Use of xenofree matrices and molecularly-defined media to control human embryonic stem cell pluripotency: effect of low physiological TGF-beta concentrations.
To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).
Centre National de la Recherche Scientifique, Institut André Lwoff, Villejuif, France. peiffeisa@yahoo.fr
Isabelle
I
Peiffer
Romain
R
Barbet
Yi-Ping
YP
Zhou
Ma-Lin
ML
Li
Marie-Noëlle
MN
Monier
Antoinette
A
Hatzfeld
Jacques A
JA
Hatzfeld
D028341:Activins / Q000494:pharmacology; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003470:Culture Media; D016207:Cytokines / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007621:Karyotyping; D055458:Left-Right Determination Factors; D051379:Mice; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D000418:Albumins; C514249:CER1 protein, human; D003470:Culture Media; D016207:Cytokines; C486399:GREM1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C526161:LEFTY1 protein, human; D055458:Left-Right Determination Factors; D016212:Transforming Growth Factor beta; C422405:activin A; D028341:Activins