Transcriptome-Wide Analysis of Human Chondrocyte Expansion on Synoviocyte Matrix
Transcriptome-Wide Analysis of Human Chondrocyte Expansion on Synoviocyte Matrix
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DOI：
10.3390/cells8020085
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发表时间：
2019-02-01
期刊：
影响因子：
6
通讯作者：
Dennis, James E.
中科院分区：
文献类型：
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作者：
Kean, Thomas J.;Ge, Zhongqi;Dennis, James E.
人软骨细胞被扩展并用于自体软骨细胞植入技术，并且已知在培养中迅速去分化。当在组织培养塑料（TCP）上培养时，这些软骨细胞发生表型和形态变化，并迅速失去再分化产生透明样基质的能力。滑膜细胞衍生的细胞外基质（SDECM）上的生长减少了这种去分化，允许两倍以上的群体加倍（PD）数量，同时保持软骨生成能力。本研究的目的是应用RNA测序（RNA- seq）分析来检查tcp扩展和sdecm扩展的人软骨细胞之间的差异。将三个供体的人软骨细胞从原代储备中解冻，在TCP烧瓶或sdecm包覆烧瓶上以生理氧张力（5%）培养4代。在对数扩增过程中，在传代1和4时从细胞层（70-90%合流）中提取RNA。在质控分析和下一代RNA测序之前，对总RNA进行柱纯化和dna处理。培养表面和传代数对基因表达均有显著影响。这些结果有助于深入了解SDECM如何为细胞扩增提供更有利于软骨形成的环境的机制，培养过程中发生的转录组范围内的变化，以及进一步增强保护软骨形成生长的潜在机制。
Human chondrocytes are expanded and used in autologous chondrocyte implantation techniques and are known to rapidly de-differentiate in culture. These chondrocytes, when cultured on tissue culture plastic (TCP), undergo both phenotypical and morphological changes and quickly lose the ability to re-differentiate to produce hyaline-like matrix. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation, allowing for more than twice the number of population doublings (PD) whilst retaining chondrogenic capacity. The goal of this study was to apply RNA sequencing (RNA-Seq) analysis to examine the differences between TCP-expanded and SDECM-expanded human chondrocytes. Human chondrocytes from three donors were thawed from primary stocks and cultured on TCP flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. During log expansion, RNA was extracted from the cell layer (70-90% confluence) at passages 1 and 4. Total RNA was column-purified and DNAse-treated before quality control analysis and next-generation RNA sequencing. Significant effects on gene expression were observed due to both culture surface and passage number. These results offer insight into the mechanism of how SDECM provides a more chondrogenesis-preserving environment for cell expansion, the transcriptome-wide changes that occur with culture, and potential mechanisms for further enhancement of chondrogenesis-preserving growth.