Differentiation of human umbilical cord mesenchymal stem cells into chondrocytes induced by direct co-culture with chondrocytes

doi:10.3969/j.issn.2095-4344.2017.01.004

Abstract

Abstract:

BACKGROUND: Both chondrocytes and mesenchymal stem cells (MSCs) can be used to construct tissue-engineered cartilage. Chondrocytes cultured in vitro however are prone to dedifferentiation and difficult to maintain phenotypes, and accordingly, their clinical application is limited.
OBJECTIVE: To explore the effect of human articular chondrocytes and human umbilical cord mesenchymal stem cells (hUC-MSCs) co-culture in vitro on the chondrogenic differentiation of hUC-MSCs and to optimize the co-culture ratio.
METHODS: The hUC-MSCs surface marker was identified by flow cytometry. Human articular chondrocytes and hUC-MSCs were co-cultured at the ratio of 1:1, 3:1 and 5:1. The hUC-MSCs induced by transforming growth factor-beta 1 were set as a positive control group. Human articular chondrocytes and hUC-MSCs cultured alone were set as two negative control groups. The expression level of type II collagen (COL2) was analyzed by immunofluorescence staining. The protein expression of SRY-box9 (SOX9), type I collagen (COL1) and COL2 were determined by western blot. The mRNA levels of SOX9, Col1a1 and Col2a1 were detected by quantitative real-time PCR.
RESULTS AND CONCLUSION: The hUC-MSCs were isolated from the human umbilical cord and identified with flow cytometry. After 28 days of culture, both the co-culture group and the positive control group were observed with positive staining under the immunofluorescence microscope. The Col2a1 mRNA expression level of the positive control group was higher than that of the co-culture group, but the total COL2 protein expression was lower. The Col2a1 mRNA expression level of the co-culture group in 1:1 was higher than that of the co-culture group in 3:1 or 5:1. Col1a1 mRNA and COL1 protein expression levels of the positive control group and co-culture group were lower than those of human articular chondrocyte negative control group. To conclude, the co-culture of hUC-MSCs and human articular chondrocytes significantly induces the hUC-MSC differentiation into chondrocytes and effectively restrains cell fibrosis. The optimal cell ratio in the co-culture system is demonstrated to be 5:1 (hUC-MSCs:chondrocytes). Therefore, the direct co-culture can be an economic way for inducing hUC-MSC differentiation into chondrocytes, which provides reliable seeding cells for cartilage tissue engineering.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Umbilical Cord, Mesenchymal Stem Cells, Chondrocytes, Tissue Engineering

CLC Number:

R394.2

Li Xing-fu, Duan Li, Liang Yu-jie, Zhu Wei-min, Wang Da-ping. Differentiation of human umbilical cord mesenchymal stem cells into chondrocytes induced by direct co-culture with chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 18-24.

Figures/Tables 2

2.1 人脐带间充质干细胞表面抗原标志物检测结果流式细胞仪检测细胞表面抗原的结果显示，人脐带间充质干细胞高表达CD105和CD73，低表达CD34和CD45(图1)，符合间充质干细胞的免疫学特性。 2.2 Ⅱ型胶原的定性检测结果人脐带间充质干细胞空白对照组免疫荧光染色呈阴性，而人关节软骨细胞空白对照组、阳性对照组和共培养组的细胞染色均呈阳性，胞质及细胞外基质均呈现红色荧光(图2)，共培养组的荧光信号最强，且组内无明显差异。 2.3 实时定量PCR检测SOX9、Col1a1和Col2a1 mRNA表达共培养组的SOX9 mRNA表达量均高于阳性对照组(P < 0.05)，3∶1共培养组相对表达量最高(P < 0.05)。另外，空白对照组的SOX9 mRNA表达量均低于阳性对照组(P < 0.05)，人脐带间充质干细胞空白对照组的表达量最低(P < 0.05)(图3A)。 1∶1和5∶1共培养组的Col1a1 mRNA表达量与阳性对照组相比无明显差异(P > 0.05)，但3∶1共培养组的Col1a1 mRNA表达量比阳性对照组高(P < 0.05)，人脐带间充质干细胞空白对照组的Col1a1 mRNA表达量最低(P < 0.05)，人关节软骨细胞空白对照组的Col1a1 mRNA表达量最高(P < 0.05)(图3B)。与空白对照组相比，共培养组和阳性对照组的Col2a1 mRNA表达量较高(P < 0.05)，其中阳性对照组表达最高，人脐带间充质干细胞空白对照组表达量最低(P < 0.05)。共培养组中人脐带间充质干细胞比例增加，Col2a1 mRNA表达量降低(P < 0.05)(图3C)。 2.4 Western-blot检测SOX9、Ⅰ型胶原和Ⅱ型胶原蛋白表达共培养组SOX9 蛋白表达量高于阳性对照组，且组内无明显差异。人关节软骨细胞空白对照组SOX9 蛋白表达量最高，而人脐带间充质干细胞空白对照组SOX9 蛋白表达量最低(图4)。共培养组Ⅰ型胶原蛋白表达量略高于阳性对照组，且组内无明显差异。阳性对照组和人脐带间充质干细胞空白对照组无明显Ⅰ型胶原蛋白表达，而人关节软骨细胞空白对照组Ⅰ型胶原蛋白表达量最高。与空白对照组和阳性对照组相比，共培养组Ⅱ型胶原蛋白表达量较高，且组内无明显差异。"

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