title: Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.
abstract: 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.
authors.affiliation: WiCell Research Institute, Madison, Wisconsin, USA. markl@cdibios.com
authors.forename: Mark E
authors.initials: ME
authors.lastname: Levenstein
authors.forename: W Travis
authors.initials: WT
authors.lastname: Berggren
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Conard
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Llanas
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Wagner
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: 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
chemical_list: D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2
references.citation: Biochem J. 2000 Jan 1;345 Pt 1:107-13
references.pmid: 10600645
references.citation: Nature. 2007 Aug 30;448(7157):1015-21
references.pmid: 17625568
references.citation: J Clin Invest. 2001 Aug;108(3):357-61
references.pmid: 11489926
references.citation: Nat Biotechnol. 2001 Oct;19(10):971-4
references.pmid: 11581665
references.citation: J Biol Chem. 2002 Apr 26;277(17):14657-65
references.pmid: 11847221
references.citation: Front Biosci. 1998;3:d781-94
references.pmid: 9683641
references.citation: Anal Chem. 2004 Jul 15;76(14):4193-201
references.pmid: 15253663
references.citation: J Biol Chem. 1989 Feb 25;264(6):3514-23
references.pmid: 2521637
references.citation: Biochemistry. 1992 Sep 22;31(37):8876-83
references.pmid: 1390674
references.citation: J Cell Biol. 1992 Nov;119(4):961-75
references.pmid: 1385449
references.citation: J Biol Chem. 1994 Jan 14;269(2):931-5
references.pmid: 8288646
references.citation: J Biol Chem. 1994 Feb 11;269(6):3976-84
references.pmid: 8307953
references.citation: J Biol Chem. 1998 Sep 25;273(39):24979-82
references.pmid: 9737951
references.citation: Science. 1998 Nov 6;282(5391):1145-7
references.pmid: 9804556
references.citation: Langmuir. 2005 Jan 4;21(1):266-71
references.pmid: 15620313
references.citation: Stem Cells. 2005 Mar;23(3):315-23
references.pmid: 15749926
references.citation: Stem Cells. 2005 Apr;23(4):489-95
references.pmid: 15790770
references.citation: Nat Methods. 2005 Mar;2(3):185-90
references.pmid: 15782187
references.citation: Lancet. 2005 May 7-13;365(9471):1636-41
references.pmid: 15885296
references.citation: Biotechnol Bioeng. 2005 Sep 20;91(6):688-98
references.pmid: 15971228
references.citation: Nat Biotechnol. 2006 Feb;24(2):185-7
references.pmid: 16388305
references.citation: Stem Cells. 2006 Mar;24(3):568-74
references.pmid: 16282444
references.citation: Biochem Soc Trans. 2006 Jun;34(Pt 3):442-5
references.pmid: 16709182
references.citation: J Proteome Res. 2006 Jul;5(7):1580-5
references.pmid: 16823965
references.citation: Nat Methods. 2006 Aug;3(8):637-46
references.pmid: 16862139
references.citation: J Cell Biol. 2006 Jul 31;174(3):323-7
references.pmid: 16880267
references.citation: J Cell Biochem. 2006 Nov 1;99(4):1040-51
references.pmid: 16767693
references.citation: Nature. 2000 Oct 26;407(6807):1029-34
references.pmid: 11069186