Tracking hematopoietic precursor division ex vivo in real time

Abstract

Background: Deciphering molecular mechanisms underlying the division of hematopoietic stem cells (HSCs) and malignant precursors would improve our understanding of the basis of stem cell-fate decisions and oncogenic transformation. Methods: Using a novel reporter of hematopoietic precursor, Evi1-GFP, we tracked the division of hematopoietic precursors in culture in real time. Results: 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 cytokine and growth factor 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 with the wild-type control. Intriguingly, OP9 stromal cells reverse the phenotype of Tet2 knockout HSCs ex vivo. Furthermore, we demonstrated that Tet2–/–;Flt3ITD acute myeloid leukemia (AML) precursors primarily underwent symmetric renewal divisions in culture. Mechanistically, we demonstrated that inhibiting DNA methylation can reverse the aberrant division phenotypes of Tet2–/– and Tet2–/–;FLT3ITD precursors, suggesting that abnormal DNA methylation plays an important role in controlling (pre-)leukemic precursor fate decision ex vivo. Conclusions: Our study exploited a new system to explore the molecular mechanisms of the regulation of benign and malignant hematopoietic precursor division ex vivo. The knowledge learned from these studies will provide new insights into the molecular mechanisms of HSC fate decision and leukemogenesis.