title: Maintenance of human embryonic stem cells in media conditioned by human mesenchymal stem cells obviates the requirement of exogenous basic fibroblast growth factor supplementation.
abstract: Despite the improvements in the human embryonic stem cell (hESC) culture systems, very similar conditions to those used to maintain hESCs on mouse feeders are broadly applied to culture methods based on human feeders. Indeed, basic fibroblast growth factor (bFGF), a master hESC-sustaining factor, is still added in nearly all medium formulations for hESC propagation. Human foreskin fibroblasts (HFFs) and mesenchymal stem cells (MSCs) used as feeders have recently been reported to support hESC growth without exogenous bFGF. However, whether hESCs may be maintained undifferentiated without exogenous bFGF using media conditioned (CM) by human feeders remains elusive. We hypothesize that HFFs and hMSCs are likely to be functionally different and therefore the mechanisms by which HFF-CM and MSC-CM support undifferentiated growth of hESCs may differ. We have thus determined whether HFF-CM and/or MSC-CM sustain feeder-free undifferentiated growth of hESC without exogenous supplementation of bFGF. We report that hMSCs synthesize higher levels of endogenous bFGF than HFFs. Accordingly and in contrast to HFF-CM, MSC-CM produced without the addition of exogenous bFGF supports hESC pluripotency and culture homeostasis beyond 20 passages without the need of bFGF supplementation. hESCs maintained without exogenous bFGF in MSC-CM retained hESC morphology and expression of pluripotency surface markers and transcription factors, formed teratomas, and showed spontaneous and lineage-directed in vitro differentiation capacity. Our data indicate that MSC-CM, but not HFF-CM, provides microenvironment cues supporting feeder-free long-term maintenance of pluripotent hESCs and obviates the requirement of exogenous bFGF at any time.
authors.affiliation: GENYO-Centre Pfizer-University of Granada-Government of Andalucía for Genomic and Oncological Research, Avda de la Ilustración, Granada, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Sánchez
authors.forename: Iván
authors.initials: I
authors.lastname: Gutierrez-Aranda
authors.forename: Gertrudis
authors.initials: G
authors.lastname: Ligero
authors.forename: Miguel
authors.initials: M
authors.lastname: Martín
authors.forename: Verónica
authors.initials: V
authors.lastname: Ayllón
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Real
authors.forename: Verónica
authors.initials: V
authors.lastname: Ramos-Mejía
authors.forename: Clara
authors.initials: C
authors.lastname: Bueno
authors.forename: Pablo
authors.initials: P
authors.lastname: Menendez
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D052816:Foreskin / Q000166:cytology; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*
chemical_list: D016222:Fibroblast Growth Factor 2