Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

Abstract Purpose With multiple groups generating methods for differentiating induced pluripotent stem cells (hiPSCs) into endothelial cells/endothelial progenitors (ECs/EPs), there is a need to better understand the specific endothelial subtypes that different differentiation protocols produce. This is especially important for accurate tissue modeling as researchers continue to incorporate the endothelium into engineered tissues. Methods We illustrated the heterogeneity of cells produced from different differentiation protocols by focusing on two selected protocols, one driving differentiation exclusively using small molecules (SM Protocol) and one driving differentiation primarily using growth factors (GF Protocol). We characterized the cells through a combination of vasculogenic computational analysis following encapsulation in 3D hydrogels, RNA sequencing, and measurement of soluble factor release. Results Vasculogenic computational analysis indicates that cells from the GF protocol formed more dense and interconnected vasculature compared to cells from the SM protocol. Likewise, RNA-seq analysis showed GF-derived cell enrichment in pathways involved in cell migration and angiogenesis. In addition, GF-derived cells favor differentiation to arterial endothelial cells as well as predisposition to undergoing a partial endothelial-to-mesenchymal transition, whereas SM-derived cells resemble immature progenitors based on higher proliferation rates as well as ECM remodeling. These trends also persisted following extended 3D culture. Conclusions The results demonstrate that despite using the same starting hiPSC population and isolating EPs using the same surface marker, the two differentiation protocols yield highly distinct cell populations. This work highlights the importance of understanding the specific endothelial subtypes produced by different differentiation protocols for the creation of more accurate tissue models. Lay Summary Although multiple groups have created protocols for differentiating human induced pluripotent stem cells (hiPSCs) to endothelial progenitors (EPs), it is not clear if different differentiation protocols generate different endothelial subtypes, matching the variance seen in vivo To that end, we performed an in-depth characterization of two selected hiPSC-EP differentiation protocols, primarily focusing on cells cultured in 3D hydrogels, to better understand the specific endothelial subtypes produced. We found significant differences in both cell maturity and cell phenotype that persisted even after extended culture. Future Work Future work will involve extending the existing study to include additional hiPSC-EP differentiation protocols as well as conducting single-cell RNAseq to better understand the makeup of different cell populations both before and after isolating CD34 + cells. Because smooth muscle cells play an important role in vessel stabilization, we will also incorporate hiPSC-derived smooth muscle cells into the system.