Differentiation of Human Embryonic Stem Cells to Endothelial Progenitor Cells on Laminins in Defined and Xeno-free Systems
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ABSTRACT: A major hurdle for in vitro culturing of primary endothelial cells (ECs) is that they readily de-differentiate, hampering their use for therapeutic applications. Human embryonic stem cells (hESCs) may provide an unlimited cell source; however, most current protocols deriving endothelial progenitor cells (EPCs) from hESCs use direct differentiation approaches albeit on undefined matrices, yet final yields are insufficient. We developed a method to culture monolayer hESCs on stem-cell niche laminin (LN) LN511 or LN521 matrix. Here, we report a chemically defined, xeno-free protocol for differentiation of hESCs to EPCs using LN521 as the main culture substrate. We could generate ~95% functional EPCs defined as VEGFR2+CD34+CD31+VE-Cadherin+. RNA sequencing analyses of hESCs, EPCs and primary HUVECs showed differentiation-related ECs expression signatures, regarding basement membrane composition, cell-matrix interactions, and changes in endothelial lineage markers. Our results may facilitate production of stable ECs for treatment of vascular diseases and in vitro cell modeling.
Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.
Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs. Six samples were analyzed. The gene expression profile of four iPS clones were compared to the H9 human embryonic stem cell line and the parent endothelial progenitor cell line.
Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xenofree culture condition has significantly limited their wide applications. We developed a chemically defined and xeno-free culture condition for culturing and deriving human EPS cells, which can be long-term stably propagated in vitro, as well as preserve their embryonic and extraembryonic developmental potentials.
Project description:Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. In this study, we developed an extracellular matrix culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). (Note: Please see our publication, PMID 20046878, which includes analysis of array data from GSM348001-GSM348004 in conjunction with this series).
Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xenofree culture condition has significantly limited their wide applications. We developed a chemically defined and xeno-free culture condition for culturing and deriving human EPS cells, which can be long-term stably propagated in vitro, as well as preserve their embryonic and extraembryonic developmental potentials.
Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xenofree culture condition has significantly limited their wide applications. We developed a chemically defined and xeno-free culture condition for culturing and deriving human EPS cells, which can be long-term stably propagated in vitro, as well as preserve their embryonic and extraembryonic developmental potentials.
Project description:Developing an effective xeno-free and defined system for human keratinocyte culture has been one of the fundamental measures in current cellular therapy. For keratinocytes, in particular, such system will not only fulfill clinical safety and quality criteria, it will allow for the management of less severe burns and other skin injuries such as chronic wounds. To date, the expansion of autologous keratinocytes in the clinical settings still relies on 3T3 feeder co-culture system. Here we report a completely xeno-free and defined culture system by using human recombinant laminins as feeder replacement. We have thoroughly characterized the cells cultured in this new system both in vitro and in vivo. We found that laminin-511/-521, and the unanticipated laminin-411/-421, but not -332, -111, -121, -211, or LN-221 support adult human skin keratinocytes and could maintain their self-renewal properties comparable to the 3T3 system. We believe our new culture method should facilitate broader use of cultured epithelial cell products for today’s regenerative medicine.
Project description:Analysis of regulators of endothelial progenitor cells (EPCs) differentiation at gene expression level. Results provide information of gene expression pattern and critical biological processing during EPCs differentiation. Taken together, we define the network of regulators of cord blood-derived EPCs during EPCs differentiation, which can be used to identify genes involved in vascular pathology. Furthermore, we select the critical regulators and then observe these selected regulator's functionality in vitro and in vivo. Total RNA obtained from isolated cord blood-derived EPCs at different time points after the beginning of the EPCs culture under endothelial differentiation condition.
Project description:In this study, we sought to examine whether an extracellular matrix (ECM)-based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are (1) the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and (2) the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2-0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.
Project description:Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and hemostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Although human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs, the differentiation and purification processes have not been optimized. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83-01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-β]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3β [GSK3β] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.