Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Abstract Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood. Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time. CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors. To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. Disclosures No relevant conflicts of interest to declare.