In Utero Chorionic Villus Stem Cell Transplantation for the Treatment of Hemophilia A

Pregnancy epitomizes graft tolerance, where the intrauterine allogeneic fetus is protected from rejection by the maternal immune system. Fetal cells are able to enter the maternal circulation as early as six weeks of gestation, and persist for decades without graft rejection. We hypothesize that a window of opportunity exists prior to maturation of T cells where in utero transplantation of donor cells may be recognized as self, therefore inducing tolerance permissive for long term engraftment. Hemophilia A (HA) is an X-linked recessive coagulation disorder that results in bleeding diathesis. Molecular diagnosis for HA can be performed prenatally using Chorionic Villus Sampling (CVS). Cells from CVS can be manipulated in vitro as a source of autologous stem cells. We have established a robust protocol to isolate and expand human placental chorionic tissue-derived multi-potent stem cells (PMSCs). We have genetically modified these cells to express human factor VIII (hFVIII) using a lentiviral vector. Here we report results in a murine model to assess the safety and survivability of in utero xenogeneic transplantation of gene modified human PMSCs that express hFVIII. PMSCs isolated from preterm human placenta (GA 10-12 weeks) were plastic adherent, showed spindle-shaped morphology and demonstrated expression of mesenchymal stem cell markers CD105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD31. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs). PMSCs were transduced for FVIII transgene expression via lentiviral vector with B-domain deleted hFVIII, GFP and luciferase, and driven by MNDU₃ promoter (pCCLc-MNDU3-EGFP/LUC-PGK-Fb-WPRE). At an MOI (Multiplicity of infection) of 3, lentiviral vector transduction resulted in GFP expression of 90-95% of PMSCs, assessed via immunofluorescence and flow cytometry, without alteration in morphology or proliferative capacity. Immunocytochemistry, using an antibody specific to hFVIII and HUVECs as negative control, showed that transduced PMSCs expressed hFVIII. Furthermore, functional hFVIII was detected in cell supernatant by chromogenic assay: 4.5 IU/10⁶ cells (~150% factor activity / 10⁶ cells) and FVIII protein was measured using an enzyme-linked immunosorbent assay (ELISA): 24 ng/10⁶ cells. PMSCs were transplanted intraperitoneally into murine fetuses (embryonic age 12.5 days) with a 33 gauge non-coring needle. Bioluminescence imaging analysis revealed focal density and transgene expression in the fetuses in utero 1-3 days after transplantation and subsequently in the postnatal pups 12-15 days after transplantation. Our data demonstrate that intrauterine transplantation of gene modified human PMSCs via a lentiviral vector may be a reasonable approach for cell therapy, and further suggest the potential of in utero approaches for tolerance induction. Additionally, our data showed sustained hFVIII transgene expression with cell survivability and safety in the transplanted mice, suggesting that PMSCs are a potential stem cell source for the prenatal phenotypic correction of hemophilia A.