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Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment, at the single-cell level over time. We established and evaluated the use of a novel U2-Net based network for automated cell segmentation and applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes. Current approaches to derive transfusable amounts of erythrocytes and platelets fall short of producing physiologically relevant amounts of each cell type. Thrombopoietin (TPO), commonly used in culture systems to increase platelet yield from cultured megakaryocytes, increases megakaryocyte (Mk) numbers and promotes platelet production. With the goal of generating adequate quantities of transfusion products in mind, establishing differentiation cultures that start with highly purified progenitors with Mk and E lineage potential as well as expansion potential is a very attractive option. However, a pervasive misconception exists that supplementing MEP cultures with TPO will increase the frequency with which the MEP produce megakaryocyte-committed daughter cells, thereby increasing the pool of lineage-restricted progenitor cells with high expansion capacity that ultimately yield higher numbers of transfusable platelets. We investigated whether TPO plays a role in instructing Megakaryocytic-Erythroid Progenitor (MEP) [1] commitment to the Mk lineage utilizing our previously presented time-lapse imaged colony forming unit assay and cell tracking approach [2]. This allowed us to calculate the frequency with which MEP give rise to lineage restricted progenitors in control conditions (with SCF, IL3, IL6, EPO, and TPO) compared to conditions lacking TPO to test the hypothesis that absence of TPO may diminish the frequency with which MEP give rise to Mk-restricted progenitors. We found that in the absence of TPO, MEP colony forming efficiency significantly (p Time-lapse imaging also confirmed that MEP cultured without TPO underwent significantly (p Cell death rates were at least 5-fold higher in the absence of TPO compared to control culture conditions containing TPO, and this effect was observed in both E and Mk-destined cells, demonstrating that TPO supports survival of both E and Mk-destined progenitors (p Our results confirm the hypothesis that TPO allows for greater self-renewal of MEP, as well as the survival of both the E and Mk-destined progenitors, however TPO is not instructive in MEP lineage commitment as evidenced by no change in the frequency of colony types. These findings also support the use of TPO in cultures of less committed progenitors to promote expansion leading to higher production of transfusable cell types. Further uses of this approach can include measuring kinetics of cellular processes such as division rate and motility, which may correlate with cell state and permit predictions of the probability of lineage choices downstream of specific progenitor populations. References: 1. Sanada and Xavier-Ferrucio et al. 2016 2. Scanlon, Kochugaeva et al. ASH. 2019 3. Fox, Priestley et al. 2002 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.