TOMA Advanced Biomedical Assays S.p.A., Laboratorio di Ricerca e Sviluppo, Busto Arsizio, Italy.
Cytotherapy. 2013 Nov;15(11):1340-51. doi: 10.1016/j.jcyt.2013.06.019.
First-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.
We set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.
Growth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.
Our findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.
妊娠早期绒毛（CV）是人类间充质基质细胞（hMSC）的理想来源，可用于细胞治疗和再生医学。我们监测了来自 CV 的 hMSC 在长期培养中的遗传稳定性。
我们为小量的原始 CV 样本建立了良好的生产规范冷冻保存程序。分离后，hMSC 在体外培养并分析其生物学终点。通过核型分析、全基因组微阵列比较基因组杂交和微卫星基因分型探索不同传代数目的基因组稳定性。
生长曲线分析显示 CV 来源细胞具有高增殖潜力。免疫表型分析显示表达典型 MSC 标志物，缺乏造血标志物。多能性分析表明可有效分化为脂肪细胞、成骨细胞、软骨细胞，并诱导神经胶质细胞的定向分化。在血管生成实验中，通过血管内皮生长因子刺激后的体外基质胶实验检测到分化为内皮细胞。核型分析、比较基因组杂交微阵列和微卫星基因分型的数据比较早期和晚期 DNA 传代，至少在传代 10 之前没有显示任何基因组变异。在最新的传代中，有 4 例（14 例中的 4 例）出现了非整倍体克隆，就在培养物生长停滞之前。
我们的研究结果表明，hCV-MSC 在长期培养中至少在传代 10 代内遗传稳定，即使经过几轮扩增也有可能获得临床相关数量的 hCV-MSC。在较高的传代中偶尔会出现基因组异常，且总是与自发生长停滞相关。在这种情况下，hCV-MSC 可能适合治疗目的。
