Isolation, culture and differentiation of human urine-derived stem cells into smooth muscle cells

doi:10.12307/2025.073

Abstract

Abstract: BACKGROUND: Traditional methods of urinary tract reconstruction are limited by donor scarcity, high complication rates, and suboptimal functional recovery. Tissue engineering strategies offer new directions in this field. Since the urinary tract is mainly composed of muscle tissue, the key is to find suitable seed cells and efficiently induce them to differentiate into smooth muscle cells. Comparative studies on the efficacy of different smooth muscle cell induction regimens are still lacking.
OBJECTIVE: To isolate, culture, and identify human urine-derived stem cells, and to compare the effects of two different induction protocols.
METHODS: Human urine-derived stem cells were isolated from urine samples of 11 healthy adult volunteers by multiple centrifugations. Surface markers were identified by flow cytometry. The multi-directional differentiation potential of human urine-derived stem cells was verified through osteogenic and adipogenic differentiation. Differentiation was induced by transforming growth factor-β1 or transforming growth factor-β1 combined with platelet derived growth factor for 14 days. Immunofluorescence staining and western blot assay were employed to compare the expression differences of smooth muscle-specific proteins (α-SMA and SM22).
RESULTS AND CONCLUSION: (1) Urine-derived stem cells were successfully isolated from the eight urine samples of healthy people. These cells exhibit a “rice grain”-like morphology and possess a robust proliferative capacity. (2) Urine-derived stem cells exhibited high expression of mesenchymal stem cell surface markers (CD73, CD90, and CD44) and extremely low expression of hematopoietic stem cell surface markers (CD34 and CD45). These cells did not express CD19, CD105, and HLA-DR. (3) After osteogenic and adipogenic differentiation, the formation of calcium nodules and lipid droplets was observed, with positive staining results from Alizarin Red S and Oil Red O staining. (4) After 14 days of smooth muscle induction culture, immunofluorescence staining revealed that the smooth muscle differentiation rate of urine-derived stem cells treated with a combination of transforming growth factor-β1 and platelet derived growth factor was significantly higher compared to those treated with transforming growth factor-β1 alone (P < 0.005). (5) After 14 days of smooth muscle induction culture, western blot assay further demonstrated that the expression levels of α-SMA and SM22 in the transforming growth factor-β1/platelet derived growth factor group were significantly elevated compared to those in the transforming growth factor-β1 only group (P < 0.005). These findings confirm that urine-derived stem cells can be non-invasively isolated using multiple rounds of centrifugation. Compared with transforming growth factor-β1 alone, the combination of transforming growth factor-β1 and platelet derived growth factor can improve the efficiency of inducing urine-derived stem cells to differentiate into smooth muscle cells.

Key words: urine-derived stem cell, induced differentiation, smooth muscle cell, transforming growth factor-β1, platelet derived growth factor, tissue engineering

CLC Number:

Chen Jiahui, Dai Xiaoqi, Xu Yangang, Li Yuanchao, Huang Mei, Zhan Yifei, Du Yuxuan, Li Liuqiang, Guo Yaochuan, Bian Jun, Lai Dehui. Isolation, culture and differentiation of human urine-derived stem cells into smooth muscle cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4076-4082.

Figures/Tables 2

2.3 尿源性干细胞的生长曲线尿源性干细胞第1，2天生长缓慢，为潜伏期；第3天开始生长加速，第3-8天分裂迅速为对数生长期；第9天细胞生长开始放缓，细胞分裂活动变慢，进入平台期；第10天逐渐进入衰退期，细胞数量稍有下降，见图3A。 2.4 尿源性干细胞成骨、成脂诱导分化结果在成脂诱导分化培养基中培养14 d后，对尿源性干细胞进行油红O染色，显微镜下观察可见大量脂滴广泛分布于细胞内被染成红色，见图3B，证实尿源性干细胞已成功实现了成脂分化。在成骨诱导分化培养基中培养21 d后，对尿源性干细胞进行茜素红S染色，显微镜下观察可见细胞内出现了明显的钙结节并被染成橘红色，见图3C，证实尿源性干细胞已成功实现了成骨分化。 2.5 尿源性干细胞成平滑肌诱导分化形态学变化尿源性干细胞分别在两种成平滑肌诱导分化培养基中诱导分化14 d，显微镜下观察到细胞形态由规则的“米粒状”“椭圆形”逐渐变为“长梭形”，细胞开始出现一些触角。TGF-β1/PDGF组诱导后的细胞形态呈较规则的均一性改变，细胞呈紧密平行排列，见图4A；TGF-β1组诱导后的细胞排列较松散，部分细胞未呈现均一性改变，见图4B。 2.6 尿源性干细胞诱导分化后平滑肌特异性蛋白的表达免疫细胞化学染色结果显示，TGF-β1组、TGF-β1/PDGF组均可见α-SMA和SM22蛋白阳性表达，TGF-β1/PDGF组荧光信号明显强于TGF-β1组，见图4A，B。通过统计两组诱导后细胞的荧光信号表达来反映细胞分化比率，结果显示TGF-β1/PDGF组细胞分化比率明显高于TGF-β1组(P < 0.005)，见图4C。Western blot结果显示TGF-β1/PDGF组α-SMA和SM22蛋白表达水平显著高于TGF-β1组(P < 0.005)，见图4D，E。"

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