title: Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium.
abstract: BACKGROUND
  Bone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation.
  
  
  METHODS
  BMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses.
  
  
  RESULTS
  The ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses.
  
  
  CONCLUSIONS
  This study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. zhenzhen_li_82@163.com.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. shichaohan_burns@163.com.
authors.forename: Shichao
authors.initials: S
authors.lastname: Han
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, No. 1 Xinsi Road, Xi'an, 710038, Shaanxi, China. chinahenry001@163.com.
authors.forename: Xingqin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. fuhan_fmmu@163.com.
authors.forename: Fu
authors.initials: F
authors.lastname: Han
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. xiongxiang_zhu@sina.com.
authors.forename: Xiongxiang
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. zhaozheng_burns@sina.com.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. hongtaowang_xijing@126.com.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. qinzhou_burns@126.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. yunchuan_wang@126.com.
authors.forename: Yunchuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. linlinsu_burns@163.com.
authors.forename: Linlin
authors.initials: L
authors.lastname: Su
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. jihong_shi@sohu.com.
authors.forename: Jihong
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. chaowu_tang@126.com.
authors.forename: Chaowu
authors.initials: C
authors.lastname: Tang
authors.affiliation: Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi'an, 710032, Shaanxi, China. dahaihu_burns@163.com.
authors.forename: Dahai
authors.initials: D
authors.lastname: Hu
mesh_terms: D000328:Adult; D000577:Amides / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D011725:Pyridines / Q000494:pharmacology; D055815:Young Adult; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000577:Amides; D015415:Biomarkers; D017077:Culture Media, Conditioned; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases