem Cell-derived Human Serotonergic Neurons

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Lining Cao

2017-05-01

Full Text Available In the brain, the serotonergic neurons located in the raphe nucleus are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the regulation of brain development and functions. Dysfunction of the serotonin system is present in many psychiatric disorders. Lack of in vitro functional human model limits the understanding of human central serotonergic system and its related diseases and clinical applications. Previously, we have developed a method generating human serotonergic neurons from induced pluripotent stem cells (iPSCs. In this study, we analyzed the features of these human iPSCs-derived serotonergic neurons both in vitro and in vivo. We found that these human serotonergic neurons are sensitive to the selective neurotoxin 5, 7-Dihydroxytryptamine (5,7-DHT in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the host’s cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.

DNA methylation dynamics in human induced pluripotent stem cells over time.

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Koichiro Nishino

2011-05-01

Full Text Available Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs. Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell and five human embryonic stem cell (ESC lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.

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...

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.

CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs.

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Wang, Lixia; Yi, Fei; Fu, Lina; Yang, Jiping; Wang, Si; Wang, Zhaoxia; Suzuki, Keiichiro; Sun, Liang; Xu, Xiuling; Yu, Yang; Qiao, Jie; Belmonte, Juan Carlos Izpisua; Yang, Ze; Yuan, Yun; Qu, Jing; Liu, Guang-Hui

2017-05-01

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts of familial ALS patients bearing SOD1 +/A272C and FUS +/G1566A mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq) analysis of motor neurons derived from SOD1 +/A272C and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.

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.

Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

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Ivana Acimovic

2014-01-01

Full Text Available Human pluripotent stem cells (hPSCs, namely, embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs, with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs.

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.

CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs

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Lixia Wang

2017-04-01

Full Text Available Abstract Amyotrophic lateral sclerosis (ALS is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs from fibroblasts of familial ALS patients bearing SOD1 +/A272C and FUS +/G1566A mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq analysis of motor neurons derived from SOD1 +/A272C and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.

Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

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Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

2013-09-01

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

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Soraia Martins

2018-01-01

Full Text Available Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were 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 transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

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.

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

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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.

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells.

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Wang, Linli; Chen, Yuehua; Guan, Chunyan; Zhao, Zhiju; Li, Qiang; Yang, Jianguo; Mo, Jian; Wang, Bin; Wu, Wei; Yang, Xiaohui; Song, Libing; Li, Jun

2017-11-02

Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system's feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application.

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.

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.

Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology.

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Zhang, Hanrui; Reilly, Muredach P

2017-11-01

Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases. © 2017 American Heart Association, Inc.

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.

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.

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.

Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature.

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Samantha M Thomas

2015-05-01

Full Text Available Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs, have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10-15. Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.

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.

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

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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.

Multistage switched inductor boost converter for renewable energy application

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Maroti, Pandav Kiran; Padmanaban, Sanjeevikumar; Bhaskar, Mahajan Sagar

2017-01-01

In this paper Multistage Switched Inductor Boost Converter (Multistage SIBC) is uttered for renewable energy applications. The projected converter is derived from an amalgamation of the conventional step-up converter and inductor stack. The number of inductor and duty ratio decides the overall...

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

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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.

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

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Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

2017-11-15

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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...

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 iPSC-derived

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

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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

One-step derivation of mesenchymal stem cell (MSC-like cells from human pluripotent stem cells on a fibrillar collagen coating.

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Yongxing Liu

Full Text Available Controlled differentiation of human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs into cells that resemble adult mesenchymal stem cells (MSCs is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

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Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish; Wu, Joseph C

2018-03-08

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling. © 2018. Published by The Company of Biologists Ltd.

Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

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Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

2015-01-01

Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P ketamine (100 μM) decreased the ATP level (22%, P ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

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

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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.

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.

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.

Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

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Daisuke Doi

2014-03-01

Full Text Available Human induced pluripotent stem cells (iPSCs can provide a promising source of midbrain dopaminergic (DA neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

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.

Electrophysiological properties of neurons derived from human stem cells and iNeurons in vitro.

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Halliwell, Robert F

2017-06-01

Functional studies of neurons have traditionally used nervous system tissues from a variety of non-human vertebrate and invertebrate species, even when the focus of much of this research has been directed at understanding human brain function. Over the last decade, the identification and isolation of human stem cells from embryonic, tissue (or adult) and induced pluripotent stem cells (iPSCs) has revolutionized the availability of human neurons for experimental studies in vitro. In addition, the direct conversion of terminally differentiated fibroblasts into Induced neurons (iN) has generated great excitement because of the likely value of such human stem cell derived neurons (hSCNs) and iN cells in drug discovery, neuropharmacology, neurotoxicology and regenerative medicine. This review addresses the current state of our knowledge of functional receptors and ion channels expressed in neurons derived from human stem cells and iNeurons and identifies gaps and questions that might be investigated in future studies; it focusses almost exclusively on what is known about the electrophysiological properties of neurons derived from human stem cells and iN cells in vitro with an emphasis on voltage and ligand gated ion channels, since these mediate synaptic signalling in the nervous system and they are at the heart of neuropharmacology. Copyright © 2016 Elsevier Ltd. All rights reserved.

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....

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

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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.

One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection

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Yali Liu

2017-03-01

Full Text Available Monogenic disorders (MGDs, which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB gene. The technologies of induced pluripotent stem cells (iPSCs and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs, high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.

Generation of Integration-Free Induced Pluripotent Stem Cells from Urine-Derived Cells Isolated from Individuals with Down Syndrome.

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M Lee, Young; Zampieri, Bruna L; Scott-McKean, Jonah J; Johnson, Mark W; Costa, Alberto C S

2017-06-01

Down syndrome (DS) is a genetic disorder caused by trisomy 21 (T21). Over the past two decades, the use of mouse models has led to significant advances in the understanding of mechanisms underlying various phenotypic features and comorbidities secondary to T21 and even informed the design of clinical trials aimed at enhancing the cognitive abilities of persons with DS. In spite of its success, this approach has been plagued by all the typical limitations of rodent modeling of human disorders and diseases. Recently, several laboratories have succeeded in producing T21 human induced pluripotent stem cells (T21-iPSCs) from individuals with DS, which is emerging as a promising complementary tool for the study of DS. Here, we describe the method by which we generated 10 T21-iPSC lines from epithelial cells in urine samples, presumably from kidney epithelial origin, using nonintegrating episomal vectors. We also show that these iPSCs maintain chromosomal stability for well over 20 passages and are more sensitive to proteotoxic stress than euploid iPSCs. Furthermore, these iPSC lines can be differentiated into glutamatergic neurons and cardiomyocytes. By culturing urine-derived cells and maximizing the efficiency of episomal vector transfection, we have been able to generate iPSCs noninvasively and effectively from participants with DS in an ongoing clinical trial, and thus address most shortcomings of previously generated T21-iPSC lines. These techniques should extend the application of iPSCs in modeling DS and other neurodevelopmental and neurodegenerative disorders, and may lead to future human cell-based platforms for high-throughput drug screening. Stem Cells Translational Medicine 2017;6:1465-1476. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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.

Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development

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Samuel Sances

2018-04-01

Full Text Available Summary: Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition. : Sances et al. combine Organ-Chip technology with human induced pluripotent stem cell-derived spinal motor neurons to study the maturation effects of Organ-Chip culture. By including microvascular cells also derived from the same patient line, the authors show enhancement of neuronal function, reproduction of vascular-neuron pathways, and specific gene activation that resembles in vivo spinal cord development. Keywords: organ-on-chip, spinal cord, iPSC, disease modeling, amyotrophic lateral sclerosis, microphysiological system, brain microvascular endothelial cells, spinal motor neurons, vasculature, microfluidic device

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.

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.

Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

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Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Nakamura, Ryosuke; Otsuki, Koshi; Murono, Shigeyuki; Omori, Koichi

2017-07-01

Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

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Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

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Yu Takahashi

2018-01-01

Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

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.

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...

Antiarrhythmic Effects of Dantrolene in Patients with Catecholaminergic Polymorphic Ventricular Tachycardia and Replication of the Responses Using iPSC Models.

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Kirsi Penttinen

Full Text Available Catecholaminergic polymorphic ventricular tachycardia (CPVT is a highly malignant inherited arrhythmogenic disorder. Type 1 CPVT (CPVT1 is caused by cardiac ryanodine receptor (RyR2 gene mutations resulting in abnormal calcium release from sarcoplasmic reticulum. Dantrolene, an inhibitor of sarcoplasmic Ca(2+ release, has been shown to rescue this abnormal Ca(2+ release in vitro. We assessed the antiarrhythmic efficacy of dantrolene in six patients carrying various RyR2 mutations causing CPVT. The patients underwent exercise stress test before and after dantrolene infusion. Dantrolene reduced the number of premature ventricular complexes (PVCs on average by 74% (range 33-97 in four patients with N-terminal or central mutations in the cytosolic region of the RyR2 protein, while dantrolene had no effect in two patients with mutations in or near the transmembrane domain. Induced pluripotent stem cells (iPSCs were generated from all the patients and differentiated into spontaneously beating cardiomyocytes (CMs. The antiarrhythmic effect of dantrolene was studied in CMs after adrenaline stimulation by Ca(2+ imaging. In iPSC derived CMs with RyR2 mutations in the N-terminal or central region, dantrolene suppressed the Ca(2+ cycling abnormalities in 80% (range 65-97 of cells while with mutations in or near the transmembrane domain only in 23 or 32% of cells. In conclusion, we demonstrate that dantrolene given intravenously shows antiarrhythmic effects in a portion of CPVT1 patients and that iPSC derived CM models replicate these individual drug responses. These findings illustrate the potential of iPSC models to individualize drug therapy of inherited diseases.Trial Registration: EudraCT Clinical Trial Registry 2012-005292-14.

Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking.

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Tan, Hong-Kee; Toh, Cheng-Xu Delon; Ma, Dongrui; Yang, Binxia; Liu, Tong Ming; Lu, Jun; Wong, Chee-Wai; Tan, Tze-Kai; Li, Hu; Syn, Christopher; Tan, Eng-Lee; Lim, Bing; Lim, Yoon-Pin; Cook, Stuart A; Loh, Yuin-Han

2014-05-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a "do-it-yourself" basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.

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.

Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells.

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Choi, Hye Yeon; Lee, Tae-Jin; Yang, Gwang-Mo; Oh, Jaesur; Won, Jihye; Han, Jihae; Jeong, Gun-Jae; Kim, Jongpil; Kim, Jin-Hoi; Kim, Byung-Soo; Cho, Ssang-Goo

2016-08-10

Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings. Copyright © 2016. Published by Elsevier B.V.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Ming-Wai Poon

Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

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Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

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Lorenz, Carmen; Lesimple, Pierre; Bukowiecki, Raul; Zink, Annika; Inak, Gizem; Mlody, Barbara; Singh, Manvendra; Semtner, Marcus; Mah, Nancy; Auré, Karine; Leong, Megan; Zabiegalov, Oleksandr; Lyras, Ekaterini-Maria; Pfiffer, Vanessa; Fauler, Beatrix; Eichhorst, Jenny; Wiesner, Burkhard; Huebner, Norbert; Priller, Josef; Mielke, Thorsten; Meierhofer, David; Izsvák, Zsuzsanna; Meier, Jochen C; Bouillaud, Frédéric; Adjaye, James; Schuelke, Markus; Wanker, Erich E; Lombès, Anne; Prigione, Alessandro

2017-05-04

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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.

Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

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Heather E Wheeler

Full Text Available There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN, the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05. The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011. The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05, demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

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Liao, Mei-Chen; Muratore, Christina R; Gierahn, Todd M; Sullivan, Sarah E; Srikanth, Priya; De Jager, Philip L; Love, J Christopher; Young-Pearse, Tracy L

2016-02-03

Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we

Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.

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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

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 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.

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

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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.

New type of Sendai virus vector provides transgene-free iPS cells derived from chimpanzee blood.

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Yasumitsu Fujie

Full Text Available Induced pluripotent stem cells (iPSCs are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

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Maffioletti, Sara Martina; Sarcar, Shilpita; Henderson, Alexander B H; Mannhardt, Ingra; Pinton, Luca; Moyle, Louise Anne; Steele-Stallard, Heather; Cappellari, Ornella; Wells, Kim E; Ferrari, Giulia; Mitchell, Jamie S; Tyzack, Giulia E; Kotiadis, Vassilios N; Khedr, Moustafa; Ragazzi, Martina; Wang, Weixin; Duchen, Michael R; Patani, Rickie; Zammit, Peter S; Wells, Dominic J; Eschenhagen, Thomas; Tedesco, Francesco Saverio

2018-04-17

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling

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May Shin Yap

2015-01-01

Full Text Available Human pluripotent stem cells (hPSCs derived from either blastocyst stage embryos (hESCs or reprogrammed somatic cells (iPSCs can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

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Saito, Taku; Yano, Fumiko; Mori, Daisuke; Kawata, Manabu; Hoshi, Kazuto; Takato, Tsuyoshi; Masaki, Hideki; Otsu, Makoto; Eto, Koji; Nakauchi, Hiromitsu; Chung, Ung-il; Tanaka, Sakae

2015-01-01

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

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.

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

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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

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family

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Eriona Hysolli

2016-07-01

Full Text Available Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs have been shown to be highly similar to embryonic stem cells (ESCs. However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells

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Xiugong Gao

2017-12-01

Full Text Available Induced pluripotent stem cells (iPSCs offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs derived from umbilical cord blood (CB or adult peripheral blood (PB afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5–7 days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications.

Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells.

Science.gov (United States)

Gao, Xiugong; Yourick, Jeffrey J; Sprando, Robert L

2017-12-01

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs) and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5-7days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications. Published by Elsevier B.V.

HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

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Kuan-Teh Jeang

2012-07-01

Full Text Available A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

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Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A; Itoh, Munenari; Christiano, Angela M

2015-01-01

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

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Karl Gledhill

Full Text Available The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

Induced pluripotent stem cell-derived gamete-associated proteins incite rejection of induced pluripotent stem cells in syngeneic mice.

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Kim, Eun-Mi; Manzar, Gohar; Zavazava, Nicholas

2017-06-01

The safety of induced pluripotent stem cells (iPSCs) in autologous recipients has been questioned after iPSCs, but not embryonic stem cells (ESCs), were reported to be rejected in syngeneic mice. This important topic has remained controversial because there has not been a mechanistic explanation for this phenomenon. Here, we hypothesize that iPSCs, but not ESCs, readily differentiate into gamete-forming cells that express meiotic antigens normally found in immune-privileged gonads. Because peripheral blood T cells are not tolerized to these antigens in the thymus, gamete-associated-proteins (GAPs) sensitize T cells leading to rejection. Here, we provide evidence that GAPs expressed in iPSC teratomas, but not in ESC teratomas, are responsible for the immunological rejection of iPSCs. Furthermore, silencing the expression of Stra8, 'the master regulator of meiosis', in iPSCs, using short hairpin RNA led to significant abrogation of the rejection of iPSCs, supporting our central hypothesis that GAPs expressed after initiation of meiosis in iPSCs were responsible for rejection. In contrast to iPSCs, iPSC-derivatives, such as haematopoietic progenitor cells, are able to engraft long-term into syngeneic recipients because they no longer express GAPs. Our findings, for the first time, provide a unifying explanation of why iPSCs, but not ESCs, are rejected in syngeneic recipients, ending the current controversy on the safety of iPSCs and their derivatives. © 2017 John Wiley & Sons Ltd.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

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Dorota Jeziorowska

2017-06-01

Full Text Available Human induced pluripotent stem cells (iPSCs represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D or, more recently, on monolayer culture (2D. We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response or IWP2 (inhibitor of Wnt production. We firstly found that the level of Troponin T (TNNT2 expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.

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.

Multi-stage decoding for multi-level block modulation codes

Science.gov (United States)

Lin, Shu

1991-01-01

In this paper, we investigate various types of multi-stage decoding for multi-level block modulation codes, in which the decoding of a component code at each stage can be either soft-decision or hard-decision, maximum likelihood or bounded-distance. Error performance of codes is analyzed for a memoryless additive channel based on various types of multi-stage decoding, and upper bounds on the probability of an incorrect decoding are derived. Based on our study and computation results, we find that, if component codes of a multi-level modulation code and types of decoding at various stages are chosen properly, high spectral efficiency and large coding gain can be achieved with reduced decoding complexity. In particular, we find that the difference in performance between the suboptimum multi-stage soft-decision maximum likelihood decoding of a modulation code and the single-stage optimum decoding of the overall code is very small: only a fraction of dB loss in SNR at the probability of an incorrect decoding for a block of 10(exp -6). Multi-stage decoding of multi-level modulation codes really offers a way to achieve the best of three worlds, bandwidth efficiency, coding gain, and decoding complexity.

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

Science.gov (United States)

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.

Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells: A novel approach to study metabolism in human neurons.

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Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog; Bak, Lasse K; Nielsen, Jørgen E; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K; Waagepetersen, Helle S

2017-06-01

particularly similar to the profile of mouse cortical neurons, important differences between the metabolic profile of human and mouse neurons were observed. The results of the present investigation establish hallmarks of cellular metabolism in human neurons derived from iPSC. Copyright © 2017. Published by Elsevier Ltd.

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.

RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders.

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Mingyan Lin

Full Text Available Genome-wide expression analysis using next generation sequencing (RNA-Seq provides an opportunity for in-depth molecular profiling of fundamental biological processes, such as cellular differentiation and malignant transformation. Differentiating human neurons derived from induced pluripotent stem cells (iPSCs provide an ideal system for RNA-Seq since defective neurogenesis caused by abnormalities in transcription factors, DNA methylation, and chromatin modifiers lie at the heart of some neuropsychiatric disorders. As a preliminary step towards applying next generation sequencing using neurons derived from patient-specific iPSCs, we have carried out an RNA-Seq analysis on control human neurons. Dramatic changes in the expression of coding genes, long non-coding RNAs (lncRNAs, pseudogenes, and splice isoforms were seen during the transition from pluripotent stem cells to early differentiating neurons. A number of genes that undergo radical changes in expression during this transition include candidates for schizophrenia (SZ, bipolar disorder (BD and autism spectrum disorders (ASD that function as transcription factors and chromatin modifiers, such as POU3F2 and ZNF804A, and genes coding for cell adhesion proteins implicated in these conditions including NRXN1 and NLGN1. In addition, a number of novel lncRNAs were found to undergo dramatic changes in expression, one of which is HOTAIRM1, a regulator of several HOXA genes during myelopoiesis. The increase we observed in differentiating neurons suggests a role in neurogenesis as well. Finally, several lncRNAs that map near SNPs associated with SZ in genome wide association studies also increase during neuronal differentiation, suggesting that these novel transcripts may be abnormally regulated in a subgroup of patients.

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.

Interconnected Levels of Multi-Stage Marketing

DEFF Research Database (Denmark)

Vedel, Mette; Geersbro, Jens; Ritter, Thomas

2012-01-01

different levels of multi-stage marketing and illustrates these stages with a case study. In addition, a triadic perspective is introduced as an analytical tool for multi-stage marketing research. The results from the case study indicate that multi-stage marketing exists on different levels. Thus, managers...... must not only decide in general on the merits of multi-stage marketing for their firm, but must also decide on which level they will engage in multi-stage marketing. The triadic perspective enables a rich and multi-dimensional understanding of how different business relationships influence each other...... in a multi-stage marketing context. This understanding assists managers in assessing and balancing different aspects of multi- stage marketing. The triadic perspective also offers avenues for further research....

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

Science.gov (United States)

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.

Generation of “Off-the-Shelf” Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells

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Jieming Zeng

2017-12-01

Full Text Available Summary: Current donor cell-dependent strategies can only produce limited “made-to-order” therapeutic natural killer (NK cells for limited patients. To provide unlimited “off-the-shelf” NK cells that serve many recipients, we designed and demonstrated a holistic manufacturing scheme to mass-produce NK cells from induced pluripotent stem cells (iPSCs. Starting with a highly accessible human cell source, peripheral blood cells (PBCs, we derived a good manufacturing practice-compatible iPSC source, PBC-derived iPSCs (PBC-iPSCs for this purpose. Through our original protocol that excludes CD34+ cell enrichment and spin embryoid body formation, high-purity functional and expandable NK cells were generated from PBC-iPSCs. Above all, most of these NK cells expressed no killer cell immunoglobulin-like receptors (KIRs, which renders them unrestricted by recipients' human leukocyte antigen genotypes. Hence, we have established a practical “from blood cell to stem cells and back with less (less KIRs” strategy to generate abundant “universal” NK cells from PBC-iPSCs for a wide range of patients. : To provide unlimited “off-the-shelf” NK cells that serve many recipients, Zeng and colleagues demonstrate a manufacturing scheme to mass-produce NK cells from peripheral blood cell-derived iPSCs (PBC-iPSCs. Through their original protocol, high-purity functional NK cells are generated from PBC-iPSCs. Most of these NK cells express no killer cell immunoglobulin-like receptors, which renders them unrestricted by recipients' HLA genotypes. Keywords: induced pluripotent stem cells, peripheral blood cells, natural killer cells, killer cell immunoglobulin-like receptors, cell therapy, immunotherapy, cancer, cytotoxicity

Human iPSC Derived GABA Ergic Precursor Cell Therapy for Chronic Epilepsy

Science.gov (United States)

2016-10-01

of hMGE progenitors (obtained from hPSCs expanded from human embryonic stem cells) that were transduced with DREADDs through CRISPR/Cas9 technology...regions and cell layers of the hippocampus, which include the subgranular zone and the dentate hilus in the dentate gyrus, and strata oriens and...have migrated extensively in the dentate hilus (A3) and into different layers of the CA1 subfield. DG, dentate gyrus; DH, dentate hilus; GCL, granule

Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons.

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Anna Chatzidaki

Full Text Available Neurons derived from human induced pluripotent stem cells (iPSCs represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes. In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B, in the presence of the α7-selective positive allosteric modulator (PAM PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA. In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular.

Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

Science.gov (United States)

Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

2014-12-01

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Multistage Effort and the Equity Structure of Venture Investment Based on Reciprocity Motivation

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Chuan Ding

2015-01-01

Full Text Available For venture capitals, it is a long process from an entry to its exit. In this paper, the activity of venture investment will be divided into multistages. And, according to the effort level entrepreneurs will choose, the venture capitalists will provide an equity structure at the very beginning. As a benchmark for comparison, we will establish two game models on multistage investment under perfect rationality: a cooperative game model and a noncooperative one. Further, as a cause of pervasive psychological preference behavior, reciprocity motivation will influence the behavior of the decision-makers. Given this situation, Rabin’s reciprocity motivation theory will be applied to the multistage game model of the venture investment, and multistage behavior game model will be established as well, based on the reciprocity motivation. By looking into the theoretical derivations and simulation studies, we find that if venture capitalists and entrepreneurs both have reciprocity preferences, their utility would have been Pareto improvement compared with those under perfect rationality.

Coupling methods for multistage sampling

OpenAIRE

Chauvet, Guillaume

2015-01-01

Multistage sampling is commonly used for household surveys when there exists no sampling frame, or when the population is scattered over a wide area. Multistage sampling usually introduces a complex dependence in the selection of the final units, which makes asymptotic results quite difficult to prove. In this work, we consider multistage sampling with simple random without replacement sampling at the first stage, and with an arbitrary sampling design for further stages. We consider coupling ...

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....

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

Science.gov (United States)

Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

2018-01-22

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

Science.gov (United States)

Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

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.

Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.

Science.gov (United States)

Hollmann, Emma K; Bailey, Amanda K; Potharazu, Archit V; Neely, M Diana; Bowman, Aaron B; Lippmann, Ethan S

2017-04-13

Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm 2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm 2 and fluorescein permeability below 1.95 × 10 -7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm 2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated

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.

A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

Directory of Open Access Journals (Sweden)

Kristiina Rajala

2010-04-01

Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

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Sara Martina Maffioletti

2018-04-01

Full Text Available Summary: Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. : Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons. Keywords: skeletal muscle, pluripotent stem cells, iPS cells, myogenic differentiation, tissue engineering, disease modeling, muscular dystrophy, organoids

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

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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.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

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Takata, Kazuyuki; Kozaki, Tatsuya; Lee, Christopher Zhe Wei; Thion, Morgane Sonia; Otsuka, Masayuki; Lim, Shawn; Utami, Kagistia Hana; Fidan, Kerem; Park, Dong Shin; Malleret, Benoit; Chakarov, Svetoslav; See, Peter; Low, Donovan; Low, Gillian; Garcia-Miralles, Marta; Zeng, Ruizhu; Zhang, Jinqiu; Goh, Chi Ching; Gul, Ahmet; Hubert, Sandra; Lee, Bernett; Chen, Jinmiao; Low, Ivy; Shadan, Nurhidaya Binte; Lum, Josephine; Wei, Tay Seok; Mok, Esther; Kawanishi, Shohei; Kitamura, Yoshihisa; Larbi, Anis; Poidinger, Michael; Renia, Laurent; Ng, Lai Guan; Wolf, Yochai; Jung, Steffen; Önder, Tamer; Newell, Evan; Huber, Tara; Ashihara, Eishi; Garel, Sonia; Pouladi, Mahmoud A; Ginhoux, Florent

2017-07-18

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparing ESC and iPSC?Based Models for Human Genetic Disorders

OpenAIRE

Halevy, Tomer; Urbach, Achia

2014-01-01

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 ne...

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

Fuzzy-like multiple objective multistage decision making

CERN Document Server

Xu, Jiuping

2014-01-01

Decision has inspired reflection of many thinkers since the ancient times. With the rapid development of science and society, appropriate dynamic decision making has been playing an increasingly important role in many areas of human activity including engineering, management, economy and others. In most real-world problems, decision makers usually have to make decisions sequentially at different points in time and space, at different levels for a component or a system, while facing multiple and conflicting objectives and a hybrid uncertain environment where fuzziness and randomness co-exist in a decision making process. This leads to the development of fuzzy-like multiple objective multistage decision making. This book provides a thorough understanding of the concepts of dynamic optimization from a modern perspective and presents the state-of-the-art methodology for modeling, analyzing and solving the most typical multiple objective multistage decision making practical application problems under fuzzy-like un...

Immunogenicity and in vitro Protective Efficacy of a Recombinant Multistage Plasmodium falciparum Candidate Vaccine

Science.gov (United States)

Shi, Ya Ping; Hasnain, Seyed E.; Sacci, John B.; Holloway, Brian P.; Fujioka, Hisashi; Kumar, Nirbhay; Wohlhueter, Robert; Hoffman, Stephen L.; Collins, William E.; Lal, Altaf A.

1999-02-01

Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

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.

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs, generation and characterization of HiPSC-derived neurons and astrocytes.

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Yohannes Haile

Full Text Available Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs into iPSCs (HiPSCs. Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

Energy Technology Data Exchange (ETDEWEB)

Fan Jinshui; Robert, Carine [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States); Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce [Johns Hopkins University School of Medicine, Department of Oncology, Baltimore, MD 21231-1000 (United States); Rassool, Feyruz Virgilia, E-mail: frassool@som.umaryland.edu [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States)

2011-08-01

Highlights: {yields} iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. {yields} iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. {yields} iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. {yields} iPSC however show a partial apoptotic response to DNA damage, compared to hESC. {yields} DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

International Nuclear Information System (INIS)

Fan Jinshui; Robert, Carine; Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce; Rassool, Feyruz Virgilia

2011-01-01

Highlights: → iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. → iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. → iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. → iPSC however show a partial apoptotic response to DNA damage, compared to hESC. → DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels

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.

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.

Application of biomaterials to advance induced pluripotent stem cell research and therapy

Science.gov (United States)

Tong, Zhixiang; Solanki, Aniruddh; Hamilos, Allison; Levy, Oren; Wen, Kendall; Yin, Xiaolei; Karp, Jeffrey M

2015-01-01

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation. PMID:25766254

Performance prediction method for a multi-stage Knudsen pump

Science.gov (United States)

Kugimoto, K.; Hirota, Y.; Kizaki, Y.; Yamaguchi, H.; Niimi, T.

2017-12-01

In this study, the novel method to predict the performance of a multi-stage Knudsen pump is proposed. The performance prediction method is carried out in two steps numerically with the assistance of a simple experimental result. In the first step, the performance of a single-stage Knudsen pump was measured experimentally under various pressure conditions, and the relationship of the mass flow rate was obtained with respect to the average pressure between the inlet and outlet of the pump and the pressure difference between them. In the second step, the performance of a multi-stage pump was analyzed by a one-dimensional model derived from the mass conservation law. The performances predicted by the 1D-model of 1-stage, 2-stage, 3-stage, and 4-stage pumps were validated by the experimental results for the corresponding number of stages. It was concluded that the proposed prediction method works properly.

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

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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.

An integrated practical implementation of continuous aqueous two-phase systems for the recovery of human IgG: From the microdevice to a multistage bench-scale mixer-settler device.

Science.gov (United States)

Espitia-Saloma, Edith; Vâzquez-Villegas, Patricia; Rito-Palomares, Marco; Aguilar, Oscar

2016-05-01

Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer-settler device in one stage, multistage and multistage with recirculation configuration. Single-stage pure IgG extraction with a polyethylene glycol (PEG) 3350-phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Science.gov (United States)

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.

Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias

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Maaike eHoekstra

2012-08-01

Full Text Available Cardiac arrhythmias are a major cause of morbidity and mortality. In younger patients, the majority of sudden cardiac deaths have an underlying Mendelian genetic cause. Over the last 15 years, enormous progress has been made in identifying the distinct clinical phenotypes and in studying the basic cellular and genetic mechanisms associated with the primary Mendelian (monogenic arrhythmia syndromes. Investigation of the electrophysiological consequences of an ion channel mutation is ideally done in the native cardiomyocyte environment. However, the majority of such studies so far have relied on heterologous expression systems in which single ion channel genes are expressed in non-cardiac cells. In some cases, transgenic mouse models haven been generated, but these also have significant shortcomings, primarily related to species differences.The discovery that somatic cells can be reprogrammed to pluripotency as induced pluripotent stem cells (iPSC has generated much interest since it presents an opportunity to generate patient- and disease-specific cell lines from which normal and diseased human cardiomyocytes can be obtained These genetically diverse human model systems can be studied in vitro and used to decipher mechanisms of disease and identify strategies and reagents for new therapies. Here we review the present state of the art with respect to cardiac disease models already generated using IPSC technology and which have been (partially characterized.Human iPSC (hiPSC models have been described for the cardiac arrhythmia syndromes, including LQT1, LQT2, LQT3-Brugada Syndrome, LQT8/Timothy syndrome and catecholaminergic polymorphic ventricular tachycardia. In most cases, the hiPSC-derived cardiomyoctes recapitulate the disease phenotype and have already provided opportunities for novel insight into cardiac pathophysiology. It is expected that the lines will be useful in the development of pharmacological agents for the management of these

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.

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.

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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.

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

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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.

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

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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.

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

DEFF Research Database (Denmark)

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...

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.

Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

Science.gov (United States)

Al-Ahmad, Abraham J

2017-10-01

Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells. Copyright © 2017 the American Physiological Society.

Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

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Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2011-06-01

Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

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 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.

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

Science.gov (United States)

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.

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

Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs

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Akihiro Yamashita

2015-03-01

Full Text Available Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs. We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1, and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

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.

CRISPR/Cas9 system and its applications in human hematopoietic cells.

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Hu, Xiaotang

2016-11-01

Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells.

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Hiroyuki Hirai

Full Text Available Human induced pluripotent stem cells (iPSCs can be divided into a leukemia inhibitory factor (LIF-dependent naïve type and a basic fibroblast growth factor (bFGF-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

Multistage stochastic optimization

CERN Document Server

Pflug, Georg Ch

2014-01-01

Multistage stochastic optimization problems appear in many ways in finance, insurance, energy production and trading, logistics and transportation, among other areas. They describe decision situations under uncertainty and with a longer planning horizon. This book contains a comprehensive treatment of today’s state of the art in multistage stochastic optimization.  It covers the mathematical backgrounds of approximation theory as well as numerous practical algorithms and examples for the generation and handling of scenario trees. A special emphasis is put on estimation and bounding of the modeling error using novel distance concepts, on time consistency and the role of model ambiguity in the decision process. An extensive treatment of examples from electricity production, asset liability management and inventory control concludes the book

Interconnected levels of multi-stage marketing: A triadic approach

OpenAIRE

Vedel, Mette; Geersbro, Jens; Ritter, Thomas

2012-01-01

Multi-stage marketing gains increasing attention as knowledge of and influence on the customer's customer become more critical for the firm's success. Despite this increasing managerial relevance, systematic approaches for analyzing multi-stage marketing are still missing. This paper conceptualizes different levels of multi-stage marketing and illustrates these stages with a case study. In addition, a triadic perspective is introduced as an analytical tool for multi-stage marketing research. ...

Antioxidant Supplementation Reduces Genomic Aberrations in Human Induced Pluripotent Stem Cells

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Junfeng Ji

2014-01-01

Full Text Available Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs using oncogenic transcription factors. However, this method leads to genetic aberrations in iPSCs via unknown mechanisms, which may limit their clinical use. Here, we demonstrate that the supplementation of growth media with antioxidants reduces the genome instability of cells transduced with the reprogramming factors. Antioxidant supplementation did not affect transgene expression level or silencing kinetics. Importantly, iPSCs made with antioxidants had significantly fewer de novo copy number variations, but not fewer coding point mutations, than iPSCs made without antioxidants. Our results suggest that the quality and safety of human iPSCs might be enhanced by using antioxidants in the growth media during the generation and maintenance of iPSCs.

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...

Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity.

Science.gov (United States)

Ryan, Kristen R; Sirenko, Oksana; Parham, Fred; Hsieh, Jui-Hua; Cromwell, Evan F; Tice, Raymond R; Behl, Mamta

2016-03-01

Due to the increasing prevalence of neurological disorders and the large number of untested compounds in the environment, there is a need to develop reliable and efficient screening tools to identify environmental chemicals that could potentially affect neurological development. Herein, we report on a library of 80 compounds screened for their ability to inhibit neurite outgrowth, a process by which compounds may elicit developmental neurotoxicity, in a high-throughput, high-content assay using human neurons derived from induced pluripotent stem cells (iPSC). The library contains a diverse set of compounds including those that have been known to be associated with developmental neurotoxicity (DNT) and/or neurotoxicity (NT), environmental compounds with unknown neurotoxic potential (e.g., polycyclic aromatic hydrocarbons (PAHs) and flame retardants (FRs)), as well as compounds with no documented neurotoxic potential. Neurons were treated for 72h across a 6-point concentration range (∼0.3-100μM) in 384-well plates. Effects on neurite outgrowth were assessed by quantifying total outgrowth, branches, and processes. We also assessed the number ofviable cells per well. Concentration-response profiles were evaluated using a Hill model to derive benchmark concentration (BMC) values. Assay performance was evaluated using positive and negative controls and test replicates. Compounds were ranked by activity and selectivity (i.e., specific effects on neurite outgrowth in the absence of concomitant cytotoxicity) and repeat studies were conducted to confirm selectivity. Among the 80 compounds tested, 38 compounds were active, of which 16 selectively inhibited neurite outgrowth. Of these 16 compounds, 12 were known to cause DNT/NT and the remaining 4 compounds included 3 PAHs and 1 FR. In independent repeat studies, 14/16 selective compounds were reproducibly active in the assay, of which only 6 were selective for inhibition of neurite outgrowth. These 6 compounds were

Translation of Human-Induced Pluripotent Stem Cells: From Clinical Trial in a Dish to Precision Medicine.

Science.gov (United States)

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

2016-05-10

The prospect of changing the plasticity of terminally differentiated cells toward pluripotency has completely altered the outlook for 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. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

Science.gov (United States)

Yamashita, Akihiro; Morioka, Miho; Yahara, Yasuhito; Okada, Minoru; Kobayashi, Tomohito; Kuriyama, Shinichi; Matsuda, Shuichi; Tsumaki, Noriyuki

2015-03-10

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells

Science.gov (United States)

Bershteyn, Marina; Hayashi, Yohei; Desachy, Guillaume; Hsiao, Edward C.; Sami, Salma; Tsang, Kathryn M.; Weiss, Lauren A.; Kriegstein, Arnold R.; Yamanaka, Shinya; Wynshaw-Boris, Anthony

2014-03-01

Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of `chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

Donor‐Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte‐Like Cells

Science.gov (United States)

Heslop, James A.; Kia, Richard; Pridgeon, Christopher S.; Sison‐Young, Rowena L.; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W.; Mills, John S.; Kitteringham, Neil R.; Park, Bong K.

2017-01-01

Abstract Drug‐induced liver injury is the greatest cause of post‐marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)‐derived hepatocyte‐like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this “resetting” is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte‐ and dermal fibroblast‐derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC‐derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast‐derived iPSCs. We conclude that the donor and inter‐clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC‐derived HLCs. Stem Cells Translational Medicine 2017;6:1321–1331 PMID:28456008

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.

The Use of Patient-Specific Induced Pluripotent Stem Cells (iPSCs to Identify Osteoclast Defects in Rare Genetic Bone Disorders

Directory of Open Access Journals (Sweden)

I-Ping Chen

2014-12-01

Full Text Available More than 500 rare genetic bone disorders have been described, but for many of them only limited treatment options are available. Challenges for studying these bone diseases come from a lack of suitable animal models and unavailability of skeletal tissues for studies. Effectors for skeletal abnormalities of bone disorders may be abnormal bone formation directed by osteoblasts or anomalous bone resorption by osteoclasts, or both. Patient-specific induced pluripotent stem cells (iPSCs can be generated from somatic cells of various tissue sources and in theory can be differentiated into any desired cell type. However, successful differentiation of hiPSCs into functional bone cells is still a challenge. Our group focuses on the use of human iPSCs (hiPSCs to identify osteoclast defects in craniometaphyseal dysplasia. In this review, we describe the impact of stem cell technology on research for better treatment of such disorders, the generation of hiPSCs from patients with rare genetic bone disorders and current protocols for differentiating hiPSCs into osteoclasts.

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Qiang Feng

2014-11-01

Full Text Available Human induced pluripotent stem cells (iPSCs provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Multi-stage internal gear/turbine fuel pump

Energy Technology Data Exchange (ETDEWEB)

Maier, Eugen; Raney, Michael Raymond

2004-07-06

A multi-stage internal gear/turbine fuel pump for a vehicle includes a housing having an inlet and an outlet and a motor disposed in the housing. The multi-stage internal gear/turbine fuel pump also includes a shaft extending axially and disposed in the housing. The multi-stage internal gear/turbine fuel pump further includes a plurality of pumping modules disposed axially along the shaft. One of the pumping modules is a turbine pumping module and another of the pumping modules is a gerotor pumping module for rotation by the motor to pump fuel from the inlet to the outlet.

Is Human-induced Pluripotent Stem Cell the Best Optimal?

OpenAIRE

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

2018-01-01

Objective: 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...

Derivation of induced pluripotent stem cells from a familial Alzheimer's disease patient carrying the L282F mutation in presenilin 1

DEFF Research Database (Denmark)

Poon, Anna Fong-Yee; Li, Tong; Pires, Carlota

2016-01-01

Mutations in presenilin 1 (PSEN1) lead to the most aggressive form of familial Alzheimer's disease (AD). Human induced pluripotent stem cells (hiPSCs) derived from AD patients can be differentiated and used for disease modeling. Here, we derived hiPSC from skin fibroblasts obtained from an AD...... patient carrying a L282F mutation in PSEN1. We transfected skin fibroblasts with episomal iPSC reprogramming vectors targeting human OCT4, SOX2, L-MYC, KLF4, NANOG, LIN28, and short hairpin RNA against TP53. Our hiPSC line, L282F-hiPSC, displayed typical stem cell characteristics with consistent...... expression of pluripotency genes and the ability to differentiation into the three germ layers....

Interconnected levels of Multi-Stage Marketing – A Triadic approach

DEFF Research Database (Denmark)

Vedel, Mette; Geersbro, Jens; Ritter, Thomas

2012-01-01

must not only decide in general on the merits of multi-stage marketing for their firm, but must also decide on which level they will engage in multi-stage marketing. The triadic perspective enables a rich and multi-dimensional understanding of how different business relationships influence each other......Multi-stage marketing gains increasing attention as knowledge of and influence on the customer's customer become more critical for the firm's success. Despite this increasing managerial relevance, systematic approaches for analyzing multi-stage marketing are still missing. This paper conceptualizes...... different levels of multi-stage marketing and illustrates these stages with a case study. In addition, a triadic perspective is introduced as an analytical tool for multi-stage marketing research. The results from the case study indicate that multi-stage marketing exists on different levels. Thus, managers...

40 CFR 600.316-78 - Multistage manufacture.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 29 2010-07-01 2010-07-01 false Multistage manufacture. 600.316-78 Section 600.316-78 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) ENERGY POLICY... and Later Model Year Automobiles-Labeling § 600.316-78 Multistage manufacture. Where more than one...

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

Science.gov (United States)

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

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

Experiments for Multi-Stage Processes

DEFF Research Database (Denmark)

Tyssedal, John; Kulahci, Murat

2015-01-01

Multi-stage processes are very common in both process and manufacturing industries. In this article we present a methodology for designing experiments for multi-stage processes. Typically in these situations the design is expected to involve many factors from different stages. To minimize...... the required number of experimental runs, we suggest using mirror image pairs of experiments at each stage following the first. As the design criterion, we consider their projectivity and mainly focus on projectivity 3 designs. We provide the methodology for generating these designs for processes with any...

Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors

Directory of Open Access Journals (Sweden)

Andreas Hermann

2016-01-01

Full Text Available Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC or two (OCT4, KLF4; hiPSC2F-NSC reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB or four reprogramming factors (hiPSC4F-FIB. After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

Comparing ESC and iPSC—Based Models for Human Genetic Disorders

Directory of Open Access Journals (Sweden)

Tomer Halevy

2014-10-01

Full Text Available 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.

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.

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

Science.gov (United States)

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

Split-plot designs for multistage experimentation

DEFF Research Database (Denmark)

Kulahci, Murat; Tyssedal, John

2016-01-01

at the same time will be more efficient. However, there have been only a few attempts in the literature to provide an adequate and easy-to-use approach for this problem. In this paper, we present a novel methodology for constructing two-level split-plot and multistage experiments. The methodology is based...... be accommodated in each stage. Furthermore, split-plot designs for multistage experiments with good projective properties are also provided....

Modelling urea-cycle disorder citrullinemia type 1 with disease-specific iPSCs.

Science.gov (United States)

Yoshitoshi-Uebayashi, Elena Yukie; Toyoda, Taro; Yasuda, Katsutaro; Kotaka, Maki; Nomoto, Keiko; Okita, Keisuke; Yasuchika, Kentaro; Okamoto, Shinya; Takubo, Noriyuki; Nishikubo, Toshiya; Soga, Tomoyoshi; Uemoto, Shinji; Osafune, Kenji

2017-05-06

Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

Science.gov (United States)

Heslop, James A; Kia, Richard; Pridgeon, Christopher S; Sison-Young, Rowena L; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W; Mills, John S; Kitteringham, Neil R; Goldring, Chris E; Park, Bong K

2017-05-01

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of Alpha

Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

OpenAIRE

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-01

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

Induced pluripotent stem cell technology: a decade of progress.

Science.gov (United States)

Shi, Yanhong; Inoue, Haruhisa; Wu, Joseph C; Yamanaka, Shinya

2017-02-01

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 modelling, drug discovery and cell therapy development. Novel pathological mechanisms have been elucidated, new drugs originating from iPSC screens are in the pipeline and the first clinical trial using human iPSC-derived products has been initiated. In particular, the combination of human iPSC technology with recent developments in gene editing and 3D organoids makes iPSC-based platforms even more powerful in each area of their application, including precision medicine. 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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.

Science.gov (United States)

Willmann, Charlotte A; Hemeda, Hatim; Pieper, Lisa A; Lenz, Michael; Qin, Jie; Joussen, Sylvia; Sontag, Stephanie; Wanek, Paul; Denecke, Bernd; Schüler, Herdit M; Zenke, Martin; Wagner, Wolfgang

2013-01-01

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.

A Novel Protocol for Directed Differentiation of C9orf72-Associated Human Induced Pluripotent Stem Cells Into Contractile Skeletal Myotubes.

Science.gov (United States)

Swartz, Elliot W; Baek, Jaeyun; Pribadi, Mochtar; Wojta, Kevin J; Almeida, Sandra; Karydas, Anna; Gao, Fen-Biao; Miller, Bruce L; Coppola, Giovanni

2016-11-01

: Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease, therapeutic drug screening, and transplant-based regenerative medicine. However, methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here, we present a novel, small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG + cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC), sporadic FTD, and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid, efficient, and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved

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 (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

DEFF Research Database (Denmark)

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...

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

Directory of Open Access Journals (Sweden)

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.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

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Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

2016-01-01

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Multi-stage fuzzy PID power system automatic generation controller in deregulated environments

International Nuclear Information System (INIS)

Shayeghi, H.; Shayanfar, H.A.; Jalili, A.

2006-01-01

In this paper, a multi-stage fuzzy proportional integral derivative (PID) type controller is proposed to solve the automatic generation control (AGC) problem in a deregulated power system that operates under deregulation based on the bilateral policy scheme. In each control area, the effects of the possible contracts are treated as a set of new input signals in a modified traditional dynamical model. The multi-stage controller uses the fuzzy switch to blend a proportional derivative (PD) fuzzy logic controller with an integral fuzzy logic input. The proposed controller operates on fuzzy values passing the consequence of a prior stage on to the next stage as fact. The salient advantage of this strategy is its high insensitivity to large load changes and disturbances in the presence of plant parameter variations and system nonlinearities. This newly developed strategy leads to a flexible controller with simple structure that is easy to implement, and therefore, it can be useful for the real world power systems. The proposed method is tested on a three area power system with different contracted scenarios under various operating conditions. The results of the proposed controller are compared with those of the classical fuzzy PID type controller and classical PID controller through some performance indices to illustrate its robust performance

Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

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Götz Pilarczyk

2016-01-01

Full Text Available The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds.

Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

Science.gov (United States)

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-08

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. Copyright 2010 Elsevier Inc. All rights reserved.

Bio-inspired approach to multistage image processing

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Timchenko, Leonid I.; Pavlov, Sergii V.; Kokryatskaya, Natalia I.; Poplavska, Anna A.; Kobylyanska, Iryna M.; Burdenyuk, Iryna I.; Wójcik, Waldemar; Uvaysova, Svetlana; Orazbekov, Zhassulan; Kashaganova, Gulzhan

2017-08-01

Multistage integration of visual information in the brain allows people to respond quickly to most significant stimuli while preserving the ability to recognize small details in the image. Implementation of this principle in technical systems can lead to more efficient processing procedures. The multistage approach to image processing, described in this paper, comprises main types of cortical multistage convergence. One of these types occurs within each visual pathway and the other between the pathways. This approach maps input images into a flexible hierarchy which reflects the complexity of the image data. The procedures of temporal image decomposition and hierarchy formation are described in mathematical terms. The multistage system highlights spatial regularities, which are passed through a number of transformational levels to generate a coded representation of the image which encapsulates, in a computer manner, structure on different hierarchical levels in the image. At each processing stage a single output result is computed to allow a very quick response from the system. The result is represented as an activity pattern, which can be compared with previously computed patterns on the basis of the closest match.

Exposure Control Using Adaptive Multi-Stage Item Bundles.

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Luecht, Richard M.

This paper presents a multistage adaptive testing test development paradigm that promises to handle content balancing and other test development needs, psychometric reliability concerns, and item exposure. The bundled multistage adaptive testing (BMAT) framework is a modification of the computer-adaptive sequential testing framework introduced by…

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.

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.

Rats, cats, and elephants, but still no unicorn: induced pluripotent stem cells from new species.

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Trounson, Alan

2009-01-09

Two independent studies in this issue of Cell Stem Cell (Liao et al., 2009; Li et al., 2009) derive rat induced pluripotent stem cells (iPSCs). In one report, the method used results in rat and human iPSCs that exhibit phenotypic traits similar to mouse embryonic stem cells.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

Science.gov (United States)

Huebsch, Nathaniel; Loskill, Peter; Mandegar, Mohammad A; Marks, Natalie C; Sheehan, Alice S; Ma, Zhen; Mathur, Anurag; Nguyen, Trieu N; Yoo, Jennie C; Judge, Luke M; Spencer, C Ian; Chukka, Anand C; Russell, Caitlin R; So, Po-Lin; Conklin, Bruce R; Healy, Kevin E

2015-05-01

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

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.

Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4α Transduction

OpenAIRE

Takayama, Kazuo; Inamura, Mitsuru; Kawabata, Kenji; Katayama, Kazufumi; Higuchi, Maiko; Tashiro, Katsuhisa; Nonaka, Aki; Sakurai, Fuminori; Hayakawa, Takao; Kusuda Furue, Miho; Mizuguchi, Hiroyuki

2012-01-01

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is kn...

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.

Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells

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Lee, Han-Kyu; Velazquez Sanchez, Clara; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.

2016-01-01

The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug. PMID:27684569

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

Science.gov (United States)

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.

Mapping transcriptome profiles of in vitro iPSC-derived cardiac differentiation to in utero heart development

Directory of Open Access Journals (Sweden)

Xing Li

2016-03-01

Full Text Available The dataset includes microarray data (Affymetrix Mouse Genome 430 2.0 Array from WT and Nos3−/− mouse embryonic heart ventricular tissues at 14.5 days post coitum (E14.5, induced pluripotent stem cells (iPSCs derived from WT and Nos3−/− mouse tail tip fibroblasts, iPSC-differentiated cardiomyocytes at Day 11, and mouse embryonic stem cells (mESCs and differentiated cardiomyocytes as positive controls for mouse iPSC differentiation. Both in utero (using embryonic heart tissues and in vitro (using iPSCs and differentiated cells microarray datasets were deposited to the NCBI Gene Expression Omnibus (GEO database. The deposited data in GEO include raw microarray data, metadata for sample source information, experimental design, sample and data processing, and gene expression matrix. The data are available under GEO Access Number GSE69317 (GSE69315 for tissue sample microarray data, GSE69316 for iPSCs microarray data, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE69317. Keywords: Induced pluripotent stem cell, Cardiac development, Nos3 knockout, Disease modeling, Microarray analysis

Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

Science.gov (United States)

Rathod, Reena; Surendran, Harshini; Battu, Rajani; Desai, Jogin; Pal, Rajarshi

2018-02-12

Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized. Copyright © 2018 Elsevier B.V. All rights reserved.

Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

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Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

2015-01-01

Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

Novel methodology for wide-ranged multistage morphing waverider based on conical theory

Science.gov (United States)

Liu, Zhen; Liu, Jun; Ding, Feng; Xia, Zhixun

2017-11-01

This study proposes the wide-ranged multistage morphing waverider design method. The flow field structure and aerodynamic characteristics of multistage waveriders are also analyzed. In this method, the multistage waverider is generated in the same conical flowfield, which contains a free-stream surface and different compression-stream surfaces. The obtained results show that the introduction of the multistage waverider design method can solve the problem of aerodynamic performance deterioration in the off-design state and allow the vehicle to always maintain the optimal flight state. The multistage waverider design method, combined with transfiguration flight strategy, can lead to greater design flexibility and the optimization of hypersonic wide-ranged waverider vehicles.

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...

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Engineering of Pulsatile Conduits from Human Pluripotent Stem Cell Derived Cardiomyocytes

Science.gov (United States)

2017-06-01

Stem cells . Cardiovascular . Regenerative medicine . Induced pluripotent stem cells . Embryonic stem ...lineage-specific marker ex- pression. The advent of these induced pluripotent stem cells (iPSCs) generated a large interest in the stem cell and regen ... pluripotent stem cells : macro- andmicrostructures for disease modeling, drug screening , and

Induced pluripotent stem cells and their implication for regenerative medicine.

Science.gov (United States)

Csobonyeiova, Maria; Polak, Stefan; Koller, Jan; Danisovic, Lubos

2015-06-01

In 2006 Yamanaka's group showed that stem cells with properties similar to embryonic stem cells could be generated from mouse fibroblasts by introducing four genes. These cells were termed induced pluripotent stem cells (iPSCs). Because iPSCs avoid many of ethical concerns associated with the use of embryonic material, they have great potential in cell-based regenerative medicine. They are suitable also for other various purposes, including disease modelling, personalized cell therapy, drug or toxicity screening and basic research. Moreover, in the future, there might become possible to generate organs for human transplantation. Despite these progresses, several studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to immunogenicity of some cells differentiated from iPSCs. Recent methodological improvements are increasing the ease and efficacy of reprogramming, and reducing the genomic modification. However, to minimize or eliminate genetic alternations in the derived iPSC line creation, factor-free human iPSCs are necessary. In this review we discuss recent possibilities of using iPSCs for clinical applications and new advances in field of their reprogramming methods. The main goal of present article was to review the current knowledge about iPSCs and to discuss their potential for regenerative medicine.

Hepatic differentiation of human iPSCs in different 3D models: A comparative study.

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Meier, Florian; Freyer, Nora; Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A; Beilmann, Mario; Zeilinger, Katrin

2017-12-01

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.

Cost-effective master cell bank validation of multiple clinical-grade human pluripotent stem cell lines from a single donor.

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Devito, Liani; Petrova, Anastasia; Miere, Cristian; Codognotto, Stefano; Blakely, Nicola; Lovatt, Archie; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

2014-10-01

Standardization guidelines for human pluripotent stem cells are still very broadly defined, despite ongoing clinical trials in the U.S., U.K., and Japan. The requirements for validation of human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) in general follow the regulations for other clinically compliant biologics already in place but without addressing key differences between cell types or final products. In order to realize the full potential of stem cell therapy, validation criteria, methodology, and, most importantly, strategy, should address the shortfalls and efficiency of current approaches; without this, hESC- and, especially, iPSC-based therapy will not be able to compete with other technologies in a cost-efficient way. We addressed the protocols for testing cell lines for human viral pathogens and propose a novel strategy that would significantly reduce costs. It is highly unlikely that the multiple cell lines derived in parallel from a tissue sample taken from one donor would have different profiles of endogenous viral pathogens; we therefore argue that samples from the Master Cell Banks of sibling lines could be safely pooled for validation. We illustrate this approach with tiered validation of two sibling clinical-grade hESC lines, KCL033 and KCL034 (stage 1, sterility; stage 2, specific human pathogens; and stage 3, nonspecific human pathogens). The results of all tests were negative. This cost-effective strategy could also be applied for validation of Master Cell Banks of multiple clinical-grade iPSC lines derived from a single donor. ©AlphaMed Press.

Phase-I monitoring of standard deviations in multistage linear profiles

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Kalaei, Mahdiyeh; Soleimani, Paria; Niaki, Seyed Taghi Akhavan; Atashgar, Karim

2018-03-01

In most modern manufacturing systems, products are often the output of some multistage processes. In these processes, the stages are dependent on each other, where the output quality of each stage depends also on the output quality of the previous stages. This property is called the cascade property. Although there are many studies in multistage process monitoring, there are fewer works on profile monitoring in multistage processes, especially on the variability monitoring of a multistage profile in Phase-I for which no research is found in the literature. In this paper, a new methodology is proposed to monitor the standard deviation involved in a simple linear profile designed in Phase I to monitor multistage processes with the cascade property. To this aim, an autoregressive correlation model between the stages is considered first. Then, the effect of the cascade property on the performances of three types of T 2 control charts in Phase I with shifts in standard deviation is investigated. As we show that this effect is significant, a U statistic is next used to remove the cascade effect, based on which the investigated control charts are modified. Simulation studies reveal good performances of the modified control charts.

Study on multi-stage hydropyrolysis of coal in fixed-bed reactor

Energy Technology Data Exchange (ETDEWEB)

Wang, N.; Li, W.; Li, B.-Q. [Chinese Academy of Sciences, Taiyuan (China). State Key Lab of Coal Conversion

1999-07-01

The composition and quantity of the oil in hydropyrolysis (HyPy) and multi-stage HyPy with high and slow heating rate were compared and the effect of multistage HyPy process on desulfurization was investigated. Multistage HyPy of lignite and high sulphur coal were investigated and the effects of residence time, heating rate and pressure on product yields were studied. 6 refs., 4 figs., 2 tabs.

Multi-Stage Recognition of Speech Emotion Using Sequential Forward Feature Selection

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Liogienė Tatjana

2016-07-01

Full Text Available The intensive research of speech emotion recognition introduced a huge collection of speech emotion features. Large feature sets complicate the speech emotion recognition task. Among various feature selection and transformation techniques for one-stage classification, multiple classifier systems were proposed. The main idea of multiple classifiers is to arrange the emotion classification process in stages. Besides parallel and serial cases, the hierarchical arrangement of multi-stage classification is most widely used for speech emotion recognition. In this paper, we present a sequential-forward-feature-selection-based multi-stage classification scheme. The Sequential Forward Selection (SFS and Sequential Floating Forward Selection (SFFS techniques were employed for every stage of the multi-stage classification scheme. Experimental testing of the proposed scheme was performed using the German and Lithuanian emotional speech datasets. Sequential-feature-selection-based multi-stage classification outperformed the single-stage scheme by 12–42 % for different emotion sets. The multi-stage scheme has shown higher robustness to the growth of emotion set. The decrease in recognition rate with the increase in emotion set for multi-stage scheme was lower by 10–20 % in comparison with the single-stage case. Differences in SFS and SFFS employment for feature selection were negligible.

Enhanced antioxidant capacity of dental pulp-derived iPSC-differentiated hepatocytes and liver regeneration by injectable HGF-releasing hydrogel in fulminant hepatic failure.

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Chiang, Chih-Hung; Wu, Wai-Wah; Li, Hsin-Yang; Chien, Yueh; Sun, Cho-Chin; Peng, Chi-Hsien; Lin, Alex Tong-Long; Huang, Chi-Shuan; Lai, Ying-Hsiu; Chiou, Shih-Hwa; Hung, Shuen-Iu; Chang, Yuh-Lih; Lan, Yuan-Tzu; Liu, Dean-Mo; Chien, Chian-Shiu; Huo, Teh-Ia; Lee, Shou-Dong; Wang, Chien-Ying

2015-01-01

Acute hepatic failure (AHF) is a severe liver injury leading to sustained damage and complications. Induced pluripotent stem cells (iPSCs) may be an alternative option for the treatment of AHF. In this study, we reprogrammed human dental pulp-derived fibroblasts into iPSCs, which exhibited pluripotency and the capacity to differentiate into tridermal lineages, including hepatocyte-like cells (iPSC-Heps). These iPSC-Heps resembled human embryonic stem cell-derived hepatocyte-like cells in gene signature and hepatic markers/functions. To improve iPSC-Heps engraftment, we next developed an injectable carboxymethyl-hexanoyl chitosan hydrogel (CHC) with sustained hepatocyte growth factor (HGF) release (HGF-CHC) and investigated the hepatoprotective activity of HGF-CHC-delivered iPSC-Heps in vitro and in an immunocompromised AHF mouse model induced by thioacetamide (TAA). Intrahepatic delivery of HGF-CHC-iPSC-Heps reduced the TAA-induced hepatic necrotic area and rescued liver function and recipient viability. Compared with PBS-delivered iPSC-Heps, the HGF-CHC-delivered iPSC-Heps exhibited higher antioxidant and antiapoptotic activities that reduced hepatic necrotic area. Importantly, these HGF-CHC-mediated responses could be abolished by administering anti-HGF neutralizing antibodies. In conclusion, our findings demonstrated that HGF mediated the enhancement of iPSC-Hep antioxidant/antiapoptotic capacities and hepatoprotection and that HGF-CHC is as an excellent vehicle for iPSC-Hep engraftment in iPSC-based therapy against AHF.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells

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Takanori Takebe

2017-12-01

Full Text Available Summary: Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC. By conducting massive “reverse” screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108. Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. : With the goal of clinical translation of liver bud transplant therapy, Takebe et al. established a massive organoid production platform from endoderm, endothelial, and mesenchymal progenitor populations specified entirely from human iPSCs, reproducibly demonstrating functionality both in vitro and in vivo. Keywords: iPSC, liver bud, organoid, transplantation, self-organization, endothelial, mesenchymal, liver failure, clinical grade

Modeling Kidney Disease with iPS Cells

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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

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

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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

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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

Multi-Stage System for Automatic Target Recognition

Science.gov (United States)

Chao, Tien-Hsin; Lu, Thomas T.; Ye, David; Edens, Weston; Johnson, Oliver

2010-01-01

A multi-stage automated target recognition (ATR) system has been designed to perform computer vision tasks with adequate proficiency in mimicking human vision. The system is able to detect, identify, and track targets of interest. Potential regions of interest (ROIs) are first identified by the detection stage using an Optimum Trade-off Maximum Average Correlation Height (OT-MACH) filter combined with a wavelet transform. False positives are then eliminated by the verification stage using feature extraction methods in conjunction with neural networks. Feature extraction transforms the ROIs using filtering and binning algorithms to create feature vectors. A feedforward back-propagation neural network (NN) is then trained to classify each feature vector and to remove false positives. The system parameter optimizations process has been developed to adapt to various targets and datasets. The objective was to design an efficient computer vision system that can learn to detect multiple targets in large images with unknown backgrounds. Because the target size is small relative to the image size in this problem, there are many regions of the image that could potentially contain the target. A cursory analysis of every region can be computationally efficient, but may yield too many false positives. On the other hand, a detailed analysis of every region can yield better results, but may be computationally inefficient. The multi-stage ATR system was designed to achieve an optimal balance between accuracy and computational efficiency by incorporating both models. The detection stage first identifies potential ROIs where the target may be present by performing a fast Fourier domain OT-MACH filter-based correlation. Because threshold for this stage is chosen with the goal of detecting all true positives, a number of false positives are also detected as ROIs. The verification stage then transforms the regions of interest into feature space, and eliminates false positives using an

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

Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

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Budd A Tucker

2011-04-01

Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

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 using non-synthetic mRNA.

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Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

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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

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

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Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

2014-01-01

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

Health and Human Rights in Chin State, Western Burma: A Population-Based Assessment Using Multistaged Household Cluster Sampling

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Sollom, Richard; Richards, Adam K.; Parmar, Parveen; Mullany, Luke C.; Lian, Salai Bawi; Iacopino, Vincent; Beyrer, Chris

2011-01-01

Background The Chin State of Burma (also known as Myanmar) is an isolated ethnic minority area with poor health outcomes and reports of food insecurity and human rights violations. We report on a population-based assessment of health and human rights in Chin State. We sought to quantify reported human rights violations in Chin State and associations between these reported violations and health status at the household level. Methods and Findings Multistaged household cluster sampling was done. Heads of household were interviewed on demographics, access to health care, health status, food insecurity, forced displacement, forced labor, and other human rights violations during the preceding 12 months. Ratios of the prevalence of household hunger comparing exposed and unexposed to each reported violation were estimated using binomial regression, and 95% confidence intervals (CIs) were constructed. Multivariate models were done to adjust for possible confounders. Overall, 91.9% of households (95% CI 89.7%–94.1%) reported forced labor in the past 12 months. Forty-three percent of households met FANTA-2 (Food and Nutrition Technical Assistance II project) definitions for moderate to severe household hunger. Common violations reported were food theft, livestock theft or killing, forced displacement, beatings and torture, detentions, disappearances, and religious and ethnic persecution. Self reporting of multiple rights abuses was independently associated with household hunger. Conclusions Our findings indicate widespread self-reports of human rights violations. The nature and extent of these violations may warrant investigation by the United Nations or International Criminal Court. Please see later in the article for the Editors' Summary PMID:21346799

Human Pluripotent Stem Cells to Engineer Blood Vessels.

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Chan, Xin Yi; Elliott, Morgan B; Macklin, Bria; Gerecht, Sharon

2018-01-01

Development of pluripotent stem cells (PSCs) is a remarkable scientific advancement that allows scientists to harness the power of regenerative medicine for potential treatment of disease using unaffected cells. PSCs provide a unique opportunity to study and combat cardiovascular diseases, which continue to claim the lives of thousands each day. Here, we discuss the differentiation of PSCs into vascular cells, investigation of the functional capabilities of the derived cells, and their utilization to engineer microvascular beds or vascular grafts for clinical application. Graphical Abstract Human iPSCs generated from patients are differentiated toward ECs and perivascular cells for use in disease modeling, microvascular bed development, or vascular graft fabrication.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

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Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

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

Derivation of porcine pluripotent stem cells for biomedical research.

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Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

2016-07-01

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

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Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

Effects of the Post-Spinal Cord Injury Microenvironment on the Differentiation Capacity of Human Neural Stem Cells Derived from Induced Pluripotent Stem Cells.

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López-Serrano, Clara; Torres-Espín, Abel; Hernández, Joaquim; Alvarez-Palomo, Ana B; Requena, Jordi; Gasull, Xavier; Edel, Michael J; Navarro, Xavier

2016-10-01

Spinal cord injury (SCI) causes loss of neural functions below the level of the lesion due to interruption of spinal pathways and secondary neurodegenerative processes. The transplant of neural stem cells (NSCs) is a promising approach for the repair of SCI. Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) is expected to provide an autologous source of iPSC-derived NSCs, avoiding the immune response as well as ethical issues. However, there is still limited information on the behavior and differentiation pattern of transplanted iPSC-derived NSCs within the damaged spinal cord. We transplanted iPSC-derived NSCs, obtained from adult human somatic cells, into rats at 0 or 7 days after SCI, and evaluated motor-evoked potentials and locomotion of the animals. We histologically analyzed engraftment, proliferation, and differentiation of the iPSC-derived NSCs and the spared tissue in the spinal cords at 7, 21, and 63 days posttransplant. Both transplanted groups showed a late decline in functional recovery compared to vehicle-injected groups. Histological analysis showed proliferation of transplanted cells within the tissue and that cells formed a mass. At the final time point, most grafted cells differentiated to neural and astroglial lineages, but not into oligodendrocytes, while some grafted cells remained undifferentiated and proliferative. The proinflammatory tissue microenviroment of the injured spinal cord induced proliferation of the grafted cells and, therefore, there are possible risks associated with iPSC-derived NSC transplantation. New approaches are needed to promote and guide cell differentiation, as well as reduce their tumorigenicity once the cells are transplanted at the lesion site.

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...

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future

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Mingyue Luo

2018-01-01

Full Text Available As a constituent of blood-retinal barrier and retinal outer segment (ROS scavenger, retinal pigmented epithelium (RPE is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

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Luo, Mingyue; Chen, Youxin

2018-01-01

As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

Enhanced Multistage Homotopy Perturbation Method: Approximate Solutions of Nonlinear Dynamic Systems

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Daniel Olvera

2014-01-01

Full Text Available We introduce a new approach called the enhanced multistage homotopy perturbation method (EMHPM that is based on the homotopy perturbation method (HPM and the usage of time subintervals to find the approximate solution of differential equations with strong nonlinearities. We also study the convergence of our proposed EMHPM approach based on the value of the control parameter h by following the homotopy analysis method (HAM. At the end of the paper, we compare the derived EMHPM approximate solutions of some nonlinear physical systems with their corresponding numerical integration solutions obtained by using the classical fourth order Runge-Kutta method via the amplitude-time response curves.

Progress, problems and prospects of porcine pluripotent stem cells

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Hanning WANG,Yangli PEI,Ning LI,Jianyong HAN

2014-02-01

Full Text Available Pluripotent stem cells (PSCs, including embryonic stem cells (ESCs and induced PSCs (iPSCs, can differentiate into cells of the three germ layers, suggesting that PSCs have great potential for basic developmental biology research and wide applications for clinical medicine. Genuine ESCs and iPSCs have been derived from mice and rats, but not from livestock such as the pig─an ideal animal model for studying human disease and regenerative medicine due to similarities with human physiologic processes. Efforts to derive porcine ESCs and iPSCs have not yielded high-quality PSCs that can produce chimeras with germline transmission. Thus, exploration of the unique porcine gene regulation network of preimplantation embryonic development may permit optimization of in vitro culture systems for raising porcine PSCs. Here we summarize the recent progress in porcine PSC generation as well as the problems encountered during this progress and we depict prospects for generating porcine naive PSCs.

Translating chimpanzee personality to humans: Investigating the transportability of chimpanzee-derived personality scales to humans.

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Latzman, Robert D; Sauvigné, Katheryn C; Hopkins, William D

2016-06-01

There is a growing interest in the study of personality in chimpanzees with repeated findings of a similar structure of personality in apes to that found in humans. To date, however, the direct translational value of instruments used to assess chimpanzee personality to humans has yet to be explicitly tested. As such, in the current study we sought to determine the transportability of factor analytically-derived chimpanzee personality scales to humans in a large human sample (N = 301). Human informants reporting on target individuals they knew well completed chimpanzee-derived and human-derived measures of personality from the two most widely studied models of human personality: Big Five and Big Three. The correspondence between informant-reported chimpanzee- and human-derived personality scales was then investigated. Results indicated high convergence for corresponding scales across most chimpanzee- and human-derived personality scales. Findings from the current study provide evidence that chimpanzee-derived scales translate well to humans and operate quite similarly to the established human-derived personality scales in a human sample. This evidence of transportability lends support to the translational nature of chimpanzee personality research suggesting clear relevance of this growing literature to humans. Am. J. Primatol. 78:601-609, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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

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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.

Micro-Raman Imaging for Biology with Multivariate Spectral Analysis

KAUST Repository

Malvaso, Federica

2015-05-05

Raman spectroscopy is a noninvasive technique that can provide complex information on the vibrational state of the molecules. It defines the unique fingerprint that allow the identification of the various chemical components within a given sample. The aim of the following thesis work is to analyze Raman maps related to three pairs of different cells, highlighting differences and similarities through multivariate algorithms. The first pair of analyzed cells are human embryonic stem cells (hESCs), while the other two pairs are induced pluripotent stem cells (iPSCs) derived from T lymphocytes and keratinocytes, respectively. Although two different multivariate techniques were employed, ie Principal Component Analysis and Cluster Analysis, the same results were achieved: the iPSCs derived from T-lymphocytes show a higher content of genetic material both compared with the iPSCs derived from keratinocytes and the hESCs . On the other side, equally evident, was that iPS cells derived from keratinocytes assume a molecular distribution very similar to hESCs.

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.

Venture Investment Incentive Mechanisms and Simulation with Venture Entrepreneurs Having Multistage Efforts Based on Fairness Preference Theory

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Kaihong Wang

2016-01-01

Full Text Available When venture capital has been invested into venture companies, venture capitalists and venture entrepreneurs form a principal-agent relationship. Take into account the fact that the venture entrepreneur’s effort is a long process, because the effort is not the same at different stage. Therefore, efforts variables are seen as the multistage dynamic variable, and venture investment principal-agent model with venture entrepreneurs having multistage efforts is constructed on the basis of the classic principal-agent theory in the paper. Further, in the later stage effort of venture entrepreneurs is affected by the size of prestage benefit with venture capitalists and venture entrepreneurs; thus the fairness preference model is improved, and venture investment principal-agent model with venture entrepreneurs having multistage efforts is constructed on the basis of fairness preference theory. Both theoretical derivation and simulation have demonstrated that, under the condition of information asymmetry, if the fairness preference of venture entrepreneurs holds, then (1 venture capitalists provide venture entrepreneurs with level higher than that without fairness preference, (2 in every single stage venture entrepreneurs make efforts higher than those without fairness preference, and (3 in two periods both venture investors and venture entrepreneurs gain total real gains higher than those in two periods without fair preference.

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

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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.

Multi-Stage Transportation Problem With Capacity Limit

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I. Brezina

2010-06-01

Full Text Available The classical transportation problem can be applied in a more general way in practice. Related problems as Multi-commodity transportation problem, Transportation problems with different kind of vehicles, Multi-stage transportation problems, Transportation problem with capacity limit is an extension of the classical transportation problem considering the additional special condition. For solving such problems many optimization techniques (dynamic programming, linear programming, special algorithms for transportation problem etc. and heuristics approaches (e.g. evolutionary techniques were developed. This article considers Multi-stage transportation problem with capacity limit that reflects limits of transported materials (commodity quantity. Discussed issues are: theoretical base, problem formulation as way as new proposed algorithm for that problem.

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

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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.

Generation of Spinal Motor Neurons from Human Pluripotent Stem Cells.

Science.gov (United States)

Santos, David P; Kiskinis, Evangelos

2017-01-01

Human embryonic stem cells (ESCs) are characterized by their unique ability to self-renew indefinitely, as well as to differentiate into any cell type of the human body. Induced pluripotent stem cells (iPSCs) share these salient characteristics with ESCs and can easily be generated from any given individual by reprogramming somatic cell types such as fibroblasts or blood cells. The spinal motor neuron (MN) is a specialized neuronal subtype that synapses with muscle to control movement. Here, we present a method to generate functional, postmitotic, spinal motor neurons through the directed differentiation of ESCs and iPSCs by the use of small molecules. These cells can be utilized to study the development and function of human motor neurons in healthy and disease states.

The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation

OpenAIRE

Youngkyun Kim; Yeri Alice Rim; Hyoju Yi; Narae Park; Sung-Hwan Park; Ji Hyeon Ju

2016-01-01

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for differentiation into diverse tissues. We report a straightforward and highly efficient method for the generation of iPSCs from PBMCs. By plating the cells serially to a newly coated plate by centrifugation, this protocol provides multiple healthy iPSC colonies even from a small number of PBMCs. The generated iPSCs expressed pluripotent markers and differentiated into all three germ layer lineages. The protocol...

Biologically based multistage modeling of radiation effects

Energy Technology Data Exchange (ETDEWEB)

William Hazelton; Suresh Moolgavkar; E. Georg Luebeck

2005-08-30

This past year we have made substantial progress in modeling the contribution of homeostatic regulation to low-dose radiation effects and carcinogenesis. We have worked to refine and apply our multistage carcinogenesis models to explicitly incorporate cell cycle states, simple and complex damage, checkpoint delay, slow and fast repair, differentiation, and apoptosis to study the effects of low-dose ionizing radiation in mouse intestinal crypts, as well as in other tissues. We have one paper accepted for publication in ''Advances in Space Research'', and another manuscript in preparation describing this work. I also wrote a chapter describing our combined cell-cycle and multistage carcinogenesis model that will be published in a book on stochastic carcinogenesis models edited by Wei-Yuan Tan. In addition, we organized and held a workshop on ''Biologically Based Modeling of Human Health Effects of Low dose Ionizing Radiation'', July 28-29, 2005 at Fred Hutchinson Cancer Research Center in Seattle, Washington. We had over 20 participants, including Mary Helen Barcellos-Hoff as keynote speaker, talks by most of the low-dose modelers in the DOE low-dose program, experimentalists including Les Redpath (and Mary Helen), Noelle Metting from DOE, and Tony Brooks. It appears that homeostatic regulation may be central to understanding low-dose radiation phenomena. The primary effects of ionizing radiation (IR) are cell killing, delayed cell cycling, and induction of mutations. However, homeostatic regulation causes cells that are killed or damaged by IR to eventually be replaced. Cells with an initiating mutation may have a replacement advantage, leading to clonal expansion of these initiated cells. Thus we have focused particularly on modeling effects that disturb homeostatic regulation as early steps in the carcinogenic process. There are two primary considerations that support our focus on homeostatic regulation. First, a number of

Being human: The role of pluripotent stem cells in regenerative medicine and humanizing Alzheimer's disease models.

Science.gov (United States)

Sproul, Andrew A

2015-01-01

Human pluripotent stem cells (PSCs) have the capacity to revolutionize medicine by allowing the generation of functional cell types such as neurons for cell replacement therapy. However, the more immediate impact of PSCs on treatment of Alzheimer's disease (AD) will be through improved human AD model systems for mechanistic studies and therapeutic screening. This review will first briefly discuss different types of PSCs and genome-editing techniques that can be used to modify PSCs for disease modeling or for personalized medicine. This will be followed by a more in depth analysis of current AD iPSC models and a discussion of the need for more complex multicellular models, including cell types such as microglia. It will finish with a discussion on current clinical trials using PSC-derived cells and the long-term potential of such strategies for treating AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

Naked Mole Rat Induced Pluripotent Stem Cells and Their Contribution to Interspecific Chimera

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Sang-Goo Lee

2017-11-01

Full Text Available Naked mole rats (NMRs are exceptionally long-lived, cancer-resistant rodents. Identifying the defining characteristics of these traits may shed light on aging and cancer mechanisms. Here, we report the generation of induced pluripotent stem cells (iPSCs from NMR fibroblasts and their contribution to mouse-NMR chimeric embryos. Efficient reprogramming could be observed under N2B27+2i conditions. The iPSCs displayed a characteristic morphology, expressed pluripotent markers, formed embryoid bodies, and showed typical differentiation patterns. Interestingly, NMR embryonic fibroblasts and the derived iPSCs had propensity for a tetraploid karyotype and were resistant to forming teratomas, but within mouse blastocysts they contributed to both interspecific placenta and fetus. Gene expression patterns of NMR iPSCs were more similar to those of human than mouse iPSCs. Overall, we uncovered unique features of NMR iPSCs and report a mouse-NMR chimeric model. The iPSCs and associated cell culture systems can be used for a variety of biological and biomedical applications.

Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine.

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Revilla, Ana; González, Clara; Iriondo, Amaia; Fernández, Bárbara; Prieto, Cristina; Marín, Carlos; Liste, Isabel

2016-11-01

Over the last few years, the generation of induced pluripotent stem cells (iPSCs) from human somatic cells has proved to be one of the most potentially useful discoveries in regenerative medicine. iPSCs are becoming an invaluable tool to study the pathology of different diseases and for drug screening. However, several limitations still affect the possibility of applying iPS cell-based technology in therapeutic prospects. Most strategies for iPSCs generation are based on gene delivery via retroviral or lentiviral vectors, which integrate into the host's cell genome, causing a remarkable risk of insertional mutagenesis and oncogenic transformation. To avoid such risks, significant advances have been made with non-integrative reprogramming strategies. On the other hand, although many different kinds of somatic cells have been employed to generate iPSCs, there is still no consensus about the ideal type of cell to be reprogrammed. In this review we present the recent advances in the generation of human iPSCs, discussing their advantages and limitations in terms of safety and efficiency. We also present a selection of somatic cell sources, considering their capability to be reprogrammed and tissue accessibility. From a translational medicine perspective, these two topics will provide evidence to elucidate the most suitable combination of reprogramming strategy and cell source to be applied in each human iPSC-based therapy. The wide variety of diseases this technology could treat opens a hopeful future for regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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...

A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

Science.gov (United States)

Son, Mi-Young; Kwak, Jae Eun; Seol, Binna; Lee, Da Yong; Jeon, Hyejin; Cho, Yee Sook

2015-09-01

GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal β-galactosidase (β-gal) gene. Insufficient β-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective β-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multi-stage mixing in subduction zone: Application to Merapi volcano, Indonesia

Science.gov (United States)

Debaille, V.; Doucelance, R.; Weis, D.; Schiano, P.

2003-04-01

Basalts sampling subduction zone volcanism (IAB) often show binary mixing relationship in classical Sr-Nd, Pb-Pb, Sr-Pb isotopic diagrams, generally interpreted as reflecting the involvement of two components in their source. However, several authors have highlighted the presence of minimum three components in such a geodynamical context: mantle wedge, subducted and altered oceanic crust and subducted sediments. The overlying continental crust can also contribute by contamination and assimilation in magma chambers and/or during magma ascent. Here we present a multi-stage model to obtain a two end-member mixing from three components (mantle wedge, altered oceanic crust and sediments). The first stage of the model considers the metasomatism of the mantle wedge by fluids and/or melts released by subducted materials (altered oceanic crust and associated sediments), considering mobility and partition coefficient of trace elements in hydrated fluids and silicate melts. This results in the generation of two distinct end-members, reducing the number of components (mantle wedge, oceanic crust, sediments) from three to two. The second stage of the model concerns the binary mixing of the two end-members thus defined: mantle wedge metasomatized by slab-derived fluids and mantle wedge metasomatized by sediment-derived fluids. This model has been applied on a new isotopic data set (Sr, Nd and Pb, analyzed by TIMS and MC-ICP-MS) of Merapi volcano (Java island, Indonesia). Previous studies have suggested three distinct components in the source of indonesian lavas: mantle wedge, subducted sediments and altered oceanic crust. Moreover, it has been shown that crustal contamination does not significantly affect isotopic ratios of lavas. The multi-stage model proposed here is able to reproduce the binary mixing observed in lavas of Merapi, and a set of numerical values of bulk partition coefficient is given that accounts for the genesis of lavas.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

Science.gov (United States)

Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

2018-03-15

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically

Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.

Science.gov (United States)

Takayama, Naoya; Eto, Koji

2012-10-01

Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.

Optimization of organic Rankine cycle power systems considering multistage axial turbine design

DEFF Research Database (Denmark)

Meroni, Andrea; Andreasen, Jesper Graa; Persico, Giacomo

2018-01-01

Organic Rankine cycle power systems represent a viable and efficient solution for the exploitation of medium-to-low temperature heat sources. Despite the large number of commissioned units, there is limited literature on the design and optimization of organic Rankine cycle power systems considering...... multistage turbine design. This work presents a preliminary design methodology and working fluid selection for organic Rankine cycle units featuring multistage axial turbines. The method is then applied to the case of waste heat recovery from a large marine diesel engine. A multistage axial turbine model...

Optimization of organic Rankine cycle power systems considering multistage axial turbine design

DEFF Research Database (Denmark)

Meroni, Andrea; Andreasen, Jesper Graa; Persico, Giacomo

2017-01-01

Organic Rankine cycle power systems represent a viable and efficient solution for the exploitation of medium-to-low temperature heat sources. Despite the large number of commissioned units, there is limited literature on the design and optimization of organic Rankine cycle power systems considering...... multistage turbine design. This work presents a preliminary design methodology and working fluid selection for organic Rankine cycle units featuring multistage axial turbines. The method is then applied to the case of waste heat recovery from a large marine diesel engine. A multistage axial turbine model...

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.

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

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Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

2016-07-30

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

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

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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.

Multi-stage decoding of multi-level modulation codes

Science.gov (United States)

Lin, Shu; Kasami, Tadao; Costello, Daniel J., Jr.

1991-01-01

Various types of multi-stage decoding for multi-level modulation codes are investigated. It is shown that if the component codes of a multi-level modulation code and types of decoding at various stages are chosen properly, high spectral efficiency and large coding gain can be achieved with reduced decoding complexity. Particularly, it is shown that the difference in performance between the suboptimum multi-stage soft-decision maximum likelihood decoding of a modulation code and the single-stage optimum soft-decision decoding of the code is very small, only a fraction of dB loss in signal to noise ratio at a bit error rate (BER) of 10(exp -6).

A Methodology for Optimization in Multistage Industrial Processes: A Pilot Study

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Piotr Jarosz

2015-01-01

Full Text Available The paper introduces a methodology for optimization in multistage industrial processes with multiple quality criteria. Two ways of formulation of optimization problem and four different approaches to solve the problem are considered. Proposed methodologies were tested first on a virtual process described by benchmark functions and next were applied in optimization of multistage lead refining process.

Strategies and limits in multi-stage single-point incremental forming

DEFF Research Database (Denmark)

Skjødt, Martin; Silva, M.B.; Martins, P. A. F.

2010-01-01

paths. The results also reveal that the sequence of multi-stage forming has a large effect on the location of strain points in the principal strain space. Strain paths are linear in the first stage and highly non-linear in the subsequent forming stages. The overall results show that the experimentally......Multi-stage single-point incremental forming (SPIF) is a state-of-the-art manufacturing process that allows small-quantity production of complex sheet metal parts with vertical walls. This paper is focused on the application of multi-stage SPIF with the objective of producing cylindrical cups......-limit curves and fracture forming-limit curves (FFLCs), numerical simulation, and experimentation, namely the evaluation of strain paths and fracture strains in actual multi-stage parts. Assessment of numerical simulation with experimentation shows good agreement between computed and measured strain and strain...

A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.

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Gunhanlar, N; Shpak, G; van der Kroeg, M; Gouty-Colomer, L A; Munshi, S T; Lendemeijer, B; Ghazvini, M; Dupont, C; Hoogendijk, W J G; Gribnau, J; de Vrij, F M S; Kushner, S A

2017-04-18

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (-58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (-50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.Molecular Psychiatry advance online publication, 18 April 2017; doi:10.1038/mp.2017.56.

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

The Power and the Promise of Cell Reprogramming: Personalized Autologous Body Organ and Cell Transplantation

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Ana Belen Alvarez Palomo

2014-04-01

Full Text Available Reprogramming somatic cells to induced pluripotent stem cells (iPSCs or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids, bone, optic vesicle-like structures (eye, cardiac muscle tissue (heart, primitive pancreas islet cells, a tooth-like structure (teeth, and functional liver buds (liver. Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1 such transplants will stimulate host immune responses; and (2 whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by iPSC-derived

Glis family proteins are differentially implicated in the cellular reprogramming of human somatic cells.

Science.gov (United States)

Lee, Seo-Young; Noh, Hye Bin; Kim, Hyeong-Taek; Lee, Kang-In; Hwang, Dong-Youn

2017-09-29

The ground-breaking discovery of the reprogramming of somatic cells into pluripotent cells, termed induced pluripotent stem cells (iPSCs), was accomplished by delivering 4 transcription factors, Oct4, Sox2, Klf4, and c-Myc, into fibroblasts. Since then, several efforts have attempted to unveil other factors that are directly implicated in or might enhance reprogramming. Importantly, a number of transcription factors are reported to retain reprogramming activity. A previous study suggested Gli-similar 1 (Glis1) as a factor that enhances the reprogramming of fibroblasts during iPSC generation. However, the implication of other Glis members, including Glis2 and Glis3 (variants 1 and 2), in cellular reprogramming remains unknown. In this study, we investigated the potential involvement of human Glis family proteins, including hGlis1-3, in cellular reprogramming. Our results demonstrate that hGlis1, which is reported to reprogram human fibroblasts, promotes the reprogramming of human adipose-derived stromal cells (hADSCs), indicating that the reprogramming activity of Glis1 is not cell type-specific. Strikingly, hGlis3 promoted the reprogramming of hADSCs as efficiently as hGlis1. On the contrary, hGlis2 showed a strong negative effect on reprogramming. Together, our results reveal clear differences in the cellular reprogramming activity among Glis family members and provide valuable insight into the development of a new reprogramming strategy using Glis family proteins.

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

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Yuncheng Zhao

2018-02-01

Full Text Available Summary: Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs. These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility. : In this article, Wang and colleagues established a feeder- and xeno-free system to robustly induce human pluripotent stem cells (PSCs into spermatogonia-like cells. This chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Keywords: pluripotent stem cells, spermatogonia, infertility, non-obstructive azoospermia

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

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Lafaille, Fabien G; Pessach, Itai M.; Zhang, Shen-Ying; Ciancanelli, Michael J.; Herman, Melina; Abhyankar, Avinash; Ying, Shui-Wang; Keros, Sotirios; Goldstein, Peter A.; Mostoslavsky, Gustavo; Ordovas-Montanes, Jose; Jouanguy, Emmanuelle; Plancoulaine, Sabine; Tu, Edmund; Elkabetz, Yechiel; Al-Muhsen, Saleh; Tardieu, Marc; Schlaeger, Thorsten M.; Daley, George Q.; Abel, Laurent; Casanova, Jean-Laurent; Studer, Lorenz; Notarangelo, Luigi D.

2012-01-01

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies. PMID:23103873

The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

Science.gov (United States)

Watanabe, Daisuke; Koyanagi-Aoi, Michiyo; Taniguchi-Ikeda, Mariko; Yoshida, Yukiko; Azuma, Takeshi; Aoi, Takashi

2018-01-01

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation

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Youngkyun Kim

2016-01-01

Full Text Available Human induced pluripotent stem cells (hiPSCs have demonstrated great potential for differentiation into diverse tissues. We report a straightforward and highly efficient method for the generation of iPSCs from PBMCs. By plating the cells serially to a newly coated plate by centrifugation, this protocol provides multiple healthy iPSC colonies even from a small number of PBMCs. The generated iPSCs expressed pluripotent markers and differentiated into all three germ layer lineages. The protocol can also be used with umbilical cord blood mononuclear cells (CBMCs. In this study, we present a simple and efficient protocol that improved the yield of iPSCs from floating cells such as PBMCs and CBMCs by serial plating and centrifugation.

Handling Imbalanced Data Sets in Multistage Classification

Science.gov (United States)

López, M.

Multistage classification is a logical approach, based on a divide-and-conquer solution, for dealing with problems with a high number of classes. The classification problem is divided into several sequential steps, each one associated to a single classifier that works with subgroups of the original classes. In each level, the current set of classes is split into smaller subgroups of classes until they (the subgroups) are composed of only one class. The resulting chain of classifiers can be represented as a tree, which (1) simplifies the classification process by using fewer categories in each classifier and (2) makes it possible to combine several algorithms or use different attributes in each stage. Most of the classification algorithms can be biased in the sense of selecting the most populated class in overlapping areas of the input space. This can degrade a multistage classifier performance if the training set sample frequencies do not reflect the real prevalence in the population. Several techniques such as applying prior probabilities, assigning weights to the classes, or replicating instances have been developed to overcome this handicap. Most of them are designed for two-class (accept-reject) problems. In this article, we evaluate several of these techniques as applied to multistage classification and analyze how they can be useful for astronomy. We compare the results obtained by classifying a data set based on Hipparcos with and without these methods.

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

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Damián Hernández

2016-01-01

Full Text Available Background. Human induced pluripotent stem cells (iPSCs are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

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Felipe Augusto Andre Ishiy

2015-01-01

Full Text Available Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED and MSC-like cells from iPSCs from SHED (iPS-SHED and from human dermal fibroblasts (iPS-FIB. MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.

Directed Differentiation of Human Pluripotent Stem Cells to Microglia

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Panagiotis Douvaras

2017-06-01

Full Text Available Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs. Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Information Overload in Multi-Stage Selection Procedures

NARCIS (Netherlands)

S.S. Ficco (Stefano); V.A. Karamychev (Vladimir)

2004-01-01

textabstractThe paper studies information processing imperfections in a fully rational decision-making network. It is shown that imperfect information transmission and imperfect information acquisition in a multi-stage selection game yield information overload. The paper analyses the mechanisms

Configuration of management accounting information system for multi-stage manufacturing

Science.gov (United States)

Mkrtychev, S. V.; Ochepovsky, A. V.; Enik, O. A.

2018-05-01

The article presents an approach to configuration of a management accounting information system (MAIS) that provides automated calculations and the registration of normative production losses in multi-stage manufacturing. The use of MAIS with the proposed configuration at the enterprises of textile and woodworking industries made it possible to increase the accuracy of calculations for normative production losses and to organize accounting thereof with the reference to individual stages of the technological process. Thus, high efficiency of multi-stage manufacturing control is achieved.

Effects of mesenchymal stem cells from human induced pluripotent stem cells on differentiation, maturation, and function of dendritic cells.

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Gao, Wen-Xiang; Sun, Yue-Qi; Shi, Jianbo; Li, Cheng-Lin; Fang, Shu-Bin; Wang, Dan; Deng, Xue-Quan; Wen, Weiping; Fu, Qing-Ling

2017-03-02

Mesenchymal stem cells (MSCs) have potent immunomodulatory effects on multiple immune cells and have great potential in treating immune disorders. Induced pluripotent stem cells (iPSCs) serve as an unlimited and noninvasive source of MSCs, and iPSC-MSCs have been reported to have more advantages and exhibit immunomodulation on T lymphocytes and natural killer cells. However, the effects of iPSC-MSCs on dendritic cells (DCs) are unclear. The aim of this study is to investigate the effects of iPSC-MSCs on the differentiation, maturation, and function of DCs. Human monocyte-derived DCs were induced and cultured in the presence or absence of iPSC-MSCs. Flow cytometry was used to analyze the phenotype and functions of DCs, and enzyme-linked immunosorbent assay (ELISA) was used to study cytokine production. In this study, we successfully induced MSCs from different clones of human iPSCs. iPSC-MSCs exhibited a higher proliferation rate with less cell senescence than BM-MSCs. iPSC-MSCs inhibited the differentiation of human monocyte-derived DCs by both producing interleukin (IL)-10 and direct cell contact. Furthermore, iPSC-MSCs did not affect immature DCs to become mature DCs, but modulated their functional properties by increasing their phagocytic ability and inhibiting their ability to stimulate proliferation of lymphocytes. More importantly, iPSC-MSCs induced the generation of IL-10-producing regulatory DCs in the process of maturation, which was mostly mediated by a cell-cell contact mechanism. Our results indicate an important role for iPSC-MSCs in the modulation of DC differentiation and function, supporting the clinical application of iPSC-MSCs in DC-mediated immune diseases.

Experience in North America Tight Oil Reserves Development. Horizontal Wells and Multistage Hydraulic Fracturing

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R.R. Ibatullin

2017-09-01

Full Text Available The accelerated development of horizontal drilling technology in combination with the multistage hydraulic fracturing of the reservoir has expanded the geological conditions for commercial oil production from tight reservoirs in North America. Geological and physical characteristics of tight reservoirs in North America are presented, as well as a comparison of the geological and physical properties of the reservoirs of the Western Canadian Sedimentary Basin and the Volga-Ural oil and gas province, in particular, in the territory of Tatarstan. The similarity of these basins is shown in terms of formation and deposition. New drilling technologies for horizontal wells (HW and multistage hydraulic fracturing are considered. The drilling in tight reservoirs is carried out exclusively on hydrocarbon-based muds The multi-stage fracturing technology with the use of sliding sleeves, and also slick water – a low-viscous carrier for proppant is the most effective solution for conditions similar to tight reservoirs in the Devonian formation of Tatarstan. Tax incentives which are actively used for the development of HW and multistage fracturing technologies in Canada are described. wells, multistage fracturing

Multistage models of carcinogenesis and their implications for dose-response models and risk projections

International Nuclear Information System (INIS)

Hoel, D.G.

1992-01-01

Multistage models are used to both describe the biological steps in developing a cancer and as a mathematical description of the relationship of exposure to tumor incidence. With the rapid development of molecular biology the stages of tumor development are becoming understood. Specifically, the effect and role of proto-oncogenes and suppressor genes are exciting developments in the field of carcinogenesis. Mathematically the field has moved from the original Armitage-Doll multistage model to the more current cell kinetic models. These latter models attempt to describe both the rate of cell mutation and the birth-death process involved in clonal expansion. This then allows modeling of both initiation and promotion or cellular proliferation. The field of radiation carcinogenesis has a considerable body of data and knowledge. Unfortunately, relatively little work has been done with the cell kinetic models as to estimation of tumor incidence. This may be due to the newness of kinetic models in general. The field holds promise and it is essential if we are to develop better human risk estimates from exposure to ionizing radiation. (author)

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

Science.gov (United States)

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 application of the consecutive-Woehler-curve-concept in computation of the life values for multi-stage creep

International Nuclear Information System (INIS)

Schott, G.

1991-01-01

It is known that at multi-stage creep load there cannot be calculated any reliable life values by means of linear damage accumulation hypotheses. A practicable non-linear statement was proposed by Pantelakis. Besides the one-stage creep life curve, results from two-stage tests are required for determining the damage exponent. With this exponent, which is a function of temperature and stress in the load stage applied first, the life values can be calculated only for two-stage sequences whose stress stages have to be identical to those of the two-stage tests. For the application of the consecutive Woehler curve concept described in the following there is required the knowledge of the one-stage creep life curve and of the creep function for increasing and decreasing stress sequences derived from two-stage tests. Then, the life values can be calculated for the most different multi-stage loads. The stress stages should lie within the stress range used in the two-stage tests. (orig.) [de

Unsteady Aerodynamics & Aeromechanics of Multi-Stage Turbomachinery Blading

National Research Council Canada - National Science Library

Fleeter, Sanford

2002-01-01

.... A benchmark-standard multistage transonic research compressor was developed by modifying the Purdue High-Speed Axial Compressor to feature new IGV and stator rows representative of modern high pressure compressors...

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

Directory of Open Access Journals (Sweden)

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.

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

Science.gov (United States)

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.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

2017-08-14

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Renata Santos

2017-06-01

Full Text Available Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.

Mitochondrial Alterations by PARKIN in Dopaminergic Neurons Using PARK2 Patient-Specific and PARK2 Knockout Isogenic iPSC Lines

Directory of Open Access Journals (Sweden)

Atossa Shaltouki

2015-05-01

Full Text Available In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction. The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism.

Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.

Science.gov (United States)

Yabe, Shigeharu G; Fukuda, Satsuki; Takeda, Fujie; Nashiro, Kiyoko; Shimoda, Masayuki; Okochi, Hitoshi

2017-02-01

Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Multistage feature extraction for accurate face alignment

NARCIS (Netherlands)

Zuo, F.; With, de P.H.N.

2004-01-01

We propose a novel multistage facial feature extraction approach using a combination of 'global' and 'local' techniques. At the first stage, we use template matching, based on an Edge-Orientation-Map for fast feature position estimation. Using this result, a statistical framework applying the Active

Assessment of Farmers' Benefits Derived from Olam Organisation's ...

African Journals Online (AJOL)

The study assessed farmers' benefits derived from Olam organization's sustainable cocoa production extension activities in Ondo state. Structured and validated interview schedule was used to collect relevant information from thirty cocoa farmers, using multistage random sampling technique from cocoa producing towns ...

Amniotic fluid-derived mesenchymal stem cells as a novel ...

African Journals Online (AJOL)

CLEMENTINA

2012-06-28

Jun 28, 2012 ... stem cells (AFMSCs) have many advantages over other stem cells: avoiding much ethical controversy ... showed that induced pluripotent stem cells (iPSCs) have ... disadvantages of ESCs, BM-MSCs and iPSCs have.

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

This report describes a gap analysis performed in the process of 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 rigorous verification, validation, and software quality requirements. The gap analyses documented in this report were are performed during an initial gap analysis to identify candidate codes and tools to support the development and integration of the Waste IPSC, and during follow-on activities that delved into more detailed assessments of the various codes that were acquired, studied, and tested. 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. The gap analysis indicates that significant capabilities may already exist in the existing THC codes although there is no single code able to fully account for all physical and chemical processes involved in a waste disposal system. Large gaps exist in modeling chemical processes and their couplings with other processes. The coupling of chemical processes with flow transport and mechanical deformation remains challenging. The data for extreme environments (e.g., for elevated temperature and high ionic strength media) that are

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

This report describes a gap analysis performed in the process of 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 rigorous verification, validation, and software quality requirements. The gap analyses documented in this report were are performed during an initial gap analysis to identify candidate codes and tools to support the development and integration of the Waste IPSC, and during follow-on activities that delved into more detailed assessments of the various codes that were acquired, studied, and tested. 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. The gap analysis indicates that significant capabilities may already exist in the existing THC codes although there is no single code able to fully account for all physical and chemical processes involved in a waste disposal system. Large gaps exist in modeling chemical processes and their couplings with other processes. The coupling of chemical processes with flow transport and mechanical deformation remains challenging. The data for extreme environments (e.g., for elevated temperature and high ionic strength media) that are

Multi-stage volcanic island flank collapses with coeval explosive caldera-forming eruptions.

Science.gov (United States)

Hunt, James E; Cassidy, Michael; Talling, Peter J

2018-01-18

Volcanic flank collapses and explosive eruptions are among the largest and most destructive processes on Earth. Events at Mount St. Helens in May 1980 demonstrated how a relatively small (300 km 3 ), but can also occur in complex multiple stages. Here, we show that multistage retrogressive landslides on Tenerife triggered explosive caldera-forming eruptions, including the Diego Hernandez, Guajara and Ucanca caldera eruptions. Geochemical analyses were performed on volcanic glasses recovered from marine sedimentary deposits, called turbidites, associated with each individual stage of each multistage landslide. These analyses indicate only the lattermost stages of subaerial flank failure contain materials originating from respective coeval explosive eruption, suggesting that initial more voluminous submarine stages of multi-stage flank collapse induce these aforementioned explosive eruption. Furthermore, there are extended time lags identified between the individual stages of multi-stage collapse, and thus an extended time lag between the initial submarine stages of failure and the onset of subsequent explosive eruption. This time lag succeeding landslide-generated static decompression has implications for the response of magmatic systems to un-roofing and poses a significant implication for ocean island volcanism and civil emergency planning.

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

Energy Technology Data Exchange (ETDEWEB)

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com [Molecular Devices, LLC, Sunnyvale, CA (United States); Grimm, Fabian A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Ryan, Kristen R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Iwata, Yasuhiro; Chiu, Weihsueh A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Parham, Frederick [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Wignall, Jessica A. [ICF, Fairfax, VA (United States); Anson, Blake [Cellular Dynamics International, Madison, WI (United States); Cromwell, Evan F. [Protein Fluidics, Inc., Burlingame, CA (United States); Behl, Mamta [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Rusyn, Ivan [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Tice, Raymond R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States)

2017-05-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca{sup 2+} flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights: �

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

International Nuclear Information System (INIS)

Sirenko, Oksana; Grimm, Fabian A.; Ryan, Kristen R.; Iwata, Yasuhiro; Chiu, Weihsueh A.; Parham, Frederick; Wignall, Jessica A.; Anson, Blake; Cromwell, Evan F.; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R.

2017-01-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca 2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights: �

Biomedical and Clinical Promises of Human Pluripotent Stem Cells for Neurological Disorders

Directory of Open Access Journals (Sweden)

Nopporn Jongkamonwiwat

2013-01-01

Full Text Available Neurological disorders are characterized by the chronic and progressive loss of neuronal structures and functions. There is a variability of the onsets and causes of clinical manifestations. Cell therapy has brought a new concept to overcome brain diseases, but the advancement of this therapy is limited by the demands of specialized neurons. Human pluripotent stem cells (hPSCs have been promised as a renewable resource for generating human neurons for both laboratory and clinical purposes. By the modulations of appropriate signalling pathways, desired neuron subtypes can be obtained, and induced pluripotent stem cells (iPSCs provide genetically matched neurons for treating patients. These hPSC-derived neurons can also be used for disease modeling and drug screening. Since the most urgent problem today in transplantation is the lack of suitable donor organs and tissues, the derivation of neural progenitor cells from hPSCs has opened a new avenue for regenerative medicine. In this review, we summarize the recent reports that show how to generate neural derivatives from hPSCs, and discuss the current evidence of using these cells in animal studies. We also highlight the possibilities and concerns of translating these hPSC-derived neurons for biomedical and clinical uses in order to fight against neurological disorders.

Some design aspects of multistage flash distillation process

International Nuclear Information System (INIS)

Ahmad, Mohammad.

1975-01-01

The purpose of this paper is to examine the effect of the design variables of multistage flash (MSF) process on the performance and/or the cost of the desalting plant, and to establish certain design trends

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.

Science.gov (United States)

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.

Demand management in Multi-Stage Distribution Chain

NARCIS (Netherlands)

de Kok, T.; Janssen, F.B.S.L.P.

1996-01-01

In this paper we discuss demand management problems in a multi-stage distribution chain.We focus on distribution chains where demand processes have high variability due to a few large customer orders.We give a possible explanation, and suggest two simple procedures that help to smooth demand.It is

Reducing Delay in Diagnosis: Multistage Recommendation Tracking.

Science.gov (United States)

Wandtke, Ben; Gallagher, Sarah

2017-11-01

The purpose of this study was to determine whether a multistage tracking system could improve communication between health care providers, reducing the risk of delay in diagnosis related to inconsistent communication and tracking of radiology follow-up recommendations. Unconditional recommendations for imaging follow-up of all diagnostic imaging modalities excluding mammography (n = 589) were entered into a database and tracked through a multistage tracking system for 13 months. Tracking interventions were performed for patients for whom completion of recommended follow-up imaging could not be identified 1 month after the recommendation due date. Postintervention compliance with the follow-up recommendation required examination completion or clinical closure (i.e., biopsy, limited life expectancy or death, or subspecialist referral). Baseline radiology information system checks performed 1 month after the recommendation due date revealed timely completion of 43.1% of recommended imaging studies at our institution before intervention. Three separate tracking interventions were studied, showing effectiveness between 29.0% and 57.8%. The multistage tracking system increased the examination completion rate to 70.5% (a 52% increase) and reduced the rate of unknown follow-up compliance and the associated risk of delay in diagnosis to 13.9% (a 74% decrease). Examinations completed after tracking intervention generated revenue of 4.1 times greater than the labor cost. Performing sequential radiology recommendation tracking interventions can substantially reduce the rate of unknown follow-up compliance and add value to the health system. Unknown follow-up compliance is a risk factor for delay in diagnosis, a form of preventable medical error commonly identified in malpractice claims involving radiologists and office-based practitioners.

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

A development of time-resolved emulsion detector by multi-stage shifter

International Nuclear Information System (INIS)

Takahashi, Satoru; Aoki, Shigeki

2017-01-01

Nuclear emulsion is a powerful tracking device that can record the three-dimensional trajectory of charged particles within 1 μm spatial resolution. We are promoting GRAINE project which is 10 MeV-100 GeV cosmic γ-ray observations with a precise (0.08deg at 1-2 GeV) and polarization-sensitive large-aperture-area (∼10 m 2 ) emulsion telescope by repeating long duration balloon flights. We are developing multi-stage shifter which allows us to give a timing information to emulsion tracks with ∼seconds or below. The multi-stage shifter opened feasibilities of precise cosmic γ-ray observations, GRAINE, as well as precise measurements of ν-N interactions, J-PARC T60. ∼Millisecond time resolution in a balloon-borne experiment, ∼second time resolution for 126.7 days in an accelerator ν experiment and ∼10 6 time-resolved numbers are being achieved. New model of multi-stage shifter is also being developed for future experiments. (author)

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

Science.gov (United States)

Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

2017-06-08

Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

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.

Multistage Stochastic Programming via Autoregressive Sequences

Czech Academy of Sciences Publication Activity Database

Kaňková, Vlasta

2007-01-01

Roč. 15, č. 4 (2007), s. 99-110 ISSN 0572-3043 R&D Projects: GA ČR GA402/07/1113; GA ČR(CZ) GA402/06/0990; GA ČR GD402/03/H057 Institutional research plan: CEZ:AV0Z10750506 Keywords : Economic proceses * Multistage stochastic programming * autoregressive sequences * individual probability constraints Subject RIV: BB - Applied Statistics, Operational Research

The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells.

Science.gov (United States)

Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

2016-01-01

N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.

Physiological Aβ Concentrations Produce a More Biomimetic Representation of the Alzheimer's Disease Phenotype in iPSC Derived Human Neurons.

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Berry, Bonnie J; Smith, Alec S T; Long, Christopher J; Martin, Candace C; Hickman, James J

2018-05-22

Alzheimer's disease (AD) is characterized by slow, progressive neurodegeneration leading to severe neurological impairment, but current drug development efforts are limited by the lack of robust, human-based disease models. Amyloid-β (Aβ) is known to play an integral role in AD progression as it has been shown to interfere with neurological function. However, studies into AD pathology commonly apply Aβ to neurons for short durations at nonphysiological concentrations to induce an exaggerated dysfunctional phenotype. Such methods are unlikely to elucidate early stage disease dysfunction, when treatment is still possible, since damage to neurons by these high concentrations is extensive. In this study, we investigated chronic, pathologically relevant Aβ oligomer concentrations to induce an electrophysiological phenotype that is more representative of early AD progression compared to an acute high-dose application in human cortical neurons. The high, acute oligomer dose resulted in severe neuronal toxicity as well as upregulation of tau and phosphorylated tau. Chronic, low-dose treatment produced significant functional impairment without increased cell death or accumulation of tau protein. This in vitro phenotype more closely mirrors the status of early stage neural decline in AD pathology and could provide a valuable tool to further understanding of early stage AD pathophysiology and for screening potential therapeutic compounds.

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

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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.

Application of stem cell derived neuronal cells to evaluate neurotoxic chemotherapy

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Claudia Wing

2017-07-01

Full Text Available The generation of induced pluripotent stem cells (iPSCs and differentiation to cells composing major organs has opened up the possibility for a new model system to study adverse toxicities associated with chemotherapy. Therefore, we used human iPSC-derived neurons to study peripheral neuropathy, one of the most common adverse effects of chemotherapy and cause for dose reduction. To determine the utility of these neurons in investigating the effects of neurotoxic chemotherapy, we measured morphological differences in neurite outgrowth, cell viability as determined by ATP levels and apoptosis through measures of caspase 3/7 activation following treatment with clinically relevant concentrations of platinating agents (cisplatin, oxaliplatin and carboplatin, taxanes (paclitaxel, docetaxel and nab-paclitaxel, a targeted proteasome inhibitor (bortezomib, an antiangiogenic compound (thalidomide, and 5-fluorouracil, a chemotherapeutic that does not cause neuropathy. We demonstrate differential sensitivity of neurons to mechanistically distinct classes of chemotherapeutics. We also show a dose-dependent reduction of electrical activity as measured by mean firing rate of the neurons following treatment with paclitaxel. We compared neurite outgrowth and cell viability of iPSC-derived cortical (iCell® Neurons and peripheral (Peri.4U neurons to cisplatin, paclitaxel and vincristine. Goshajinkigan, a Japanese herbal neuroprotectant medicine, was protective against paclitaxel-induced neurotoxicity but not oxaliplatin as measured by morphological phenotypes. Thus, we have demonstrated the utility of human iPSC-derived neurons as a useful model to distinguish drug class differences and for studies of a potential neuroprotectant for the prevention of chemotherapy-induced peripheral neuropathy.

Finite Element Analysis and Optimization for the Multi-stage Deep Drawing of Molybdenum Sheet

International Nuclear Information System (INIS)

Kim, Heung-Kyu; Hong, Seok Kwan; Kang, Jeong Jin; Heo, Young-moo; Lee, Jong-Kil; Jeon, Byung-Hee

2005-01-01

Molybdenum, a bcc refractory metal with a melting point of about 2600 deg. C, has a high heat and electrical conductivity. In addition, it remains strong mechanically at high temperatures as well as at low temperatures. Therefore it is a technologically very important material for the applications operating at high temperatures. However, a multi-stage process is required due to the low drawability for making a deep drawn part from the molybdenum sheet. In this study, a multi-stage deep drawing process for a molybdenum circular cup was designed by combining the drawing with the ironing, which was effective for the low drawability materials. A parametric study by FE analysis for the multi-stage deep drawing was conducted for evaluation of the design variables effect. Based on the FE analysis result, the multi-stage deep drawing process was parameterized by the design variables, and an optimum process design was obtained by the process optimization based on the FE simulation at each stage

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.

Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.

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Byrne, Susan M; Ortiz, Luis; Mali, Prashant; Aach, John; Church, George M

2015-02-18

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

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

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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.

Multi-stage wake-field accelerator

International Nuclear Information System (INIS)

Gai, Wei.

1989-01-01

In this paper we propose a multi-stage wake field acceleration scheme to overcome the low transformer ratio problem and still provide high accelerating gradients. The idea is very simple. We use a train of several electron bunches from a linear accelerator (main linac) with well defined separations between the bunches (tens of ns) to drive wake field devices. Here we have made the assumption that the wake field devices are available, whether plasma, iris-loaded metallic or dielectric wake field structures. 10 refs

Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve: viscoelasticity characterization

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Xue-man Lv

2016-01-01

Full Text Available The optic nerve is a viscoelastic solid-like biomaterial. Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury. We hypothesized that stress relaxation and creep properties of the optic nerve change after injury. More-over, human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal. To validate this hypothesis, a rabbit model of optic nerve injury was established using a clamp approach. At 7 days after injury, the vitreous body re-ceived a one-time injection of 50 µg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells. At 30 days after injury, stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly, with patho-logical changes in the injured optic nerve also noticeably improved. These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves, and thereby contributes to nerve recovery.

Hydrogen enriched gas production in a multi-stage downdraft gasification process

International Nuclear Information System (INIS)

Dutta, A.; Jarungthammachote, S.

2009-01-01

To achieve hydrogen enriched and low-tar producer gas, multi-stage air-blown and air-steam gasification were studied in this research. Results showed that the tar content from multi-stage air-blown and air-steam gasification was lower compared to the average value of that from downdraft gasification. It was also seen that an air-steam gasification process could potentially increase the hydrogen concentration in the producer gas in the expense of carbon monoxide; however, the summation of hydrogen and carbon monoxide in the producer gas was increased. (author)

Optimal testlet pool assembly for multistage testing designs

NARCIS (Netherlands)

Ariel, A.; Veldkamp, Bernard P.; Breithaupt, Krista

2006-01-01

Computerized multistage testing (MST) designs require sets of test questions (testlets) to be assembled to meet strict, often competing criteria. Rules that govern testlet assembly may dictate the number of questions on a particular subject or may describe desirable statistical properties for the

Generation of functional neuromuscular junctions from human pluripotent stem cell lines

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Katja ePuttonen

2015-12-01

Full Text Available Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs, yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC or embryonic stem cell (ESC lines as monolayers without basic fibroblast factor (bFGF and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs. The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical MEPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Multi-stage circulating fluidized bed syngas cooling

Science.gov (United States)

Liu, Guohai; Vimalchand, Pannalal; Guan, Xiaofeng; Peng, WanWang

2016-10-11

A method and apparatus for cooling hot gas streams in the temperature range 800.degree. C. to 1600.degree. C. using multi-stage circulating fluid bed (CFB) coolers is disclosed. The invention relates to cooling the hot syngas from coal gasifiers in which the hot syngas entrains substances that foul, erode and corrode heat transfer surfaces upon contact in conventional coolers. The hot syngas is cooled by extracting and indirectly transferring heat to heat transfer surfaces with circulating inert solid particles in CFB syngas coolers. The CFB syngas coolers are staged to facilitate generation of steam at multiple conditions and hot boiler feed water that are necessary for power generation in an IGCC process. The multi-stage syngas cooler can include internally circulating fluid bed coolers, externally circulating fluid bed coolers and hybrid coolers that incorporate features of both internally and externally circulating fluid bed coolers. Higher process efficiencies can be realized as the invention can handle hot syngas from various types of gasifiers without the need for a less efficient precooling step.

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

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Koehler Karl R

2011-08-01

Full Text Available Abstract Background The use of induced pluripotent stem cells (iPSCs for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC neural induction protocol to test whether iPSCs (1 have the competence to give rise to functional neurons with similar efficiency as ESCs and (2 whether the extent of neural differentiation could be altered or enhanced by increased passaging. Results Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs. Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable. Conclusions These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

Susceptibility of Human Oral Squamous Cell Carcinoma (OSCC H103 and H376 cell lines to Retroviral OSKM mediated reprogramming

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Nalini Devi Verusingam

2017-04-01

Full Text Available Although numbers of cancer cell lines have been shown to be successfully reprogrammed into induced pluripotent stem cells (iPSCs, reprogramming Oral Squamous Cell Carcinoma (OSCC to pluripotency in relation to its cancer cell type and the expression pattern of pluripotent genes under later passage remain unexplored. In our study, we reprogrammed and characterised H103 and H376 oral squamous carcinoma cells using retroviral OSKM mediated method. Reprogrammed cells were characterized for their embryonic stem cells (ESCs like morphology, pluripotent gene expression via quantitative real-time polymerase chain reaction (RT-qPCR, immunofluorescence staining, embryoid bodies (EB formation and directed differentiation capacity. Reprogrammed H103 (Rep-H103 exhibited similar ESCs morphologies with flatten cells and clear borders on feeder layer. Reprogrammed H376 (Rep-H376 did not show ESCs morphologies but grow with a disorganized morphology. Critical pluripotency genes Oct4, Sox2 and Nanog were expressed higher in Rep-H103 against the parental counterpart from passage 5 to passage 10. As for Rep-H376, Nanog expression against its parental counterpart showed a significant decrease at passage 5 and although increased in passage 10, the level of expression was similar to the parental cells. Rep-H103 exhibited pluripotent signals (Oct4, Sox2, Nanog and Tra-1-60 and could form EB with the presence of three germ layers markers. Rep-H103 displayed differentiation capacity into adipocytes and osteocytes. The OSCC cell line H103 which was able to be reprogrammed into an iPSC like state showed high expression of Oct4, Sox2 and Nanog at late passage and may provide a potential iPSC model to study multi-stage oncogenesis in OSCC.

Simulated dynamic response of a multi-stage compressor with variable molecular weight flow medium

Science.gov (United States)

Babcock, Dale A.

1995-01-01

A mathematical model of a multi-stage compressor with variable molecular weight flow medium is derived. The modeled system consists of a five stage, six cylinder, double acting, piston type compressor. Each stage is followed by a water cooled heat exchanger which serves to transfer the heat of compression from the gas. A high molecular weight gas (CFC-12) mixed with air in varying proportions is introduced to the suction of the compressor. Condensation of the heavy gas may occur in the upper stage heat exchangers. The state equations for the system are integrated using the Advanced Continuous Simulation Language (ACSL) for determining the system's dynamic and steady state characteristics under varying operating conditions.

Inspection logistics planning for multi-stage production systems with applications to semiconductor fabrication lines

Science.gov (United States)

Chen, Kyle Dakai

Since the market for semiconductor products has become more lucrative and competitive, research into improving yields for semiconductor fabrication lines has lately received a tremendous amount of attention. One of the most critical tasks in achieving such yield improvements is to plan the in-line inspection sampling efficiently so that any potential yield problems can be detected early and eliminated quickly. We formulate a multi-stage inspection planning model based on configurations in actual semiconductor fabrication lines, specifically taking into account both the capacity constraint and the congestion effects at the inspection station. We propose a new mixed First-Come-First-Serve (FCFS) and Last-Come-First-Serve (LCFS) discipline for serving the inspection samples to expedite the detection of potential yield problems. Employing this mixed FCFS and LCFS discipline, we derive approximate expressions for the queueing delays in yield problem detection time and develop near-optimal algorithms to obtain the inspection logistics planning policies. We also investigate the queueing performance with this mixed type of service discipline under different assumptions and configurations. In addition, we conduct numerical tests and generate managerial insights based on input data from actual semiconductor fabrication lines. To the best of our knowledge, this research is novel in developing, for the first time in the literature, near-optimal results for inspection logistics planning in multi-stage production systems with congestion effects explicitly considered.

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.

The Secret Lives of Pluripotent Cells: There and Back Again

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Paolo Cinelli

2010-03-01

Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (IPSCs hold great promise for the therapeutic treatment of human diseases, but their functional similarity, their stability and especially the mechanism underlying their derivation are not yet clearly explained. [...

Modeling the Pathogenesis of Charcot-Marie-Tooth Disease Type 1A Using Patient-Specific iPSCs

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Lei Shi

2018-01-01

Full Text Available Charcot-Marie-Tooth disease type 1A (CMT1A, one of the most frequent inherited peripheral neuropathies, is associated with PMP22 gene duplication. Previous studies of CMT1A mainly relied on rodent models, and it is not yet clear how PMP22 overexpression leads to the phenotype in patients. Here, we generated the human induced pluripotent stem cell (hiPSC lines from two CMT1A patients as an in vitro cell model. We found that, unlike the normal control cells, CMT1A hiPSCs rarely generated Schwann cells through neural crest stem cells (NCSCs. Instead, CMT1A NCSCs produced numerous endoneurial fibroblast-like cells in the Schwann cell differentiation system, and similar results were obtained in a PMP22-overexpressing iPSC model. Therefore, despite the demyelination-remyelination and/or dysmyelination theory for CMT1A pathogenesis, developmental disabilities of Schwann cells may be considered as an underlying cause of CMT1A. Our results may have important implications for the uncovering of the underlying mechanism and the development of a promising therapeutic strategy for CMT1A neuropathy.

Design of intermediate die shape of multistage profile drawing for linear motion guide

International Nuclear Information System (INIS)

Lee, Sang Kon; Lee, Jae Eun; Kim, Sung Min; Kim, Byung Min

2010-01-01

The design of an intermediate die shape is very important in multistage profile drawing. In this study, two design methods for the intermediate die shape of a multistage profile drawing for producing a linear motion guide (LM) guide is proposed. One is the electric field analysis method using the equipotential lines generated by electric field analysis, and the other is the virtual die method using a virtual drawing die constructed from the initial material and the final product shape. In order to design the intermediate die shapes of a multistage profile drawing for producing LM guide, the proposed design methods are applied, and then FE analysis and profile drawing experiment are performed. As a result, based on the measurement of dimensional accuracy, it can be known that the intermediate die shape can be designed effectively

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

Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo

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Dustin R. Wakeman

2017-07-01

Full Text Available A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

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

Analysis of multi-stage open shop processing systems

NARCIS (Netherlands)

Eggermont, C.E.J.; Schrijver, A.; Woeginger, G.J.; Schwentick, T.; Dürr, C.

2011-01-01

We study algorithmic problems in multi-stage open shop processing systems that are centered around reachability and deadlock detection questions. We characterize safe and unsafe system states. We show that it is easy to recognize system states that can be reached from the initial state (where the

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

Decomposition and (importance) sampling techniques for multi-stage stochastic linear programs

Energy Technology Data Exchange (ETDEWEB)

Infanger, G.

1993-11-01

The difficulty of solving large-scale multi-stage stochastic linear programs arises from the sheer number of scenarios associated with numerous stochastic parameters. The number of scenarios grows exponentially with the number of stages and problems get easily out of hand even for very moderate numbers of stochastic parameters per stage. Our method combines dual (Benders) decomposition with Monte Carlo sampling techniques. We employ importance sampling to efficiently obtain accurate estimates of both expected future costs and gradients and right-hand sides of cuts. The method enables us to solve practical large-scale problems with many stages and numerous stochastic parameters per stage. We discuss the theory of sharing and adjusting cuts between different scenarios in a stage. We derive probabilistic lower and upper bounds, where we use importance path sampling for the upper bound estimation. Initial numerical results turned out to be promising.

Preliminary study on functional performance of compound type multistage safety injection tank

International Nuclear Information System (INIS)

Bae, Youngmin; Kim, Young In; Kim, Keung Koo

2015-01-01

Highlights: • Functional performance of compound type multistage safety injection tanks is studied. • Effects of key design parameters are scrutinized. • Distinctive flow features in compound type safety injection tanks are explored. - Abstract: A parametric study is carried out to evaluate the functional performance of a compound type multistage safety injection tank that would be considered one of the components for the passive safety injection systems in nuclear power plants. The effects of key design parameters such as the initial volume fraction and charging pressure of gas, tank elevation, vertical location of a sparger, resistance coefficient, and operating condition on the injection flow rate are scrutinized along with a discussion of the relevant flow features. The obtained results indicate that the compound type multistage safety injection tank can effectively control the injection flow rate in a passive manner, by switching the driving force for the safety injection from gas pressure to gravity during the refill and reflood phases, respectively

Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Santos, Renata; Vadodaria, Krishna C; Jaeger, Baptiste N; Mei, Arianna; Lefcochilos-Fogelquist, Sabrina; Mendes, Ana P D; Erikson, Galina; Shokhirev, Maxim; Randolph-Moore, Lynne; Fredlender, Callie; Dave, Sonia; Oefner, Ruth; Fitzpatrick, Conor; Pena, Monique; Barron, Jerika J; Ku, Manching; Denli, Ahmet M; Kerman, Bilal E; Charnay, Patrick; Kelsoe, John R; Marchetto, Maria C; Gage, Fred H

2017-06-06

Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

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Yulia Shamis

Full Text Available Human embryonic and induced pluripotent stem cells (hESC/hiPSC are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.

Multi-Stage Transportation Problem With Capacity Limit

OpenAIRE

I. Brezina; Z. Čičková; J. Pekár; M. Reiff

2010-01-01

The classical transportation problem can be applied in a more general way in practice. Related problems as Multi-commodity transportation problem, Transportation problems with different kind of vehicles, Multi-stage transportation problems, Transportation problem with capacity limit is an extension of the classical transportation problem considering the additional special condition. For solving such problems many optimization techniques (dynamic programming, linear programming, special algor...

A Humanized Mouse Model Generated Using Surplus Neonatal Tissue

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Matthew E. Brown

2018-04-01

Full Text Available Summary: Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs. Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue. : Corresponding author William Burlingham and colleagues created a humanized mouse model called the NeoThy. The NeoThy uses human neonatal, rather than fetal, tissue sources for generating a human immune system within immunocompromised mouse hosts. NeoThy mice are an attractive alternative to conventional humanized mouse models, as they enable robust and reproducible iPSC immunogenicity experiments in vivo. Keywords: NeoThy, humanized mouse, iPSC, PSC, immunogenicity, transplantation, immunology, hematopoietic stem cells, induced pluripotent stem cells, thymus

Mechanical and mathematical models of multi-stage horizontal fracturing strings and their application

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Zhanghua Lian

2015-03-01

Full Text Available Multi-stage SRV fracturing in horizontal wells is a new technology developed at home and abroad in recent years to effectively develop shale gas or low-permeability reservoirs, but on the other hand makes the mechanical environment of fracturing strings more complicated at the same time. In view of this, based on the loading features of tubing strings during the multi-stage fracturing of a horizontal well, mechanical models were established for three working cases of multiple packer setting, open differential-pressure sliding sleeve, and open ball-injection sliding sleeve under a hold-down packer. Moreover, mathematical models were respectively built for the above three cases. According to the Lame formula and Von Mises stress calculation formula for the thick-walled cylinder in the theory of elastic mechanics, a mathematical model was also established to calculate the equivalent stress for tubing string safety evaluation when the fracturing string was under the combined action of inner pressure, external squeezing force and axial stress, and another mathematical model was built for the mechanical strength and safety evaluation of multi-stage fracturing strings. In addition, a practical software was developed for the mechanical safety evaluation of horizontal well multi-stage fracturing strings according to the mathematical model developed for the mechanical calculation of the multi-packer string in horizontal wells. The research results were applied and verified in a gas well of Tahe Oilfield in the Tarim Basin with excellent effects, providing a theoretical basis and a simple and reliable technical means for optimal design and safety evaluation of safe operational parameters of multi-stage fracturing strings in horizontal wells.

Influence of perforation erosion on multiple growing hydraulic fractures in multi-stage fracturing

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Yongming Li

2018-02-01

Full Text Available In multi-stage hydraulic fracturing, the limited-entry method is widely used to promote uniform growth of multiple fractures. However, this method's effectiveness may be lost because the perforations will be eroded gradually during the fracturing period. In order to study the influence of perforation erosion on multiple growing hydraulic fractures, we combined the solid–fluid coupled model of hydraulic fracture growth with an empirical model of perforation erosion to implement numerical simulation. The simulations show clearly that the erosion of perforation will significantly deteriorate the non-uniform growth of multiple fractures. Based on the numerical model, we also studied the influences of proppant concentration and injection rates on perforation erosion in multi-stage hydraulic fracturing. The results indicate that the initial erosion rates become higher with the rising proppant concentration, but the growth of multiple hydraulic fractures is not sensitive to the varied proppant concentration. In addition, higher injection rates are beneficial significantly to the limited-entry design, leading to more uniform growth of fractures. Thus, in multi-stage hydraulic fracturing enough high injection rates are proposed to keep uniform growths. Keywords: Unconventional oil and gas reservoir, Horizontal well, Perforation friction, Perforation erosion, Multi-stage hydraulic fracturing, Numerical simulation, Mathematic model, Uniform growth of fractures

A New Feature Ensemble with a Multistage Classification Scheme for Breast Cancer Diagnosis

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Idil Isikli Esener

2017-01-01

Full Text Available A new and effective feature ensemble with a multistage classification is proposed to be implemented in a computer-aided diagnosis (CAD system for breast cancer diagnosis. A publicly available mammogram image dataset collected during the Image Retrieval in Medical Applications (IRMA project is utilized to verify the suggested feature ensemble and multistage classification. In achieving the CAD system, feature extraction is performed on the mammogram region of interest (ROI images which are preprocessed by applying a histogram equalization followed by a nonlocal means filtering. The proposed feature ensemble is formed by concatenating the local configuration pattern-based, statistical, and frequency domain features. The classification process of these features is implemented in three cases: a one-stage study, a two-stage study, and a three-stage study. Eight well-known classifiers are used in all cases of this multistage classification scheme. Additionally, the results of the classifiers that provide the top three performances are combined via a majority voting technique to improve the recognition accuracy on both two- and three-stage studies. A maximum of 85.47%, 88.79%, and 93.52% classification accuracies are attained by the one-, two-, and three-stage studies, respectively. The proposed multistage classification scheme is more effective than the single-stage classification for breast cancer diagnosis.

Thinking outside the liver: induced pluripotent stem cells for hepatic applications.

Science.gov (United States)

Subba Rao, Mekala; Sasikala, Mitnala; Nageshwar Reddy, D

2013-06-14

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

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.

Vibration based condition monitoring of a multistage epicyclic gearbox in lifting cranes

Science.gov (United States)

Assaad, Bassel; Eltabach, Mario; Antoni, Jérôme

2014-01-01

This paper proposes a model-based technique for detecting wear in a multistage planetary gearbox used by lifting cranes. The proposed method establishes a vibration signal model which deals with cyclostationary and autoregressive models. First-order cyclostationarity is addressed by the analysis of the time synchronous average (TSA) of the angular resampled vibration signal. Then an autoregressive model (AR) is applied to the TSA part in order to extract a residual signal containing pertinent fault signatures. The paper also explores a number of methods commonly used in vibration monitoring of planetary gearboxes, in order to make comparisons. In the experimental part of this study, these techniques are applied to accelerated lifetime test bench data for the lifting winch. After processing raw signals recorded with an accelerometer mounted on the outside of the gearbox, a number of condition indicators (CIs) are derived from the TSA signal, the residual autoregressive signal and other signals derived using standard signal processing methods. The goal is to check the evolution of the CIs during the accelerated lifetime test (ALT). Clarity and fluctuation level of the historical trends are finally considered as a criteria for comparing between the extracted CIs.

Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling.

Science.gov (United States)

Hirata, Shinji; Takayama, Naoya; Jono-Ohnishi, Ryoko; Endo, Hiroshi; Nakamura, Sou; Dohda, Takeaki; Nishi, Masanori; Hamazaki, Yuhei; Ishii, Ei-ichi; Kaneko, Shin; Otsu, Makoto; Nakauchi, Hiromitsu; Kunishima, Shinji; Eto, Koji

2013-09-01

Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor-mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl(-/-) mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC-derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

Trophoblast lineage cells derived from human induced pluripotent stem cells

International Nuclear Information System (INIS)

Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

2013-01-01

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

Trophoblast lineage cells derived from human induced pluripotent stem cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

2013-07-12

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

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.

Multi-stage pulsed laser deposition of aluminum nitride at different temperatures

Energy Technology Data Exchange (ETDEWEB)

Duta, L. [National Institute for Lasers, Plasma, and Radiation Physics, 409 Atomistilor Street, 077125 Magurele (Romania); Stan, G.E. [National Institute of Materials Physics, 105 bis Atomistilor Street, 077125 Magurele (Romania); Stroescu, H.; Gartner, M.; Anastasescu, M. [Institute of Physical Chemistry “Ilie Murgulescu”, Romanian Academy, 202 Splaiul Independentei, 060021 Bucharest (Romania); Fogarassy, Zs. [Research Institute for Technical Physics and Materials Science, Hungarian Academy of Sciences, Konkoly Thege Miklos u. 29-33, H-1121 Budapest (Hungary); Mihailescu, N. [National Institute for Lasers, Plasma, and Radiation Physics, 409 Atomistilor Street, 077125 Magurele (Romania); Szekeres, A., E-mail: szekeres@issp.bas.bg [Institute of Solid State Physics, Bulgarian Academy of Sciences, Tzarigradsko Chaussee 72, Sofia 1784 (Bulgaria); Bakalova, S. [Institute of Solid State Physics, Bulgarian Academy of Sciences, Tzarigradsko Chaussee 72, Sofia 1784 (Bulgaria); Mihailescu, I.N., E-mail: ion.mihailescu@inflpr.ro [National Institute for Lasers, Plasma, and Radiation Physics, 409 Atomistilor Street, 077125 Magurele (Romania)

2016-06-30

Highlights: • Multi-stage pulsed laser deposition of aluminum nitride at different temperatures. • 800 °C seed film boosts the next growth of crystalline structures at lower temperature. • Two-stage deposited AlN samples exhibit randomly oriented wurtzite structures. • Band gap energy values increase with deposition temperature. • Correlation was observed between single- and multi-stage AlN films. - Abstract: We report on multi-stage pulsed laser deposition of aluminum nitride (AlN) on Si (1 0 0) wafers, at different temperatures. The first stage of deposition was carried out at 800 °C, the optimum temperature for AlN crystallization. In the second stage, the deposition was conducted at lower temperatures (room temperature, 350 °C or 450 °C), in ambient Nitrogen, at 0.1 Pa. The synthesized structures were analyzed by grazing incidence X-ray diffraction (GIXRD), transmission electron microscopy (TEM), atomic force microscopy and spectroscopic ellipsometry (SE). GIXRD measurements indicated that the two-stage deposited AlN samples exhibited a randomly oriented wurtzite structure with nanosized crystallites. The peaks were shifted to larger angles, indicative for smaller inter-planar distances. Remarkably, TEM images demonstrated that the high-temperature AlN “seed” layers (800 °C) promoted the growth of poly-crystalline AlN structures at lower deposition temperatures. When increasing the deposition temperature, the surface roughness of the samples exhibited values in the range of 0.4–2.3 nm. SE analyses showed structures which yield band gap values within the range of 4.0–5.7 eV. A correlation between the results of single- and multi-stage AlN depositions was observed.

Stage-dependent hierarchy of criteria in multiobjective multistage decision processes

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Tadeusz Trzaskalik

2017-01-01

Full Text Available This paper will consider a multiobjective, multistage discrete dynamic process with a changeable, state-dependent hierarchy of stage criteria determined by the decision maker. The goal of this paper is to answer the question of how to control a multistage process while taking into account both the tendency to achieve multiobjective optimization of the entire process and the time-varying hierarchy of stage criteria. We consider in detail possible situations, where the hierarchy of stage criteria changes over time in individual stages and is stage dependent. We present an interactive proposal to solving the problem, where the decision maker actively participates in finding the final realization of the process. The algorithm proposed is illustrated using a numerical example.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Science.gov (United States)

Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

2013-01-01

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

Potential and Challenges of Induced Pluripotent Stem Cells in Liver Diseases Treatment

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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.

Multistage Effort and the Equity Structure of Venture Investment Based on Reciprocity Motivation

OpenAIRE

Ding, Chuan; Chen, Jiacheng; Liu, Xin; Zheng, Junjun

2015-01-01

For venture capitals, it is a long process from an entry to its exit. In this paper, the activity of venture investment will be divided into multistages. And, according to the effort level entrepreneurs will choose, the venture capitalists will provide an equity structure at the very beginning. As a benchmark for comparison, we will establish two game models on multistage investment under perfect rationality: a cooperative game model and a noncooperative one. Further, as a cause of pervasive ...

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.

Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

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He, Pengfei; Fu, Jiayin; Wang, Dong-An

2016-04-15

By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive

Few single nucleotide variations in exomes of human cord blood induced pluripotent stem cells.

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Rui-Jun Su

Full Text Available The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs reprogrammed from human cord blood (CB CD34(+ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2-14% reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS, OS and ZSCAN4 (OSZ, OS and MYC and KLF4 (OSMK. Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs, in exomes, including untranslated regions (UTR, in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.

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.

Induced pluripotent stem cell-based therapy for age-related macular degeneration.

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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.

Generation of a transplantable erythropoietin-producer derived from human mesenchymal stem cells.

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Yokoo, Takashi; Fukui, Akira; Matsumoto, Kei; Ohashi, Toya; Sado, Yoshikazu; Suzuki, Hideaki; Kawamura, Tetsuya; Okabe, Masataka; Hosoya, Tatsuo; Kobayashi, Eiji

2008-06-15

Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.

Continuous-data diagnostic tests for paratuberculosis as a multistage disease

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Toft, Nils; Nielsen, Søren Saxmose; Jørgensen, Erik

2005-01-01

We devised a general method for interpretation of multistage diseases using continuous-data diagnostic tests. As an example, we used paratuberculosis as a multistage infection with 2 stages of infection as well as a noninfected state. Using data from a Danish research project, a fecal culture...... testing scheme was linked to an indirect ELISA and adjusted for covariates (parity, age at first calving, and days in milk). We used the log-transformed optical densities in a Bayesian network to obtain the probabilities for each of the 3 infection stages for a given optical density (adjusted...... for covariates). The strength of this approach was that the uncertainty associated with a test was imposed directly on the individual test result rather than aggregated into the population-based measures of test properties (i.e., sensitivity and specificity)...

Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

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Svetlana Gavrilov

2011-01-01

Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

Progress and bottleneck in induced pluripotency

Directory of Open Access Journals (Sweden)

Zhang Zhen-Ning

2012-07-01

Full Text Available Abstract With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs, reprogrammed from somatic cells of individual patients with defined factors, have unlimited potential in cell therapy and in modeling complex human diseases. Significant progress has been achieved to improve the safety of iPSCs and the reprogramming efficiency. To avoid the cancer risk and spontaneous reactivation of the reprogramming factors associated with the random integration of viral vectors into the genome, several approaches have been established to deliver the reprogramming factors into the somatic cells without inducing genetic modification. In addition, a panel of small molecule compounds, many of which targeting the epigenetic machinery, have been identified to increase the reprogramming efficiency. Despite these progresses, recent studies have identified genetic and epigenetic abnormalities of iPSCs as well as the immunogenicity of some cells derived from iPSCs. In addition, due to the oncogenic potential of the reprogramming factors and the reprogramming-induced DNA damage, the critical tumor suppressor pathways such as p53 and ARF are activated to act as the checkpoints that suppress induced pluripotency. The inactivation of these tumor suppression pathways even transiently during reprogramming processes could have significant adverse impact on the genome integrity. These safety concerns must be resolved to improve the feasibility of the clinic development of iPSCs into human cell therapy.

Fractional Multistage Hydrothermal Liquefaction of Biomass and Catalytic Conversion into Hydrocarbons

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