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Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells
Author(s) -
Hou Luqia,
Kim Joseph J,
Wanjare Maureen J,
Patlolla Bhagat,
Coller John,
Natu Vanita,
Hastie Trevor,
Huang Ngan F
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.327.4
Subject(s) - matrigel , induced pluripotent stem cell , extracellular matrix , microbiology and biotechnology , laminin , fibronectin , basement membrane , chemistry , endothelial stem cell , cellular differentiation , stem cell , heparan sulfate , biology , embryonic stem cell , cell , biochemistry , in vitro , gene
As the basement membrane extracellular matrix (ECM) plays an important role in modulating endothelial cell function and phenotype, here we aim to systematically examine the role of combinatorial ECMs on endothelial differentiation using an arrayed microscale platform. ECM microarrays were developed by covalent conjugation of basement membrane proteins (gelatin, fibronectin, laminin, heparan sulfate proteoglycans, collagen IV, and matrigel) and the multi‐component combinations thereof, with a total of 63 unique ECM compositions. Endothelial differentiation was induced on the microarray using human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). We observed significantly higher CD31 expression when cultured on collagen IV + gelatin + heparan sulfate (C+G+H) or collagen IV + heparan sulfate + Laminin (C+H+L), compared to other combinations across three human pluripotent stem cell lines (N=13; p<0.05). This enhancement in endothelial differentiation on an arrayed microsystem was confirmed under conventional cell culture platforms in which a marked relative increase in CD31 expression was observed in C+G+H modified‐dishes (201.1±11.18%, N=5; p<0.01), and C+H+L combination (244.9±9.92%, N=5; p<0.01), compared to collagen IV‐modified dishes (100±0%, N=5). To elucidate the role of cell‐ECM interactions on endothelial differentiation, the temporal expression pattern of endothelial phenotypic genes was matched by the similar expression pattern of integrin subunits β3 (51.69±13.28 fold, N=3; p<0.01), α1 (9.62±2.11 fold, N=3; p<0.01), and αV (4.14±1.5 fold; N=3; p<0.05), relative to undifferentiated ESC. To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody abrogated the enhancement of endothelial differentiation on C+H+L modified‐dishes to 24.38±1.24% (N=3; p<0.01) comparing to control (100±0%). Together, these findings demonstrate that combinatorial ECMs C+H+L and C+G+H, promote higher levels of endothelial differentiation, compared to many single‐factor ECMs, in part through integrin β3‐mediated pathways. This work highlights the importance of combinatorial cell‐ECM interactions in modulating stem cell fate. Support or Funding Information NIH R00HL098688; R01HL127113; R21EB020235‐01; VA Merit Review Award 1I01BX002310; Cardiovascular Institute at Stanford University