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Am J Transl Res 2013;5(1):21-35
Original Article
Human induced pluripotent stem cell-derived endothelial cells exhibit
functional heterogeneity
Abdul Jalil Rufaihah, Ngan F Huang, Jeanna Kim, Joerg Herold, Katharina S Volz, Tea Soon Park, Jerry C Lee,
Elias T Zambidis, Renee Reijo-Pera, John P Cooke
Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA
94305, USA; Johns Hopkins Institute for Cell Engineering, The Johns Hopkins University School of Medicine, 733
N. Broadway, BRB 755 Baltimore, MD, 21205, USA; Institute for Stem Cell Biology & Regenerative Medicine,
Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, 300
Pasteur Drive, Stanford, CA 94305, USA. *Contributed equally to this work.
Received November 1, 2012; Accepted December 3, 2012; Epub January 21, 2013; Published January 30, 2013
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. (AJTR1211001).
Keywords: Heterogeneity, induced pluripotent stem cells, differentiation, endothelial cells, angiogenesis
Address correspondence to: Dr. John P Cooke, Division of Cardiovascular Medicine, Stanford University, 300
Pasteur Drive, Stanford, CA 94305, USA. Fax: 650-723-8392; Phone: 650-723-6459; E-mail: john.cooke@stanford.
edu
Original Article
Human induced pluripotent stem cell-derived endothelial cells exhibit
functional heterogeneity
Abdul Jalil Rufaihah, Ngan F Huang, Jeanna Kim, Joerg Herold, Katharina S Volz, Tea Soon Park, Jerry C Lee,
Elias T Zambidis, Renee Reijo-Pera, John P Cooke
Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA
94305, USA; Johns Hopkins Institute for Cell Engineering, The Johns Hopkins University School of Medicine, 733
N. Broadway, BRB 755 Baltimore, MD, 21205, USA; Institute for Stem Cell Biology & Regenerative Medicine,
Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, 300
Pasteur Drive, Stanford, CA 94305, USA. *Contributed equally to this work.
Received November 1, 2012; Accepted December 3, 2012; Epub January 21, 2013; Published January 30, 2013
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. (AJTR1211001).
Keywords: Heterogeneity, induced pluripotent stem cells, differentiation, endothelial cells, angiogenesis
Address correspondence to: Dr. John P Cooke, Division of Cardiovascular Medicine, Stanford University, 300
Pasteur Drive, Stanford, CA 94305, USA. Fax: 650-723-8392; Phone: 650-723-6459; E-mail: john.cooke@stanford.
edu
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