Scalable and stable human pluripotent stem cell-derived microvascular-like endothelial cells for cardiac regeneration and disease modelling applications
Authors: Majid Q , Talman V , Harding S E , Foldes G 
Published: November 2023

Abstract

Introduction
The microvasculature is a vital constituent of the adult human vasculature 1. Cardiac tissue engineering, in vitro models of cardiac remodelling and regeneration, and disease models of the microvasculature would benefit from the inclusion of well-characterised, phenotypically stable microvascular-like endothelial cells (ECs) that could be generated at scale without xenogeneic reagents. Primary microvascular ECs have been utilised for such applications however, they are susceptible to passaging-induced senescence². Moreover, alternative solutions including current protocols for the derivation of human pluripotent stem cell-derived ECs (hPSC-ECs) generate cells with a heterogenous vascular identity that often transdifferentiate into non-EC lineages³.

Purpose
The purpose of this study was to create a 3D, xenogeneic-free differentiation protocol that yielded stable hPSC-derived microvascular-like ECs (hPSC-MVECs) at scale.

Methods
Endothelial differentiation of hPSCs was conducted in 2D adherent monolayers and within stirred 3D bioreactors for 12 days. Thereafter, CD31pos ECs were isolated via fluorescence-activated cell sorting (FACS) and cultured for a further 14 days. Gene expression analysis of hPSCs undergoing 2D and 3D differentiation was evaluated via RT-qPCR. The day 26 hPSC-ECs were treated with VEGFA (0 – 50 ng/ml) for a further 7 days and evaluated for CD31 expression via high-content image analysis. The angiogenic profiles of 2D-, 3D-, and 3D-hPSC-ECs cultured in high concentration VEGFA (herein, 3D+V), relative to primary Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs) was assessed using the Proteome Profiler Human Angiogenesis Array. Transcriptomic analysis of these EC populations was subsequently conducted via single-cell RNA sequencing (scRNA-seq).

Results
Complex vascular clusters emerged by day 12 of the 3D differentiation protocol (fig. 1). The emergent 3D hPSC-ECs had a comparable expression of PECAM1 and CDH5 relative to 2D-derived hPSC-ECs however, VEGFA expression was significantly reduced (P&lt;0.01, fig. 2A). VEGFA treatment at 50 ng/ml yielded phenotypically stable hPSC-ECs (3D+V, fig. 2B). Principal component analysis of the angiogenic factors assayed for revealed 3D+V hPSC-ECs clustered with HMVEC-Cs (fig. 2C). scRNA-seq identified a subset of 3D-derived hPSC-ECs expressing genes associated with microvascular vessels (GMFG, PLVAP), capillary ECs (RGCC, GLUL), and angiogenic tip cells (ESM1, HSPG2).

Conclusion
The scalable and xenogeneic-free 3D protocol outlined within allows for the attainment of hPSC-derived microvascular-like ECs with high angiogenic potential. The absence of Matrigel enhances the translational capacity of the hPSC-MVECs arising from this protocol that could now be derived from patient-specific hPSC lines for evaluation in pre-clinical models of post-myocardial infarction cardiac regeneration or for in vivo models of cardiac remodelling and microvasculature disease.