title: Human Menstrual Blood-Derived Mesenchymal Cells as New Human Feeder Layer System for Human Embryonic Stem Cells.
abstract: Human embryonic stem cells (hESCs) in general require coculture 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 MBMC feeder layer capacity, hESCs were grown over MBMCs and MEFs under standard conditions. hESC 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. It is 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; mean ± SD, n = 3). Growth factor analysis revealed amplification of the transcripts for FGF-2, BMP4, TGF-β, VEGF, and PEDF by RT-PCR in MBMCs and MEFs before and after inactivation. Furthermore, similar embryoid body formation, size, and morphology were observed in both feeder layers. In addition, EBs expressed marker genes for the three germ layers cultured on both feeder cells. In conclusion, MBMCs are able to maintain hESCs in an undifferentiated state with comparable efficiency to MEFs. Therefore, MBMCs are a suitable alternative to animal-derived feeder layers for growing hESCs.
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Danúbia
authors: D
authors: Silva Dos Santos
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Vanessa Carvalho
authors: VC
authors: Coelho de Oliveira
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Karina Dutra
authors: KD
authors: Asensi
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Leandro
authors: L
authors: Vairo
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Adriana Bastos
authors: AB
authors: Carvalho
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Antonio Carlos
authors: AC
authors: Campos de Carvalho
authors: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.
authors: Regina Coeli Dos Santos
authors: RC
authors: Goldenberg
keywords: Human embryonic stem cells (hESCs); Human feeder layer; Human menstrual blood-derived mesenchymal cells; Undifferentiated state