Matrix reverses immortalization-mediated stem cell fate determination

Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40 LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. Passage 1 (P1) IPFSCs (CTR group) were transduced with lentivirus carrying either SV40 LT (SV40 group) or GFP (GFP group). P5, P10 and P15 cells from each group were compared in proliferation, chondrogenic and adipogenic capacities. P15 IPFSCs were expanded on tissue culture flasks or dECMs deposited by P5 (“young”) or P15 (“old”) IPFSCs with (SP5 and SP15, respectively) or without transduction of SV40 LT (CP5 and CP15, respectively). Cell proliferation was evaluated using population doubling time, flow cytometry for EdU incorporation, cell cycle and surface markers, and real-time quantitative PCR (qPCR) for stemness and senescence gene expression. Chondrogenic capacity was evaluated using Alcian blue staining for sulfated GAGs, immunostaining for type II collagen and qPCR for chondrogenic markers. Adipogenic capacity was evaluated using Oil Red O staining for lipid droplets, western blot and qPCR for adipogenic markers. Proteomics analysis was used to evaluate matrix components from dECMs, expanded cells and chondrogenically induced pellets. We found that SV40 transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by SV40 LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40 LT transduced P15 cells. This study provides a promising model for in-depth investigation of ECM proteins to determine how these matrix proteins affect surrounding stem cells’ differentiation preference.