Transcriptome-Wide Analysis of Human Chondrocyte Expansion on Synoviocyte Matrix

Abstract

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.

Keywords

• chondrocyte RNA-Seq
• dedifferentiation
• chondrogenesis
• synoviocyte matrix
• physioxia
• RNA-Seq
• cell senescence