Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.
Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.
Levenstein
Mark E
ME
WiCell Research Institute, Madison, Wisconsin, USA. markl@cdibios.com
Berggren
W Travis
WT
Lee
Ji Eun
JE
Conard
Kevin R
KR
Llanas
Rachel A
RA
Wagner
Ryan J
RJ
Smith
Lloyd M
LM
Thomson
James A
JA
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002845:Chromatography / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism*; D015398:Signal Transduction
D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2
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