Generation and Characterization of an immortalized human mesenchymal stromal cell line model [Illumina adipocyte differentiation]

Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use. Self-renewal and multipotent differentiation capacity are key elements in the increasing interest of this cell type. However, a disadvantage of primary hMSCs is the limited in vitro lifespan, but this can be overcome by introduction of the catalytic subunit of human telomerase reverse transcriptase (TERT). In this paper, the generation and characterization of a TERT-immortalized, non-tumorigenic human bone marrow-derived stromal mesenchymal model cell line is described. The resulting cell line, iMSC#3, maintained capacity to differentiate into osteoblasts and adipocytes, and growth characteristics comparable to primary hMSCs into after long-term culturing (155 population doublings). A detailed characterization of the mRNA- and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. Furthermore, iMSC#3 showed no major differences from hMSCs regarding surface marker expression. The cell line had a normal karyotype, and high-resolution array comparative genomic hybridization confirmed that the cells in general had normal copy number. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding methylation profiles were more diverse. In conclusion, a human bone marrow-derived stromal cell line was developed, which can be used for basic studies of mesenchymal cells and to functionally study transgenes that might play a role in oncogenic transformation. Comparison of gene expression upon adipocyte differentiation of iMSC#3

人类间充质基质细胞（human mesenchymal stromal cells, hMSCs）展现出巨大的临床与实验应用潜力。自我更新与多向分化潜能是该类细胞受到日益广泛关注的核心要素。然而，原代hMSCs存在体外增殖寿命有限的缺陷，但可通过导入人端粒酶逆转录酶（human telomerase reverse transcriptase, TERT）的催化亚基来克服这一局限。本文报道了一株经TERT永生化的非致瘤性人骨髓来源间充质基质模式细胞系的构建与鉴定。所得细胞系iMSC#3在长期培养（155次群体倍增）后，仍维持了向成骨细胞与脂肪细胞分化的能力，且生长特性与原代hMSCs相当。对脂肪细胞分化过程中的mRNA与microRNA转录组进行的详细分析显示，iMSC#3可在分子水平重现该分化过程。此外，iMSC#3的表面标志物表达与hMSCs无显著差异。该细胞系具有正常核型，高分辨阵列比较基因组杂交（high-resolution array comparative genomic hybridization）结果证实，细胞整体拷贝数处于正常范围。永生化与原代hMSCs的基因表达谱较为相似，而对应的甲基化谱则更为多样。综上，本研究成功构建了一株人骨髓来源间充质基质细胞系，可用于间充质细胞的基础研究，以及功能研究潜在参与致癌转化的转基因。对比iMSC#3在脂肪细胞分化过程中的基因表达