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Mesenchymal Stem Cells as Vehicles for Gene Delivery

Mesenchymal Stem Cells as Vehicles for Gene Delivery. Moscaeide M J, Hendricks J, Buyaner D, Davis J, Chaung L, Majumdar M, Chopra R, Barry F, Murphy M, Thiede M, et al. 2000. Clinical Orthopaedics and Related Research. 379:S71-S90. Presented by Victoria Backes. Background.

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Mesenchymal Stem Cells as Vehicles for Gene Delivery

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  1. Mesenchymal Stem Cells as Vehicles for Gene Delivery Moscaeide M J, Hendricks J, Buyaner D, Davis J, Chaung L, Majumdar M, Chopra R, Barry F, Murphy M, Thiede M, et al. 2000. Clinical Orthopaedics and Related Research. 379:S71-S90. Presented by Victoria Backes

  2. Background • Two distinct stem cell populations of different lineages • Multipotent hematopoietic • regeneration of peripheral blood • Mesenchymal stem cells (MSCs) • mesodermal tissue • marrow stromal connective tissue • supports hematopoiesis

  3. MSCs from bone marrow: • Proliferate w/o differentiation • osteogenic, chondrogenic, adipogenic, tenogenic, & marrow stromal lineages • identical phenotype

  4. Importance of this study • Support hematopoietic recovery by replacing the stroma • Efficiency of hematopoietic recovery is linked to the degree of stromal damage due to certain cancer therapies • Myeloablative marrow is unable to sustain adequate hematopoietic stem cell maintenance • Colony-forming unit-fibroblast (CFU-F) values • reduced by 60% • no recovery after 12 years

  5. Isolation of MSCs • Autologous MSCs from all species were isolated & culture expanded from marrow aspirates (except rat) • Cells were plated and fractionated (except rat and goat) • Medium was supplemented to maximize recovery & culture expansion • Cells were isolated & characterized by their ability to differentiate

  6. Construction & packaging of mouse retroviral vectors • Retroviral vectors • CD8 [alpha] (Lyt2 gene, mouse surface antigen) • surface expression->transduction efficiency • long-terminal repeats • MESV-murine embryonic stem cell virus • MSCV-murine stem cell virus • SFFVa-Friend spleen focus-forming virus anemia • SFFVp- SFFV polycythemia • different retroviral packaging cell lines • GFP-enhanced green fluorescent protein • map biodistribution of infused MSCs

  7. Retroviral vector

  8. Transduction of MSCs & flow cytometry analysis • Transduction of MSCs • Human, baboon, pig, sheep, rat, canine, goat and rabbit • comparative study • MSCs were thawed, evaluated for viability, adhesion and proliferation-->infusion • Flow cytometry analysis • CD8[alpha] and GFP-transduced cultures

  9. Other methods employed • Followup after transplant • periodic blood sampling • blood cell recovery • serum chemistry • Samples collected at necropsymonitor GFP-transduced MSCs in tissues & marrow compartments • Days 3, 14, 28, 60, and 182 • Tissue & blood samples were analyzed for presence & expression of transgenes • DNA & RT-PCR ELISA

  10. Results • Influence of retroviral long terminal repeats on transgene expression & retro viral transduction in hMSCs • detection of cell surface murine CD8 [alpha] expression measured the ability of retroviruses to transduce MSCs • 1st-all vectors were packaged in the same retroviral packaging cell line • 2nd-optimal transduction efficiency might be achieved using packaging cell lines that expressed different retroviral envelopes

  11. Retroviral transduction of canine MSCs

  12. hMSC expression & transduction efficiency

  13. Retroviral envelope-dependent transduction of MSCs using different species • using the standard MULV LTR- each species has a preference to the retroviral envelope used • Will additional exposure to a particular vector supernatant increase efficiency?

  14. MSC expression & transduction efficiency

  15. Additional exposure on MSC transduction efficiency

  16. Biodistribution • DNA PCR-ELISA • standard curve -amplifying GFP • total number of cells in each PCR was determined by same analysis for a single-copy canine gene • numbers were calculated as the number of GFP-positive cells per million canine equivalents

  17. Canine MSC biodistribution peripheral blood

  18. Canine MSC expression in bone marrow

  19. Canine MSC biodistribution positive tissues

  20. Canine MSC biodistribution bone marrow

  21. Conclusion • Parameters for optimal retroviral transduction of eight species was described • comparing 5 different LTR with same vector backbone • transduction efficiency with retroviral supernatants from 4 different packaging cell lines

  22. Demonstrated the distribution of intravenously infused genetically marked cMSCs • Shows the ease of expressing potentially therapeutic genes in MSCs • Potential of using MSCs for in vivo efficacy studies in animals • Future experiments will define the role of MSCs in supporting hematopoietic reconstitution

  23. Rreferences • Galotto M, Berisso G, Delfino L, et al. 1999. Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients. Experimental Hematology. 27:1460-1466 • Morrison S, Shah N, Anderson D. 1997. Regulatory mechanisms in stem cell biology. Cell 88:287-298. • Moscaeide M J, Hendricks J, Buyaner D, Davis J, Chaung L, Majumdar M, Chopra R, Barry F, Murphy M, Thiede M, et al. 2000. Mesenchymal stem cells as vehicles for gene delivery. Clinical Orthopaedics and Related Research. 379:S71-S90.

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