Single-Cell RNA Sequencing of Expanded Chondrocytes: MSC-Like Cells With No Evidence of Distinct Subsets.

OBJECTIVE

To investigate the heterogeneity of expanded chondrocytes used for autologous chondrocyte implantation.

METHODS

Human articular chondrocytes were expanded for 14 days, sorted into 86 single cells using fluorescence-activated cell sorting and subjected to single-cell RNA sequencing. Principal component, Cross hierarchical clustering, and differential gene expression analyses were used for data evaluation. Flow cytometry and single-cell RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) was used to validate the results of the RNA sequencing data Polyclonal chondrocyte populations from the same donor were differentiated toward the osteogenic and adipogenic lineages.

RESULTS

There was considerable variation in gene expression between individual cells, but we found no evidence for separate cell subpopulations based on principal component, hierarchical clustering, and differential gene expression analysis. Most of the cells expressed all the markers defining mesenchymal stem cells, and as polyclonal chondrocyte populations from the same donor were shown to differentiate into osteocytes and adipocytes , these cells formally qualify as mesenchymal stem cells.

CONCLUSIONS

expanded chondrocytes consist of one single population of cells with heterogeneity in gene expression between the cells. Dedifferentiated chondrocytes qualify as mesenchymal stem cells as they fulfill all the criteria suggested by the International Society for Cellular Therapy.