Matrix reverses immortalization-mediated stem cell fate determination

Abstract

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 (SV40LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins as top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of the matrix protein influence on surrounding stem cell differentiation.

Introduction

As cartilage does not readily self-heal, articular cartilage has difficulty recovering from trauma or degenerative disease. Given that autologous chondrocyte implantation, the most promising cell therapy for articular cartilage defects, has limited cell sources for clinical application [1], mesenchymal stromal/stem cells (MSCs), especially tissue-specific stem cells deposited by synovial tissue (SDSCs), have received much attention as possible cartilage repair therapies [[2], [3], [4]]. Considering the difficulty in harvesting synovium without contamination by surrounding connective tissue, the infrapatellar fat pad (IPFP), an easily accessible adjacent tissue, might serve as an alternative, large quantity autologous tissue-specific stem cell source for cartilage regeneration and repair [5,6]. However, increasing evidence shows that IPFSCs also suffer from replicative senescence after long-term ex vivo expansion [7].

To acquire a sufficient number of cells for cartilage engineering and regeneration, modification strategies, including modification of internal genomics or the external matrix microenvironment, have been proposed for cell-based therapy [8]. Simian virus 40 (SV40), a well-known oncogene, has been commonly utilized for cell immortalization. SV40 is restricted to two proteins, the large T (LT) antigen and small t antigen (ST). The former mainly influences the SV40-extended lifespan due to its ability to bind to pRb and p53 to inactivate these two tumor suppressors, causing cells to move from G₁ phase into S phase thus promoting DNA replication [9]. However, malignant transformation is one of the potential risks caused by genetic manipulation [10].

Decellularized extracellular matrix (dECM), an integral part of the external matrix microenvironment, can be prepared from cell or tissue sources but they play a different role in cell functionality [11]. Briefly, cell-derived dECM has a distinct role in rejuvenation of adult stem cells, mainly promoting adult stem cells’ proliferation and differentiation capacity [[12], [13], [14]], which is different from tissue-derived dECM that largely guides tissue-specific behaviors [15]. Meanwhile, cells isolated from patients themselves for dECM preparation provide another benefit in avoiding potential immune issues [16], despite the fact that most patients are either elderly or older adults and the isolated stem cells are prone to premature senescence.

Few reports have investigated the influence of SV40LT transduction alone or a combined dECM approach on stem cells' chondrogenic potential. SV40LT transduction in “old” IPFSCs might be a novel strategy to provide “young” autologous cells for dECM preparation, in which a patient's IPFSCs could be rejuvenated. In this study, we investigated whether SV40LT transduction could promote IPFSCs' proliferation and chondrogenic potential and whether high-passage IPFSCs could be rejuvenated after expansion on dECM deposited by SV40LT transduced IPFSCs from either early or late passage IPFSCs. Given the close relationship between chondrogenesis and adipogenesis [17], we also aimed to explore whether the influence of SV40LT transduction on chondrogenesis also applied to adipogenesis. The main purpose of this study was to identify key matrix components guiding adult stem cells' differentiation preference by using both dECM and immortalization approaches, which might facilitate engineering of smart matrix materials for cartilage engineering and regeneration in the near future.