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. 2013;5(1):21-35.
Epub 2013 Jan 21.

Human Induced Pluripotent Stem Cell-Derived Endothelial Cells Exhibit Functional Heterogeneity

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Free PMC article

Human Induced Pluripotent Stem Cell-Derived Endothelial Cells Exhibit Functional Heterogeneity

Abdul Jalil Rufaihah et al. Am J Transl Res. .
Free PMC article

Abstract

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.

Keywords: Heterogeneity; angiogenesis; differentiation; endothelial cells; induced pluripotent stem cells.

Figures

Figure 1
Figure 1
Endothelial differentiation, purification, and characterization of hiPSCs. A. Schematic of endothelial differentiation procedure. B. Efficiency of CD31+ expression for 2 different cell lines. C. Quantitative PCR analysis of endothelial markers during differentiation in 2 different hiPSC lines. D. Expression of endothelial markers in purified hiPSC-ECs. *P<0.05 relative to hiPSCs.
Figure 2
Figure 2
Characterization of purified hiPSC-ECs. A. The hiPSC-ECs showed cobblestone morphology and expressed endothelial phenotypic markers. Functionally they could take up ac-LDL, form tube-like structure in matrigel, and upregulate ICAM-1 protein levels in response to TNF-α (*P<0.05 relative to hiPSCs). B. BrdU incorporation assay showed an increase in proliferation in the presence of VEGF and/or bFGF *P<0.05 relative to untreated control group. C. Quantitative gene expression analysis of arterial, venous and lymphatic markers in purified hiPSC-ECs, in comparison to parental hiPSCs. *P<0.05 relative to hiPSCs. Scale bar: 50μm.
Figure 3
Figure 3
Arterial enrichment of hiPSC-ECs using 8Br-cAMP and VEGF-A. A. Schematic of differentiation procedure to generate arterial EC subtype (hiPSC-artECs). B-C. Gene expression of CD31 and CD144 increased throughout the period of differentiation. *P<0.05 relative to day 0 hiPSCs. D. Upregulated gene expression of arterial markers in the presence of 8Br-cAMP. *P<0.05 relative to hiPSC, #P<0.05 relative to CD31+ hiPSC-ECs generated without 8Br-cAMP. E. CD31+ hiPSC-artECs stained positive for arterial marker, EphrinB2 (red). F. Western blot showing enhancement of EphrinB2 in CD31+ hiPSC-ECs generated using 8Br-cAMP with VEGF-A. G. VEGF-Notch signaling pathway genes were significantly upregulated in the CD31+ hiPSC-artECs, in comparison to heterogeneous hiPSC-ECs. *P<0.05 relative to hiPSC-ECs (heterogeneous). H. Immunoblots validate gene expression analysis showing CD31+ hiPSC-artECs expressed Notch-related markers. Scale bar: 50μm.
Figure 4
Figure 4
Enrichment of venous EC subtype using low VEGF-A concentration. A. Schematic diagram of hiPSC differentiation procedure to generate hiPSC-derived venous EC (hiPSC-vEC) subtype. B. Comparison of the gene expression of venous markers; EphB4 and Coup-TFII in CD31+ ECs differentiated using high (50ng/ml) or low (10ng/ml) VEGF-A. *P<0.05 relative to hiPSCs induced with VEGF (50ng/ml). Gene expression of CD31 (C) and CD144 (D) increased significantly throughout the differentiation course. *P<0.05 relative to day 0.
Figure 5
Figure 5
Differentiation of hiPSCs into lymphatic EC subtype using VEGF-C and Ang-1. A. Schematic diagram of hiPSC differentiation procedure to generate hiPSC-derived lymphatic EC (hiPSC-LEC) subtype. Endothelial gene expression of CD31 (B) and CD144 (C) increased during differentiation. Lymphatic markers LYVE-1 (D) and podoplanin (E) increased in gene expression during endothelial differentiation. F. Lymphatic enriched CD31+ (green) hiPSC-LECs stained positive for podoplanin (red). Scale bar: 50μm. *P<0.05 relative to day 0 hiPSCs.
Figure 6
Figure 6
Formation of blood capillaries by hiPSC-ECs in subcutaneous matrigel plugs. A, C. Quantification of total capillary density (*P<0.05 relative to matrigel plug with bFGF only). B, D. Quantification of human-derived capillaries in hiPSC-artEC group (*P<0.05 relative to matrigel with heterogeneous hiPSC-ECs). E. The erythrocytes within neovessels were stained positive for Ter119, a mouse red blood cell marker and not for human blood cell marker glycophorin A. E. Blood capillaries formed by hiPSC-artECs were surrounded by α-SMA positive smooth muscle layer. Scale bar: 50μm.

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