Leading OpinionMatrix reverses immortalization-mediated stem cell fate determination
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.
Section snippets
IPFSC culture and SV40LT transduction
The study was approved by the Institutional Review Board. Adult human infrapatellar fat pads were collected from six young patients with acute meniscus or anterior cruciate ligament tears (four male and two female, 22 years old on average). Minced infrapatellar fat pads were digested with 0.1% trypsin (Roche, Indianapolis, IN) for 30 min followed by 0.1% collagenase P (Roche) for 2 h at 37 °C before filtration. After centrifugation, IPFSCs were cultured in alpha minimum essential medium (αMEM)
Evaluation of Cas9-SV40LT transduction and effect on IPFSC proliferation
To make an immortalized cell line of human IPFSCs, SV40LT lentiviral vector was used for transduction with GFP as a control. RT-PCR data confirmed successful transduction of Cas9-SV40LT in IPFSCs, evidenced by strong expression of SV40LT in cells from the SV40 group at both passages 5 and 15, followed by weak expression in the GFP group and negligible expression in the CTR group (Fig. 1A). To determine whether SV40LT transduction affects the proliferation ability of IPFSCs, PDN results showed,
Discussion
Donor age and long-term in vitro culture are both responsible for MSC senescence and are a challenge for stem cell-based tissue engineering and regeneration [7,27]. Previous reports indicate that SV40 transduction could immortalize primary cells but there were conflicting results in differentiation capacities [8]. In this study, we found that SV40LT transduction yielded human IPFSCs with significantly improved proliferation and adipogenic differentiation ability; however, chondrogenic capacity
Conclusion
For the first time, this study indicated that SV40LT transduction in human IPFSCs not only promoted proliferation but also increased adipogenic potential and decreased chondrogenic potential. Furthermore, we found that dECM deposited by SV40LT transduced IPFSCs reversed the differentiation preference of expanded IPFSCs by promoting chondrogenic potential but decreasing adipogenic capacity. Despite the unelucidated mystery underlying the rejuvenation effect of dECM deposited by high-passage
CRediT authorship contribution statement
Yiming Wang: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Gangqing Hu: Methodology, Software, Validation, Formal analysis, Writing - review & editing. Ryan C. Hill: Software, Validation, Formal analysis, Writing - review & editing. Monika Dzieciatkowska: Software, Validation, Formal analysis, Writing - review & editing. Kirk C. Hansen: Software, Validation, Formal analysis, Writing - review & editing. Xiao-Bing
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Suzanne Danley and Amanda Stewart for editing the manuscript. This project was supported by Research Grants from the National Institutes of Health (1R01AR067747) and the Musculoskeletal Transplant Foundation (MTF). We also would like to acknowledge the WVU Flow Cytometry & Single Cell Core Facility and the Bioinformatics Core as well as the grants that support the facility, WV-INBRE grant P20 GM103434, TME CoBRE grant P20GM131322 and the WV CTS grant 5U54 GM104942-04.
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The first two authors contributed equally.