Hematopoietic Hierarchy Under Steady-State and Stress Conditions

The maintenance of the hematopoietic system by hematopoietic stem cells (HSCs) is an important topic in both clinical and basic hematology study due to their enormous therapeutic potential. The hematopoietic hierarchy has recently garnered renewed interest following the development of single-cell assays and improved strategies for genetic labeling, resulting in new hierarchical models that challenge the classical view of hematopoiesis. However, the kinetic of hematopoiesis under steady-state and stress condition and the contribution of HSCs toward steady-state hematopoiesis remain controversial and unclear.

Using two tamoxifen-induced lineage tracing models, namely Fgd5-CreERT2/ROSA26-tdTomato and Krt18-CreERT2/ROSA26-tdTomato, we traced the contribution of HSCs to downstream blood cells. The initial labeling will occurred mostly within HSCs due to the largely restricted expression of Fgd5 and Krt18 in HSCs. Our preliminary data suggest that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward platelets and myeloid lineages compared with lymphoid lineages. Interestingly, we found that myeloid cells and platelets were labeled rapidly after the initial HSC labeling without the appearance of the previously identified intermediate populations, suggesting the potential bypassing differentiation from HSCs to these lineages.

In addition to steady-state hematopoiesis, we also investigated the response of the hematopoietic system to stress. Using the lineage tracing models mentioned above, we observed that HSCs are able to respond to stress to compensate the loss of a specific cell type, without affecting their contribution toward other lineages. For example, antibody-mediated platelet depletion resulted in a compensatory output from HSCs toward platelets. On the other hand, PHZ-induced hemolytic anemia led to an accelerated production of red blood cells. In both cases, other blood lineages remained unaffected compared to controls.

In order to acquire a more comprehensive view of steady-state hematopoiesis, we combined Fgd5-CreERT2/ROSA26-tdTomato lineage tracing model with single-cell RNA-seq. To be inclusive of known and unknown HSPC populations, we sorted Lin^-tdTomato⁺ cells at 1 week, 2 weeks and 1 month after the first tamoxifen treatment. HSCs, MPPs and HPC1/2 sorted from wild-type animals served as the reference groups. We observed the appearance of myeloid and megakaryocytic progeny as early as 1 week after the initial HSC labeling, suggesting the rapid contribution of HSCs toward these lineages. Erythroid-biased population emerged at the 2-week time point, with the expansion of myeloid progeny. Intriguingly, pseudo-time analysis revealed a relatively upstream position of erythroid-biased cluster, indicating the early emergence of erythroid progeny. The clear appearance of lymphoid progeny was absent until 1 month after the initial labeling, corresponding to the later onset of lymphoid differentiation.

In summary, our results support the active role of HSCs in steady-state hematopoiesis and rapid output toward megakaryocytic, erythroid, and myeloid lineages from HSCs. Moreover, our data also provide insight into the responses of HSCs toward different stimuli, which can potentially contribute to our understanding of hematopoietic-related diseases and uncover novel treatment avenues.