Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are 
promising for treatment of vascular diseases. However, hiPSC-ECs purified based 
on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It 
is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may 
be functional benefits of enriching for specific subtypes. Therefore, we sought 
to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate 
whether such enrichment would have functional significance. The hiPSC-ECs were 
generated from differentiation of hiPSCs using vascular endothelial growth 
factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified 
based on positive expression of CD31. Subsequently, we sought to enrich for each 
subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A 
and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower 
concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 
promoted the expression of lymphatic phenotype. Upon FACS purification based on 
CD31+ expression, the hiPSC-EC population was observed to display typical 
endothelial surface markers and functions. However, the hiPSC-EC population was 
heterogeneous in that they displayed arterial, venous, and to a lesser degree, 
lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs 
in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive 
capillary network in this model, by comparison to a heterogeneous population of 
hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs 
produces a diverse population of ECs. Refining the differentiation methods can 
enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing 
neovascularization.