Analysis of Idiopathic Myelofibrosis Initiating Cell in NOD/SCID/IL2rgKO Mice

Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM lead to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38− cells from four IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Twelve out of nineteen recipients transplanted with patient CD34+ cells or CD34+CD38− cells exhibited human hematopoietic engraftment, and the frequency of CD33+ myeloid cells (82.5+/−12.2% among the engrafted CD45+ cells) was higher than that in the recipients transplanted with normal HSCs. These CD33+ cells expressed other myelo-monocytic markers such as CD14, CD11b, CD15, and HLA-DR. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. With FISH analysis using human X probe and immuno-staining using anti-TGF-beta1 antibody, we could confirm the TGF-beta1 production of human megakaryocytes in the recipient BM. To investigate the origin of BM fibroblasts, we performed FISH analysis using human X chromosome probe and mouse centromeric probe, and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of one hundred fifty fibroblasts examined, one hundred thirty six cells (90.7%) were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38− HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.