Role of bone marrow transplantation for correcting hemophilia A in mice

Abstract

To better understand cellular basis of hemophilia, cell types capable of producing FVIII need to be identified. We determined whether bone marrow (BM)-derived cells would produce cells capable of synthesizing and releasing FVIII by transplanting healthy mouse BM into hemophilia A mice. To track donor-derived cells, we used genetic reporters. Use of multiple coagulation assays demonstrated whether FVIII produced by discrete cell populations would correct hemophilia A. We found that animals receiving healthy BM cells survived bleeding challenge with correction of hemophilia, although donor BM-derived hepatocytes or endothelial cells were extremely rare, and these cells did not account for therapeutic benefits. IntroductionHemophilia A is characterized by inability to clot blood because of FVIII gene mutations and deficiency of this coagulation factor. 1 The potential for cell and gene therapy in hemophilia A is highly attractive because even small amounts of FVIII may substantially decrease bleeding risk. This requires sound knowledge of cell types capable of replacing FVIII, especially within proximity of von Willebrand factor (vWF), which protects FVIII from degradation. 2 However, the cell-type origin of FVIII has been controversial. 3 Correction of hemophilia after orthotopic liver transplantation (OLT) but not after kidney transplantation, despite FVIII mRNA expression in both organs, 3,4 indicated that liver was a major site for FVIII production. Recently, the cell transplantation approach established that of various liver cell types, liver sinusoidal endothelial cells (LSECs) replaced FVIII in hemophilia mice. 5,6 Nonetheless, extrahepatic organs probably contributed in FVIII production, as indicated by FVIII synthesis in spleen, lungs, or pancreatic islets. [6][7][8] This was in agreement with lack of plasma FVIII deficiency after OLT with donor liver from dogs or people with hemophilia A because such donor liver cells would not have synthesized or secreted FVIII. 9,10 Therefore, whether nonendothelial cells, and cells in extrahepatic organs, could also produce FVIII was not excluded. For instance, macrophages, which originate in bone marrow (BM), and contained FVIII mRNA, 11 could be such a candidate. Although FVIII was cloned from T cells, 12 and transplantation of lymphatic tissue was thought to correct hemophilia in dogs, 13 whether lymphocytes did express FVIII was uncertain, because correction of hemophilia by transplanted organs (eg, spleen) included other cell types. 7,14,15 Because BM cells may generate multiple lineages, the potential of BM-derived cells in FVIII production seemed relevant to us. Previously, BM transplantation studies in hemophilia A, 40 years ago, were limited to just 3 dogs and 2 persons. [16][17][18][19] BM transplantation in hemophilia dogs was carefully performed, although there were limitations, such as suboptimal allograft tolerance, lack of studies showing engraftment and generation of various BM cell types, absence of FVIII expression analysis in donor BM-derived cells,...

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A

“…We also found that HA mice can be treated by reconstitution of healthy BM, indicating that BM−derived hematopoietic or mesenchymal cells can produce and secrete FVIII. 23 This study built on these possibilities by investigating whether human LSEC, hepatocytes or peripheral blood, cord blood and BM cells may be suitable for cell therapy in HA.…”

“…This confirmed previous findings of FVIII expression in myeloid cells in the mouse. 23 We, therefore, explored, in both humans and mice, which hematopoietic cell populations express FVIII (Figure 2 and Online Supplementary Figure S2). Peripheral blood monocytes and monocyte-derived macrophages expressed FVIII at the mRNA and protein levels.…”

“…22 Recently, bone marrow (BM) transplantation was demonstrated to correct the bleeding phenotype in HA mice, in part through donor-derived monocytes/macrophages and mesenchymal stromal cells. 23,24 Further investigations into the role of hematopoietic cells in FVIII expression are, therefore, appropriate. December 29, 2014.…”

The search for the origin of factor VIII synthesis and its impact on therapeutic strategies for hemophilia A

“…These include acute myeloid leukemia and acute lymphoblastic leukemias caused by translocations of the Mixed Lineage Leukemia (MLL) gene, [2][3][4] fusions of the HOXA9 gene that produce a novel HOXA9-NUP98 fusion protein in acute myeloid leukemia, 5 and T-cell acute lymphoblastic leukemias that have translocations between the TCRβ and HOXA9/A10 loci. 6 Interestingly however, despite this seemingly central role in a subset of acute leukemias, Hoxa9 expression alone is only weakly oncogenic in mouse leukemia models and usually requires a second "hit" via overexpression of Meis1, 7,8 or in some cases Pbx3. 9 Much work has been done trying to understand the molecular function of the HOXA9 protein.…”

“…8 Notably, in a series of experiments in hemophilia A mice, injection of isolated CD11 + human monocytes resulted in a transient increase in the circulating levels of factor VIII; the protein was biologically active, as demonstrated by the improved survival of animals upon hemostatic challenge to the macrocirculation (tail clip assay). Furthermore, hemophilia A mice that received cord bloodderived CD34 + cells from wild-type donors following total body irradiation experienced a dose-dependent elevation of factor VIII levels and improvement of the bleeding phenotype upon vascular injury.…”