Isolation, Characterization, and Safety Evaluation of Human Skin-Derived Precursors from an Adherent Monolayer Culture System
Isolation, Characterization, and Safety Evaluation of Human Skin-Derived Precursors from an Adherent Monolayer Culture System
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从贴壁单层培养系统中分离、表征和安全性评估人皮肤源性前体
DOI：
10.1155/2019/9194560
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发表时间：
2019-08-19
影响因子：
4.3
通讯作者：
Li, Yiming
中科院分区：
文献类型：
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作者：
Dai, Ru;Hua, Wei;Li, Yiming
背景皮肤来源的前体细胞(SKP)在再生医学领域具有广阔的应用前景。一些研究已经从单层培养扩增的成纤维细胞(FBS)中转移培养人SKP(称为tSKP)。在此，我们通过使用聚甲基丙烯酸羟乙酯(Poly-HEMA)处理烧瓶来优化工艺。方法对贴壁单层培养体系中产生的tSKPs进行蛋白表达和分化能力的检测。将表达两种不同荧光蛋白的FBS分别与BrdU或Edu阳性细胞混合培养，检测tSKP微球内的聚集细胞和增殖细胞。为了区分tSKP和FBS，我们比较了它们的表型和转录本。在我们的研究中，还评估了tSKPs和FBS的致瘤性。结果tSKPs表达Versican、FN、Vimentin、Sox2和Nestin。在适当的刺激下，tSKPs可分化为间充质或神经系。虽然这些球体是由增殖细胞和聚集细胞组成的异质群体，但增殖细胞的比率与种植密度有关。从胎牛血清中分离出的tSKP在细胞周期、标志物表达、神经分化潜能和转录谱等方面与胎牛血清不同，尽管两者在某些性质上有部分相似之处。至于肿瘤的发生，tSKPs和FBS都可以被认为是体外和体内的非致瘤性。结论tSKP是异质性群体，具有与传统SKP相似的特征，但又不同于FBS。微球中可能存在的FBS混合物不影响tSKP的生物安全性，因为两者都具有正常的核型和非致瘤性。综上所述，我们认为tSKPs在再生医学中具有潜在的应用前景。
Background Skin-derived precursors (SKPs) are promising candidates for regenerative medicine. Several studies have transcultured human SKPs (termed tSKPs) from fibroblasts (FBs) expanded in monolayer culture. Herein, we optimized the procedure by treating flasks with poly-2-hydroxyethyl methacrylate (poly-HEMA). Methods tSKPs generated from our adherent monolayer culture system were investigated for protein expression and differentiation capacity. The aggregated cells and the proliferative cells within tSKP spheres were detected by mix-culturing FBs expressing two different fluorescent proteins and BrdU- or EdU-positive cells, respectively. To distinguish tSKPs from FBs, we compared their phenotypes and transcriptomes. The tumorigenicity of tSKPs and FBs was also assessed in our study. Results tSKPs expressed Versican, Fibronectin, Vimentin, Sox2, and Nestin. Under appropriate stimuli, tSKPs could differentiate to mesenchymal or neural lineages. While these spheres were heterogeneous populations consisting of both proliferative and aggregated cells, the rate of proliferative cells correlated with a seeding density. tSKPs, isolated from FBs, were distinctive from FBs in cell cycle, marker expression, neural differentiation potential, and transcript profiles despite the two sharing partial similarity in certain properties. As for tumorigenesis, both tSKPs and FBs could be considered as nontumorigenic ex vivo and in vivo. Conclusion tSKPs were heterogeneous populations presenting similar characteristics as traditional SKPs, while being different from FBs. The potential mixture of FBs in spheres did not affect the biosafety of tSKPs, as both of which had normal karyotype and nontumorigenicity. Taken together, we suggested tSKPs had potential applications in regenerative medicine.