1226. Gene-Induced Chondrogenesis in Coagulated Bone Marrow Aspirates

GENE THERAPY APPLICATIONS TO SKIN, BONE AND CONNECTIVE TISSUE CD34 expressing MSC) leads to engraftment of gene-marked cells with mesenchymal characteristics in select baboons with high genemarking and long-term follow-up. We therefore analyzed the bone marrow of two baboons that were transplanted with green fluorescence protein (GFP)-marked CD34+ enriched autologous marrow cells, after myeloablative conditioning. Both animals showed persistent and stable GFPmarking by flow-cytrometry in peripheral blood leukocytes (J11615%, M093-30%) as well as 3% (J116) and 30% (M093) in CD34+ marrow cells at 275-300 days after transplantation, respectively. To investigate stromal cell engraftment bone marrow was obtained and adherent cells were cultured for 2 to 4 weeks in Human Dexter or MEM-MSC medium. For detection of GFP+ stromal cells the cultures were trypsinized and stained with anti-CD11a (panleukocyte-Ab) to distinguish surviving hematopoietic cells. We detected GFP+/CD11a- cells flow-cytometrically (J116- 0.01%, M093- <0.01%) and sorted these events as single cells into 96-well plates to positively identify any genetically modified GFP+ mesenchymal cells and exclude possible contamination with false positive events. Single cell sorts performed after 2 weeks of primary culture yielded only a small number of proliferating clones (7/185). These colonies were negative for GFP-expression. In a separate experiment clones sorted after 4 weeks of primary culture did not proliferate. Experiments to exclude proviral silencing in the clonal cultures are ongoing. We conclude from two independent experiments in two different animals with substantial, long-term peripheral blood marking, that genetically modified CD34+ cells do not contribute to the adherent marrow-derived mesenchymal cell population. These results are consistent with studies in allogeneic human transplantation where no contribution of donor-derived bone marrow cells to the mesenchymal cell population was observed.

natural clot created from freshly coagulated bone marrow aspirate forms a simple, but surprisingly effective, matrix for delivery and localization of gene transfer vectors or genetically modified cells. Primary aspirates of bone marrow are enriched for MSCs, and the liquid form enables mixing and uniform distribution of vector or cells throughout. By placing the mixture in a vessel of convenient dimensions, the resulting semi-solid coagulate acquires a useful size and shape. Moreover, the matrix formed is completely native to the donor and part of the natural reparative process. Mixture of fresh bone marrow aspirate from the rabbit with a suspension of recombinant adenovirus encoding GFP or luciferase resulted in transgenic expression in the clot for several weeks in vitro and in vivo following implantation into osteochondral defects. The coagulates were also found to maintain their structural integrity for several weeks in culture, providing the opportunity to evaluate gene-induced chondrogenesis in vitro within the context of the bone marrow clot. Culture of clots for 21 days in defined media without specific growth factor stimulation resulted in the generation of large regions of adipose-like tissue. Bone marrow clots cultured in the presence of 10 ng/ml TGF-β1, or clots genetically modified to express TGF-β1 within a specific range were found to result in the generation of cartilaginous formations throughout the clot. These results suggest that the application of genetically modified bone marrow clots may be an effective method for influencing the biology of MSCs in the repair of damaged cartilage or other musculoskeletal tissues.

1226. Gene-Induced Chondrogenesis in Coagulated Bone Marrow Aspirates Glyn D. Palmer,1 Arnulf Pascher,1,2 Elvire Gouze,1 Jean-Noel Gouze,1 Oliver Betz,1 Christopher H. Evans,1 Steven C. Ghivizzani.1 1 Center for Molecular Orthopaedics, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA, United States; 2 Orthopaedic Surgery, University of Graz, Graz, Austria. Articular cartilage frequently incurs damage following injury or disease, but has very limited regenerative capacity. It is thought that exposure of mesenchymal stem cells (MSCs) infiltrating osteochondral lesions to proteinaceous chondrogenic factors could enhance the synthesis and improve the quality of repair tissue. This study is based on the hypothesis that gene transfer can be used as a means to achieve sustained synthesis of specific chondrogenic proteins within an osteochondral lesion, and that this can be used to augment the differentiation of MSCs toward chondrogenesis in vivo. In initial experiments, we found that primary, monolayer cultures of MSCs isolated from rabbit bone marrow could be genetically modified using recombinant adenovirus to express a variety of marker genes and growth factors. Following condensation of the modified cells into aggregates and culture in defined media, expression of transgenes encoding TGF-β1 or BMP-2 was found to stimulate differentiation of the MSCs toward the formation of neocartilage-like tissue, as shown by histologic and immunhistochemical staining. Infection of the MSCs over a wide dose range of virus showed that the induction of chondrogenesis was dependent upon both the level and duration of transgene expression. Interestingly, both under- and overexpression of either factor were found to inhibit chondroinduction. Having demonstrated the principle of gene-induced chondrogenesis in primary MSCs, we explored various methods of gene delivery to MSCs within osteochondral defects in rabbits. We found that the Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy

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