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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med    2013, Vol. 7 Issue (3) : 345-353     DOI: 10.1007/s11684-013-0282-2
RESEARCH ARTICLE |
Capacity of human umbilical cord-derived mesenchymal stem cells to differentiate into sweat gland-like cells: a preclinical study
Siming Yang1,2, Kui Ma2, Changjiang Feng2, Yan Wu2, Yao Wang2, Sha Huang1,2, Xiaobing Fu1,2()
1. Institute of Basic Medical Sciences, PLA General Hospital, the PLA Medical College, Beijing 100853, China; 2. Burns Institute, the First Affiliated Hospital, PLA General Hospital, Beijing 100048, China
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Abstract  

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess various advantageous properties, including self-renewal, extended proliferation potential, multi-lineage differentiation potential and capacity for differentiating into sweat gland-like cells in certain conditions. However, little is known about the effect of clinical-grade culture conditions on these properties and on the differentiative potential of hUC-MSCs. In this study, we sought to investigate the properties of hUC-MSCs expanded with animal serum free culture media (ASFCM) in order to determine their potential for differentiation into sweat gland-like cells. We found that primary cultures of hUC-MSCs could be established with ASFCM. Moreover, cells cultured in ASFCM showed vigorous proliferation comparable to those of cells grown in classical culture conditions containing fetal bovine serum (FBS). Morphology of hUC-MSCs cultured in ASFCM was comparable to those of cells grown under classical culture conditions, and hUC-MSCs grown in both of the two culture conditions tested showed the typical antigen profile of MSCs—positive for CD29, CD44, CD90, and CD105, and negative for CD34 and CD45, as expected. Chromosomal aberration assay revealed that the cells were stable after long-term culture under both culture conditions. Like normal cultured MSCs, hUC-MSCs induced under ASFCM conditions exhibited expression of the same markers (CEA, CK14 and CK19) and developmental genes (EDA and EDAR) that are characteristic of normal sweat gland cells. Taken together, our findings indicate that the classical culture medium used to differentiate hUC-MSCs into sweat gland-like cells can be replaced safely by ASFCM for clinical purposes.

Keywords umbilical cord      mesenchymal stem cells      sweat gland      preclinical     
Corresponding Authors: Fu Xiaobing,Email:fuxb@cgw.net.cn; fuxiaobing@vip.sina.com   
Issue Date: 05 September 2013
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-013-0282-2     OR     http://academic.hep.com.cn/fmd/EN/Y2013/V7/I3/345
Fig.1  Panel I: Morphology of hUC-MSCs of two culture groups. (A) Cells of group 1 at passage 1; (B) Cells of group 2 at passage 2; (C) Cells of group 1 at passage 3; (D) Cells of group 2 at passage 3; = 3 for each group; scale bar= 50 μm. Panel II: Expression of surface markers by flow cytometry analysis. The mean percentages of each phenotype were compared between group 1 and group 2. Both the positive percentages of hUC-MSCs for mesenchymal antigens (CD29, CD44, CD90, and CD105) and the mean negative percentages for hematopoietic stem cells (CD34, CD45) showed no significant difference between group 1 and group 2. Markers of sweat gland cells (CEA, CK14) were all negative in group 1 and group 2 ( = 3 for each group).
Fig.2  Proliferative potency of hUC-MSCs in different culture groups determined by MTT analysis (data shown as mean±SEM). Induction groups 1 and 2 represent previous culture conditions groups 1 and 2, respectively; control group represent cells cultured in basic medium. Proliferation of hUC-MSCs in both induction groups was distinctively lower than that in the control group. (*<0.05, = 3 for each group). No distinct difference in the proliferative potency was observed between induction group 1 and group 2.
Fig.3  Morphology of hUC-MSCs in different conditioned induction media. (A) Cells of induction group 1 were cultured for 1 week and their morphology changed gradually; (B) Cells of induction group 2 were also cultured for 1 week and also changed gradually; (C) Cells of induction group 1 differentiated into sweat gland-like cells after 3 weeks of induction; (D) Cells of induction group 2 differentiated into sweat gland-like cells after 3 weeks of induction. ( = 3 for each group; bar= 50 μm.)
Fig.4  Expression of CEA, CK14 and CK19 in different induction groups (mean±SEM) after 3 weeks. The mean expression percentages of sweat gland markers between induction group 1 and 2 showed no significant difference; >0.05, unpaired post-Bonferroni -test; = 5 for each group.
Fig.5  Expression of EDA and EDAR in different induction groups and normal SGCs. (A) RT-PCR analysis showed gene expression profiles (EDA and EDAR) of sweat gland-like cells relative to uninduced hUC-MSCs and normal sweat gland cells. (B) Western blotting analysis was conducted to assess the expression level of EDA and EDAR in each group. (C) Greyscale analysis of RT-PCR bands; expression of EDA and EDAR between groups 1 and 2 revealed no significant difference; >0.05, unpaired post-Bonferroni -test; = 3 for each group. hUC-MSCs were taken as negative control, while normal SGCs were taken as positive control. SGCs, sweat gland cells; hUC-MSCs, human umbilical cord Wharton’s jelly-derived mesenchymal stem cells; EDA, anhidrotic ectodermal dysplasia; EDAR, anhidrotic ectodermal dysplasia receptor.
Fig.6  Karyotype analysis showed no chromosomal alteration. (A) The 10th passage cells in induction group 1; (B)The 10th passage cells in induction group 2.
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