Abstract
Background: Mesenchymal stem cells (MSCs) and their chondrogenic differentiation have been extensively investigated in vitro as MSCs provide an attractive source besides chondrocytes for cartilage repair therapies. Here we established prototype foamyviral vectors (FVV) that are derived from apathogenic parent viruses and are characterized by a broad host range and a favorable integration pattern into the cellular genome. As the inflammatory cytokine interleukin 1 beta (IL1β) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1β.Materials and Methods: We generated different recombinant FVVs encoding enhanced green fluorescent protein (EGFP) or IL1RA and examined their transduction efficiencies and transgene expression profiles using different cell lines and human primary MSCs derived from bone marrow-aspirates. Transgene expression was evaluated by fluorescence microscopy (EGFP), flow cytometry (EGFP), and ELISA (IL1RA). For evaluation of the functionality of the IL1RA transgene to block the inhibitory effects of IL1β on chondrogenesis of primary MSCs and an immortalized MSC cell line (TERT4 cells), the cells were maintained following transduction as aggregate cultures in standard chondrogenic media in the presence or absence of IL1β. After 3 weeks of culture, pellets were harvested and analyzed by histology and immunohistochemistry for chondrogenic phenotypes.Results: The different FVV efficiently transduced cell lines as well as primary MSCs, thereby reaching high transgene expression levels in 6-well plates with levels of around 100 ng/ml IL1RA. MSC aggregate cultures which were maintained in chondrogenic media without IL1β supplementation revealed a chondrogenic phenotype by means of strong positive staining for collagen type II and matrix proteoglycan (Alcian blue). Addition of IL1β was inhibitory to chondrogenesis in untreated control pellets. In contrast, foamyviral mediated IL1RA expression rescued the chondrogenesis in pellets cultured in the presence of IL1β. Transduced MSC pellets reached thereby very high IL1RA transgene expression levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21, while IL1RA concentrations of controls were permanently below 200 pg/ml.Conclusion: Our results indicate that FVV are capable of efficient gene transfer to MSCs, while reaching IL1RA transgene expression levels, that were able to efficiently block the impacts of IL1β in vitro. FVV merit further investigation as a means to study the potential as a gene transfer tool for MSC based therapies for cartilage repair.
Highlights
The repair capacity of articular cartilage is very limited, among others due to the lack of vascularization that could provide progenitor cells to the injured tissue (Caplan et al, 1997; Patra and Sandell, 2012; Orth et al, 2014)
For cloning of the foamy vector (FV) vector plasmid construct NA4 that expresses enhanced green fluorescent protein (EGFP) via an internal ribosomal entry site (IRES) of the encephalomyocarditis virus driven by the human elongation factor 1α (EF1α) promoter (Uetsuki et al, 1989), EF1α was PCR amplified with specific primers using the pEF-GW-51-lacZ plasmid as PCR template (Gateway Vektor System, Invitrogen) and inserted into the pBF014 plasmid via the AfeI and AscI restriction site (Armbruster et al, 2014) (Figure 1A)
The EGFP transgene in the foamyviral vectors (FVV) was under the control of either the constitutively active spleen focus forming virus (SFFV)-U3 promoter, the cytomegalovirus immediate-early promoter (CMV) or the human EF1α promoter via an IRES site (Figure 1A)
Summary
The repair capacity of articular cartilage is very limited, among others due to the lack of vascularization that could provide progenitor cells to the injured tissue (Caplan et al, 1997; Patra and Sandell, 2012; Orth et al, 2014). MSCs can be isolated readily from several sources, like for instance bone marrow, blood, and mesenchymal tissues (Nöth et al, 2010) They are able to self-renew and differentiate into multiple tissues, which makes ex vivo expanded MSCs an attractive alternative cell source to chondrocytes (Pittenger et al, 1999). MSCs are already intensively investigated and applied in clinical trials for regenerative therapies in the musculoskeletal system (Steinert et al, 2012) Such demands failed so far and did not result in the desired sustained regeneration of hyaline cartilage in vivo, as the newly formed tissue resulted widely in fibrocartilage like structures (Steinert et al, 2007; Orth et al, 2014). As the inflammatory cytokine interleukin 1 beta (IL1β) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1β
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