Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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In utero Transplantation of Gene Modified Human Adipose Stem Cells for Sustained Factor VIII Transgene Expression
Maricel G. Miguelino, M.D., Christopher D. Pivetti, M.S., David E. Sahar, M.D., Jerry S. Powell, M.D..
University of California, Davis, Sacramento, CA, USA.

PURPOSE: The paradoxical nature of pregnancy is demonstrated by the ability of the mother to nourish and sustain for weeks or months the antigenic fetus [1]. Gestation presents an opportunity where the immunologically immature fetal environment may be amenable to allogeneic or xenogeneic transplantation where fetal cells are able to enter circulation as early as six weeks and persist for weeks without graft rejection [2]. Preclinical studies using in utero stem cell transplantation (IUSCT) are promising. However, low engraftment and safety concerns preclude its clinical utility. Hemophilia A (HA) is an X-linked recessive coagulation disorder caused by a mutation of the factor VIII (FVIII) gene that results in bleeding diathesis. Availability of prenatal diagnosis for HA allows treatment of HA before birth. Our laboratory has developed a robust protocol in isolating and gene modified human adipose derived mesenchymal stem cells (hASCs) to express FVIII via lentiviral transduction. We hypothesize that IUSCT of gene modified hASCs will result in sustained FVIII transgene expression. The success of the first human IUSCT has been shown in a severe combined immunodeficiency patient that utilized paternal bone marrow (BM) derived stem cells [3]. However, BM stem cell isolation is invasive and has a low quantitative yield. Adipose derived stem cell harvesting is minimally invasive and has a high stem cell count per gram. Here we report the safety and survivability of IUSCT of hASCs into a C57BL/6 murine model.
METHODS: hASCs cells were characterized by morphologic and immunophenotypic analysis. The differentiation ability of mesenchymal lineages was detected by exposure to specific media to persuade differentiation of cells into desired lineages. hASCs were transduced using an MNDU3 promoter (pCCLc-MNDU3-eGFP/LUC-PGK-F8-WPRE) tagged with green fluorescent protein (GFP) and luciferase at an MOI (Multiplicity of infection) of 3. Transduction efficacy was assessed via detection of GFP immunofluorescence imaging and flow cytometry. Prior to IUSCT, luciferase assay was conducted. hASCs were transplanted into fetuses (embryonic age 12-14 days) via the intraperitoneal (IP) route using a 33 gauge non-coring needle. Bioluminescence imaging (BLI) was conducted in the pregnant mice one day after transplantation and in the fetuses 12 days after IUSCT.
RESULTS: Isolated hASCs showed a homogeneous population of spindle-shaped cells, expressed CD90, CD105, CD73, CD44 but not CD45, CD14, CD34 and demonstrated tri-lineage differentiation potential analogous to BM mesenchymal stem cells. Immunofluorescence of transduced hASCs revealed 90-95% efficacy without phenotypic alteration throughout several passages. Sustained transgene expression and focal density of transplanted hASCs was observed in the dam one day after IUSCT and in the fetuses 12 days post IUSCT via BLI (Figure 1).
CONCLUSION: Our study demonstrates the biologic safety and survivability of hASCs after prenatal transplantation suggesting that IUSCT is a feasible approach for long term engraftment of cells. Further, the sustained transgene expression of FVIII suggests hASCs as an alternative cell source for the treatment of HA before birth.


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