Investigating cellular identity and manipulating cell fate using induced pluripotent stem cells.
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ABSTRACT:
SUBMITTER: Sugawara T
PROVIDER: S-EPMC3392777 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
Investigating cellular identity and manipulating cell fate using induced pluripotent stem cells.
Sugawara Tohru T Nishino Koichiro K Umezawa Akihiro A Akutsu Hidenori H
Stem cell research & therapy 20120308 2
Induced pluripotent stem (iPS) cells, obtained from reprogramming somatic cells by ectopic expression of a defined set of transcription factors or chemicals, are expected to be used as differentiated cells for drug screening or evaluations of drug toxicity and cell replacement therapies. As pluripotent stem cells, iPS cells are similar to embryonic stem (ES) cells in morphology and marker expression. Several types of iPS cells have been generated using combinations of reprogramming molecules and ...[more]
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Project description:PurposeTo model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method.MethodsBoth normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A "self-cleaving" peptides, using a single polycistronic lentiviral vector coexpressing four transcription factors (Oct 4, Sox2, Klf4, and Myc) to yield iPSC. These iPS cells were characterized by immunofluorescence detection using of stem cell markers (SSEA4, Oct4, and Sox2). The mRNA sequencing was performed and the datasets were analyzed using ingenuity pathways analysis (IPA) software.ResultsThe generated stem cell-like clones expressed the pluripotency markers, SSEA4, Oct4, Sox2, Tra-1-60, and also expressed pax6. Our transcriptome analysis showed 4300 genes, which had 2-fold change and 870 genes with a q-value of <0.05 in keratoconus iPSC compared to normal iPSC. One of the genes that showed difference in KC iPSC was FGFR2 (down-regulated by 2.4 fold), an upstream target of Pi3-Kinase pathway, was further validated in keratoconus corneal sections and also KC iPSC-derived keratocytes (down regulated by 2.0-fold). Both normal and KC-derived keratocytes expressed keratocan, signature marker for keratocytes. KC iPSC-derived keratocytes showed adverse growth and proliferation and was further confirmed by using Ly2924002, a PI3k inhibitor, which severely affected the growth and differentiation in normal iPSC.ConclusionsBased on our result, we propose a model for KC in which inhibition FGFR2-Pi3-Kinase pathway affects the AKT phosphorylation, and thus affecting the keratocytes survival signals. This inhibition of the survival signals could be a potential mechanism for the KC-specific decreased cell survival and apoptosis of keratocytes.
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Project description:Studies of human germ cell development are limited in large part by inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells (PSCs) in vitro, differentiation of human germ cells from PSCs in vivo has not been reported. Here, we tested whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem cells (iPSCs). For this purpose, we derived integration-free iPSCs via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell-specific mRNA, VASA (OSKMV). All iPSC lines met classic criteria of pluripotency. Moreover, global gene expression profiling did not distinguish large differences between undifferentiated OSKM and OSKMV iPSCs; however, some differences were observed in expression of pluripotency factors and germ cell-specific genes, and in epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPSCs directly into the seminiferous tubules of germ cell-depleted immunodeficient mice revealed divergent fates of iPSCs produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation may contribute to tools for genetic analysis of human germ cell development.
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Project description:Induced pluripotent stem cells (iPSCs) offer an effective platform for studies of human physiology and have revealed new possibilities for disease modeling at the cellular level. These cells also have the potential to be leveraged in the practice of precision medicine, including personalized drug testing. Aortic diseases result in significant morbidity and mortality and pose a global burden to healthcare. Their pathogenesis is mostly associated with functional alterations of vascular components, such as endothelial cells and vascular smooth muscle cells. Drugs that have been proven to be effective in animal models often fail to protect patients from adverse aortic events in clinical studies, provoking researchers to develop reliable in vitro models using human cells. In this review, we summarize the patient iPSC-derived aortic cells that have been utilized to model aortic diseases in vitro. In advanced models, hemodynamic factors, such as blood flow-induced shear stress and cyclic strain, have been added to the systems to replicate cellular microenvironments in the aortic wall. Examples of the utility of such factors in modeling various aortopathies, such as Marfan syndrome, Loeys-Dietz syndrome, and bicuspid aortic valve-related aortopathy, are also described. Overall, the iPSC-based in vitro cell models have shown the potential to promote the development and practice of precision medicine in the treatment of aortic diseases.
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Project description:Parkinson's disease (PD) is an age-related neurodegenerative disease caused by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. As DA neurons degenerate, PD patients gradually lose their ability of movement. To date no effective therapies are available for the treatment of PD and its pathogenesis remains unknown. Experimental models that appropriately mimic the development of PD are certainly needed for gaining mechanistic insights into PD pathogenesis and identifying new therapeutic targets. Human induced pluripotent stem cells (iPSCs) could provide a promising model for fundamental research and drug screening. In this review, we summarize various iPSCs-based PD models either derived from PD patients through reprogramming technology or established by gene-editing technology, and the promising application of iPSC-based PD models for mechanistic studies and drug testing.
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