Hematopoietic and Endothelial Differentiation of Human Induced Pluripotent Stem Cells

ArticleinStem Cells 27(3):559-67 · April 2009with45 Reads
DOI: 10.1634/stemcells.2008-0922 · Source: PubMed
Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.
    • "Recently, a new appreciation of the possibility to create simultaneously different cell types needed for generation of complete blood vessel, emerged. Using human PSCs, ECs were generated together with smooth muscle cells [27][28][29], pericytes [30] or hematopoietic progenitors [6]. However, regardless of the protocol used, one of the most prominent problem in generating iPS-ECs is the rather low percentage of differentiated cells and the need for cell sorting on certain endothelial marker for enrichment and purification of the population. "
    [Show abstract] [Hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSCs) have shown great potential in regenerative medicine and research applications like disease modeling or drug discovery. Endothelium is indispensable for vascular homeostasis, whereas endothelial dysfunction could lead to different diseases. Therefore, generating autologous cells, able to restore the endothelial lining, can be crucial for slowing or reversing certain pathological processes. In the current study we show efficient differentiation of murine iPSCs into endothelial cells (ECs) with stable CD34+/Tie-2+/Sca-1+/CD45- phenotype and proven functionality. iPS-derived ECs (iPS-ECs) were positive for phospho-eNOS and von Willebrand factor, and responded to shear stress with up-regulation of KLF-2, KDR, HO-1, and increased nitric oxide and VEGF production. These cells reacted to cytokine stimulation through increase in VCAM-1 and inflammatory cytokine secretion. iPS-ECs showed also certain progenitor features, like expression of progenitor markers (CD34, Sca-1, c-kit) and high clonogenic potential. The angiogenic capacity of iPS-ECs in spheroid sprouting assay was similar to primary ECs, whereas on Matrigel, tube structures could be formed only in the presence of other support cells. Angiogenic potential of iPS-ECs in vivo, was similar to murine endothelial cell line MS-1. Summarizing, our approach enabled generation of functional progenitor-like ECs, which can be used as a research model.
    Full-text · Article · Aug 2016
    • "The precise timing of specification mechanisms of these presumptive precursors remains poorly understood, and no protocols have reported efficient generation of functional endocardial cells from PSCs to date. In contrast, many protocols have been developed to generate endothelial cells from both mouse and human PSCs (Nourse et al., 2010; Choi et al., 2009; Orlova et al., 2014; Kane et al., 2010; White et al., 2013), which closely resemble endocardial cells both functionally and phenotypically. The basic approach to generating endothelial cells relies on inducing mesoderm via BMP signaling, followed by specification of the endothelial lineages via high levels of VEGF. "
    [Show abstract] [Hide abstract] ABSTRACT: Scientists have studied organs and their development for centuries, and along that path described models and mechanisms explaining the developmental principles of organogenesis. In particular, with respect to the heart, new fundamental discoveries are reported continuously, that keep changing the way we think about early cardiac development. These discoveries are driven by the need to answer long-standing questions regarding the origin of the earliest cells specified to the cardiac lineage, the differentiation potential of distinct cardiac progenitor cells and, importantly, the molecular mechanisms underlying these specification events. As evidenced by numerous examples the wealth of developmental knowledge collected over the years has had an invaluable impact on establishing efficient strategies to generate cardiovascular cell types ex vivo, from either pluripotent stem cells or via direct reprogramming approaches. The ability to generate functional cardiovascular cells in an efficient and reliable manner will contribute to therapeutic strategies aimed at alleviating the increasing burden of cardiovascular disease and morbidity. Here we will discuss the recent discoveries in the field of cardiac progenitor biology and their translation to the pluripotent stem cell model to illustrate how developmental concepts have instructed regenerative model systems in the past and promise to do so in the future. This article is protected by copyright. All rights reserved.
    Full-text · Article · Aug 2016
    • "Generation of ex-vivo multipotent hematopoietic stem cells from ES and iPS cells may serve as an alternative source for long-term in vivo hematopoietic reconstitution and for understanding early stages of hematopoietic development in normal and pathological contexts. Many ES cell lines have been characterized for their hematopoietic potential in different studies but only few iPS cell lines have been characterized in detail [3,5,7] . Lineage-specific differentiation potential varies among different pluripotent stem cells (PSC) [5,9–12] however variations in hematopoietic differentiation among iPS cell lines have not been widely addressed. "
    [Show abstract] [Hide abstract] ABSTRACT: Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.
    Full-text · Article · Mar 2016
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