bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium

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• 作者：Xiaoling Guo ; Ruiling Lian ; Yonglong Guo ; Qing Liu ; Qingshan Ji ; Jiansu Chen
• 关键词：Single iPS cells ; Survival ; Conditioned medium culture ; bFGF ; Activin A
• 刊名：Human Cell
• 出版年：2015
• 出版时间：July 2015
• 年：2015
• 卷：28
• 期：3
• 页码：122-132
• 全文大小：1,438 KB
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• 作者单位：Xiaoling Guo (1)
  Ruiling Lian (3)
  Yonglong Guo (1)
  Qing Liu (3)
  Qingshan Ji (4)
  Jiansu Chen (1) (2) (3) (5)
  
  1. Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China
  3. Department of Ophthalmology, the First Clinical Medical College of Jinan University, Guangzhou, China
  4. Department of Ophthalmology, Affiliated Anhui Provincial Hospital of Anhui Medical University, Hefei, China
  2. Eye Institute, Medical College of Jinan University, Guangzhou, China
  5. Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, 510632, China
  
• 刊物主题：Cell Biology; Embryology; Oncology; Stem Cells; Reproductive Medicine; Cell Culture;
• 出版者：Springer Japan
• ISSN：1749-0774

文摘

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?×?106 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.