Guo Xiaoling, Lian Ruiling, Guo Yonglong, Liu Qing, Ji Qingshan, Chen Jiansu
Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China.
Hum Cell. 2015 Jul;28(3):122-32. doi: 10.1007/s13577-015-0113-7. Epub 2015 Mar 10.
Human induced pluripotent stem (iPS) cells can be well maintained by clonal growth. The pluripotent growth of single iPS cells is limited by low survival. To facilitate robust single iPS cells cultured in vitro, half-exchange mTeSR1 medium (HM), whole-exchange medium (WM) and iPS cell-derived conditioned medium (iPS-CM) culture were used. The effects of bFGF and Activin A on the growth of single iPS cells were explored. The dissociation and propagation of single iPS cells also included Accutase enzymatic isolation, Rho-associated protein kinase (ROCK) inhibitor Y27632 protection and high-density single-cell seeding (1 × 10(6) cells/well). CCK-8 assays demonstrated that the viability of clonal iPS cells in mTeSR1 medium and single iPS cells in HM, iPS-CM or WM supplemented with 100 ng/ml bFGF and 10 ng/ml Activin A was significantly higher than that in WM. Annexin v and propidium iodide (PI) assay, Calcein AM and EthD-III double staining also confirmed the similar results. ELISA assays showed that the levels of bFGF and Activin A of single iPS cells in HM and iPS-CM were higher than single iPS cells in WM. Meanwhile, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), quantitative Polymerase Chain Reaction (qPCR), Western Blotting (WB), Immunofluorescence (IF) and karyotype analysis revealed that HM culture was able to maintain undifferentiated markers of Nanog, Klf4, Sox2, Oct4, and did not affect the karyotype of iPS cells. Undifferentiated single iPS cells in HM displayed homogenized growth. These findings demonstrate that bFGF and Activin A are important for the survival and growth of single iPS cells. HM culture system combined Accutase, Y27632 and high-density single-cell seeding can facilitate short-term growth of single iPS cells in vitro.
人诱导多能干细胞（iPS细胞）可通过克隆生长得到良好维持。单个iPS细胞的多能性生长受低存活率限制。为便于在体外培养健壮的单个iPS细胞，采用了半换液的mTeSR1培养基（HM）、全换液培养基（WM）和iPS细胞来源的条件培养基（iPS-CM）培养。探究了碱性成纤维细胞生长因子（bFGF）和激活素A对单个iPS细胞生长的影响。单个iPS细胞的解离和增殖还包括使用Accutase酶解法分离、Rho相关蛋白激酶（ROCK）抑制剂Y27632保护以及高密度单细胞接种（1×10⁶个细胞/孔）。CCK-8检测表明，在mTeSR1培养基中的克隆iPS细胞以及在添加100 ng/ml bFGF和10 ng/ml激活素A的HM、iPS-CM或WM中的单个iPS细胞的活力显著高于在WM中的细胞。膜联蛋白v和碘化丙啶（PI）检测、钙黄绿素AM和EthD-III双重染色也证实了类似结果。酶联免疫吸附测定（ELISA）显示，HM和iPS-CM中单个iPS细胞的bFGF和激活素A水平高于WM中的单个iPS细胞。同时，逆转录-聚合酶链反应（RT-PCR）、定量聚合酶链反应（qPCR）、蛋白质免疫印迹（WB）、免疫荧光（IF）和核型分析表明，HM培养能够维持Nanog、Klf4、Sox2、Oct4的未分化标记，且不影响iPS细胞的核型。HM中未分化的单个iPS细胞呈现均匀生长。这些发现表明，bFGF和激活素A对单个iPS细胞的存活和生长很重要。HM培养系统结合Accutase、Y27632和高密度单细胞接种可促进单个iPS细胞在体外的短期生长。
