Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

Coelho de Oliveira,Vanessa Carvalho; Silva dos Santos,Danúbia; Vairo,Leandro; Kasai Brunswick,Tais Hanae; Pimentel,Luiz Alberto Soares; Carvalho,Adriana Bastos; Campos de Carvalho,Antonio Carlos; Goldenberg,Regina Coeli dos Santos

doi:10.3892/etm.2017.4195

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells

Authors:
- Vanessa Carvalho Coelho de Oliveira
- Danúbia Silva dos Santos
- Leandro Vairo
- Tais Hanae Kasai Brunswick
- Luiz Alberto Soares Pimentel
- Adriana Bastos Carvalho
- Antonio Carlos Campos de Carvalho
- Regina Coeli dos Santos Goldenberg
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Affiliations: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941‑902, Brazil, Clínica Luiz Pimentel, Niterói, Rio de Janeiro, RJ 24210‑480, Brazil
Published online on: March 8, 2017 https://doi.org/10.3892/etm.2017.4195
Pages: 1779-1788
Copyright: © Coelho de Oliveira et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F‑12 and low‑glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co‑culture with H9‑hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific ‘undifferentiation’ markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population‑doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription‑quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer‑binding transcription factor 4 (Oct4), Nanog and sex determining region Y‑box 2], and immunofluorescence assays of markers (stage‑specific embryonic antigen‑4 and Oct4) in hESCs co‑cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor‑2, vascular endothelial growth factor, Pigment epithelium‑derived factor and transforming growth factor‑β1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.

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