School of Life Science and Engineering, Foshan University, Foshan 528231, China.
Faculty of Science, Kafrelsheikh University, Kafr el-Sheikh 33516, Egypt.
Int J Mol Sci. 2019 Mar 25;20(6):1485. doi: 10.3390/ijms20061485.
Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.
间充质干细胞（MSCs）是细胞治疗最有前途的种子细胞。比较不同来源的 MSCs 的生物学和转录组基因特征，为筛选临床应用的细胞提供了重要依据。本实验主要目的是建立从五种不同犬源（脂肪组织、骨髓、脐带、羊膜和胎盘）分离和培养 MSCs 的方法，并比较 MSCs 的生物学和转录组特征，为犬 MSCs 的临床应用提供依据。我们从中国牧羊犬中分离 MSCs，并进行以下实验：（1）分别计数第 3、6 和 9 代细胞，绘制生长曲线，计算 MSC 群体倍增时间；（2）通过免疫荧光研究 CD34 和 CD44 表面标志物的表达；（3）第 3 代细胞进行成骨和成脂分化实验；（4）使用 RNA 测序进行 MSC 转录组谱分析。所有五种类型的 MSCs 均表现出成纤维细胞样贴壁生长，细胞表面表达 CD44 而不表达 CD34；第 3 代 MSCs 具有最高的增殖活性。脂肪间充质干细胞（AD-MSCs）、胎盘间充质干细胞（P-MSCs）、骨髓间充质干细胞（BM-MSCs）、脐带间充质干细胞（UC-MSCs）和羊膜间充质干细胞（AM-MSCs）的平均群体倍增时间分别为 15.8 h、21.2 h、26.2 h、35 h 和 41.9 h。所有五种类型的 MSCs 均可在体外诱导分化为成脂细胞和成骨细胞，AD-MSC 诱导后 8 天出现脂滴，5 天出现骨形成。然而，与 AD-MSCs 相比，其余 MSC 的多能分化时间更长。MSC 转录组谱分析表明，AD-MSC 和 BM-MSCs 具有最高的同源性，而 P-MSCs 与其他四种类型的 MSCs 差异显著。所有分离的 MSC 均具有 MSC 的主要生物学特征。AD-MSCs 增殖、成脂分化和成骨分化时间最短。
