Recent advances in single-cell technologies have permitted the investigation of heterogeneous cell populations at previously unattainable resolution. Here we apply such approaches to resolve the molecular mechanisms driving disease in mouse hematopoietic stem cells (HSCs), using JAK2V617F mutant myeloproliferative neoplasms (MPNs) as a model. Single-cell gene expression and functional assays identified a subset of JAK2V617F mutant HSCs that display defective self-renewal. This defect is rescued at the single HSC level by crossing JAK2V617F mice with mice lacking TET2, the most commonly comutated gene in patients with MPN. Single-cell gene expression profiling of JAK2V617F-mutant HSCs revealed a loss of specific regulator genes, some of which were restored to normal levels in single TET2/JAK2 mutant HSCs. Of these, Bmi1 and, to a lesser extent, Pbx1 and Meis1 overexpression in JAK2-mutant HSCs could drive a disease phenotype and retain durable stem cell self-renewal in functional assays. Together, these single-cell approaches refine the molecules involved in clonal expansion of MPNs and have broad implications for deconstructing the molecular network of normal and malignant stem cells.MS is the recipient of a BBSRC Industrial CASE PhD Studentship, and CAO and JF are recipients of Wellcome Trust PhD Studentships. Work in the Kent lab is supported by a Bloodwise Bennett Fellowship (15008), a European Hematology Association Non-Clinical Advanced Research Fellowship, and an ERC Starting Grant (ERC-2016-STG–715371). Work in the Green Lab is supported by the Wellcome Trust, Bloodwise, Cancer Research UK, the Kay Kendall Leukaemia Fund, and the Leukemia and Lymphoma Society of America. Dr. Kent, Professor Göttgens, and Professor Green are all supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute the National Institute for Health Research Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre
