In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response

Abstract

In vitro expansion of mesenchymal stem cells (MSCs) has been implicated in loss of multipotency, leading to impaired chondrogenic potential and an eventual therapeutic effect, as reported in our previous study. However, the precise regulatory mechanism is still unclear. Here, we demonstrate that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) were involved in transformation of MSCs induced by in vitro culture based on the comparative profiling of in vitro cultured bone marrow MSCs at passage 3 (P3 BMSCs) vs. fresh P0 BMSCs by microarray analysis. Indeed, RT-PCR and Western blot analysis showed significantly lower expression levels of three key UPR-related molecules, ATF4, ATF6 and XBP1, in P3 BMSCs than P0 BMSCs. Further, we found that UPR suppression by 4-phenylbutyrate (4-PBA) reduced the chondrogenic potential of P0 BMSCs and further cartilage regeneration. Conversely, UPR induction by tunicamycin (TM) enhanced the chondrogenic differentiation of P3 BMSCs and the therapeutic effect on cartilage repair. Thus, the decline in the chondrogenic potential of stem cells after in vitro culture and expansion may be due to changes in ER stress and the UPR pathway.

Articular chondrocyte-derived extracellular vesicles promote cartilage differentiation of human umbilical cord mesenchymal stem cells by activation of autophagy

“…The western blot analysis was performed as previously described 40 . Brie y, collected cells were washed with cold PBS and lysed in lysis RIPA buffer containing PMSF (1 mM phenylmethylsulfonyl uoride).…”

“…Immuno uorescence examination was performed as previously described 40 . For cell immuno uorescence preparation, planted cells were xed by 4% paraformaldehyde, successively treated with 3% H 2 O 2 for 10 minutes and blocked with goat serum at room temperature for 10 minutes.…”

Articular chondrocyte-derived exosomes promote cartilage differentiation of human umbilical cord mesenchymal stem cells by activation of autophagy

“…The western blot analysis was performed as previously described 39 . Brie y, collected cells were washed with cold PBS and lysed in lysis RIPA buffer containing PMSF (1 mM phenylmethylsulfonyl uoride).…”

“…Immuno uorescence examination was performed as previously described 39 . For cell immuno uorescence preparation, planted cells were xed by 4% paraformaldehyde, successively treated with 3% H 2 O 2 for 10 minutes and blocked with goat serum at room temperature for 10 minutes.…”

Mesenchymal stem cells for cartilage regeneration in dogs

“…Therefore, the initial cell selection may improve the chondrogenic potential of MSCs. The in vitro expansion of human MSCs, rabbit MSCs, and cMSCs in culture also impairs chondrogenic differentiation potential[44,81-83]. Moreover, senescence is reportedly more rapid in cMSCs than in human MSCs, particularly after passage 3[60].…”