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1
El-Sayes, Abdullah. "Induced Pluripotent Stem Cells." Sciential - McMaster Undergraduate Science Journal, no. 1 (November 25, 2018): 16–22. http://dx.doi.org/10.15173/sciential.v1i1.1908.
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The isolation of human embryonic stem cells in 1998 has since fueled the ideology that stem cells may eventually be used for human disease therapies as well as the regeneration of tissues and organs. The transformation of somatic cells to a pluripotent state via somatic nuclear transfer and embryonic stem cell fusion brought the scientific community nearer to understanding the molecular mechanisms that govern cellular pluripotency. In 2006, the first induced pluripotent stem (iPS) cell was reported, where a mouse somatic cell was successfully converted to a pluripotent state via transduction of four essential factors. This cellular breakthrough has allowed for robust scientific investigations of human diseases that were once extremely difficult to study. Scientists and pharmaceuticals now use iPS cells as means for disease investigations, drug development and cell or tissue transplantation. There is little doubt that scientific progress on iPS cells will change many aspects of medicine in the next couple of decades.
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Cruvinel, Estela, Isabella Ogusuku, Rosanna Cerioni, Sirlene Rodrigues, Jéssica Gonçalves, Maria Elisa Góes, Juliana Morais Alvim, et al. "Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity." SAGE Open Medicine 8 (January 2020): 205031212096645. http://dx.doi.org/10.1177/2050312120966456.
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Objectives: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency. Methods: Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. Results: Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm. Conclusions: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.
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Loh, Yuin-Han, Suneet Agarwal, In-Hyun Park, Achia Urbach, Hongguang Huo, Garrett C. Heffner, Kitai Kim, Justine D. Miller, Kitwa Ng, and George Q. Daley. "Generation of induced pluripotent stem cells from human blood." Blood 113, no. 22 (May 28, 2009): 5476–79. http://dx.doi.org/10.1182/blood-2009-02-204800.
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Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.
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Dinnyes, A., M. K. Pirity, E. Gocza, P. Osteil, N. Daniel, Zs Tancos, Zs Polgar, et al. "GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES." Reproduction, Fertility and Development 24, no. 1 (2012): 286. http://dx.doi.org/10.1071/rdv24n1ab246.
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Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.
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Yuan, Liyun, Xiaoyan Tang, Binyan Zhang, and Guohui Ding. "Cell Pluripotency Levels Associated with Imprinted Genes in Human." Computational and Mathematical Methods in Medicine 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/471076.
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Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs.
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Go, Young-Hyun, Jumee Kim, Ho-Chang Jeong, Seong-Min Kim, Yun-Jeong Kim, Soon-Jung Park, Sung-Hwan Moon, and Hyuk-Jin Cha. "Luteolin Induces Selective Cell Death of Human Pluripotent Stem Cells." Biomedicines 8, no. 11 (October 27, 2020): 453. http://dx.doi.org/10.3390/biomedicines8110453.
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Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.
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Salloum-Asfar, Salam, Rudolf Engelke, Hanaa Mousa, Neha Goswami, I. Richard Thompson, Freshteh Palangi, Kamal Kamal, et al. "Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells." Stem Cells International 2021 (October 15, 2021): 1–19. http://dx.doi.org/10.1155/2021/8274936.
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Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.
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Unzu, Carmen, Marc Friedli, Alexis Bosman, Marisa E. Jaconi, Barbara E. Wildhaber, and Anne-Laure Rougemont. "Human Hepatocyte-Derived Induced Pluripotent Stem Cells: MYC Expression, Similarities to Human Germ Cell Tumors, and Safety Issues." Stem Cells International 2016 (2016): 1–16. http://dx.doi.org/10.1155/2016/4370142.
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Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogenemycshowed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such asmycmight be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases.
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Cantone, Irene, and Amanda G. Fisher. "Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1733 (September 25, 2017): 20160358. http://dx.doi.org/10.1098/rstb.2016.0358.
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X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro . Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell–cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the ‘primed’ and ‘naive’ states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.
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Palladino, Antonio, Isabella Mavaro, Carmela Pizzoleo, Elena De Felice, Carla Lucini, Paolo de Girolamo, Paolo A. Netti, and Chiara Attanasio. "Induced Pluripotent Stem Cells as Vasculature Forming Entities." Journal of Clinical Medicine 8, no. 11 (October 25, 2019): 1782. http://dx.doi.org/10.3390/jcm8111782.
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Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine.
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Vallier, Ludovic, Thomas Touboul, Stephanie Brown, Candy Cho, Bilada Bilican, Morgan Alexander, Jessica Cedervall, et al. "Signaling Pathways Controlling Pluripotency and Early Cell Fate Decisions of Human Induced Pluripotent Stem Cells." STEM CELLS 27, no. 11 (August 17, 2009): 2655–66. http://dx.doi.org/10.1002/stem.199.
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Talan, Jamie. "Investigators Induce Human Pluripotent Stem Cells into Astrocytes." Neurology Today 11, no. 12 (June 2011): 1. http://dx.doi.org/10.1097/01.nt.0000399611.11026.9e.
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Romito, Antonio, and Gilda Cobellis. "Pluripotent Stem Cells: Current Understanding and Future Directions." Stem Cells International 2016 (2016): 1–20. http://dx.doi.org/10.1155/2016/9451492.
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Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells.
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Sun, Guoqiang, Chelsea Fu, Caroline Shen, and Yanhong Shi. "Histone Deacetylases in Neural Stem Cells and Induced Pluripotent Stem Cells." Journal of Biomedicine and Biotechnology 2011 (2011): 1–6. http://dx.doi.org/10.1155/2011/835968.
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Stem cells have provided great hope for the treatment of a variety of human diseases. However, the molecular mechanisms underlying stem cell pluripotency, self-renewal, and differentiation remain to be unveiled. Epigenetic regulators, including histone deacetylases (HDACs), have been shown to coordinate with cell-intrinsic transcription factors and various signaling pathways to regulate stem cell pluripotency, self-renewal, and fate determination. This paper focuses on the role of HDACs in the proliferation and neuronal differentiation of neural stem cells and the application of HDAC inhibitors in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). It promises to be an active area of future research.
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Lian, Qizhou, Yenyen Chow, Miguel Esteban, Duanqing Pei, and Hung-Fat Tse. "Future perspective of induced pluripotent stem cells for diagnosis, drug screening and treatment of human diseases." Thrombosis and Haemostasis 104, no. 07 (2010): 39–44. http://dx.doi.org/10.1160/th10-05-0269.
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SummaryRecent advances in stem cell biology have transformed the understanding of cell physiology and developmental biology such that it can now play a more prominent role in the clinical application of stem cell and regenerative medicine. Success in the generation of human induced pluripotent stem cells (iPS) as well as related emerging technology on the iPS platform provide great promise in the development of regenerative medicine. Human iPS cells show almost identical properties to human embryonic stem cells (ESC) in pluripotency, but avoid many of their limitations of use. In addition, investigations into reprogramming of somatic cells to pluripotent stem cells facilitate a deeper understanding of human stem cell biology. The iPS cell technology has offered a unique platform for studying the pathogenesis of human disease, pharmacological and toxicological testing, and cell-based therapy. Nevertheless, significant challenges remain to be overcome before the promise of human iPS cell technology can be realised.
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Sp, Nipin, Dong Young Kang, Eun Seong Jo, Alexis Rugamba, Wan Seop Kim, Yeong-Min Park, Dae-Yong Hwang, et al. "Tannic Acid Promotes TRAIL-Induced Extrinsic Apoptosis by Regulating Mitochondrial ROS in Human Embryonic Carcinoma Cells." Cells 9, no. 2 (January 23, 2020): 282. http://dx.doi.org/10.3390/cells9020282.
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Human embryonic carcinoma (EC; NCCIT) cells have self-renewal ability and pluripotency. Cancer stem cell markers are highly expressed in NCCIT cells, imparting them with the pluripotent nature to differentiate into other cancer types, including breast cancer. As one of the main cancer stem cell pathways, Wnt/β-catenin is also overexpressed in NCCIT cells. Thus, inhibition of these pathways defines the ability of a drug to target cancer stem cells. Tannic acid (TA) is a natural polyphenol present in foods, fruits, and vegetables that has anti-cancer activity. Through Western blotting and PCR, we demonstrate that TA inhibits cancer stem cell markers and the Wnt/β-catenin signaling pathway in NCCIT cells and through a fluorescence-activated cell sorting analysis we demonstrated that TA induces sub-G1 cell cycle arrest and apoptosis. The mechanism underlying this is the induction of mitochondrial reactive oxygen species (ROS) (mROS), which then induce the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptosis pathway instead of intrinsic mitochondrial apoptosis pathway. Moreover, ribonucleic acid sequencing data with TA in NCCIT cells show an elevation in TRAIL-induced extrinsic apoptosis, which we confirm by Western blotting and real-time PCR. The induction of human TRAIL also proves that TA can induce extrinsic apoptosis in NCCIT cells by regulating mROS.
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Hall, Vanessa Jane. "Early development of the porcine embryo: the importance of cell signalling in development of pluripotent cell lines." Reproduction, Fertility and Development 25, no. 1 (2013): 94. http://dx.doi.org/10.1071/rd12264.
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Understanding the cell signalling events that govern cell renewal in porcine pluripotent cells may help improve culture conditions and allow for establishment of bona fide porcine embryonic stem cells (pESC) and stable porcine induced pluripotent stem cells (piPSC). This review investigates cell signalling in the porcine preimplantation embryo containing either the inner cell mass or epiblast, with particular emphasis on fibroblast growth factor, SMAD, WNT and Janus tyrosine kinases/signal transducers and activators of transcription signalling. It is clear that key differences exist in the cell signalling events that govern pluripotency in this species compared with similar embryonic stages in mouse and human. The fact that bona fide pESC have still not been produced and that piPSC cannot survive in culture following the silencing or downregulation of the reprogramming transgenes suggest that culture conditions are not optimal. Unravelling the factor/s that regulate pluripotency in porcine embryos will pave the way for future establishment of stable pluripotent stem cell lines.
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Aprihati, Aprihati, B. S. Pikir, and Andrianto Andrianto. "Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells using Small-Molecule Compound VC6TFZ." Open Access Macedonian Journal of Medical Sciences 8, A (May 6, 2020): 250–55. http://dx.doi.org/10.3889/oamjms.2020.3862.
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BACKGROUND: Induced pluripotent stem cells (iPSCs) were generated from somatic cells through reprogramming process. Peripheral blood mononuclear cells (PBMNCs) were an attractive source cells due to the ease of accessibility, need minimal invasive procedure, and can be stored frozen. Small-molecule compound VC6TFZ has been successfully reprogrammed iPSCs from mouse fibroblast, but it has not been proven in human.
AIM: This study aims to determine whether the small-molecule compound VC6TFZ can induce pluripotency of PBMNC to generate iPSCs.
METHODS: Mononuclear cells were isolated from peripheral venous blood using centrifugation gradient density method. Mononuclear cells were cultured for 6 days in expansion medium and 48 h using hanging drop method. Pluripotency induction process using small-molecule compound VC6TFZ was done in 14 days then the medium changed to 2i medium for 7 days. Identification of iPSCs based on colony morphology and expression of pluripotency marker OCT4 and SOX2.
RESULTS: Colonies appeared on day 9 of reprogramming process. These colonies had round, large, and cobble stone morphology like embryonic stem cell. These colonies had positive expression of pluripotency markers OCT4 and SOX2. All experimental groups had significantly higher expression of OCT4 and SOX2 than control group.
CONCLUSION: Small-molecule compound VC6TFZ could induce pluripotency of human PBMNC to generate iPSCs.
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Naaman, Hila, Tatiana Rabinski, Avi Yizhak, Solly Mizrahi, Yonat Shemer Avni, Ran Taube, Bracha Rager, et al. "Measles Virus Persistent Infection of Human Induced Pluripotent Stem Cells." Cellular Reprogramming 20, no. 1 (February 2018): 17–26. http://dx.doi.org/10.1089/cell.2017.0034.
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Yang, Yu-Hua, Ru-Zhi Zhang, Sai Cheng, Bin Xu, Ting Tian, Hai-Xia Shi, Li Xiao, and Ren-He Chen. "Generation of Induced Pluripotent Stem Cells from Human Epidermal Keratinocytes." Cellular Reprogramming 20, no. 6 (December 2018): 356–64. http://dx.doi.org/10.1089/cell.2018.0035.
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Nemade, Harshal, Aviseka Acharya, Umesh Chaudhari, Erastus Nembo, Filomain Nguemo, Nicole Riet, Hinrich Abken, Jürgen Hescheler, Symeon Papadopoulos, and Agapios Sachinidis. "Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells." Cells 9, no. 3 (February 27, 2020): 554. http://dx.doi.org/10.3390/cells9030554.
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Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.
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Pekkanen-Mattila, Mari, Marisa Ojala, Erja Kerkelä, Kristiina Rajala, Heli Skottman, and Katriina Aalto-Setälä. "The Effect of Human and Mouse Fibroblast Feeder Cells on Cardiac Differentiation of Human Pluripotent Stem Cells." Stem Cells International 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/875059.
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Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) are commonly used as feeder cells to maintain the pluripotent state of stem cells. The aim of the present study was to evaluate the effect of MEF and hFF feeders on the cardiac differentiation. Two human embryonic and two induced pluripotent stem cell lines were cultured on MEF and hFF before cardiac differentiation. The expression of Brachyury T was higher in cell lines cultured on MEF, than if cultured on hFF, suggesting enhanced mesoderm formation. However, significant positive influence of MEF feeders on cardiac differentiation was only seen with one cell line. Further, the ability of hFF to maintain pluripotency of stem cells originally cultured on MEF was quite poor. In conclusion, the cells behaved differently whether cultured on hFF or MEF feeders. However, the influence of the feeder cells on differentiation was less than the difference observed between the cell lines.
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Park, J. K., K. H. Choi, D. C. Son, J. I. Oh, and C. K. Lee. "294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS." Reproduction, Fertility and Development 25, no. 1 (2013): 294. http://dx.doi.org/10.1071/rdv25n1ab294.
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A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells. This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.
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Thomson, James A., and Junying Yu. "Human Embryonic and Human Induced Pluripotent Stem Cell Lines." Journal of Medical Sciences 1, no. 3 (November 25, 2008): 106–13. http://dx.doi.org/10.2174/1996327000801030106.
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Rehakova, Daniela, Tereza Souralova, and Irena Koutna. "Clinical-Grade Human Pluripotent Stem Cells for Cell Therapy: Characterization Strategy." International Journal of Molecular Sciences 21, no. 7 (March 31, 2020): 2435. http://dx.doi.org/10.3390/ijms21072435.
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Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans’ lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and ′for information only′ characterization methods with release criteria for the establishment of clinical-grade hPSC lines.
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Coco-Martin, Rosa M., Salvador Pastor-Idoate, and Jose Carlos Pastor. "Cell Replacement Therapy for Retinal and Optic Nerve Diseases: Cell Sources, Clinical Trials and Challenges." Pharmaceutics 13, no. 6 (June 11, 2021): 865. http://dx.doi.org/10.3390/pharmaceutics13060865.
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The aim of this review was to provide an update on the potential of cell therapies to restore or replace damaged and/or lost cells in retinal degenerative and optic nerve diseases, describing the available cell sources and the challenges involved in such treatments when these techniques are applied in real clinical practice. Sources include human fetal retinal stem cells, allogenic cadaveric human cells, adult hippocampal neural stem cells, human CNS stem cells, ciliary pigmented epithelial cells, limbal stem cells, retinal progenitor cells (RPCs), human pluripotent stem cells (PSCs) (including both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs). Of these, RPCs, PSCs and MSCs have already entered early-stage clinical trials since they can all differentiate into RPE, photoreceptors or ganglion cells, and have demonstrated safety, while showing some indicators of efficacy. Stem/progenitor cell therapies for retinal diseases still have some drawbacks, such as the inhibition of proliferation and/or differentiation in vitro (with the exception of RPE) and the limited long-term survival and functioning of grafts in vivo. Some other issues remain to be solved concerning the clinical translation of cell-based therapy, including (1) the ability to enrich for specific retinal subtypes; (2) cell survival; (3) cell delivery, which may need to incorporate a scaffold to induce correct cell polarization, which increases the size of the retinotomy in surgery and, therefore, the chance of severe complications; (4) the need to induce a localized retinal detachment to perform the subretinal placement of the transplanted cell; (5) the evaluation of the risk of tumor formation caused by the undifferentiated stem cells and prolific progenitor cells. Despite these challenges, stem/progenitor cells represent the most promising strategy for retinal and optic nerve disease treatment in the near future, and therapeutics assisted by gene techniques, neuroprotective compounds and artificial devices can be applied to fulfil clinical needs.
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Eguizabal, C., N. Montserrat, R. Vassena, M. Barragan, E. Garreta, L. Garcia-Quevedo, F. Vidal, A. Giorgetti, A. Veiga, and J. C. Izpisua Belmonte. "Complete Meiosis from Human Induced Pluripotent Stem Cells." STEM CELLS 29, no. 8 (July 26, 2011): 1186–95. http://dx.doi.org/10.1002/stem.672.
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Kim, Eun-Mi, Gohar Manzar, and Nicholas Zavazava. "Human iPS cell-derived CD34+ hematopoietic progenitor cells induce T cell anergy in alloreactive CD8+ T cells (P2188)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 69.32. http://dx.doi.org/10.4049/jimmunol.190.supp.69.32.
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Abstract Human induced pluripotent stem (iPS) cells have emerged as an alternative source of pluripotent stem cells that can be used in regenerative medicine. However, knowledge about the immunological characteristics of their derivatives remains enigmatic. Here, human iPS cells were differentiated into CD34+ hematopoietic progenitor cells (HPCs).These iPS cell-derived HPCs poorly express MHC class-I antigens, and are MHC class-II negative. They also lack expression of costimulatory molecules CD80 and CD86, but show relatively high expression of PD-L1. To investigate whether these HPCs stimulate CTLs, we generated CD8+ T cells against HLA-A2. Subsequently, HPCs were used as target cells for the CTLs. Interestingly HPCs were not susceptible to alloreactive CTL killing in a cytotoxicity assay. HPCs also failed to trigger release of IL-2 by CTLs as a sign of their activation. This lack of CTL activation was partially due to low MHC class I expression by the HPCs because HPC cell lysis was partially restored after MHC class I upregulation by treatment with IFN-γ. Interestingly, the CTLs became anergic when incubated with HPCs. As expected, T cell anergy was abrogated by treatment with rIL-2. Collectively, anergy was a result of low expression of CD80 and CD86 and high expression of PD-L1. These data indicate for the first time that human iPS cell-derived CD34+ HPCs induce T cell anergy, a property that could be exploited for cellular allo-transplantation of iPS cell-derived tissues.
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Grauer, Matthias, Martina Konantz, Nina I. Niebuhr, Lothar Kanz, In-Hyun Park, George Q. Daley, and Claudia Lengerke. "Hematopoietic Development From Human Induced Pluripotent Stem Cells." Blood 114, no. 22 (November 20, 2009): 2530. http://dx.doi.org/10.1182/blood.v114.22.2530.2530.
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Abstract Abstract 2530 Poster Board II-507 A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generate pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) human pluripotent stem cell lines (Park et al, 2008). Current research is focusing on improving reprogramming protocols (e.g. circumventing the use of retroviral technology and oncoproteins) and methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. We have asked whether these signaling pathways patterning blood fate are conserved during hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data showed robust differentiation of iPS cells to mesoderm and to blood lineages, comparable to reports on differentiation of human ESC in this system. We detected robust formation of CD34+ (28.9±12), CD45+ (26.8±13.4) and CD34+CD45+ (16.1±13.7) cells, and a high incidence of CFU-initiating cells in functional colony assays, predominantly displaying myeloid but also some mixed CFU-GEMM activity. Similar to our findings in mouse ESC, mesodermal and hematopoietic genes were expressed in waves, and expression was augmented by supplementation of cultures with BMP4. Mesodermal markers (e.g. BRACHYURY ) were induced at day 2, and declined after day 9, when hematopoietic markers (SCL) appeared, indicating conversion of mesoderm to progenitors of the blood lineage. Expression of all three human CDX genes (CDX1, CDX2 and CDX4) peaked at day 6, suggesting that the function of CDX genes to pattern preformed mesoderm to blood fate may be conserved in human embryogenesis. Ongoing experiments in our laboratory focus on genetic modification of human iPS cells to study effects of specific genes during human emrbyonic hematopoiesis. Furthermore we have succeeded in transducing iPS cells with lentiviruses that allow GFP expression and puromycin selection, thus indicating feasibility for genetic modification. Taken together, our results show robust hematopoietic differentiation of human iPS cells and suggest that genetically modified in vitro differentiating iPS cells can be used to study human developmental hematopoiesis. Characterizing genetic pathways governing human embryonic blood formation will direct differentiation of induced pluripotent stem cells into repopulating hematopoietic stem cells, enabling generation of isogenic cell replacement therapies. Moreover, this experimental approach enables modeling of hematologic diseases, opening up a novel platform for gradual studies of genetic mechanisms during disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.
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Watanabe, Katsuhito, Takashi Nakamura, Shoko Onodera, Akiko Saito, Takahiko Shibahara, and Toshifumi Azuma. "A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors." Tumor Biology 42, no. 9 (September 2020): 101042832096258. http://dx.doi.org/10.1177/1010428320962588.
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A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 ( GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS-mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNASR201H -mutated ( GNASR201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS-mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell–derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNASR201H-mutated induced pluripotent stem cells and GNASR201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNASR201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS-mutated diseases.
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Zhu, Qian, Qiqi Lu, Rong Gao, and Tong Cao. "Prospect of Human Pluripotent Stem Cell-Derived Neural Crest Stem Cells in Clinical Application." Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/7695836.
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Neural crest stem cells (NCSCs) represent a transient and multipotent cell population that contributes to numerous anatomical structures such as peripheral nervous system, teeth, and cornea. NCSC maldevelopment is related to various human diseases including pigmentation abnormalities, disorders affecting autonomic nervous system, and malformations of teeth, eyes, and hearts. As human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can serve as an unlimited cell source to generate NCSCs, hESC/hiPSC-derived NCSCs can be a valuable tool to study the underlying mechanisms of NCSC-associated diseases, which paves the way for future therapies for these abnormalities. In addition, hESC/hiPSC-derived NCSCs with the capability of differentiating to various cell types are highly promising for clinical organ repair and regeneration. In this review, we first discuss NCSC generation methods from human pluripotent stem cells and differentiation mechanism of NCSCs. Then we focus on the clinical application potential of hESC/hiPSC-derived NCSCs on peripheral nerve injuries, corneal blindness, tooth regeneration, pathological melanogenesis, Hirschsprung disease, and cardiac repair and regeneration.
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Gallegos-Cárdenas, Amalia, Robin Webb, Erin Jordan, Rachel West, Franklin D. West, Jeong-Yeh Yang, Kai Wang, and Steven L. Stice. "Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Developmentally Mimic Human Pluripotent Stem Cell Neural Differentiation." Stem Cells and Development 24, no. 16 (August 15, 2015): 1901–11. http://dx.doi.org/10.1089/scd.2015.0025.
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Ivanova, Julia S., Natalia A. Pugovkina, Irina E. Neganova, Irina V. Kozhukharova, Nikolay N. Nikolsky, and Olga G. Lyublinskaya. "Cell Cycle-Coupled Changes in the Level of Reactive Oxygen Species Support the Proliferation of Human Pluripotent Stem Cells." Stem Cells 39, no. 12 (September 21, 2021): 1671–87. http://dx.doi.org/10.1002/stem.3450.
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Abstract The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G2/M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G1-phase or mitosis. Cells exposed to antioxidants in the early-G1-phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase—CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied by activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants.
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Choi, Kyung-Dal, Junying Yu, Kimberly Smuga-Otto, Jessica Dias, Giorgia Salvagiotto, Maxim Vodyanik, James Thomson, and Igor Slukvin. "Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells." Blood 112, no. 11 (November 16, 2008): 731. http://dx.doi.org/10.1182/blood.v112.11.731.731.
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Abstract Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In the present study, we employed an OP9 differentiation system to characterize the hematopoietic differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC; H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs in coculture with OP9 generated all types of colony forming cells (CFCs) as well as CD34+ cells that can be separated into distinct subsets based on differential expression of CD43 and CD31. CD34+CD31+CD43− cells obtained from all iPSCs expressed molecules present on endothelial cells and readily formed a monolayer when placed in endothelial conditions, while hematopoietic CFC potential was restricted to CD43+ cells. iPSC-derived CD43+ cells could be separated into three major subsets based on differential expression of CD235a/CD41a and CD45: CD235a+CD41a+/− (erythro-megakaryocytic progenitors), and lin-CD34+CD43+CD45− (multipotent), and lin-CD34+CD43+CD45+ (myeloid-skewed) primitive hematopoietic cells. Both subsets of primitive hematopoietic cells expressed genes associated with myeloid and lymphoid development, although myeloid genes were upregulated in CD45+ cells, which are skewed toward myeloid differentiation. Cytogenetic analysis demonstrated that iPSCs and derived from them CD43+ cells maintained normal karyotype. In addition short tandem repeat analysis of CFCs generated from IMR90-1 cells has been performed to confirm that blood cells are in fact derived from reprogrammed IMR90 cells, and not from contaminating hESCs. While we observed some variations in the efficiency of hematopoietic differentiation between different iPSCs, the pattern of differentiation was very similar in all seven tested iPSC and five hESC lines. Using different cytokine combinations and culture conditions we were able to expand iPSC-derived myeloid progenitors and induce their differentiation toward red blood cells, neutrophils, eosinophils, macrophages, ostoeclasts, dendritic and Langerhans cells. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and to identify molecules that can correct affected genetic networks.
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Arnold, Antje, Yahaira M. Naaldijk, Claire Fabian, Henry Wirth, Hans Binder, Guido Nikkhah, Lyle Armstrong, and Alexandra Stolzing. "Reprogramming of Human Huntington Fibroblasts Using mRNA." ISRN Cell Biology 2012 (December 7, 2012): 1–12. http://dx.doi.org/10.5402/2012/124878.
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The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.
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Bayzigitov, Daniel R., Sergey P. Medvedev, Elena V. Dementyeva, Sevda A. Bayramova, Evgeny A. Pokushalov, Alexander M. Karaskov, and Suren M. Zakian. "Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling." Cardiology Research and Practice 2016 (2016): 1–17. http://dx.doi.org/10.1155/2016/3582380.
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Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.
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Isogai, Sumito, Naoki Yamamoto, Noriko Hiramatsu, Yasuhiro Goto, Masamichi Hayashi, Masashi Kondo, and Kazuyoshi Imaizumi. "Preparation of Induced Pluripotent Stem Cells Using Human Peripheral Blood Monocytes." Cellular Reprogramming 20, no. 6 (December 2018): 347–55. http://dx.doi.org/10.1089/cell.2018.0024.
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Pierson, Tyler Mark, Yogesh K. Kushwaha, Maria Gabriela Otero, Phillip J. Kenny, Fabian David Nonis, and Jaemin Kim. "Human induced pluripotent stem cell models for CLN6." Molecular Genetics and Metabolism 132, no. 2 (February 2021): S86—S87. http://dx.doi.org/10.1016/j.ymgme.2020.12.206.
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Zhang, Jue, Li-Fang Chu, Zhonggang Hou, Michael P. Schwartz, Timothy Hacker, Vernella Vickerman, Scott Swanson, et al. "Functional characterization of human pluripotent stem cell-derived arterial endothelial cells." Proceedings of the National Academy of Sciences 114, no. 30 (July 10, 2017): E6072—E6078. http://dx.doi.org/10.1073/pnas.1702295114.
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Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with anEFNB2-tdTomato/EPHB4-EGFPdual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.
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Pouyanfard, Somayeh, Nairika Meshgin, Luisjesus S. Cruz, Karin Diggle, Hamidreza Hashemi, Timothy V. Pham, Manuel Fierro, et al. "Human Induced Pluripotent Stem Cell-Derived Macrophages Ameliorate Liver Fibrosis." Stem Cells 39, no. 12 (October 1, 2021): 1701–17. http://dx.doi.org/10.1002/stem.3449.
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Abstract With an increasing number of patients with degenerative hepatic diseases, such as liver fibrosis, and a limited supply of donor organs, there is an unmet need for therapies that can repair or regenerate damaged liver tissue. Treatment with macrophages that are capable of phagocytosis and anti-inflammatory activities such as secretion of matrix metalloproteinases (MMPs) provide an attractive cellular therapy approach. Human induced pluripotent stem cells (iPSCs) are capable of efficiently generating a large-scale, homogenous population of human macrophages using fully defined feeder- and serum-free differentiation protocol. Human iPSC-macrophages exhibit classical surface cell markers and phagocytic activity similar to peripheral blood-derived macrophages. Moreover, gene and cytokine expression analysis reveal that these macrophages can be efficiently polarized to pro-inflammatory M1 or anti-inflammatory M2 phenotypes in presence of LPS + IFN-γ and IL-4 + IL-13, respectively. M1 macrophages express high level of CD80, TNF-α, and IL-6 while M2 macrophages show elevated expression of CD206, CCL17, and CCL22. Here, we demonstrate that treatment of liver fibrosis with both human iPSC-derived macrophage populations and especially M2 subtype significantly reduces fibrogenic gene expression and disease associated histological markers including Sirius Red, αSMA and desmin in immunodeficient Rag2−/−γc−/− mice model, making this approach a promising cell-based avenue to ameliorate fibrosis.
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Maysubara, Hiroyuki, Akira Niwa, Tatsutoshi Nakahata, and Megumu K. Saito. "NK Cells from Human Pluripotent Stem Cells for Immunotherapy." Blood 132, Supplement 1 (November 29, 2018): 4955. http://dx.doi.org/10.1182/blood-2018-99-115499.
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Abstract Natural Killer (NK) cells are a one of innate lymphocytes and show cytotoxicity against tumour cells without prior antigen specific stimulation. . NK cells can demonstrate stronger cytotoxicity than T cells in the absence of MHC Class I, and survive short lifespan from several weeks to one month. It suggested that NK cells show low risk of cytokine long-term secretion inside patient's body. Previous studies have developed peripheral blood mononuclear cells (PBMC) derived NK cells expansions or NK cells differentiation from cord blood (CB) cells for immunotherapy. Expansion trial using K562 tumor cell line, or with IL-15, or an anti-tumor antibody dasatinib is not sufficient to obtain NK cells with high cytotoxicity.More recently, NK cells induction from human pluripotent stem cells (hPSCs), taking the advantage of their unlimited growth potential, has been reported. Although previous studies regarding hPSC-derived NK cells seems impressive and successful, most systems used a bovine and human serum, which might result in the unstable yield and efficiency in the production of Hematopoietic progenitor cells (HPCs) and NK cells for immunotherapy. To resolve those problems, we tried to induce functional NK cells from hPSCs in xeno and serum free condition. This study used three hPSC cell lines; human ES cell (cell line: KhES1) and iPS cells (cell line: 409B2 and CB-A11) to check reproducibility. To differentiate hPSCs into hematopoietic cells, changed cytokine combinations and chemically defined medium in step-wise manner. We first induced HPC from hPSCs over 90% purity by 12 days culture. At this point, we selected two media to induce NK cells. We compared serum-containing medium that previous report used (Medium A) and chemically-defined medium (Medium B) by evaluating the differentiation efficiency and function of NK cells. NK cell marker CD56 (NCAM) was gradually expressed after additional 16 days culture (28 days of differentiation). Until hPSC-derived NK cells were maturated, we traced the expression of NK specific markers and transcriptional factors. On day48, the frequency of CD56 positive cells showed no significant differences between medium A (79.15 ± 5.30%) and medium B (80.90 ± 1.27%). In both conditions, NK cells expressed specific receptors such as CD161, NKG2D, killer immunoglobulin-like receptors (KIRs), NKG2a (CD94/CD159a heterodimeric inhibitory receptor), NKp44 and NKp46. hPSC-derived NK cells showed the compatible size and morphology to NK cells isolated from peripheral blood NK (PB-NK) cells: their nucleus was kidney-like shape and cytoplasm contained azurophilic granules. For functional assay, leukemia cell line K562 was incubated with 51 chromium (51Cr) for 1 hour at 37 degrees. After that, K562 was co-cultured with purified CD56 positive hPSC-derived NK cells for 4 hours at 37 degrees. The cytotoxic activity of NK cells was confirmed by 51Cr release from K562. PBMC-NK cells showed 49.65 ± 3.46% of killing activity against K562 target cells, while the killing potential of PSC-derived NK cell showed killing potential against K562 cells (Medium A: 25.4 ± 5.52%, Medium B: 23.25 ± 9.26%) which was slightly lower than that of PB-NK cells. Next trial, we are going to transplant hPSC-derived NK cells into immune deficiency mice. In detail, this mice was infected luciferase expressed K562. Using IVIS imaging system to detect intensity of luciferase, we characterized hPSC-derived NK cells potential in vivo. Here we have developed a novel and robust method to facilitate efficient NK cells differentiation in serum and xeno-free condition in all clones. They showed similar phenotypes compare to PBMC derived NK cells in terms of morphology, surface markers, translational factors and cytotoxicity against leukemia cell line K562 in vitro. This technology expected to be applicable not only to immunotherapy but also to model studies of the NK cells associating diseases. Disclosures No relevant conflicts of interest to declare.
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Sato, Takahiko. "Induction of Skeletal Muscle Progenitors and Stem Cells from human induced Pluripotent Stem Cells." Journal of Neuromuscular Diseases 7, no. 4 (September 18, 2020): 395–405. http://dx.doi.org/10.3233/jnd-200497.
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Induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells and tissues including skeletal muscle. The approach to convert these stem cells into skeletal muscle cells offers hope for patients afflicted with skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). Several methods have been reported to induce myogenic differentiation with iPSCs derived from myogenic patients. An important point for generating skeletal muscle cells from iPSCs is to understand in vivo myogenic induction in development and regeneration. Current protocols of myogenic induction utilize techniques with overexpression of myogenic transcription factors such as Myod1(MyoD), Pax3, Pax7, and others, using recombinant proteins or small molecules to induce mesodermal cells followed by myogenic progenitors, and adult muscle stem cells. This review summarizes the current approaches used for myogenic induction and highlights recent improvements.
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Wang, Ping, Tao Ma, Dong Guo, Kevin Hu, Yan Shu, Hockin H. K. Xu, and Abraham Schneider. "Metformin induces osteoblastic differentiation of human induced pluripotent stem cell‐derived mesenchymal stem cells." Journal of Tissue Engineering and Regenerative Medicine 12, no. 2 (August 11, 2017): 437–46. http://dx.doi.org/10.1002/term.2470.
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Lewandowski, Jarosław, and Maciej Kurpisz. "Techniques of Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derivation." Archivum Immunologiae et Therapiae Experimentalis 64, no. 5 (March 3, 2016): 349–70. http://dx.doi.org/10.1007/s00005-016-0385-y.
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Petkov, Stoyan. "THE QUEST FOR PORCINE PLURIPOTENT STEM CELLS." Reproduction, Fertility and Development 25, no. 1 (2013): 319. http://dx.doi.org/10.1071/rdv25n1ab342.
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The isolation of embryonic stem cells (ESC) and embryonic germ cells (EGC) from early embryos is a major milestone in modern science and holds a great potential for human medicine. In 2007, Shinia Yamanaka and co-workers reprogrammed somatic cells to pluripotency by induced expression of pluripotency transcription factors. These so-called induced pluripotent stem cells (iPSC) are equivalent to ESC in terms of pluripotency and have the same potential for use in regenerative therapies. However, before the use of pluripotent cells or their derivatives in humans, potential therapies need to be tested in suitable animal models to ensure their safety. In this respect, the domestic pig is particularly suited for the testing of stem cell-based therapies intended for humans, since in general physiology and metabolism are similar in human and pigs. Since the isolation of the different types of pluripotent cells in human and mouse, there have been reports of derivation of ESC-like and EGC-like cell lines from porcine embryos. Despite the significant progress that has been reported in these studies, none of the described porcine cell lines have fulfilled all of the criteria for pluripotency, such as long-term maintenance and the ability to differentiate into all of the cells in the organism, including the germ line. This has prevented the use of these cells in the genetic engineering of livestock as well as their therapeutic application in animal models for human diseases. The derivation of the first porcine cell lines with iPSC characteristics (Ezashi et al. 2009 PNAS 27, 10 993–10 998) has provided a viable alternative to the ESC/EGC, and some major successes have been already achieved. The majority of the putative iPSC described in the literature have demonstrated pluripotent characteristics such as expression of various pluripotency markers and an ability to differentiate into the three primary germ layers in vivo by forming teratomas in immunodeficient mice. One group has reported the derivation of iPSC lines that have been capable to generate chimeras with germline contribution (West et al. 2011 Stem Cells 29, 1640–1643), which is the first fully confirmed report of successfully produced porcine germ line chimera to date. Additionally, the differentiation of putative iPSC into rod photoreceptors and their integration into the retinas of recipient pigs has been reported (Zhou et al. 2011 Stem Cells 29, 972–980). Despite these major achievements, some challenges remain to be overcome in order to make porcine iPSC more widely applicable in disease models and in the transgenic technology. Due to some variations in the morphological and molecular characteristics of the reported putative iPSC lines, it needs to be determined which markers are the hallmarks of truly pluripotent porcine iPSC. Second, it is still not clear which are the optimal culture conditions for derivation and long-term culture of these cells. Since the culture conditions used today have been proven ineffective to maintain pluripotency in porcine ESC and EGC, the question remains whether the continuous expression of the transgenes is an important factor in the long-term culture of iPSC. Finally, it needs to be determined whether putative porcine iPSC derived from cell types other than multipotent stem cells (such as mesenchymal stem cells used by West et al., 2011) possess full pluripotency, which should be demonstrated by germ line chimera production via blastocyst injection or tetraploid complementation.
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Resar, Linda, Sandeep N. Shah, Candace Kerr, Leslie Cope, Elias Zambidis, Amy Belton, and David L. Huso. "HMGA1, a Factor Enriched in Hematopoietic Stem Cells, Embryonic Stem Cells, and Hematologic Malignancy, Enhances Cellular Reprogramming to a Pluripotent Stem-Like Cell." Blood 120, no. 21 (November 16, 2012): 2323. http://dx.doi.org/10.1182/blood.v120.21.2323.2323.
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Abstract Abstract 2323 Although recent studies have identified genes important in hematopoietic stem cells (HSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), the molecular underpinnings of normal stem cell function are unclear. A better understanding of key stem cell pathways will be essential for the safe use of stem cells in regenerative medicine and should also uncover novel therapeutic targets in aggressive hematologic malignancies and other stem-like cancer cells. To elucidate the molecular underpinnings of “stemness”, we investigated transcriptional networks in pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. These proteins bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. HMGA1 is highly expressed during embryogenesis with low or undetectable levels in adult, differentiated tissues. HMGA1 is also enriched in HSCs, hESCs, iPSCs, refractory leukemia, and poorly differentiated solid tumors. Our group discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive leukemia in transgenic mice. We also found that high levels of HMGA1 expression correlate with relapse in acute lymphoblastic leukemia. Together, these findings suggest that HMGA1 drives a stem cell phenotype during normal development, hematopoiesis, and malignant transformation. To further investigate the role of HMGA1 in a stem cell state, we compared its expression in iPSCs, hESCs, HSCs, and cancer cells. HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in HSCs and cancer cells, and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation in hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells (mesenchymal stem cells, HSCs, and fetal lung fibroblasts) to an iPSC together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC or OSKM). HMGA1 results in an increase in the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign, teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In summary, our findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in hematologic and other malignancies or exploited in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.
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Illing, Anett, Marianne Stockmann, Narasimha Swamy Telugu, Leonhard Linta, Ronan Russell, Martin Müller, Thomas Seufferlein, Stefan Liebau, and Alexander Kleger. "Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation." Stem Cells International 2013 (2013): 1–13. http://dx.doi.org/10.1155/2013/360573.
Full textAbstract:
Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs) generated from plucked human hair keratinocytes have been exploitedin vitroto recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa) subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.
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Gajbhiye, Virendra, Leah Escalante, Guojun Chen, Alex Laperle, Qifeng Zheng, Benjamin Steyer, Shaoqin Gong, and Krishanu Saha. "Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives." Nanoscale 6, no. 1 (2014): 521–31. http://dx.doi.org/10.1039/c3nr04794f.
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Stebbins, Matthew J., Benjamin D. Gastfriend, Scott G. Canfield, Ming-Song Lee, Drew Richards, Madeline G. Faubion, Wan-Ju Li, Richard Daneman, Sean P. Palecek, and Eric V. Shusta. "Human pluripotent stem cell–derived brain pericyte–like cells induce blood-brain barrier properties." Science Advances 5, no. 3 (March 2019): eaau7375. http://dx.doi.org/10.1126/sciadv.aau7375.
Full textAbstract:
Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte–like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte–like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte–like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte–like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.
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Gao, Jinghui, Sophia Petraki, Xingshen Sun, Leonard A. Brooks, Thomas J. Lynch, Chih-Lin Hsieh, Reem Elteriefi, et al. "Derivation of induced pluripotent stem cells from ferret somatic cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 318, no. 4 (April 1, 2020): L671—L683. http://dx.doi.org/10.1152/ajplung.00456.2019.
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Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.