A Novel Mouse Xenograft Model for Generation of JAK2 V617F Driven Leukemic Stem Cells From Human Embryonic Stem Cells.

Many myeloproliferative neoplasms (MPN) arise in the hematopoietic stem cell (HSC) population upon acquisition of a somatic mutation leading to an aberrantly activated tyrosine kinase, JAK2 V617F. Identification of cell type and context specific genetic and epigenetic events driving leukemic transformation of MPNs has been hampered by the limited supply of the MPN patient blood and marrow samples. Recent publications demonstrate that human embryonic stem cells (hESC) can be differentiated to HSCs efficiently upon coculture with stromal cell lines derived from the murine aorta-gonad-mesonephros (AGM) region. These methods can provide a potentially limitless supply of self-renewing stem cells. We have utilized two hES cell lines, H9 and Hues9 cells, to differentiate toward HSC. We developed novel in vitro and in vivo model systems involving lentivirally enforced expression of genes, such as JAK2V617F, wild type JAK2 and/or activated beta-catenin, in an attempt to generate MPN stem cells.

To generate MPN stem cells in initial experiments (n=4), karyotypically normal Hues9 cells were lentivirally transduced with luciferase-GFP and constitutively active beta-catenin (Beta-Cat) alone or in combination with 1) JAK2 V617F, 2) wild-type JAK2, or 3) backbone lentiviral vector. The engraftment capacity of these genetically modified Hues9 cells was compared following intrahepatic transplantation (105-106 cells/mouse) of neonatal RAG2-/-gamma c-/- mice. Lentiviral transduction efficiency was 50-60%. Bioluminescent imaging demonstrated human engraftment at 6 and 11 weeks in all subgroups. The greatest proportion of human cells was noted in the thymus at week 6 and 11, and in the spleens at week 6. There was a 2.4 fold increase in thymic engraftment of CD34+CD45+ cells derived from JAK2 V617F transduced Hues9 cells and a 1.7-fold increase in CD38+CD45+ cells in the JAK2-wild type subset of mice compared with backbone controls. However, overall engraftment rates were low. To determine if hESC differentiation into CD34+ cells improved engraftment, both H9 and Hues9 hES cells were plated in 6 well plates onto mitomycin treated stromal cell lines AM20.1B4 (AM) and UG26.1B6 (UG) that were derived from aorta/mesenchyme and the urogenital ridge of the AGM region of 10 dpc murine embryos respectively. Peak CD34 expression was observed on day 10-12 (n=4) with a sharp decline in CD34 expression by day 18 (n=2). Notably, CD34+ cells began to express CD31 by day 18 with over 50% of CD34+ cells expressing CD31 by day 21 (n=2). Both undifferentiated H9 and Hues9 hES cells as well as CD34+ derivatives expressed CD90. RUNX-1 and GATA-1 expression was higher on day 12 compared to day 18 by Q-RT-PCR in H9 cultured on UG stroma. Plating of 100,000 hESC from day 10-12 cultures with UG stroma generated colonies in hematopoietic progenitor assays with in vitro replating potential while day 18-24 hESC cultures did not. In vitro derived CD34+ cells from day 13 H9 cultures with UG stroma were double selected for CD34 using immunomagnetic beads. The cells were transduced for 6 hr with lentiviral luciferase GFP, Beta-Cat alone (N=4), or Beta-Cat and lentiviral backbone vector (LV) (N=4), wild-type JAK2 (N=4) or JAK2 V617F. The cells were transplanted intrahepatically (5×104 cells/mouse) as above and monitored for engraftment by bioluminescent imaging. Human engraftment was detected at 6 weeks in 2 out of 3 JAK2 V617F/Beta-Cat, 1 of 4 wild-type JAK2/ Beta-Cat, and 0 of 4 Beta-Cat transplanted mice.

Co-culturing of hES cell lines, H9 and Hues9, on AGM stromal cell lines appears to generate replatable colonies in hematopoietic progenitor assays. Furthermore, engraftment of lentiviral luciferase transduced hESC in RAG2-/-gamma c-/- mouse hematopoietic organs can be monitored by non-invasive bioluminescent imaging. Although enhanced by lentivirally enforced expression of genes that promote hematopoietic differentiation, hematopoietic engraftment is not robust suggesting a relative dearth of environmental cues that promote human hematopoietic differentiation. Transplantation of genetically engineered hESC together with supportive stroma as well as secondary transplantation will be used to monitor changes in hESC derived HSC self-renewal capacity upon introduction of oncogenes in order to provide further insights into MPN stem cell generation.

Barroga: Wintherix: Employment. Jamieson: Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding; Coronado Biosciences: Research Funding.