Journal articles on the topic 'IPSC derivation'
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Tan, Yu-Ting, Lin Ye, Fei Xie, Ashley I. Beyer, Marcus O. Muench, Jiaming Wang, Zhu Chen, Han Liu, Sai-Juan Chen, and Yuet Wai Kan. "Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor." Proceedings of the National Academy of Sciences 115, no. 9 (January 31, 2018): 2180–85. http://dx.doi.org/10.1073/pnas.1718446115.
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Derivation of human hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) offers considerable promise for cell therapy, disease modeling, and drug screening. However, efficient derivation of functional iPSC-derived HSCs with in vivo engraftability and multilineage potential remains challenging. Here, we demonstrate a tractable approach for respecifying iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells (HSPCs) through transient expression of a single transcription factor, MLL-AF4. These induced HSPCs (iHSPCs) derived from iPSCs are able to fully reconstitute the human hematopoietic system in the recipient mice without myeloid bias. iHSPCs are long-term engraftable, but they are also prone to leukemic transformation during the long-term engraftment period. On the contrary, primary HSPCs with the same induction sustain the long-term engraftment without leukemic transformation. These findings demonstrate the feasibility of activating the HSC network in human iPSC-derived blood cells through expression of a single factor and suggest iHSPCs are more genomically instable than primary HSPCs, which merits further attention.
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Miki, Toshio, Ludivina Vazquez, Lisa Yanuaria, Omar Lopez, Irving M. Garcia, Kazuo Ohashi, and Natalie S. Rodriguez. "Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model." Stem Cells International 2019 (April 1, 2019): 1–10. http://dx.doi.org/10.1155/2019/6978303.
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Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.
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Lee, Hyelim, Hyeonjin Cha, and Ju Hyun Park. "Derivation of Cell-Engineered Nanovesicles from Human Induced Pluripotent Stem Cells and Their Protective Effect on the Senescence of Dermal Fibroblasts." International Journal of Molecular Sciences 21, no. 1 (January 5, 2020): 343. http://dx.doi.org/10.3390/ijms21010343.
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Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.
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Yu, Yang, Xuechun Li, Yimei Li, Renyue Wei, Hai Li, Zhonghua Liu, and Yu Zhang. "Derivation and Characterization of Endothelial Cells from Porcine Induced Pluripotent Stem Cells." International Journal of Molecular Sciences 23, no. 13 (June 24, 2022): 7029. http://dx.doi.org/10.3390/ijms23137029.
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Although the study on the regulatory mechanism of endothelial differentiation from the perspective of development provides references for endothelial cell (EC) derivation from pluripotent stem cells, incomplete reprogramming and donor-specific epigenetic memory are still thought to be the obstacles of iPSCs for clinical application. Thus, it is necessary to establish a stable iPSC-EC induction system and investigate the regulatory mechanism of endothelial differentiation. Based on a single-layer culture system, we successfully obtained ECs from porcine iPSCs (piPSCs). In vitro, the derived piPSC-ECs formed microvessel-like structures along 3D gelatin scaffolds. Under pathological conditions, the piPSC-ECs functioned on hindlimb ischemia repair by promoting blood vessel formation. To elucidate the molecular events essential for endothelial differentiation in our model, genome-wide transcriptional profile analysis was conducted, and we found that during piPSC-EC derivation, the synthesis and secretion level of TGF-β as well as the phosphorylation level of Smad2/3 changed dynamically. TGF-β-Smad2/3 signaling activation promoted mesoderm formation and prevented endothelial differentiation. Understanding the regulatory mechanism of iPSC-EC derivation not only paves the way for further optimization, but also provides reference for establishing a cardiovascular drug screening platform and revealing the molecular mechanism of endothelial dysfunction.
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Assen, Lars, Annelien Bredenoord, Karin Jongsma, Marianna Tryfonidou, and Rosario Isasi. "iPS Cells: Don’t Forget about the Soft Impacts." Studia Universitatis Babeş-Bolyai Bioethica 66, Special Issue (September 9, 2021): 26–27. http://dx.doi.org/10.24193/subbbioethica.2021.spiss.08.
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"Induced pluripotent stem cells (iPSCs) have been praised for overcoming some of the ethical challenges of embryonic stem cell research, including oocyte donation for research and the destruction of human embryos. However, iPSC-research and iPSC-based interventions are not morally neutral alternatives and have their own ethical implications that are not fully understood yet. While there is some understanding of ethical issues surrounding the derivation, storage and use of human tissue, there is less understanding of how iPSC-research affects our society and morality. Consequentially, it is difficult to fully anticipate those implications. The notion of hard and soft impacts could benefit the understanding and anticipation of ethical implications of iPSC-research and interventions. Hard impacts are those direct physical and financial effects of iPSCs that are quantifiable and measurable. So-called soft impacts have a different focus. They consider how a technology or intervention affects our psychology, societal structures, morality and our behavior, hereby influencing the uptake, effects and evaluation of technology. So far, academic literature and researchers focus primarily on hard impacts of iPSC-research. Soft impacts are similarly important and therefore require more academic and regulatory attention. This talk focuses upon these understudied aspects of iPSC-research and technology. The goal is to show that for researchers and ethicists it is important to become aware of the soft impacts of iPSC-research and technology. This awareness could contribute to a broader understanding of the social value of stem cell research, anticipating ethical challenges of iPSC-research and in formulating new virtues for stem cell researchers. "
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Pinto, A., Y. Shamis, L. McDonnell, A. Chambon, M. Jung, J. Valls Cuevas, R. Chaffoo, M. Samberg, C. Sumen, and A. Terskikh. "Derivation of folliculogenic organoids from human iPSC." Cytotherapy 23, no. 5 (May 2021): S17. http://dx.doi.org/10.1016/s1465324921002711.
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Srimasorn, Sumitra, Matthias Kirsch, Susanne Hallmeyer-Ellgner, Dirk Lindemann, Alexander Storch, and Andreas Hermann. "Increased Neuronal Differentiation Efficiency in High Cell Density-Derived Induced Pluripotent Stem Cells." Stem Cells International 2019 (December 4, 2019): 1–8. http://dx.doi.org/10.1155/2019/2018784.
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Human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs), provide access to hard-to-obtain cells for studies under physiological and disease conditions. For the study of neurodegenerative diseases, especially sporadic cases where the “disease condition” might be restricted towards the neuroectodermal lineage, obtaining the affected neurons is important to help unravel the underlying molecular mechanism leading to the diseases. Although differentiation of iPSCs to neural lineage allows acquisition of cell types of interest, the technology suffers from low efficiency leading to low yield of neurons. Here, we investigated the potential of adult neuroprogenitor cells (aNPCs) for iPSC derivation and possible confounders such as cell density of infected NPCs on their subsequent neuronal differentiation potential from reprogrammed cells under isogenic conditions. Characterized hiPSCs of defined cell densities generated from aNPCs were subjected to neuronal differentiation on PA6 stromal cells. The results showed that hiPSC clones obtained from low seeding density (iPSC-aNPCLow) differentiated less efficiently compared to those from higher density (iPSC-aNPCHigh). Our findings might help to further improve the yield and quality of neurons for in vitro modelling of neurodegenerative diseases.
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Petkov, Stoyan, Ralf Dressel, Ignacio Rodriguez-Polo, and Rüdiger Behr. "Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules." Cells 9, no. 11 (November 5, 2020): 2422. http://dx.doi.org/10.3390/cells9112422.
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Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.
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Pouyanfard, Somayeh, Manuel Fierro, and Dan S. Kaufman. "Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity." Blood 138, Supplement 1 (November 5, 2021): 1693. http://dx.doi.org/10.1182/blood-2021-148687.
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Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing. Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.
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Schuening, Friedrich, Michail Zaboikin, Tatiana Zaboikina, and Narasimhachar Srinivasakumar. "Derivation of Hematopoietic Progenitor Cells From Vector-Free Human Induced Pluripotent Stem Cells." Blood 120, no. 21 (November 16, 2012): 4746. http://dx.doi.org/10.1182/blood.v120.21.4746.4746.
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Abstract Abstract 4746 Induced pluripotent stem cells (iPSCs), due to their self-renewal and differentiation capability, have tremendous potential in regenerative medicine. Differentiation of IPSCs in vitro to obtain sufficient number of hematopoietic stem cells (HSCs) and their progenitors (HPCs) from iPSCs for therapeutic purposes is a holy grail of cellular therapy. To this end, we are comparing different in vitro differentiation approaches for generation of HSCs/HPCs from IPSCs. We have generated iPSCs from human adult dermal fibroblasts using two different reprogramming methods: 1) Transduction with retroviral vectors encoding human Klf4, Oct3/4, Sox2 and cMyc or 2) Electroporation with Epstein–Barr virus (EBV) based episomal plasmid vectors encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA. The transduced/electroporated cells were reprogrammed on SNL5 mouse feeder cells. Putative iPSC-like colonies were cloned and adapted to grow under feeder-free conditions on Matrigel (BD) in mTeSR1 (Stem Cell Technologies) medium. From over 30 individual clones isolated, six were further characterized for: 1) expression of pluripotency markers (Tra-1–60, SSEA-3, SSEA-4, Nanog and Oct3/4) by immunofluorescence; 2) endogenous and total mRNA expression by quantitative real-time reverse-transcriptase PCR (RT-qPCR) for Klf4, Oct3/4, Sox2 and cMyc to distinguish between cellular and vector derived expression of reprogramming factors; 3) RT-qPCR to determine expression of other markers of pluripotency such as Nanog and DNA methyl transferease; 4) karyotype analysis to determine chromosomal anomalies. The vector-free IPSC clones were also tested for residual integrated EBV plasmid DNA by qPCR. Trilineage differentiation ability of the clones was determined through embryoid body formation in suspension cultures, and subsequent staining of resulting embryoid bodies after adherence to gelatin coated dishes for makers of ectoderm, mesoderm and endoderm. HSCs/HPCs were obtained from IPSCs by 1) coculture with OP9 stromal cells, or 2) step-wise differentiation in feeder-free conditions on Matrigel under defined conditions in the presence of appropriate growth factors [Niwa A et al. PLoS One. (2011); 6(7):e22261.]. The resultant HSCs/HPCs were subjected to colony forming assays in semi-solid medium containing hematopoietic cytokines. Both erythroid and myelomonocytic colonies could be readily identified. The influence of ambient oxygen concentration on the HSC/HPC derivation procedure is being investigated. The results of these studies will be presented. Disclosures: No relevant conflicts of interest to declare.
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Chen, Guokai, Daniel R. Gulbranson, Zhonggang Hou, Jennifer M. Bolin, Victor Ruotti, Mitchell D. Probasco, Kimberly Smuga-Otto, et al. "Chemically defined conditions for human iPSC derivation and culture." Nature Methods 8, no. 5 (April 10, 2011): 424–29. http://dx.doi.org/10.1038/nmeth.1593.
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Podkalicka, Paulina, Jacek Stępniewski, Olga Mucha, Neli Kachamakova-Trojanowska, Józef Dulak, and Agnieszka Łoboda. "Hypoxia as a Driving Force of Pluripotent Stem Cell Reprogramming and Differentiation to Endothelial Cells." Biomolecules 10, no. 12 (November 29, 2020): 1614. http://dx.doi.org/10.3390/biom10121614.
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Inadequate supply of oxygen (O2) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling. Endothelial cells (ECs), multifunctional components of vascular structures, may be obtained from iPSCs and subsequently used in various (hypoxia-related) disease models to investigate vascular dysfunctions. Although iPSC-ECs demonstrated functionality in vitro and in vivo, ongoing studies are conducted to increase the efficiency of differentiation and to establish the most productive protocols for the application of patient-derived cells in clinics. In this review, we highlight recent discoveries on the role of hypoxia in the derivation of ESCs and the generation of iPSCs. We also summarize the existing protocols of hypoxia-driven differentiation of iPSCs toward ECs and discuss their possible applications in disease modeling and treatment of hypoxia-related disorders.
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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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Kwon, Yoo-Wook, Jae-Seung Paek, Hyun-Jai Cho, Choon-Soo Lee, Ho-Jae Lee, In-Hyun Park, Tae-Young Roh, et al. "Role of Zscan4 in secondary murine iPSC derivation mediated by protein extracts of ESC or iPSC." Biomaterials 59 (August 2015): 102–15. http://dx.doi.org/10.1016/j.biomaterials.2015.03.031.
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Terrenoire, Cecile, Kai Wang, Kelvin W. Chan Tung, Wendy K. Chung, Robert H. Pass, Jonathan T. Lu, Jyh-Chang Jean, et al. "Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics." Journal of General Physiology 141, no. 1 (December 31, 2012): 61–72. http://dx.doi.org/10.1085/jgp.201210899.
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Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na+ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na+ channel current (INaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of INaL such that increasing the pacing rate markedly reduces INaL and, in addition, increases its inhibition by the Na+ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.
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Petkov, Stoyan, Zoltan Ivics, and Heiner Niemann. "PROGRESS TOWARDS THE DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS." Reproduction, Fertility and Development 24, no. 1 (2012): 284. http://dx.doi.org/10.1071/rdv24n1ab243.
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Porcine induced pluripotent cells (iPSC) are considered an important large animal model for developing personalized stem cell therapies. Since the derivation of the first mouse and human iPSC, there have been relatively few reports regarding the reprogramming of pig somatic cells into pluripotency, exclusively with the use of transcription factors from human and mouse origin. To investigate whether using species-specific transcription factors would allow for an efficient reprogramming of porcine somatic cells, we have developed a Sleeping Beauty (SB) transposon system based on the porcine OCT4, SOX2, C-MYC, and KLF4 sequences and have tested it in the reprogramming of mouse and porcine fetal fibroblasts and pig EGC-like cells. Transfection of mouse embryonic fibroblasts with a multicistronic SB-tetO-pOCT4-F2A-pSOX2-T2A-pC-MYC-E2A-pKLF4-ires-Tomato vector resulted in the formation of iPSC colonies, which inactivated the exogenous transcription factors and upregulated their endogenous pluripotency genes. These cells maintained mESC-like morphology, formed embryoid bodies, and differentiated into different cell types in culture, including rhythmically contracting cardiac myocytes. In contrast, porcine fibroblasts and EGC-like cells transfected with the same transposon vector did not proliferate in culture and did not form any iPSC colonies. We then transfected these cells with multiple bi-cistronic vectors SB-Ef1a-pOCT4-ires-Tomato, SB-Ef1a-pSOX2-ires-Neo, SB-Ef1a-pC-MYC-ires-Puro, and SB-Ef1a-pKLF4-ires-Puro. As a result, both cell types formed multiple colonies with mouse ESC-like morphology. Clones established from individually picked colonies from transfected fetal fibroblasts maintained this morphology for 5-6 passages, after which they became flat and epithelial-like. They expressed endogenous SOX2, C-MYC, KLF4, and E-Cadherin, but not OCT4. At the same time, clones derived from EGC-like cells proliferated at accelerated rate and maintained their morphology for over 10 passages at the time of this writing. While the exogenous genes were expressed continuously during this period, the cells expressed also endogenous OCT4, SOX2, TERT, STELLA, TDH, and CHD1; however, C-MYC, KLF4, NANOG, and E-Cadherin expression was relatively low. These cells are currently being characterized for pluripotency. Despite the use of porcine transcription factors, the overall reprogramming of porcine cells appears to be still less efficient compared with mouse fibroblasts. Our results suggest that the species and tissue origin of the somatic cells may play a more important role in the reprogramming to pluripotency than the origin of the transcription factors used. In this respect, optimization of culture conditions may be necessary in order to allow for efficient and complete reprogramming of porcine somatic cells. This work is supported by a research grant by the Deutsche Forschungsgemeinschaft.
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Nijak, Aleksandra, Johan Saenen, Alain J. Labro, Dorien Schepers, Bart L. Loeys, and Maaike Alaerts. "iPSC-Cardiomyocyte Models of Brugada Syndrome—Achievements, Challenges and Future Perspectives." International Journal of Molecular Sciences 22, no. 6 (March 10, 2021): 2825. http://dx.doi.org/10.3390/ijms22062825.
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Brugada syndrome (BrS) is an inherited cardiac arrhythmia that predisposes to ventricular fibrillation and sudden cardiac death. It originates from oligogenic alterations that affect cardiac ion channels or their accessory proteins. The main hurdle for the study of the functional effects of those variants is the need for a specific model that mimics the complex environment of human cardiomyocytes. Traditionally, animal models or transient heterologous expression systems are applied for electrophysiological investigations, each of these models having their limitations. The ability to create induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), providing a source of human patient-specific cells, offers new opportunities in the field of cardiac disease modelling. Contemporary iPSC-CMs constitute the best possible in vitro model to study complex cardiac arrhythmia syndromes such as BrS. To date, thirteen reports on iPSC-CM models for BrS have been published and with this review we provide an overview of the current findings, with a focus on the electrophysiological parameters. We also discuss the methods that are used for cell derivation and data acquisition. In the end, we critically evaluate the knowledge gained by the use of these iPSC-CM models and discuss challenges and future perspectives for iPSC-CMs in the study of BrS and other arrhythmias.
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Shen, Mengcheng, Thomas Quertermous, Michael P. Fischbein, and Joseph C. Wu. "Generation of Vascular Smooth Muscle Cells From Induced Pluripotent Stem Cells." Circulation Research 128, no. 5 (March 5, 2021): 670–86. http://dx.doi.org/10.1161/circresaha.120.318049.
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The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.
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Huang, Zhong, Rebecca Powell, James B. Phillips, and Kirsten Haastert-Talini. "Perspective on Schwann Cells Derived from Induced Pluripotent Stem Cells in Peripheral Nerve Tissue Engineering." Cells 9, no. 11 (November 17, 2020): 2497. http://dx.doi.org/10.3390/cells9112497.
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Schwann cells play a crucial role in successful peripheral nerve repair and regeneration by supporting both axonal growth and myelination. Schwann cells are therefore a feasible option for cell therapy treatment of peripheral nerve injury. However, sourcing human Schwann cells at quantities required for development beyond research is challenging. Due to their availability, rapid in vitro expansion, survival, and integration within the host tissue, stem cells have attracted considerable attention as candidate cell therapies. Among them, induced pluripotent stem cells (iPSCs) with the associated prospects for personalized treatment are a promising therapy to take the leap from bench to bedside. In this critical review, we firstly focus on the current knowledge of the Schwann cell phenotype in regard to peripheral nerve injury, including crosstalk with the immune system during peripheral nerve regeneration. Then, we review iPSC to Schwann cell derivation protocols and the results from recent in vitro and in vivo studies. We finally conclude with some prospects for the use of iPSCs in clinical settings.
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Cheung, Hoi-Hung, Xiaozhuo Liu, and Owen M. Rennert. "Apoptosis: Reprogramming and the Fate of Mature Cells." ISRN Cell Biology 2012 (April 17, 2012): 1–8. http://dx.doi.org/10.5402/2012/685852.
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Apoptosis is essential for embryogenesis, organ metamorphosis, and tissue homeostasis. In embryonic stem cells, self-renewal is balanced with proliferative potential, inhibition of differentiation, and prevention of senescence and apoptosis. Growing evidence supports the role of apoptosis in self-renewal, differentiation of pluripotent stem cells, and dedifferentiation (reprogramming) of somatic cells. In this paper we discuss the multiple roles of apoptosis in embryonic stem cells (ESCs) and reprogramming of differentiated cells to pluripotency. The role of caspases and p53 as key effectors in controlling the generation of iPSC is emphasized. Remarkably, the complication of apoptosis arising during reprogramming may provide insights into technical improvements for derivation of iPSC from senescent cells as a tool for modeling aging-related diseases.
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Cornelius, Victoria A., Hojjat Naderi-Meshkin, Sophia Kelaini, and Andriana Margariti. "RNA-Binding Proteins: Emerging Therapeutics for Vascular Dysfunction." Cells 11, no. 16 (August 11, 2022): 2494. http://dx.doi.org/10.3390/cells11162494.
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Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.
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Roberts, R. Michael, Ye Yuan, and Toshihiko Ezashi. "Exploring early differentiation and pluripotency in domestic animals." Reproduction, Fertility and Development 29, no. 1 (2017): 101. http://dx.doi.org/10.1071/rd16292.
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This short review describes some general features of the origins of the pluripotent inner cell mass and epiblast during the early development of eutherian mammals and the two kinds of embryonic stem cell (ESC), naïve and primed type, that have been produced from these structures. We point out that the derivation of pluripotent stem cells from domesticated species continues to be fraught with difficulties, most likely because the culture requirements of these cells are distinct from those of mouse and human ESCs. Generation of induced pluripotent stem cells (iPSCs) from the domesticated species has been more straightforward, although the majority of the iPSC lines remain dependent on the continued expression of one or more integrated reprogramming genes. Although hope for the potential usefulness of these cells in genetic modification of livestock and other domestic species has dimmed, ESCs and iPSCs remain our best source of self-renewing populations of pluripotent cells, with potential usefulness in preserving and propagating valuable animal breeds and making contributions to fields such as regenerative medicine, toxicology and even laboratory meat production.
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Ni, Zhenya, David A. Knorr, Christine L. Clouser, Peter Southern, Louis M. Mansky, In-Hyun Park, and Dan S. Kaufman. "Natural Killer Cells Derived From Human Pluripotent Stem Cells Provide a Novel Method to Treat HIV-1 Infection." Blood 114, no. 22 (November 20, 2009): 280. http://dx.doi.org/10.1182/blood.v114.22.280.280.
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Abstract Abstract 280 Natural killer (NK) cells are known to be key components of the innate immune system with the ability to kill diverse tumor cells and virally infected cells. Our group has previously demonstrated derivation of CD45+CD56+ natural killer (NK) cells from human embryonic stem cells (hESCs)-derived hematopoietic CD34+CD45+ progenitor cells. These hESC-derived NK cells demonstrate potent killing of various tumor cells both in vitro and in vivo. More recently, we have also successfully generated NK cells from similar CD34+CD45+ hematopoietic progenitor cells derived from human induced pluripotent stem cells (iPSCs). Again, we find that these iPSC-derived NK cells also have effective anti-tumor activity in vitro. Notably, both the hESC and iPSC-derived NK cells are uniformly CD94+CD117−, corresponding to a more mature and cytotoxic NK cell population. This is in contrast to NK cells derived from umbilical cord blood (UCB) progenitor cells that produce a mixture of CD94+CD117− and CD94−CD117+ NK cells that are more heterogeneous in their cytotoxic activity. Previous studies of NK cells isolated from peripheral blood indicate they have activity against HIV-1-infected cells. Therefore, we hypothesized that both hESC and iPSC-derived NK cells would be able to kill HIV-1-infected targets. We have applied multiple complementary systems to test this hypothesis using both HIV-1-infected cell lines and HIV-1-infected primary T cells. First, we used a chronically infected cell line (H9/HTLV IIIB) to demonstrate specific cytolytic activity of hESC-derived NK cells. Here, we found CD107a expression (a marker of NK cell functional activity) was significantly upregulated on hESC-derived effectors stimulated by the HIV-1-infected targets compared to uninfected targets (13.7% vs. 4.3%). Next, we utilized primary human CD4 T cells infected with primary patient isolate HIV96-480 as targets to demonstrate the same effect to specifically activate both hESC and iPSC-derived NK cells. In both of these studies, control NK cell populations derived from human umbilical cord blood progenitor cells were significantly less active against HIV than the hESC and iPSC-derived NK cells. In addition to the cytolytic function against HIV-1-infected targets, we demonstrate hESC and iPSC-derived NK cells also suppress HIV-1 replication by producing CC-chemokines to competitively inhibit CCR5 co-receptor binding. CCL4 (MIP1b), a CCR5 ligand, is greatly induced in both hESC and iPSC-derived NK cells after incubation with HIV-1-infected targets compared to uninfected targets: 37.2% (HIV-infected) vs. 24.8% (uninfected) for hESC-NK cells and 32.5% (HIV-infected) vs. 21.6% (uninfected) for iPSC-NK cells. Lastly, we have also tested suppression of acute HIV-1 infection by hESC-derived NK cells in vitro. Here, the T-cell line CEM-GFP was infected with HIV-1 NL4-3 and cocultured with hESC-derived NK cells for two weeks. HIV-1-infected targets detected by flow cytometry for GFP expression were strongly decreased in comparison to controls in absence of NK cells (% of GFP+ cells: 0.74 vs. 26.4). In these analyses, expression of CD107a and CCL4 on the effectors again correlates with the inhibition of HIV-1 replication. Currently, as hESC and iPSC-derived NK cells express the Fc receptor CD16, we are using anti-envelope protein antibodies against HIV-1 infected primary human CD4 T cells to determine if antibody-dependent cellular cytotoxicity provides another mechanism of lytic activity for these novel NK cells. Collectively, our results demonstrate that NK cells derived from hESCs and iPSCs provide an effective novel cellular immunotherapy for HIV-1 infection. Disclosures: No relevant conflicts of interest to declare.
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Zaslavsky, K., W. Zhang, E. Deneault, M. Zhao, DC Rodrigues, F. McReady, PJ Ross, et al. "P.135 Autism-associated mutations in SHANK2 increase synaptic connectivity and dendrite complexity in human neurons." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 45, s2 (June 2018): S52. http://dx.doi.org/10.1017/cjn.2018.237.
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Background: Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). However, their impact on the function of human neurons is unknown. Derivation of induced pluripotent stem cells (iPSC) from affected individuals permits generation of live neurons to answer this question. Methods: We generated iPSCs by reprogramming dermal fibroblasts of neurotypic and ASD-affected donors. To isolate the effect of SHANK2, we used CRISPR/Cas9 to knock out SHANK2 in control iPSCs and correct a heterozygous nonsense mutation in ASD-affected donor iPSCs. We then derived cortical neurons from SOX1+ neural precursor cells differentiated from these iPSCs. Using a novel assay that overcomes line-to-line variability, we compared neuronal morphology, total synapse number, and electrophysiological properties between SHANK2 mutants and controls. Results: Relative to controls, SHANK2 mutant neurons have increased dendrite complexity, dendrite length, total synapse number (1.5-2-fold), and spontaneous excitatory postsynaptic current (sEPSC) frequency (3-7.6-fold). Conclusions: ASD-associated heterozygous loss-of-function mutations in SHANK2 increase synaptic connectivity among human neurons by increasing synapse number and sEPSC frequency. This is partially supported by increased dendrite length and complexity, providing evidence that SHANK2 functions as a suppressor of dendrite branching during neurodevelopment.
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Guo, Hao, Zhe-long Jin, Xuerui Yao, Jeehun Park, Yong-Pil Gwon, Sungmin Kim, Kee-Pyo Kim, et al. "Derivation of iPSC lines from two idiopathic ASD patients (OFi001-A, OFi002-A)." Stem Cell Research 56 (October 2021): 102510. http://dx.doi.org/10.1016/j.scr.2021.102510.
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Najar, Ashaq H., K. M. Sneha, Aparna Ashok, Swathy Babu, Anand G. Subramaniam, Ramkrishnan Kannan, Biju Viswanath, et al. "Derivation of iPSC lines from two patients with familial Alzheimer's disease from India." Stem Cell Research 34 (January 2019): 101370. http://dx.doi.org/10.1016/j.scr.2018.101370.
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Wang, Wei, Tiansu Wang, Andriana G. Kotini, Camelia Iancu-Rubin, Ronald Hoffman, and Eirini P. Papapetrou. "Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells." Blood 132, Supplement 1 (November 29, 2018): 4319. http://dx.doi.org/10.1182/blood-2018-99-111512.
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Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.
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Zhao, Chengzhu, and Makoto Ikeya. "Generation and Applications of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells." Stem Cells International 2018 (July 31, 2018): 1–8. http://dx.doi.org/10.1155/2018/9601623.
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Mesenchymal stem cells (MSCs) are adult stem cells with fibroblast-like morphology and isolated from the bone marrow via plastic adhesion. Their multipotency and immunoregulatory properties make MSCs possible therapeutic agents, and an increasing number of publications and clinical trials have highlighted their potential in regenerative medicine. However, the finite proliferative capacity of MSCs limits their scalability and global dissemination as a standardized therapeutic product. Furthermore, adult tissue provenance could constrain accessibility, impinge on cellular potency, and incur greater exposure to disease-causing pathogens based on the donor. These issues could be circumvented by the derivation of MSCs from pluripotent stem cells. In this paper, we review methods that induce and characterize MSCs derived from induced pluripotent stem cells (iPSCs) and introduce MSC applications to disease modeling, pathogenic mechanisms, and drug discovery. We also discuss the potential applications of MSCs in regenerative medicine including cell-based therapies and issues that should be overcome before iPSC-derived MSC therapy will be applied in the clinic.
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Szepes, Monika, Anna Melchert, Julia Dahlmann, Jan Hegermann, Christopher Werlein, Danny Jonigk, Axel Haverich, Ulrich Martin, Ruth Olmer, and Ina Gruh. "Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro." International Journal of Molecular Sciences 21, no. 23 (November 25, 2020): 8947. http://dx.doi.org/10.3390/ijms21238947.
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Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types—i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.
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Sui, Dandan, Zhaolin Sun, Chunlong Xu, Yuanyuan Wu, Mario R. Capecchi, Sen Wu, and Ning Li. "Fine-Tuning of iPSC Derivation by an Inducible Reprogramming System at the Protein Level." Stem Cell Reports 2, no. 5 (May 2014): 721–33. http://dx.doi.org/10.1016/j.stemcr.2014.03.013.
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Kolagar, Tannaz Akbari, Maryam Farzaneh, Negin Nikkar, and Seyed Esmaeil Khoshnam. "Human Pluripotent Stem Cells in Neurodegenerative Diseases: Potentials, Advances and Limitations." Current Stem Cell Research & Therapy 15, no. 2 (March 26, 2020): 102–10. http://dx.doi.org/10.2174/1574888x14666190823142911.
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Neurodegenerative diseases are progressive and uncontrolled gradual loss of motor neurons function or death of neuron cells in the central nervous system (CNS) and the mechanisms underlying their progressive nature remain elusive. There is urgent need to investigate therapeutic strategies and novel treatments for neural regeneration in disorders like Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Currently, the development and identification of pluripotent stem cells enabling the acquisition of a large number of neural cells in order to improve cell recovery after neurodegenerative disorders. Pluripotent stem cells which consist of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their ability to indefinitely self-renew and the capacity to differentiate into different types of cells. The first human ESC lines were established from donated human embryos; while, because of a limited supply of donor embryos, human ESCs derivation remains ethically and politically controversial. Hence, hiPSCs-based therapies have been shown as an effective replacement for human ESCs without embryo destruction. Compared to the invasive methods for derivation of human ESCs, human iPSCs has opened possible to reprogram patient-specific cells by defined factors and with minimally invasive procedures. Human pluripotent stem cells are a good source for cell-based research, cell replacement therapies and disease modeling. To date, hundreds of human ESC and human iPSC lines have been generated with the aim of treating various neurodegenerative diseases. In this review, we have highlighted the recent potentials, advances, and limitations of human pluripotent stem cells for the treatment of neurodegenerative disorders.
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Duran, Ana, Olivia Reidell, Harald Stachelscheid, Kristin Klose, Manfred Gossen, Volkmar Falk, Wilhelm Röll, and Christof Stamm. "Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells." Thoracic and Cardiovascular Surgeon 66, no. 01 (December 7, 2017): 053–62. http://dx.doi.org/10.1055/s-0037-1608761.
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AbstractFor more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation (“cellular cardiomyoplasty”), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.
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Petkov, S., M. Nowak-Imialek, P. Hyttel, and H. Niemann. "307 REPROGRAMMING OF PIG SOMATIC CELLS TO PLURIPOTENCY WITH SLEEPING BEAUTY TRANSPOSON VECTORS CONTAINING THE PORCINE TRANSCRIPTION FACTOR SEQUENCES." Reproduction, Fertility and Development 25, no. 1 (2013): 300. http://dx.doi.org/10.1071/rdv25n1ab307.
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Induced pluripotent stem cells (iPSC), developed by Yamanaka and co-workers (Takahashi et al., 2006), hold significant potential for the development of regenerative therapies due to the possibilities of deriving patient-specific pluripotent cells. In this aspect, the pig is an important animal model for testing iPSC-based applications for the human medicine. However, even though significant progress has been made, the derivation of porcine iPSC lines fully equivalent to those from mouse and human has been elusive. To date, most of the reported putative pig iPSC lines have been derived with the use of lentiviral or retroviral vectors harboring the mouse or human transcription factor sequences. Here, we report the construction of Sleeping Beauty (SB) transposon vectors with porcine cDNA sequences coding for OCT4, SOX2, NANOG, C-MYC, and KLF4, in addition to the human LIN28. By using standard cloning techniques, we produced 2 polycistronic SB-CAG-pOSMK-ires-Tomato and SB-Ef1a-pNANOG-ires-hLIN28 transposon vectors and we transfected them together with the SB100X transposase into pig fetal fibroblasts (pFF) harboring a mouse OCT4-GFP reporter construct (Nowak-Imialek et al., 2010). Both the basic transposon and transposase vectors were generously provided by Dr. Zoltan Ivics from Paul Ehrlich Institute, Langen, Germany. In each experiment, 2 × 106 pFF were electroporated with 3 µg of each transposon together with 0.5 µg of SB100X. Two days after transfection, the cells were transferred to mouse embryonic fibroblast (MEF) feeders and cultured with iPSC medium [DMEM with antibiotics, nonessential amino acids, 20% Knockout serum replacement, 5 ng mL–1 human recombinant basic fibroblast growth factor (bFGF), and 1000 U mL–1 ESGRO]. Two weeks post-transfection, multiple compact colonies were apparent (mean = 2195; SEM = 166; n = 3), which were 95% alkaline phosphatase-positive and ~80% expressed the OCT4-GFP reporter. Reverse transcription-PCR showed that these colonies expressed high levels of endogenous OCT4, SOX2, NANOG, REX1, UTF1, CDH1, and TDH. The cultures were passaged by trypsin disaggregation, followed by seeding on fresh feeders at density 10 × 103 cells cm–2. The established cell lines proliferated as compact, mouse iPSC-like colonies that retained their OCT4 reporter expression as well as the expression of the endogenous pluripotency genes for at least 30 passages. The expression of the transgenes was persistent and showed that no silencing had occurred, even in long-term culture. When subjected to in vitro differentiation protocols, the putative iPSC formed mainly large trophectodermal (TE) vesicles (positive for TE markers CDX2, PAG, and HAND1), fibroblast-like, and neuronal-like cells. These cells still expressed the transgenes as well as most endogenous pluripotency markers, demonstrating limited differentiation capacity. Because the stable transgene expression and the suboptimal culture conditions are the most likely causes of this limited differentiation potential, we are currently working on generating transgene-free iPSC lines under improved cell culture conditions.
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Dykxhoorn, Derek M., Xiaoying Tong, Nicholas C. Gosstola, and Xue Zhong Liu. "Derivation of iPSC line UMi029-A bearing a hearing-loss associated variant in the SMPX gene." Stem Cell Research 54 (July 2021): 102405. http://dx.doi.org/10.1016/j.scr.2021.102405.
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Rozelle, Sarah S., Brenden W. Smith, Efthymia Melista, Ehimen Aneni, Paola Sebastiani, Clinton T. Baldwin, Abdulrahman Alsultan, et al. "Induced Pluripotent Stem Cell Modeling of Sickle Cell Anemia." Blood 120, no. 21 (November 16, 2012): 3233. http://dx.doi.org/10.1182/blood.v120.21.3233.3233.
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Abstract Abstract 3233 As they can be generated from the somatic cells of any individual, induced pluripotent stem cells (iPSC) represent renewable, potentially unlimited cell sources that circumvent the possibility of inappropriate immune response and open the door to the advent of patient-specific, personalized medicine. Disease-specific iPSCs have the potential to elucidate disease mechanisms, revolutionize drug discovery, and improve patient care. We have built a large library of sickle cell disease-specific iPSCs containing more than 100 individual lines from both African American and Saudi Arab patients with different HbS gene haplotypes and HbF-modulating quantitative trait loci (QTL) genotypes. The differentiation of these lines into the erythroid lineage offers a novel opportunity to study erythroid development, the regulation of globin switching, small molecule drug development and the modeling of red blood cell linked diseases in vitro. Although several teams have published proof-of-principle examples for the derivation of hematopoietic cells from pluripotent stem cells, these protocols are technically demanding and result in the production of limited numbers of cells. Our conceptual approach has been to mimic the natural sequences of development in vitro in order to derive the range and number of cell types needed for the creation of a robust iPSC-based platform. We have developed a novel, chemically defined and feeder-free methodology for the production of large numbers of functionally mature red blood cells (RBCs) from both normal and disease-specific human iPSCs. This protocol utilizes a 2D/adherent approach and eliminates the need for embryoid body formation or xenogeneic agents resulting in a shorter production time (∼10 days). Large numbers of clinically relevant, high purity hematopoietic cells can be generated such that 15,000 cells yield 1 billion cells in two weeks. This protocol produces bipotential megakaryocyte-erythroid progenitors (MEPs) that co-express the surface markers CD235 (red cells) and CD41 (megakaryocytes) and demonstrate expression of accepted panels of both erythroid and megakaryocyte-specific genes. Use of an erythroid maturation media results in efficient maturation of MEPs to erythrocytes. Due to this novel approach and the robust nature of the methodology, we are able to generate large numbers of functionally mature RBCs that produce hemoglobin, respond to oxygen deprivation, and enucleate. Furthermore, these human iPSC-derived directly differentiated erythroid-lineage cells engraft robustly in Nod-SCID-Gamma (NSG) immunocompromised mice and demonstrate detectable chimerism in peripheral blood. Importantly, these cells respond to hydroxyurea (HU), the only FDA approved drug that increases HbF levels in sickle cell anemia. Our goals are to use these cells to further understand hemoglobin switching in carriers of varied HbS haplotypes and to harness our library of sickle cell disease-specific lines in combination with the developed differentiation protocol in order to create correlations between genetics and response to new and available HbF inducing agents, furthering the clinician's capability to personalize treatment plans for each patient. Disclosures: No relevant conflicts of interest to declare.
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Blanchette, Alexander D., Sarah D. Burnett, Fabian A. Grimm, Ivan Rusyn, and Weihsueh A. Chiu. "A Bayesian Method for Population-wide Cardiotoxicity Hazard and Risk Characterization Using an In Vitro Human Model." Toxicological Sciences 178, no. 2 (October 20, 2020): 391–403. http://dx.doi.org/10.1093/toxsci/kfaa151.
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Abstract Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes are an established model for testing potential chemical hazards. Interindividual variability in toxicodynamic sensitivity has also been demonstrated in vitro; however, quantitative characterization of the population-wide variability has not been fully explored. We sought to develop a method to address this gap by combining a population-based iPSC-derived cardiomyocyte model with Bayesian concentration-response modeling. A total of 136 compounds, including 54 pharmaceuticals and 82 environmental chemicals, were tested in iPSC-derived cardiomyocytes from 43 nondiseased humans. Hierarchical Bayesian population concentration-response modeling was conducted for 5 phenotypes reflecting cardiomyocyte function or viability. Toxicodynamic variability was quantified through the derivation of chemical- and phenotype-specific variability factors. Toxicokinetic modeling was used for probabilistic in vitro-to-in vivo extrapolation to derive population-wide margins of safety for pharmaceuticals and margins of exposure for environmental chemicals. Pharmaceuticals were found to be active across all phenotypes. Over half of tested environmental chemicals showed activity in at least one phenotype, most commonly positive chronotropy. Toxicodynamic variability factor estimates for the functional phenotypes were greater than those for cell viability, usually exceeding the generally assumed default of approximately 3. Population variability-based margins of safety for pharmaceuticals were correctly predicted to be relatively narrow, including some below 10; however, margins of exposure for environmental chemicals, based on population exposure estimates, generally exceeded 1000, suggesting they pose little risk at current general population exposures even to sensitive subpopulations. Overall, this study demonstrates how a high-throughput, human population-based, in vitro-in silico model can be used to characterize toxicodynamic population variability in cardiotoxic risk.
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Mohamed, Aisha, Theresa Chow, Jennifer Whiteley, Amanda Fantin, Kersti Sorra, Ryan Hicks, and Ian M. Rogers. "Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions." Cells 10, no. 1 (December 31, 2020): 49. http://dx.doi.org/10.3390/cells10010049.
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The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.
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Zhang, Wei, Hanning Wang, Shaopeng Zhang, Liang Zhong, Yanliang Wang, Yangli Pei, Jianyong Han, and Suying Cao. "Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway." International Journal of Molecular Sciences 19, no. 2 (February 8, 2018): 509. http://dx.doi.org/10.3390/ijms19020509.
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Ma, Dongrui, Murni Tio, Shin Hui Ng, Li Zeng, Christina Ying Yan Lim, Yi Zhao, and Eng King Tan. "Derivation of human induced pluripotent stem cell (iPSC) line with LRRK2 gene R1398H variant in Parkinson's disease." Stem Cell Research 18 (January 2017): 48–50. http://dx.doi.org/10.1016/j.scr.2016.12.014.
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Maddineni, Sainiteesh, John L. Silberstein, and John B. Sunwoo. "Emerging NK cell therapies for cancer and the promise of next generation engineering of iPSC-derived NK cells." Journal for ImmunoTherapy of Cancer 10, no. 5 (May 2022): e004693. http://dx.doi.org/10.1136/jitc-2022-004693.
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Adoptive cell therapy is a rapidly advancing approach to cancer immunotherapy that seeks to facilitate antitumor responses by introducing potent effector cells into the tumor microenvironment. Expanded autologous T cells, particularly T cells with engineered T cell receptors (TCR) and chimeric antigen receptor-T cells have had success in various hematologic malignancies but have faced challenges when applied to solid tumors. As a result, other immune subpopulations may provide valuable and orthogonal options for treatment. Natural killer (NK) cells offer the possibility of significant tumor clearance and recruitment of additional immune subpopulations without the need for prior antigen presentation like in T or B cells that could require removal of endogenous antigen specificity mediated via the T cell receptor (TCR and/or the B ecll receptor (BCR). In recent years, NK cells have been demonstrated to be increasingly important players in the immune response against cancer. Here, we review multiple avenues for allogeneic NK cell therapy, including derivation of NK cells from peripheral blood or umbilical cord blood, the NK-92 immortalized cell line, and induced pluripotent stem cells (iPSCs). We also describe the potential of engineering iPSC-derived NK cells and the utility of this platform. Finally, we consider the benefits and drawbacks of each approach and discuss recent developments in the manufacturing and genetic or metabolic engineering of NK cells to have robust and prolonged antitumor responses in preclinical and clinical settings.
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Lesteven, Elodie, Doriane Pognant, Valerie Vanneaux, Jérome Larghero, Olivier Feraud, Annelise Bennaceur-Griscelli, Emmanuelle Verger, et al. "Impact of the JAK2V617F Mutation on the Hemato-Endothelial Differentiation in an Induced Pluripotent Stem Cells (iPSC) Model." Blood 124, no. 21 (December 6, 2014): 4570. http://dx.doi.org/10.1182/blood.v124.21.4570.4570.
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Abstract Background: Philadelphia-negative Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by the acquisition of genetic alterations, the most frequent being the JAK2V617F mutation. Clinical complications include thrombo-hemorrhagic events and progression to either myelofibrosis or acute leukemia. MPNs are also associated with extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. Such neo-angiogenesis has been reported to involve the development of new microvessels through the local expansion of endothelial cells (ECs). Though JAK2V617F mutation was detected in ECs of hepatic venules of MPN patients with Budd-Chiari syndrome and in ECs derived from splenic capillaries, the presence of JAK2V617F in the endothelial lineage remains debatable and demonstration of hematopoietic and endothelial differentiation from progenitor cells harboring this mutation is lacking. We used a JAK2V617F iPSC line to assess the impact of this mutation on hematopoietic and endothelial differentiation. Methods: Hematopoietic and endothelial differentiation of an iPSC line harboring heterozygous JAK2V617F mutation were performed in two protocols: after derivation of embryoid bodies or with a MatrigelTMmatrix based protocol. Endothelial differentiation was performed after CD34+ or CD144+ cell sorting in classical endothelial conditions. Cells collected after differentiation in endothelial condition were evaluated for endothelial lineage surface markers, von Willebrand factor (vWF) expression, response to TNFα, and microvessel formation. Hematopoietic differentiation was assessed by flow cytometry and by clonogenic assay in methylcellulose with or without rh-EPO and rh-TPO. Results: After 6 days of differentiation in both protocols the emergence of CD34+ cells harboring the characteristics of an hemato-endothelial bipotent stem cell was observed: the phenotype of these cells was CD34+/CD90hi/CD38neg with partial expression of hematopoietic stem cell (HSC) markers like CD49f, CD117 (c-kit receptor), CXCR-4 but also of endothelial markers including CD31 (PECAM-1), CD309 (KDR) and CD144 (VE-cadherin); markers of hematopoietic commitment (CD43 or CD135) were absent. When transferred in an endothelial medium, CD34+ or CD144+ sorted derived cells were able to produce endothelial cells expressing CD54 (ICAM-1), CD31, CD144 and CD309, whereas the same cells produced the different hematopoietic lineages when maintained in hematopoietic differentiation protocol. PCR analyses confirmed that both cell types (endothelial and hematopoietic) harbored the JAK2V617F mutation and that the JAK2gene was expressed. Functionally, JAK2V617F-mutated endothelial cells derived from iPSCs expressed vWF in Weibel Palade bodies, responded normally to TNFα with the induction of CD106 (VCAM-1) expression and were able to generate micro-vessels on a Matrigel layer. Taken together these features indicate that the presence of the JAK2V617F mutation does not hamper endothelial specification or endothelial cells functionality. In contrast, the hematopoietic cells derived from the same JAK2V617F iPSC presented with functional abnormalities reminiscent of MPN such as cytokine-independent growth of erythroid and megakaryocytic colonies. Conclusion: This iPSC model provides new evidence for the possibility to generate a bipotent stem cell, able to produce both functional endothelial and hematopoietic lineages harboring the JAK2V617F mutation, from a common JAK2-mutated pluripotent stem cell. Our iPSC differentiation protocol also provides a new model to study the impact of the presence of JAK2V617F in ECs and could represent an effective tool for the screening of molecules antagonizing the pro-thrombotic or pro-hemorrhagic events in a JAK2V617F context. Disclosures No relevant conflicts of interest to declare.
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de la Cruz, Berta Marcó, Yicheng Ding, Veronica McInerney, Janusz Krawczyk, Yin Lu, Guangming Yang, Xiaohong Qian, et al. "Derivation of two iPSC lines from a sporadic ASD patient (NUIGi033-A) and a paternal control (NUIGi034-A)." Stem Cell Research 44 (April 2020): 101722. http://dx.doi.org/10.1016/j.scr.2020.101722.
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Chhabra, Arvind. "Derivation of Human Induced Pluripotent Stem Cell (iPSC) Lines and Mechanism of Pluripotency: Historical Perspective and Recent Advances." Stem Cell Reviews and Reports 13, no. 6 (September 16, 2017): 757–73. http://dx.doi.org/10.1007/s12015-017-9766-9.
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Ding, Yicheng, Berta Marcó de la Cruz, Veronica McInerney, Yin Lu, Guangming Yang, Xiaohong Qian, Weidong Li, et al. "Derivation of iPSC lines from three young healthy donors of Caucasian origin (NUIGi035-A; NUIGi036-A; NUIGi037-A)." Stem Cell Research 49 (December 2020): 102101. http://dx.doi.org/10.1016/j.scr.2020.102101.
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Zimmer, Bastian, Osefame Ewaleifoh, Oliver Harschnitz, Yoon-Seung Lee, Camille Peneau, Jessica L. McAlpine, Becky Liu, et al. "Human iPSC-derived trigeminal neurons lack constitutive TLR3-dependent immunity that protects cortical neurons from HSV-1 infection." Proceedings of the National Academy of Sciences 115, no. 37 (August 28, 2018): E8775—E8782. http://dx.doi.org/10.1073/pnas.1809853115.
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Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/β– and -λ–inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti–HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-β but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/β receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.
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Kondrateva, Ekaterina, Anna Demchenko, Yana Slesarenko, Matvey Yasinovsky, Elena Amelina, Viacheslav Tabakov, Ekaterina Voronina, Alexander Lavrov, and Svetlana Smirnikhina. "Derivation of iPSC line (RCMGi002-A) from dermal fibroblasts of a cystic fibrosis female patient with homozygous F508del mutation." Stem Cell Research 53 (May 2021): 102251. http://dx.doi.org/10.1016/j.scr.2021.102251.
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Zhang, Shaokun, Lidi Liu, Yang Hu, Zhenshan Lv, Qiao Li, Weiquan Gong, Hui Sha, and Hong Wu. "Derivation of human induced pluripotent stem cell (iPSC) line from a 79 year old sporadic male Parkinson's disease patient." Stem Cell Research 19 (March 2017): 43–45. http://dx.doi.org/10.1016/j.scr.2016.12.025.
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Fernandes, Sophia, Prajakta Shinde, Nikhat Khan, Sanjay Singh, Shakti Vardhan, Velu Nair, Vaijayanti Kale, and Lalita Limaye. "Derivation of human iPSC line NCCSi002-A from umbilical cord blood (UCB) CD34 + cells of donor from Indian ethnicity." Stem Cell Research 26 (January 2018): 80–83. http://dx.doi.org/10.1016/j.scr.2017.12.006.
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Cichocki, Frank, Ryan Bjordahl, Svetlana Gaidarova, Sajid Mahmood, Ramzey Abujarour, Hongbo Wang, Katie Tuininga, et al. "iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti–PD-1 therapy." Science Translational Medicine 12, no. 568 (November 4, 2020): eaaz5618. http://dx.doi.org/10.1126/scitranslmed.aaz5618.
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The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti–PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an “off-the-shelf” source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a “cold” tumor “hot” by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.
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Smith, Brenden W., Darrell N. Kotton, Gustavo Mostoslavsky, and George J. Murphy. "Pluripotent Stem Cell Modeling of Normal and Abnormal Megakaryocyte Development and Platelet Production." Blood 118, no. 21 (November 18, 2011): 1276. http://dx.doi.org/10.1182/blood.v118.21.1276.1276.
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Abstract Abstract 1276 Thrombocytopenia is a multi-factorial blood disorder characterized by an abnormally low number of circulating platelets that can have devastating effects upon a wide swath of patients independent of age, race, or socioeconomic group. The two major reasons for thrombocytopenia are increased turnover in immune thrombocytopenia purpura (ITP) and decreased production due to bone marrow failure as a result of chemotherapy, aging, or drugs. Even in ITP, there is some evidence that decreased production may play a role in the etiology of the disease. Thus, patients not making enough platelets are usually treated with platelet transfusions, which carry risks of allergic reactions, infections, and eventually sensitization to allo-antigens making patients refractory to transfusions. With these facts in mind, there is a clear need for the development of novel, autologous sources of mature platelets, and the ability to produce patient-specific megakaryocytes from pluripotent stem cells would have a potential therapeutic role. We have developed a novel, excisable reprogramming vector (STEMCCA) capable of generating ‘clinical grade’ induced Pluripotent Stem Cells (iPSC) free of any residual reprogramming transgenes, and have employed this vector in the derivation of both normal and megakaryocyte disease-specific cell lines. To develop a novel source of platelet precursors for hematopoietic and cell-based therapy studies, we have established conditions for the efficient directed differentiation of these lines into a virtually unlimited supply of functional megakaryocyte-lineage cells that express a constellation of accepted megakaryocyte markers, appropriate Wright-Giemsa stained morphology, expected polyploidy via endoreduplication, and both normal and aberrant platelet production. iPSC-derived megakaryocytes were subsequently tagged with viral vectors expressing fluorescent proteins (for quantification of platelet contribution in peripheral blood) and/or luciferase (for in vivo imaging studies) and administered to mouse models via the retro orbital sinus. Transplanted mice were monitored for the presence of the transferred megakaryocytes and resulting platelets via Ly 5.1/5.2 chimerism as well as for the presence of GFP positive cells using FACS analysis. Peripheral blood from these mice was screened at 1 day post transplantation for chimerism and expression of GFP, and at subsequent 2 day time periods when GFP positive cells were noted in order to track the continued viability or death of the megakaryocyte-lineage cells and resulting platelets. Following these cell transfer experiments, the presence of green platelets in the peripheral blood of these mice indicated that the megakaryocyte-lineage cells produced from the directed differentiation of iPSC are indeed viable in vivo and are capable of the production of platelets. The duration of reconstitution and the functionality of the platelets derived from the iPSC generated megakaryocytes as well as those generated from embryonic stem cell (ESC) controls are currently being assessed by quantifying the labeled platelets over time, and carrying out tests of platelet function in vivo (bleeding time) and in vitro (platelet aggregation studies). Our current work focuses on the hypothesis that an iPSC-based system is capable of producing sufficient numbers of fully functional megakaryocytes to ameliorate thrombocytopenia in vivo. The implications of successfully testing this hypothesis are profound, for they suggest that early megakaryocyte and platelet development can be directly evaluated in vitro and, moreover, that megakaryocyte-lineage cells produced from patient-specific, directly differentiated iPSC lines can become a potent source for transfusion studies and regenerative medicine. Disclosures: No relevant conflicts of interest to declare.