Tracking Hematopoietic Precursor Division Ex Vivo in Real Time

Deciphering molecular mechanisms underlying division of hematopoietic stem cells (HSCs) and malignant precursors would improve our understanding of basis of stem cell-fate decisions and oncogenic transformation. Using a novel reporter of hematopoietic precursor Evi1-GFP, we track the division of hematopoietic precursors in culture in real time. First, we confirmed that Evi1-GFP is a faithful reporter of HSC activity and identified three dividing patterns of HSCs: symmetric renewal, symmetric differentiation, and asymmetric division. Moreover, we found that the cytokines and growth factors combination (STIF) promotes symmetric renewal, whereas OP9 stromal cells balance symmetric renewal and differentiation of HSCs ex vivo .Interestingly, we found that Tet2 knockout HSCs underwent more symmetric differentiation in culture compared to wild type control. Intriguingly, OP9 stromal cells reverse the phenotype of Tet2 knockout HSCs ex vivo . Furthermore, we demonstrated that Tet2-/- ; Flt3^ITD acute myeloid leukemia (AML) precursors primarily underwent symmetric renewal divisions in culture. Our study establishes a new system to explore the molecular mechanisms of the regulation of benign and malignant hematopoietic precursor division ex vivo . Theknowledge learned from these studies will provide new insights into the molecular mechanisms of HSC fate decision and leukemogenesis.