In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A

Abstract

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.

Keywords: chorionic villus sampling; factor VIII; hemophilia A; in utero transplantation (IUT); placenta-derived mesenchymal stromal cells (PMSCs).

Figure 1.

Characterization of first trimester PMSCs. Representative phase contrast image showing first trimester PMSCs spindle-shaped morphology (A) and differentiation into osteogenic (B), adipogenic (C), and chondrogenic (D) lineages. Flow cytometric analysis of MSC immunophenotype for expression of surface markers (E) and quantitative analysis (F) are shown. n = 4 cell lines. 10× magnification, scale bar = 100 µm. PMSC, placenta-derived mesenchymal stromal cell; MSC, mesenchymal stromal cell.

Figure 2.

Characterization of PMSCs transduced with lentiviral vectors. Representative images of phase contrast (A), GFP (B), and overlay of phase contrast and GFP (C). GFP flow cytometry analysis of PMSCs transduced with GFP-LUC vector for in vivo cell tracking (D). Trilineage differentiation of transduced PMSCs into osteogenic (E), adipogenic (F), and chondrogenic lineages (G). Flow cytometric analysis of MSC immunophenotype for expression of surface markers after transduction with both BDD-FVIII and GFP-LUC vectors (H) and quantitative analysis (I) are shown. n = 4 transduced PMSC cell lines. 10× magnification, scale bar = 100 µm. PMSC, placenta-derived mesenchymal stromal cell; MSC, mesenchymal stromal cell; GFP, green fluorescent protein; LUC, luciferase; BDD-FVIII, B-domain-deleted factor VIII.

Figure 3.

Assessment of FVIII expression by transduced PMSCs. RT-PCR (A) and Western blot (B) analysis of expression of BDD-FVIII by transduced PMSCs (A: lane 2 and B: lane 2, respectively), compared to PMSCs transduced with control vector (A: lane 1 and B: lane 1, respectively). Comparison of human FVIII secretion by ELISA (C) and activity by chromogenic assay (D) between PMSCs transduced with BDD-FVIII vector and PMSCs transduced with control vector. n = 4 pairs of PMSC cell lines. PMSC, placenta-derived mesenchymal stromal cell; BDD-FVIII, B-domain-deleted factor VIII; FVIII, factor VIII; RT-PCR, reverse transcription PCR; ELISA, enzyme-linked immunosorbent assay.

Figure 4.

IUT of BDD-FVIII expressing PMSCs. Representative images of GFP (A, a to e) and luciferase (A, f to j) signaling in heart, lung, liver, spleen, and kidney tissues, respectively, in pups 4 d after birth which was 10 d after IUT of BDD-FVIII-transduced PMSCs and quantitation of GFP signaling as described in Methods (B). Quantitative real-time PCR analysis of EGFP expression in the tissues (C). n = 4 pups. PMSC, placenta-derived mesenchymal stromal cell; BDD-FVIII, B-domain-deleted factor VIII; FVIII, factor VIII; IUT, in utero transplantation; GFP, green fluorescent protein.