In this study we have developed a three step protocol

In this study, we have developed a three-step protocol to differentiate hESCs to endothelial progenitor endothelin receptor antagonist (EPCs) using human recombinant laminins that are biologically relevant (LN521, LN511, and LN421) to mimic the in vivo endothelial substrata. First, vascular cell lineages emerge from mesoderm during embryogenesis (Huber et al., 2004). The next phase directs these mesoderm-committed cells toward the endothelial lineage (CD34+vascular endothelial growth factor receptor [VEGFR2]+CD31+VE-Cadherin+) (Sahara et al., 2014). At the last stage, to obtain a pure population of ECs, a CD31+ population is purified using CD31-coupled magnetic beads. With this three-step protocol, we were able to obtain a 95% CD34+VEGFR2+CD31+VE-Cadherin+ population after 15 days. Using RNA-sequencing (RNA-seq) technology, we compared transcriptomes of our hESC-derived EPCs with that of human umbilical vein endothelial cells (HUVECs), which are fully mature and the most commonly used human primary ECs. The results confirmed expression of markers in the early endothelial lineage, as well as the presence of some mature markers. In short, our differentiation protocol allows efficient generation of pure EPCs in a chemically defined and xeno-free system, which can be applicable for therapeutic purposes as well as modeling human diseases.
Results
Discussion Our laboratory has produced multiple laminin isoforms as recombinant proteins, and developed chemically defined and xeno-free hESC derivation and culture systems using biologically relevant substrata LN511 or LN521. Both of these laminins, which are developmentally regulated, are also present in other BMs that, additionally, may contain other tissue-specific laminin isoforms (Domogatskaya et al., 2012; Miner et al., 1998). The results of this study demonstrate the possibility to produce high-quality, homogeneous cultures of EPCs from pluripotent hESCs that are cultured on these two laminins found in all subendothelial BM. Our hESC-derived cells were functionally demonstrated by tube formation and DiIAcLDL uptake assays, and expressed specific markers for the endothelial lineage and vasculogenesis. Global transcriptome analysis shed light on the expression of genes involved in cell-cell and cell-matrix interactions, as well as expression signatures during differentiation toward maturity of the endothelial cell lineage (Figure 6). The data revealed that pluripotent hESCs do not produce high amounts of BM components, emphasizing the importance of providing the initial relevant supporting matrix, such as LN521, for hESCs to survive and maintain their pluripotency (Rodin et al., 2014a). hESCs, EPCs, and mature HUVECs did not produce Fibronectin (FN1), Fibulin-1 (FBLN1), Fibrillin-1 (FBN1), or Vitronectin (VTN), suggesting that these proteins are not biologically relevant coating substrata when culturing these cells in vitro. Interestingly, the subendothelium-specific laminin α4 chain did not appear until cells became fully mature. Along the endothelial lineage, our EPCs expressed collagen type IV as the main structural BM component, which is likely to provide structural support, while laminin α5 chain expression was low, possibly because it was added exogenously. When we allowed our cells to mature further after purification, important endothelial-specific BM markers, such as von Willebrand factor, laminin α4 chain, and matrix metallopeptidase MMP1 had an increase in mRNA levels over the 6-week period, reaching levels similar to those of HUVECs. Our results suggested that a switch in ECM composition might occur alongside a faster turnover as the cells progress toward maturation. For example, Perlecan was the main BM component expressed in HUVECs, indicating that this proteoglycan bearing a high affinity for growth factors is needed for EC proliferation during vascular development (Zoeller et al., 2009). Both laminin α4 chain and matrix metallopeptidases MMP1, MMP2, and MMP14 were highly expressed, suggesting that the main laminins LN411 and LN421 in the HUVEC BM undergo a constant turnover.