Human Menstrual Blood Derived Mesenchymal Cells As New Human Feederlayer System For Human Embryonic Stem Cells
Abstract:Human embryonic stem cells (hESCs) ingeneral require co-culture with feeder-layers in order to remain undifferentiated. However, the use of animal-derived feeder-layers is incompatible with the clinical setting. The objective of this work was to investigate whether human menstrual blood-derived mesenchymal cells(MBMCs) can substitute mouse embryonic fibroblasts (MEFs) as a feeder-layer for H9-hESCs. Both feeder cell types were isolated and cultured in DMEM F-12 and high glucose DMEM, respectively. After three passages, they were inactivated with Mitomycin C. To test MBMCs feeder-layer capacity, hESCs were grown over MBMCs and MEFs under standard conditions. hESCs growth, proliferation, survival and maintenance of the undifferentiated state were evaluated. hESCs grown over MBMCs preserved their undifferentiated state presenting standard morphology, expressing Alkaline Phosphatase, transcription factors Oct3/4, Sox2 and Nanog by RT-PCR and SSEA-4 and Oct3/4 by immunofluorescence assays. Itis noteworthy that none of the feeder cells expressed these proteins. The average colony size of the hESCs on MBMCs was higher when compared to MEFs (p<0.05; M±SD, n=3). Growth factors analysis revealed amplification of the transcripts for FGF-2, BMP4, TGF-β1, VEGF and PEDF by RT-PCR in MBMCs and MEFs beforeand after inactivation. Furthermore, similar embryoid body formation, size and morphology were observed in both feederlayers. In addition, EBs expressed marker genes for the three germ layers cultured on both feeder cells. In conclusion, MBMCs are able tomaintain hESCs in an undifferentiated state with comparable efficiency to MEFs. Therefore, MBMCs are a suitable alternative to animal-derivedfeeder-layers for growing hESCs.
Appeared or available online: March 3, 2014