Induction of cells with osteo-chondrogenic potential by transcription factor-mediated reprogramming process

Abstract

Skeletal system plays a crucial role in our life. Skeletal diseases and disorders unlike cancer, are not fatal, but affect the quality of our life. Cell-based therapeutic strategies to generate targeted desired cell types for repair or replacement of damaged skeletal tissues are ideal regenerative medicines. Because of the heterogeneous cell types generated from embryonic and mesenchymal stem cells, the ability of progenitor population to differentiate into a target cell type appear to be a better alternative for tissue regeneration. Osteo-chondroprogenitors uniquely co-expressing Sox9 and Runx2 with dual differentiation potential to become chondrocytes and osteoblasts is a progenitor cell which is suitable for cell based therapy of bone disease. Therefore, developing effective strategies to generate sufficient quantities of osteo-chondroprogenitors are essential. Toward this, we took advantage of two lineage conversion approaches. The first strategy was to interrogate the ability of osteoblasts to be reprogrammed into induced pluripotent stem (iPS) cells and another one was to use defined transcription factors to induce chondrocyte lineage from skin fibroblasts. The selection of osteoblasts is based on the fact that it is originally derived from osteo-chondroprogenitor lineage and the stochastic events of iPS induction might revert osteoblasts first to their progenitor state before becoming pluripotent. The second approach is based on a previous report using three transcription factors (Sox9, Klf4 and c-Myc) to reprogramme skin fibroblasts into chondrocyte lineage. Our aim is to examine whether osteo-chondroprogenitors would be formed during the two reprogramming processes using Sox9-EGFP knock-in mice as a reporter. We reasoned that osteoblasts can be reprogrammed into iPS cells by four Yamanaka’s factors with pluripotency as shown by their ability to form teratomas and contribute to chimeric embryos. However base on the limitation of selector marker of osteo-chondroprogenitor we still cannot capture this progenitor during iPS reprogramming. And because of the pluripotency potential, pluripotent reprogramming approach also brings high risk of teratoma formation. Therefore our second objective was performed to examine whether osteo-chondroprogenitors would be formed during lineage reprogramming. Transient appearance of Sox9-EGFP/Runx2+ve cells was observed in the intermediate stage of over 14 days of chondrocyte lineage induction from skin fibroblasts by Sox9, klf4 and c-Myc. Cells expressing Sox9-EGFP/Runx2+ve showed typical molecular markers of osteo-chondroprogenitors. In vitro and in vivo differentiation assays demonstrated that Sox9-EGFP/Runx2+ve cells can differentiate predominantly into osteoblasts and chondrocytes. Taken together our data indicate that cells with osteo-chondrogenic potential could be generated by defined transcription factors-mediated reprogramming processes.