Lentiviral Transduction of Human Postnatal Skeletal (Stromal, Mesenchymal) Stem Cells: In Vivo Transplantation and Gene Silencing

Abstract

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow

“…Note, however, that an increase in cell death was observed for BM MSCs at MOI = 20 and for HEK 293T cells at MOI = 40. A similar effect was reported for MOI = 20 or higher in the case of transduction of MSCs from embryonic tissues and BM [32,35]. Mass cell death is observed at high MOI val ues possibly because gene integrity in target cells is impaired by multiple integrations of virus genome copies [36].…”

“…The total transduction efficiency of HEK 293T cells was 1.5 fold (p = 0.00691) higher than that of BM MSCs at MOI = 10 and 1.1 fold higher (p = 0.00427) at MOI = 20. The difference in transduction efficiency can be explained by the fact that HEK 293T cells have Lentivirus transduction is known to lack a substan tial effect on the proliferative potential of a BM MSC culture [14,[32][33][34]. Note, however, that an increase in cell death was observed for BM MSCs at MOI = 20 and for HEK 293T cells at MOI = 40.…”

Fibrous Dysplasia as a Stem Cell Disease

“…In skeletal stem cells, different genes, including the Gs␣ gene, can effectively and stably be silenced by lentiviral vectors encoding short hairpin precursors of RNA interfering sequences, driven by PolIII-dependent promoters. (47,54) Compared with other strategies (e.g., gene targeting in skeletal stem cells (55) ), this approach seems simpler and at least experimentally feasible. Selective silencing of the mutated allele, however, may present additional difficulties.…”

Human mesenchymal stem cells: from basic biology to clinical applications

“…This assay has also been employed to demonstrate the ability of the multipotenital MSC cells to exhibit selfrenewal and maintenance of 'stemness' capacity during serial implantations. 22 …”