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Formation of a human hematopoietic stem cell niche in the murine bone marrow

 

Complex biological processes like hematopoiesis require in vivo analysis, but the study of human hematopoietic stem cell (HSC) biology in vivo is severely limited by ethical and technical constraints. Reconstituting mice with human hematopoietic stem cells (HSC) allows the analysis of human stem cell biology and hematopoiesis, but also permits the study of immune responses including autoimmunity, infectious diseases, cancer biology and regenerative medicine in vivo (reviewed in (1, 2)). Durable engraftment of human HSCs in the mouse stem cell niche is a vital step in the continuous generation of human blood cells. However, information on mechanisms and conditions of human HSC engraftment in the mouse bone marrow (BM) is incomplete, and HSC engraftment is often of transient nature (reviewed in (1, 2)).

In mice, engraftment levels after transplantation between major histocompatibility complex (MHC) - matched (syngeneic) donor and host depends on available HSC niche ‘space’, usually created by conditioning (e.g. irradiation), and on the number of donor HSC. Transplantation across full MHC mismatches (allogeneic transfer) or across species (xeno-transplantation) additionally requires the prevention of graft rejection by the immune system of the host (3). Thus, conditioning of recipient mice provides myeloablation and immune-suppression, and makes allogeneic and xeno-HSC transplantation feasible to a limited extent (reviewed in (1, 2)).

However, conditioning by irradiation is highly unspecific and significantly damages non-HSC hematopoietic cells and non-hematopoietic tissues, induces a state of severe inflammation characterized by the release of cytokines, and the transfer of very high cell numbers are required to ensure engraftment of allogeneic HSCs. Therefore, non-irradiated recipients provide the most physiological setting to reveal HSC function following adoptive cell transfer. Based on these considerations, HSC recipients that could accept human HSC without irradiation, or with only low doses of irradiation, would be advantageous, and may ensure regeneration of human HSC in mice.

We recently generated a mouse mutant that, even when unconditioned, accepts transplantation of allogeneic HSCs, suggesting that we defined all required parameters involved in complete and sustained inter-murine HSC transplantation. We combined immunodeficiency with an impaired HSC compartment mediated by mutant signaling of the receptor kinase Kit, which renders endogenous HSC uncompetitive compared to wild type donor HSCs. Kit is not only one of the most widely used HSC markers but is also of crucial importance for HSC functions. Mice bearing natural Kit-mutant alleles (KitW encoding a cell surface null protein; KitWv encoding a kinase weak receptor) have multiple hematopoietic defects. BM cells from compound heterozygous mutants (KitW/Wv) and the more recently established viable Kit null mice (KitW/W) (Waskow et al., 2004) are highly radiosensitive (4, 5), lack radioprotective activity, and fail to induce macroscopic spleen colonies (CFU-S) after transfer into irradiated recipients (4).

We combined null mutations in genes encoding for Rag-enzymes and the common cytokine receptor gamma chain (c) subunit with compound mutations in Kit (KitW/Wv). We could show that our newly generated mouse mutant (Rag-c-KitW/Wv) has a ‘weak’ endogenous HSC compartment, providing space for donor HSC, and that lack of T-, B-, and NK-cells is sufficient to prevent graft rejection. The hematopoietic stem cell compartment in Rag-c-KitW/Wv mice is efficiently reconstituted in the long-term by syngeneic, but also by allogeneic BM cells without a requirement for conditioning (Fig. 1). Thus, both effects of irradiation, myeloablation and immune-suppression are compensated for by the genetic modification of the mouse mutant, and our newly generated triple mutant mice are ‘universal’ HSC recipients (6).

We now plan to test the hypothesis that genetic ‘weakening’ of endogenous mouse HSCs, combined with immuno-deficiency and strain-background-specific modifiers, facilitates the efficient generation of a human HSC niche in mice.

 

 B9_Fig_1.jpg-

Fig. 1 Donor-cell chimerism in immature, lineage negative (Lin-) BM cells 7 weeks after injection of 2000 allogeneic HSC (Balb/c, d haplotype). Histograms are gated on Lin- BM cells and expression of the allogeneic donor marker (H-2Kd) is analyzed. Only Rag-c-KitW/Wv mice (bottom), but not wild type mice (B6, top), Rag-c- mice (2nd from top) or KitW/Wv mice (2nd from bottom) accept allogeneic donor cells without previous myeloablation.

 

References:

1. Shultz, L.D., F. Ishikawa, and D.L. Greiner. 2007. Humanized mice in translational biomedical research. Nat Rev Immunol 7:118-130.

 

2. Guezguez, B., and M. Bhatia. 2008. Transplantation of human hematopoietic repopulating cells: mechanisms of regeneration and differentiation using human-mouse xenografts. Curr Opin Organ Transplant 13:44-52.

 

3. Shlomchik, W.D. 2007. Graft-versus-host disease. Nat Rev Immunol 7:340-352.

 

4. Waskow, C., S. Paul, C. Haller, M. Gassmann, and H. Rodewald. 2002. Viable c-Kit(W/W) mutants reveal pivotal role for c-kit in the maintenance of lymphopoiesis. Immunity 17:277-288.

 

5. Agosti, V., S. Corbacioglu, I. Ehlers, C. Waskow, G. Sommer, G. Berrozpe, H. Kissel, C.M. Tucker, K. Manova, M.A. Moore, H.R. Rodewald, and P. Besmer. 2004. Critical Role for Kit-mediated Src Kinase But Not PI 3-Kinase Signaling in Pro T and Pro B Cell Development. J Exp Med 199:867-878.

 

6. Waskow, C., V. Madan, S. Bartels, C. Costa, R. Blasig, and H.R. Rodewald. 2009. Hematopoietic stem cell transplantation without irradiation. Nat Methods 6:267-269.

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Funding program:

DFG