Investigating The Pool Of Hematopoietic Stem Cells In Bone Marrow

Controlling proliferation and differentiation of stem cells is one of the important features of homeostasis. Hematology is an advanced field of stem cell research, and studies of the hematopoiesis have described the details of the system behind it.

Hematopoietic stem cells (HSCs) in bone marrow maintain the life-long production of all blood cells in mice and humans. The pool of HSCs exists in two cell cycle states, i.e. hypoxic, autophagy-active cells in the G₀ phase of cell cycle, and actively proliferating cells in the G₁-S-G₂-M phases.

Resting HSCs are thought to localize to hypoxic microenvironments in the bone marrow resulting in maintenance of the HSC pool size. The balance of HSC quiescence, the capacity of self-renewal after activation into proliferation and the decision to differentiate strongly depend on interactions of HSCs with their environments that provide signals through cytokines.

The cytokine Flt3 ligand (Flt3L) has an important role in hematopoiesis. It acts on Flt3, its receptor, which is expressed on early hematopoietic progenitors. Flt3L induces the differentiation of these progenitors. Flt3L-deficient mice show deficient hematopoiesis, reduced leukocyte cellularity in bone marrow, lymph nodes and spleen. All of these studies suggest that Flt3L plays an important role in the expansion of early hematopoietic progenitors and the establishment of myeloid and B lymphoid cell lineages.

However, for the maintenance and post-transplantation expansion of HSCs, Flt3L and its receptor are dispensable. Moreover, when HSCs express Flt3, they lose self-renewal capacity. This suggests that Flt3L-signalling via Flt3 terminates (long-term) LT-HSC status and induce differentiation to short-term (ST)-HSCs, multipotent progenitors, and more differentiated progenitors.

Since we would like to see the first point of differentiation of HSC to lymphoid cells, we started to analyze Flt3L expression by using Flt3L-eGFP reporter mice.

Like IL-7, which is another important cytokine for B cell development, we have expected that Flt3L expressed in stromal cells. Interestingly, the LT-HSC population includes Flt3L-expressing cells. Experiments of transplanting each population in other mice showed that Flt3L-expressing cells lost the capacity in long-term repopulation but were still short-term HSC phenotype.

Analysis of gene expression on Flt3L-non-expressing cells is identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-expressing cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L non-expressing cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-expressing HSCs upregulate genes involved in HSC differentiation.

From the results, we hypothesize that ST-HSCs would control hematopoiesis by paracrine as an initiator of further differentiation of multipotent progenitors of hematopoietic cells.

This study, Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150+ long-term repopulating murine hematopoietic stem cells was recently published in the European Journal of Immunology.