Abstract
Abstract 74
Hematopoietic stem cells (HSCs) are used therapeutically in bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) to correct hematolymphoid abnormalities. Upon intravenous transplantation, HSCs can home to specialized bone marrow niches, self-renew and differentiate and thus generate a new, complete hematolymphoid system. Unfortunately BMT has had limited applications, due to the risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy, that are deemed necessary for HSC engraftment. Elimination of these toxic conditioning regimens could expand the potential applications of BMT to include many non-malignant hematologic disorders, a wide variety of autoimmune disorders such as diabetes and multiple sclerosis, as well as in the facilitation of organ transplantation. We have previously shown that one important barrier to HSC engraftment is availability of HSC niche space. In the absence of pre-transplant conditioning, >99% of HSC niches are occupied with host HSCs and thus donor HSC engraftment under these conditions is minimal. We have shown in mouse models, that elimination of host mouse HSCs using anti-mouse-ckit monoclonal antibody ACK2 allows for >90% donor HSC engraftment with minimal toxicity in immunodeficient animals, which is sufficient to cure most hematolymphoid disorders.
To examine the effects of inhibition of ckit-signaling in human HSCs, we obtained and created various monoclonal antibodies to human ckit and verified that anti-human-ckit monoclonal antibody SR-1 uniquely inhibits SCF binding. To examine the dependence of human HSC on ckit-signaling we cultured purified human bone marrow HSC and umbilical cord blood HSC (CD34+CD38-CD90+CD45RA-Lin-) in the presence of StemSpan media supplemented with human cytokines SCF, TPO, Flt-3, IL-3, IL-6 and either 10ug/ml of SR-1 or 4F7 (an anti-human-ckit clone that did not inhibit SCF-binding). Here we show that SR-1 uniquely inhibited both human bone marrow and cord blood HSC proliferation in vitro. Interestingly, SR-1 did not induce human HSC cell death via apoptosis, as addition of Z-VAD-FMK caspase inhibitor did not abrogate the effects of SR-1. Additionally, the lack of PI+ and Annexin V+ populations day 3 and 7 days post addition of SR-1 failed to provide evidence of HSC death. However, careful examination revealed a shift in the differentiation profile of HSC cultured in the presence of SR-1. Whereas 4F7 did not change the in vitro output of human HSC, cord blood HSC cultured in the presence of SR-1 showed increased propensity to give rise to Glycophorin A+ and CD41+ cells (RBC and platelets respectively), and decreased output of CD14, CD13, and CD33 cells (macrophages/myeloid cells).
To examine the depleting capability of SR-1 in vivo, robust human-mouse hematopoietic chimeras were generated. Newborn immune deficient NOD/SCID/IL-2Rγnull (NOG) mice received 100cGy radiation and subsequently were transplanted intravenously via facial vein with ∼1000 purified human cord blood HSC. Mice with >15% human CD45+ chimerism in the peripheral blood at 12 wks post-transplant were selected for further experimentation. Bone marrow aspirates were obtained from these animals and assayed for human chimerism by FACS. Average pre-treatment baseline level of total human bone marrow engraftment (%hCD45) was 58.6%, whereas myeloid chimerism (%hCD13/33) was 25.4%. The mice were then treated intravenously with 500ug of SR-1 every other day for 1 week. Human total and myeloid engraftment 8 weeks post treatment decreased by 92.4% and 96.9% respectively, most likely due to depletion of human HSCs that maintain these populations. The remaining persistent human cells in these animals were primarily composed of mature, long-lived B and T-cells that do not need to be regenerated by HSC and are therefore unaffected by this therapy.
In summary, we have shown that human HSCs depend on SCF for proliferation, and inhibition of SCF-signaling via anti-ckit monoclonal antibody SR-1 resulted in non-toxic, specific, in vitro and in vivo HSC depletion. Through this method we hope to deplete host HSCs in humans, thereby increasing available human HSC niches for engraftment and providing effective yet mild conditioning prior to transplantation. This work could enable efficient transplantation of HSC that cannot cause GvH, resulting in a potential curative therapy for almost any hematologic or immunologic disease.
Disclosures:
Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow
