Balducci Luigi, Blasi Antonella, Saldarelli Marilisa, Soleti Antonio, Pessina Augusto, Bonomi Arianna, Coccè Valentina, Dossena Marta, Tosetti Valentina, Ceserani Valentina, Navone Stefania Elena, Falchetti Maria Laura, Parati Eugenio Agostino, Alessandri Giulio
Stem Cell Res Ther. 2014 May 6;5(3):63. doi: 10.1186/scrt452.
INTRODUCTION: Human adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors. METHODS: hASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells. RESULTS: We found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitro although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene. CONCLUSIONS: Here we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a "hybridoma-like model" of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up).
引言：人脂肪来源的间充质干细胞（hASCs）因其相对易于分离以及分泌大量血管生成因子的能力，正被用于再生医学评估。然而，它们有限的培养寿命可能对临床前研究和治疗应用都构成障碍。为克服这一问题，进行了hASCs永生化处理，以获得体外寿命延长但仍保持其间充质标记物表达及分泌血管生成因子能力的细胞。 方法：hASCs单独用人类端粒酶逆转录酶（hTERT）基因转导，或与SV - 40或HPV E6/E7基因联合转导。通过流式细胞术（FC）确认永生化hASCs系中间充质标记物的表达，通过免疫细胞化学评估分化潜能，并使用ELISA试剂盒评估血管生成因子。利用绿色荧光蛋白（GFP）基因转导获得荧光细胞。 结果：我们发现单独的hTERT未能使hASCs永生化（hASCs - T），而hTERT/SV40（hASCs - TS）或hTERT/HPV E6/E7（hASCs - TE）共转导成功使细胞永生化。hASCs - TS和hASCs - TE均培养长达一年，群体倍增水平（PDL）高达100。未转导的亲本细胞（hASCs - M）与永生化细胞系之间的比较研究表明，hASCs - TS和hASCs - TE均保持间充质表型特征，而分化特性有所降低，尤其是在hASCs - TS中。有趣的是，hASCs - TS和hASCs - TE显示出分泌大量肝细胞生长因子（HGF）和血管内皮生长因子（VEGF）的能力。此外，hASCs - TS和hASCs - TE在体外未显示致瘤特性，尽管检测到一些染色体畸变。最后，hASCs - TS和hASCs - TE系在用GFP基因转导后呈稳定荧光。 结论：在此我们首次证明，hASCs永生化后保持分泌强效血管生成分子的强大能力。通过将hASCs永生化与其旁分泌特性相结合，我们开发了一种hASCs的“杂交瘤样模型”，其在发现和生产用于再生医学（工艺放大）的分子方面可能具有潜在应用。
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