title: Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II.
abstract: A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-beta and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-beta family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-beta and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-beta in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-beta and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.
authors.affiliation: Andalusian Stem Cell Bank, Instituto de Investigación Biomédica, University of Granada, Granada, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Montes
authors.forename: Gertrudis
authors.initials: G
authors.lastname: Ligero
authors.forename: Laura
authors.initials: L
authors.lastname: Sanchez
authors.forename: Purificación
authors.initials: P
authors.lastname: Catalina
authors.forename: Teresa
authors.initials: T
authors.lastname: de la Cueva
authors.forename: Ana
authors.initials: A
authors.lastname: Nieto
authors.forename: Gustavo J
authors.initials: GJ
authors.lastname: Melen
authors.forename: Ruth
authors.initials: R
authors.lastname: Rubio
authors.forename: Javier
authors.initials: J
authors.lastname: García-Castro
authors.forename: Clara
authors.initials: C
authors.lastname: Bueno
authors.forename: Pablo
authors.initials: P
authors.lastname: Menendez
mesh_terms: D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D007335:Insulin-Like Growth Factor II