Liver Tropism And Factor Viii Transgene Expression After Intrauterine Chorionic Villus Stem Cell Transplantation

INTRODUCTION: Gestation is characterized by rapid mitosis and histogenesis in an immune naive environment. Growth and development of vital organs are occurring continuously at different time points throughout pregnancy under the regulation of paracrine signals, transcription and growth factors. Appropriate timing to target the developing organ of interest during in utero stem cell transplantation (IUT) may result in high level engraftment in that organ as well as recognition of foreign cells as self. Hemophilia A, is an X-linked recessive bleeding disorder caused by a mutation of the gene that encodes Factor VIII (FVIII) which is primarily produced by the liver. We hypothesize that introduction of stem cells in utero during hepatogenesis in mice (e9.5-15 days) will result in localization and persistence of stem cells in the liver. METHODOLOGY: We reported isolation and characterization of early preterm chorionic villus derived mesenchymal stem cells (PMSCs) at GA 9-13 weeks. PMSCs ( n =5) were transduced with a lentiviral vector encoding B-domain deleted FVIII, GFP and luciferase reporter genes (pCCLc-MNDU3-eGFP/LUC-PGK-F8-WPRE) at an MOI (Multiplicity of Infection) of 3. Chromogenic assay was performed on cell supernatant of transduced PMSCs 72 hours after transduction. Prior to transplantation, PMSCs were plated at different densities ranging from 250,000 cells to 2 x10^6 cells and subjected to bioluminescence imaging. Intraperitoneal transplantation of PBS (control group) or with 1 x10^6 PMSCs (experimental group) was conducted in fetal mice at e12-14 days using a 33 gauge non-coring needle. Bioluminescence analysis was performed 24 hours after IUT on dams, and 12-15 days after transplantation. Mice were euthanized and liver tissue was harvested from all animals. Expression of FVIII was analyzed by RT-PCR from both groups. RESULTS: Bioluminescence imaging revealed focal density and transgene expression for transduced cells in vitro , in vivo at 24 hours and in postnatal pups 12-14 days after transplantation. Postmortem bioluminescence analysis of IUT transplanted mice showed positive bioluminescence in the livers of pups injected with transduced PMSCs. RT-PCR analysis of the liver samples using human specific FVIII primers revealed FVIII expression in liver tissues in PMSC treated mice while negative for PBS treated mice. CONCLUSION: These results demonstrate the functional survival and localization of genetically modified PMSCs in the liver after intrauterine transplantation for up to two weeks. This study provide better understanding of the cell fate of stem cells after IUT and suggest that appropriate timing of stem cell transplantation in utero may lead to immunological tolerance. Disclosures Powell: CSL Behring: Employment.