Characterizing Human Pluripotent-Stem-Cell-Derived Vascular Cells for Tissue Engineering Applications

Abstract

Tissue-engineered constructs are rendered useless without a functional vasculature owing to a lack of nutrients and oxygen. Cell-based approaches to reconstruct blood vessels can yield structures that mimic native vasculature and aid transplantation. Vascular derivatives of human induced pluripotent stem cells (hiPSCs) offer opportunities to generate patient-specific therapies and potentially provide unlimited amounts of vascular cells. To be used in engineered vascular constructs and confer therapeutic benefit, vascular derivatives must exhibit additional key properties, including extracellular matrix (ECM) production to confer structural integrity and growth factor production to facilitate integration. In this study, we examine the hypothesis that vascular cells derived from hiPSCs exhibit these critical properties to facilitate their use in engineered tissues. hiPSCs were codifferentiated toward early vascular cells (EVCs), a bicellular population of endothelial cells (ECs) and pericytes, under varying low-oxygen differentiation conditions; subsequently, ECs were isolated and passaged. We found that EVCs differentiated under low-oxygen conditions produced copious amounts of collagen IV and fibronectin as well as vascular endothelial growth factor and angiopoietin 2. EVCs differentiated under atmospheric conditions did not demonstrate such abundant ECM expression, but exhibited greater expression of angiopoietin 1. Isolated ECs could proliferate up to three passages while maintaining the EC marker vascular endothelial cadherin. Isolated ECs demonstrated an increased propensity to produce ECM compared with their EVC correlates and took on an arterial-like fate. These findings illustrate that hiPSC vascular derivates hold great potential for therapeutic use and should continue to be a preferred cell source for vascular construction.

Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

“…There are several methods in the literature that describe the induction of differentiation of ES and iPS cells into ECs (29-37). Depending upon the main objectives of the downstream experiments, the methods for inducing ES and iPS cells could be different (29-37).…”

“…Depending upon the main objectives of the downstream experiments, the methods for inducing ES and iPS cells could be different (29-37). To minimize contamination of unknown cells, we use monoclonal antibodies that recognize the extracellular domains of Flk-1 and VE-cadherin.…”

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells

“…S3 A and B). EFNB2 and EPHB4 were the first identified and most widely used markers for AECs and VECs, respectively (6)(7)(8)(9)(10)(11)(12)(13)17). Specific targeting of the EFNB2 and EPHB4 locus was confirmed by junction PCR and Southern blot analysis (SI Appendix, Evaluation of Candidate Factors for Arteriovenous Specification.…”

“…Previous studies have shown progress in AEC differentiation (6)(7)(8)(9)(10)(11)(12)(13)(14), but the arterial-specific functions in vitro and the protection of ischemic tissue in vivo have not been well demonstrated in the resulting cells. Here, we use single-cell RNA sequencing (RNA-seq) of early mouse AECs and a CRISPR/Cas9-generated EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to develop a protocol for differentiating human pluripotent stem cells into AECs using fully defined culture conditions.…”

Paving the Way Toward Complex Blood-Brain Barrier Models Using Pluripotent Stem Cells

“…Differentiation into vascular cell types is enhanced by low oxygen conditions, which can be used to produce early vascular cells that can be further matured into pericytes and ECs [59,60]. A recent study found that increasing cell seeding density for spontaneous differentiation of EBs from PSCs resulted in cells with a mesenchymal-like morphology, which were supposedly perivascular precursors [61].…”