Expansion of the Endosteal Hematopoietic Stem Cell Niche Following Myeloablative and Reduced Intensity Conditioning Is Triggered By Hematopoietic Cell Loss
Authors: Olson Timothy S. , Otsuru Satoru , Hofmann Ted J. , Dominici Massimo , Horwitz Edwin M. 
Published: October 2019

Abstract
The efficiency and durability of donor hematopoietic stem cell (HSC) engraftment at specialized niches within recipient bone marrow (BM) are critical determinants of successful clinical stem cell transplant (SCT). Osteolineage elements of BM niches play active, critical roles in regulating HSC engraftment, differentiation, and self-renewal both during homeostasis and post-SCT. We have previously shown that SCT preparative regimens consisting of myeloablative total body irradiation (MB-TBI) result in expansion of osteolineage niche cells at the endosteal surface of metaphyseal bone. This expansion is facilitated by IGF-1 and megakaryocyte-derived PDGF-BB signaling, and is required for optimal HSC engraftment. However, the pathway that integrates these signals to induce expansion remains undefined, and whether expansion is unique to conditioning with MB-TBI due to direct effects on osteolineage cells is also not known. We now present data using chemotherapy-based and reduced intensity conditioning (RIC) models, demonstrating that niche expansion is triggered by hematopoietic cell loss, and not by direct effects of conditioning agents on the niche.
In vitro, MB-TBI (1125 cGy) inhibited primary murine osteoblast (OB) growth and increased apoptosis (Annexin V+ OB at 72 hours: TBI 17% versus control 4%, p &lt; 0.001). In contrast, busulfan treatment caused significantly less OB growth inhibition versus MB-TBI, with no increased apoptosis compared to sham-treated controls. We therefore conditioned wildtype (WT) C57BL/6 mice with myeloablative busulfan (Day -7 to -4) and cyclophosphamide (Day -3 to -2) (BuCy) in a schedule similar to clinical SCT regimens. Endosteal niche expansion increased over this time course, correlating with the extent of hematopoietic ablation, with most expansion occurring prior to cyclophosphamide administration. Maximal expansion occurred by the end of the treatment course, and based on a quantitative scoring index, was not significantly different than maximal MB-TBI-induced niche expansion, demonstrating that niche expansion is not specific to radiation-based conditioning. As with MB-TBI treatment, BuCy-treated recipients of GFP+donor BM consistently exhibited &gt;95% donor chimerism.
We next investigated whether RIC regimens can mediate niche expansion, using an anti-cKit antibody (clone ACK2) known to enable donor engraftment when administered alone in immunodeficient mice or in combination with low dose irradiation (LD-TBI, 300 cGy) in WT mice. ACK2 treatment alone resulted in modest endosteal cell expansion (33% of MB-TBI induced expansion), correlating with transient reductions in host BM cellularity, but absence of definitive donor engraftment in ACK2-treated WT mice. In contrast, LD-TBI alone or in combination with ACK2 produced 58% and 74%, respectively, of the endosteal expansion seen following MB-TBI. Interestingly, while these regimens reduced total BM cellularity by 85% (LD-TBI) and 93% (LD-TBI + ACK2), and led to clearance of BM Lin-Sca1+cKit+(LSK) progenitors, this lack of full niche expansion correlated with incomplete and inconsistent donor chimerism in SCT recipients.
Finally, to prove that endosteal niche expansion results from signals triggered specifically by hematopoietic cell loss, we crossed mice expressing Cre-recombinase under control of the CD45 promoter (CD45Cre) with mice expressing inducible diphtheria toxin receptor (iDTRlox). When treated with diphtheria toxin (DT), BM cellularity in these mice was reduced by 90%, and this targeted ablation of hematopoietic cells was sufficient to induce similar expansion of endosteal mesenchymal cells as seen with radiation or chemotherapy-based conditioning regimens.
Taken together, our results demonstrate that endosteal niche expansion occurs in response to both radiation- and chemotherapy-based SCT conditioning, and that the degree of expansion correlates with both conditioning intensity and with the subsequent degree of donor cell engraftment/chimerism in SCT recipients. Importantly, expansion is triggered not by direct effects of the preparative regimen on mesenchymal niche cells, but rather by loss of hematopoietic cells. These findings provide important insights into how SCT conditioning modulates niche function, and suggests that therapeutic strategies to enhance niche function may be effective in improving engraftment outcomes following RIC SCT.

Disclosures
No relevant conflicts of interest to declare.