Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.
Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Aiming at better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic- and two human fibroblast-derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency, including (i) tight colony formation with typical stem cell morphology; (ii) positive expression of alkaline phosphatase, (iii) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses; (iv) POU5F1, NANOG and SOX2 mRNA expression; and (v) in vitro differentiation and expression of germ-layer-specific markers. Our studies also reveal that although HSPG by itself-does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.
Abraham
Sheena
S
Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA 23284-3028, USA.
Riggs
Marion J
MJ
Nelson
Kristina
K
Lee
Vladimir
V
Rao
Raj R
RR
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002853:Chromatography, Liquid; D003114:Colony-Forming Units Assay; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D040901:Proteomics; D015203:Reproducibility of Results
D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D000469:Alkaline Phosphatase