Expansion and Biased Commitment of Hematopoietic Stem Cells Towards Erythroid Cells upon Acute Anemia Induction

The majority of adult hematopoietic stem cells (HSCs) are maintained in a dormant state under homeostatic conditions. In contrast, under stressed conditions such as myeloablation and infection, HSCs are known to proliferate and rapidly give rise to downstream progeny. However, it is unclear whether and how HSCs respond to severe anemic conditions. Here we report that HSCs rapidly expand with a biased differentiation towards erythroid cells upon the induction of acute anemia.

Injection of 60 mg/kg of phenylhydrazine (PHZ) was used to induce hemolytic anemia, after which the peripheral blood (PB), bone marrow (BM) and spleen of the mice were analyzed for the blood profiles and stem/progenitor cell content. The red blood cell (RBC) count of the PHZ treated mice was at its lowest at day 6 post injection. BM analysis showed that the number of HSCs (CD150⁺CD34^-c-kit⁺Sca-I⁺Lineage^-) immediately started increasing, as well as megakaryocyte-erythroid progenitors (MEP, CD34^-FcγIII/IIR^-c-kit⁺Sca-I^-Lineage^-) with a peak at day 3-4 (3.0 and 3.4 fold increase, respectively). Interestingly, the number of common myeloid progenitors (CMP, CD34⁺FcγIII/IIR^-c-kit⁺Sca-I^-Lineage^-) did not show a clear increase over time and the number of erythroid progenitors (Ter119⁺) started increasing at a later time point than the HSC/MEP expansion, suggesting that the expansion of primitive cells is a primary response to the anemic condition that possibly skips some of the regular stages that are observed in the normal differentiation towards erythrocytes.

In contrast to the BM, in the spleen HSC expansion was modest while MEP and CMP were robustly expanded (5.7 and 6.6 fold increase, respectively). These findings indicate that the BM and spleen have distinct roles in the response to the anemic conditions.

In order to accurately evaluate the lineage potential of HSCs in vitro, we developed a combined assay utilizing colony formation and flow cytometry analysis (CFU-FACS), with which all generated colonies were analyzed for the morphology and the frequency of each lineage. The result showed that HSCs isolated from control mice had a balanced differentiation towards megakaryocyte and erythroid cells with 20-25% of the colonies containing only granulocytes/macrophages and megakaryocytes, but not erythroid cells (GMMk colonies). In contrast, HSCs isolated from PHZ treated mice showed significantly increased the number of colonies containing a higher content of erythroid cells, whereas the ratio of GMMk colonies was decreased. Furthermore, 3-dimensional analysis of the three lineage potentials (myeloid, megakaryocyte and erythroid) in the colonies revealed an imbalanced lineage potential of HSCs from anemic mice, showing higher erythroid potential instead of the megakaryocyte potential.

As an alternative method, phlebotomy was performed to induce acute anemia. Although phlebotomized mice did not display a clear expansion of the HSC population, CFU-FACS analysis showed an erythroid-biased lineage potential of the HSCs, indicating that the HSC expansion and the lineage bias may be caused by independent mechanisms.

To demonstrate if the alterations in the HSCs affect the in vivo function of these cells, 50 HSCs isolated from control or PHZ injected Kusabira Orange (KuO) mice were transplanted into lethally irradiated mice. Two weeks after the transplantation, the ratio of KuO⁺ RBCs against KuO⁺ platelets was higher in the PHZ-HSC transplanted mice than control-HSC transplanted mice. This difference was not seen four weeks after transplantation and the long-term reconstitution (>12 weeks) levels did not differ between both groups, suggesting that the enhanced erythropoiesis is a transient event that does not reduce the stem cell capacity.

In summary, we demonstrated that not only progenitor cells but also HSCs respond to severe anemic conditions and contribute to erythropoiesis through rapid expansion and a transient fate change, depicting a novel model of stress response.