Single cell RNA sequencing analysis of stem cells after tenogenic differentiation

Single cell RNA sequencing was used to compare cells cultured using a tenogenic differentiation protocol. Cells included primary fibroblasts extracted from a tenolysis tissue specimen, mesenchymal progenitor cells (iMPCs) differentiated from induced pluripotent stem cells that were reprogrammed from the primary fibroblasts, as well as primary bone-marrow mesenchymal stromal cells (MSCs) from a different donor. For each cell type, cells were cultured for 21 days using a tenogenic differentiation protocol from STEMCELL Technologies. An undifferentiated negative control of each original cell type was cultured in parallel for 21 days along with a shorter culture of only 7 days. After culture, the gene expression profile of each sample was compared to assess tenogenic differentiation and heterogeneity in the cultures. It was found that tenogenic differentiation of iMPCs resulted in a small population of tenocyte-like cells with positive expression of tenogenic markers TNMD, SCX, and MKX. However, the cultures were heterogenous and had significant populations of non-tenocyte like cells. Furthermore, negative control samples also exhibited low expression of tendon markers in the abscence of tenogenic differentiation media. This study demonstrates that culture of mesenchymal progenitor cells for 21 days at high density alone is enough to promote some tenogenic differentiation. While addition of the tenogenic differentiation media led to more differentiation, the improvement was subtle and there is significant off-target differentiation that can be improved with alteration of the protocol.