Zhao Guifang, Liu Feilin, Lan Shaowei, Li Pengdong, Wang Li, Kou Junna, Qi Xiaojuan, Fan Ruirui, Hao Deshun, Wu Chunling, Bai Tingting, Li Yulin, Liu Jin Yu
Department of Pathobiology, Key Laboratory of Ministry of Education, College of Basic Medicine, Jilin University, Changchun, 130021, P.R. China.
Department of Toxicology, School of Public Health, Jilin University, Changchun, 130021, P.R. China.
Stem Cell Res Ther. 2015 Mar 19;6(1):38. doi: 10.1186/s13287-015-0031-3.
Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing.
hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments.
hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing.
Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.
成功的干细胞治疗依赖于大规模生成干细胞并将其维持在增殖性多能状态。本研究旨在建立一种用于大规模生成人脐带华通氏胶间充质干细胞（hWJ-MSC）的三维培养系统，并研究此类hWJ-MSC在促进皮肤伤口愈合方面的自我更新活性、基因组稳定性和多谱系分化潜能。
将hWJ-MSC接种于明胶微珠上，并在转瓶中培养（三维培养）。通过体内和体外实验，研究并比较了细胞增殖、核型分析、表面标志物表达、多能分化（成脂、成软骨和成骨潜能）、核心转录因子（OCT4、SOX2、NANOG和C-MYC）的表达，以及它们在加速皮肤伤口愈合方面的功效，并与平板培养（二维培养）获得的hWJ-MSC进行比较。
在三维培养中，hWJ-MSC附着于明胶微珠并在其上增殖，在第8至14天，0.5克微珠上的细胞数量最多可达1.1 - 1.30×10⁷个；相比之下，二维培养获得的hWJ-MSC在第6至10天，24孔板中每孔最多可达6.5 - 11.5×10⁵个细胞。三维培养获得的hWJ-MSC掺入的EdU显著更多（P<0.05），增殖指数也显著更高（P<0.05）。免疫荧光染色、实时PCR、流式细胞术分析和多能性检测表明，三维培养获得的hWJ-MSC保留了与二维培养获得的细胞相似的间充质干细胞表面标志物和多能性潜能。三维培养获得的hWJ-MSC的核心转录因子表达水平也与二维培养的相当。将三维或二维培养获得的hWJ-MSC直接注射到动物体内，在促进皮肤伤口愈合方面显示出相似的功效。
因此，hWJ-MSC可在明胶微珠中显著扩增，同时保留间充质干细胞表面标志物表达、多能分化潜能和核心转录因子表达。这些细胞在体内也能有效地促进皮肤伤口愈合，其效果与二维培养获得的hWJ-MSC相当。
