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Stem Cell Research & Therapy Jun 2019Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In...
BACKGROUND
Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.
METHODS
iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.
RESULTS
In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.
CONCLUSIONS
Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.
Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Embryo Transfer; Embryoid Bodies; Fibroblasts; Humans; Immunohistochemistry; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Mice; Pericytes; Pluripotent Stem Cells; Swine
PubMed: 31248457
DOI: 10.1186/s13287-019-1303-0 -
Nature Biotechnology Aug 2017Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients....
Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
Topics: Animals; Female; Graft Rejection; HLA Antigens; Humans; Killer Cells, Natural; Mice; Pluripotent Stem Cells; Transplants
PubMed: 28504668
DOI: 10.1038/nbt.3860 -
Experimental Biology and Medicine... Apr 2021The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more... (Review)
Review
The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.
Topics: Cell Differentiation; Gene Editing; Genome, Human; Humans; Induced Pluripotent Stem Cells; Organoids; Pluripotent Stem Cells
PubMed: 33467883
DOI: 10.1177/1535370220985808 -
Cell Proliferation Aug 2022The human plutiripotent stem cell registry (hPSCreg) is a global database for human embryonic and induced pluripotent stem cells (hESC, hiPSC). The publicly accessible... (Review)
Review
The human plutiripotent stem cell registry (hPSCreg) is a global database for human embryonic and induced pluripotent stem cells (hESC, hiPSC). The publicly accessible Registry (https://hpscreg.eu) was set up to provide a transparent resource of quality-assessed hPSC lines as well as to increase reproducibility of research and interoperability of data. OBJECTIVES: In this review, we describe the establishment of the Registry and its mission, its development into a knowledgebase for hPSC and the current status of hPSC-focussed databases. The data categories available in hPSCreg are detailed. In addition, sharing and hurdles to data sharing on a global level are described. CONCLUSIONS: An outlook is provided on the establishment of digital representatives of donors using hybrids of data and hPSC-based biological models, and how this can also be used to reposition databases as mediators between donors and researchers.
Topics: Cell Differentiation; Databases, Factual; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells; Registries; Reproducibility of Results
PubMed: 35522426
DOI: 10.1111/cpr.13238 -
Cell Stem Cell Apr 2018The liver, lung, pancreas, and digestive tract all originate from the endoderm germ layer, and these vital organs are subject to many life-threatening diseases affecting... (Review)
Review
The liver, lung, pancreas, and digestive tract all originate from the endoderm germ layer, and these vital organs are subject to many life-threatening diseases affecting millions of patients. However, primary cells from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells thus hold great promise for generating endoderm cells and their derivatives as tools for the development of new therapeutics against a variety of global healthcare challenges. Here we describe recent advances in methods for generating endodermal cell types from human pluripotent stem cells and their use for disease modeling and cell-based therapy.
Topics: Endoderm; Humans; Models, Biological; Pluripotent Stem Cells
PubMed: 29625066
DOI: 10.1016/j.stem.2018.03.016 -
Seminars in Cell & Developmental Biology Jul 2022Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues... (Review)
Review
Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.
Topics: Bone and Bones; Cartilage; Cell Differentiation; Humans; Mesoderm; Neural Crest; Pluripotent Stem Cells
PubMed: 34949507
DOI: 10.1016/j.semcdb.2021.11.024 -
Cell Stem Cell Jul 2019Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in... (Review)
Review
Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.
Topics: Cell Differentiation; Cellular Reprogramming; Embryonic Development; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells; Regenerative Medicine; Transcriptome
PubMed: 31271751
DOI: 10.1016/j.stem.2019.06.010 -
Stem Cells Translational Medicine Nov 2021Brain degeneration and damage is difficult to cure due to the limited endogenous repair capability of the central nervous system. Furthermore, drug development for... (Review)
Review
Brain degeneration and damage is difficult to cure due to the limited endogenous repair capability of the central nervous system. Furthermore, drug development for treatment of diseases of the central nervous system remains a major challenge. However, it now appears that using human pluripotent stem cell-derived neural cells to replace degenerating cells provides a promising cell-based medicine for rejuvenation of brain function. Accordingly, a large number of studies have carried out preclinical assessments, which have involved different neural cell types in several neurological diseases. Recent advances in animal models identify the transplantation of neural derivatives from pluripotent stem cells as a promising path toward the clinical application of cell therapies [Stem Cells Transl Med 2019;8:681-693; Drug Discov Today 2019;24:992-999; Nat Med 2019;25:1045-1053]. Some groups are moving toward clinical testing in humans. However, the difficulty in selection of valuable critical quality criteria for cell products and the lack of functional assays that could indicate suitability for clinical effect continue to hinder neural cell-based medicine development [Biologicals 2019;59:68-71]. In this review, we summarize the current status of preclinical studies progress in this area and outline the biological characteristics of neural cells that have been used in new developing clinical studies. We also discuss the requirements for translation of stem cell-derived neural cells in examples of stem cell-based clinical therapy.
Topics: Animals; Cell- and Tissue-Based Therapy; Induced Pluripotent Stem Cells; Neurodegenerative Diseases; Neurons; Pluripotent Stem Cells; Stem Cell Transplantation
PubMed: 34724724
DOI: 10.1002/sctm.20-0522 -
Cell Stem Cell Nov 2015Embryonic pluripotency can be recapitulated in vitro by a spectrum of pluripotent stem cell states stabilized with different culture conditions. Their distinct... (Review)
Review
Embryonic pluripotency can be recapitulated in vitro by a spectrum of pluripotent stem cell states stabilized with different culture conditions. Their distinct spatiotemporal characteristics provide an unprecedented tool for the study of early human development. The newly unveiled ability of some stem cell types for crossing xeno-barriers will facilitate the generation of interspecies chimeric embryos from distant species, including humans. When combined with efficient zygote genome editing technologies, xenogeneic human pluripotent stem cells may also open new frontiers for regenerative medicine applications, including the possibility of generating human organs in animals via interspecies chimeric complementation.
Topics: Animals; Cell Culture Techniques; Chimera; Humans; Mice; Pluripotent Stem Cells
PubMed: 26544113
DOI: 10.1016/j.stem.2015.10.009 -
Cellular & Molecular Immunology Jan 2014The ever-improving technology to generate induced pluripotent stem cells (iPSCs) has increased their potential use as novel candidates for disease modeling, drug... (Review)
Review
The ever-improving technology to generate induced pluripotent stem cells (iPSCs) has increased their potential use as novel candidates for disease modeling, drug screening, regenerative medicine and cell therapy. Indeed, iPSCs offer extensive capacity for self-renewal without the ethical concerns faced by embryonic stem cells (ESCs). With respect to potential applications in the immune system, many studies provide evidence to support that there are exclusive advantages to using iPSCs over other systems. Both hematopoietic stem cells and several types of mature immune cells have successfully been reprogrammed to iPSCs and vice versa, paving a path toward our ability to effectively model patient-specific diseases and provide potentially alternative cell sources for transfusion medicine. Despite these potential advances, some limitations regarding the use of iPSCs in the clinic still remain, including the immunogenicity of iPSCs and their derivatives, which is currently under debate in the field. In this review, we mainly focus on discussing the recent progress being made in the latest differentiation methods and clinical implications of iPSCs with respect to the immune system. Additionally, current issues regarding the clinical application of iPSCs are addressed, especially the controversy surrounding immunogenicity, along with various other perspectives.
Topics: Animals; Cell- and Tissue-Based Therapy; Humans; Immunotherapy; Induced Pluripotent Stem Cells
PubMed: 24336163
DOI: 10.1038/cmi.2013.62