Establishment of Mouse PMF Model by the Introduction of Constitutive STAT5a Into Purified Hematopoietic Stem Cells.

Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofiblosis (PMF) are pathologically related and now classified under myeloproliferative neoplasm (MPN). Subsequent studies revealed that MPN is a group of clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. The somatic activating mutation in the JAK2 tyrosine kinase, JAK2V617F, is now broadly recognized as a mutation responsible for MPN (Levine R.L. and Gilliland D.G. Blood 2008). Indeed, Most of PV patients, and half of patients with ET or PMF possess this mutation. Recent studies revealed that PV phenotype can be generated in homozygous JAK2V617F transgenic mice, while ET or atypical CML-like marked leukothrombocytosis with mild myelofibrosis can be observed in heterozygous JAK2V617F mice (Tiedt et al, Blood 2008, Shide et al Leukemia 2008). These results indicate that expression levels of JAK2V617F may influence PV and ET phenotypes. On the other hand, typical PMF phenotype has not been generated by the introduction of JAK2V617F. According to the WHO criteria, PMF could be defined as “spent phase of hematopoiesis” with fibrosis formation followed by increased bone marrow cellularity as consequences of granulocytic proliferation and megakaryocyte changes with ineffective hematopoiesis. In this study, we focused on STAT5a, a direct downstream molecule of JAK2, because we previously reported that upon transplantation, purified CD34^- lineage^- sca-1⁺ c-Kit⁺ (CD34-KSL) hematopoietic stem cells (HSCs) transduced with constitutive active form of STAT5A acted as MPN initiating cells causing granulocytosis without erythrocytosis/thrombocytosis (Kato Y. et al, J Exp Med 2005). Based on these observations, we attempted to make PMF model through mimicking typical PMF dynamics; hyper proliferation of HSCs by the introduction of constitutive active STAT5a and following early HSC exhaustion.

CD34-KSL HSCs or CD34+KSL hematopoietic progenitor cells (HPCs) were purified from bone marrow (BM) of C57BL/6 (B6)-Ly5.1 mice. Then, the cells were retrovirally transduced with STAT5a wild-type (wt) or its constitutive active mutant, STAT5a(1*6). The prepared cells were used for methylcellulose assay and were transplanted into lethally irradiated B6-Ly5.2 recipient mice together with 5 × 10⁵ B6-Ly5.1/5.2 competitor BM cells. Peripheral blood (PB) of transplanted mice was monitored biweekly for donor chimerism and lineage deviation using flow cytometry. Subsequently, histrogical analyses of bone marrow and spleen were performed to determine myelofiblosis grade and detecting extramedullar hematopoiesis. Finally, immunohistochemical staining of bone marrow with anti-TGF-b antibody was performed to detect effector cells of myelofibrosis.

Transplantation of STAT5a (1*6) transduced HSCs resulted in generation of 57 MPN mice (total 83 mice), while no MPN mouse was obtained by STAT5a (1*6) transduced HPCs (total 12 mice). Pathological analysis revealed that majority (70%) of MPN mice had PMF phenotype as defined by leukoerythroblastosis and dacryocytosis without leukothrombocytosis. These mice with PMF phenotype showed marked splenomegaly with extramedullary hematopoiesis, and granulocytic proliferation with megakaryocyte change. In BM, granulocytic proliferation advanced to severe myelofibrosis and osteomyelosclerosis in very short period of time (4 to 8 weeks). Those mice died of hemorrhage induced by pancytopenia within a few months, much faster than the mice with JAK2V617F based PV/ET models. Immunohistological analysis revealed that dominance of Gr-1 / Mac-1 positive granulocytes and CD41 positive small megakaryocytes strongly expressing TGF-beta, a putative inducer of fibroblastosis in BM of PMF mice.

By transplanting STAT5a(1*6) transduced HSCs, we were able to develop mice with phenotype closely resembling human PMF. Because PMF is rare disease, this animal model should be useful for understanding etiology of PMF, for evaluating existing treatment, and for developing therapeutics targeting STAT5a or its downstream pathway.

No relevant conflicts of interest to declare.