Establishment of a feeder and serum-free culture system for human embryonic stem cells
| Autor: | Xianghui Zeng, Liyun Wang, Ronghua Ma, Xue Bai, RuiNa Zhang, Binye Li, ZhenZhen Lv, Gong-Xue Jia, Chen Li, Zhengfang Xiong, Shengyan Jian |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Homeobox protein NANOG Fetal Proteins Pluripotent Stem Cells Brachyury Population Blotting Western Human Embryonic Stem Cells Cell Culture Techniques Gene Expression Biology Regenerative medicine Culture Media Serum-Free Cell Line Nestin 03 medical and health sciences 0302 clinical medicine SOX2 Humans education reproductive and urinary physiology Cell Proliferation education.field_of_study Reverse Transcriptase Polymerase Chain Reaction SOXB1 Transcription Factors Feeder Cells Cell Differentiation Cell Biology Nanog Homeobox Protein Embryonic stem cell Cell biology 030104 developmental biology embryonic structures alpha-Fetoproteins Stem cell T-Box Domain Proteins Octamer Transcription Factor-3 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | Zygote (Cambridge, England). 28(3) |
| ISSN: | 1469-8730 |
| Popis: | SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cellsin vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintainedin vitrodifferentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs. |
| Databáze: | OpenAIRE |
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