ALBERT

Description: Cytokine-induced expansion of hematopoietic stem and progenitor cells (HSPCs) is not fully understood. In the present study, we show that whereas steady-state hematopoiesis is normal in basic fibroblast growth factor (FGF-2)–knockout mice, parathyroid hormone stimulation and myeloablative treatments failed to induce normal HSPC proliferation and recovery. In vivo FGF-2 treatment expanded stromal cells, including perivascular Nestin+ supportive stromal cells, which may facilitate HSPC expansion by increasing SCF and reducing CXCL12 via mir-31 up-regulation. FGF-2 predominantly expanded a heterogeneous population of undifferentiated HSPCs, preserving and increasing durable short- and long-term repopulation potential. Mechanistically, these effects were mediated by c-Kit receptor activation, STAT5 phosphorylation, and reduction of reactive oxygen species levels. Mice harboring defective c-Kit signaling exhibited abrogated HSPC expansion in response to FGF-2 treatment, which was accompanied by elevated reactive oxygen species levels. The results of the present study reveal a novel mechanism underlying FGF-2–mediated in vivo expansion of both HSPCs and their supportive stromal cells, which may be used to improve stem cell engraftment after clinical transplantation.

Description: Lung diseases are the second leading cause of death worldwide. There is a growing understanding that lung stem cells, rather than being distributed throughout the tissue, are concentrated in specialized niches. Thus, in our previous study we demonstrated that in analogy to bone marrow (BM) transplantation, lung progenitors could navigate through the blood to their appropriate niches, provided that the niches were cleared of endogenous cell populations (Nature Medicine 2015). Lung injury was induced by Naphthalene, and lung progenitors of all lineages were further depleted by 6GY total body irradiation (TBI) 48 hrs after Naphthalene exposure; at this time, endogenous lung progenitors proliferated extensively. In the present study, we further established the feasibility of our approach in an allogeneic setting. To that end, we induced central immune tolerance towards donor cells, with no need for chronic immune suppression, by virtue of hematopoietic stem cells also present at high levels in fetallung alongside epithelial progenitors. The possibility that the E16 lung might contain high levels of hematopoietic progenitors was first suggested by examination of the peripheral blood of C57BL/6 mice transplanted with GFP+ E16 lung cells, or of RAG-SCID mice (H2Kb) receiving C3H (H2Kk) E16 lung cells. Robust chimerism was also documented in the BM and spleen of all transplanted mice. Based on this initial observation, we attempted to define by FACS the level of putative hematopoietic progenitors in E16 lungs. We thus evaluated two commonly used phenotypes, namely, LSK (lineage-, SCA-1+, C-KIT+) and SLAM (lineage-, CD48-, CD150+). Indeed, we found a similar level of LSK and SLAM cells in the fetal liver and lung, representing about 20-40% of the levels found in the adult BM. A competitive chimerism assay in which normal adult BM cells from a CD45.1+ C57BL/6 donor compete with E16 lung cells from a GFP+CD45.2+ C57BL/6 donor, revealed marked capacity of the E16 lung cells to induce robust chimersim following infusion of a 1:1 mixture of these cells into lethally irradiated CD45.1+ C57BL/6 recipients. Thus, at 9 months post-transplant, 4/4 mice exhibited blood cells derived from the lung donor. In 2 out of 4 mice, levels ofchimerism were above 30%, strongly indicating the robust capacity of the lung hematopoietic progenitors for self-renewal. Based on our ability to induce durable hematopoietic chimeras in syngeneic recipients following transplantation of E16 lung cells, we next developed a sub-lethal transplantation protocol enablingchimerism induction of both non-hematopoeitic cells in the lung, and hematopoietic cells in the blood, liver, spleen and thymus ofmis-matched recipients. The protocol used was based on recent work in haploidenticalhematopoietic stem cell transplantation (HSCT), and comprised transient T cell debulkingof host CD4 and CD8 T cells, megadoseT cell depleted HSCT, and post-transplant cyclophosphamide (CY). This conditioning was coupled with NA, and 6GY TBI to vacate lung progenitor niches (Fig.1). T cells, already present at E16 lungs, were removed from the donor lung preparation by magnetic beads. Similar to our results in haploidenticalBMT we found that chimerisminduction required the use of a 'megadose' of stem cells (5x106 compared to 1x106 in the syngeneic model). When tested at 2 months post-transplant, 4 out of 5 mice exhibited substantial hematopoieticchimerism in the BM, liver, thymus, spleen and blood with multi lineage expression, including B cells (B220), T cells (CD4/CD8) and myeloid cells (CD11b) (Fig. 2). Furthermore, 3 months post-transplant, donor-derived lung "patches" were present, exhibiting marked lungchimerism within both functional epithelial lineages (AEC1/2, marked by AQP5/SPC markers) and mesenchymal/endothelial lineages (marked byNestin/CD31 markers) (Fig. 3 a-d), confirming that the hematopoieticchimerisminduced tolerance towards donor non-hematopoietic lung cell lineages. The high level of hematopoietic progenitors with capacity for self-renewal in thefetallung, alongside non-hematopoietic progenitors, offers a novel approach for allogeneic stem cell transplantation without any need for chronic immune suppression. Further fine tuning is needed to replace NA with clinically approved agents and to define the minimal TBI dose required for effective conditioning. *C.H.K and C.R. contributed equally Disclosures Hillel Karniel: Yeda LTD: Patents & Royalties. Rosen:Yeda LTD: Patents & Royalties. Bachar Lustig:Yeda LTD: Patents & Royalties. Shezen:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Description: Abstract 840 Hematopoietic stem and progenitor cell (HSPC) regulation involves the integration of signals emanating from the nervous system and various bone marrow (BM) cells. Stress signals are produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis via secretion of corticosterone (Cort in mice is the equivalent to cortisol in humans). The major stress hormone, Cort, is secreted in a circadian manner and is known to cause bone loss in supraphysiologic levels. However, whether Cort plays a role in regulation of HSPC and their interactions with the BM microenvironment is currently unknown. Moreover, potentially undesired effects of clinical glucocorticoid administration on HSPC function have not been investigated. We hypothesized that Cort acts in a circadian and dose dependent manner to orchestrate and integrate HSPC function and bone turnover, which are essential for host defense and homeostasis. To determine the roles of circadian Cort oscillations and effects of aberrant Cort regulation on HSPC biology, we utilized CRFR1-/- (R1KO) mice, which lack corticotropin releasing factor receptor 1, a known HPA axis receptor. These mice lack circadian Cort rhythms and have only residual plasma Cort levels. We found that R1KO mice have higher levels of hematopoietic progenitors and the more primitive SKL cells in their BM and blood compared to WT mice. Cort levels measured in the BM of R1KO mice were continuously low, contrasted to the normal and circadially oscillating levels detected in WT mice. As previously established, interactions of the chemokine SDF-1 (CXCL12), the pivotal regulator of stem cell migration and quiescence, with its major receptor CXCR4, are perturbed by various immune-mediated stress signals. Interestingly, the levels of SDF-1 were constantly high in the BM of R1KO mice with no typical circadian peaks. Furthermore, SKL cell release to the blood, expected to follow BM SDF-1-circadian oscillations, as previously shown by Frenette's group, was also constantly increased. Concomitantly, CXCR4 expression in R1KO SKL cells was reduced and the cells demonstrated higher proliferation rates with superior long-term repopulation capacity over WT transplanted cells. Rescue treatment of R1KO mice attained by adding Cort to their drinking water restored their BM SKL and SDF-1 levels to the basal values of WT mice. Increasing Cort levels continuously in the drinking water of WT mice diminished their BM phenotype, including long-term engraftment potential. In vitro cultures of normal BM cells yielded higher colony formation capacity when stimulated with low Cort levels (10-8M). Conversely, high Cort levels (10-5M) inhibited colony formation. Next, we examined microenvironmental components with HSPC support potential. R1KO mice had more primitive stromal cells (CD45-/CD11b-/CD29+/Sca-1+) in their BM. Expression of osteocalcin and osterix, genes characterizing mature osteoblasts, was reduced. Osteoclast maturation was also attenuated, as indicated by increased Gr-1-/CD11b-/c-Kit+ monocyte precursors and reduced transcription of the genes RANK and cathepsin K, both associated with mature osteoclasts and bone resorption. R1KO mice had fewer active osteoclasts in their bones and lower bone turnover rates as indicated by calcein/alizarin bone labeling. In addition, μCT analyses demonstrated a dramatic reduction in femoral trabecules and bone volume. Looking for mediating mechanisms, we found that R1KO BM mononuclear cells highly express Notch1 and its target gene Hes1, known to be involved in regulation of stem cell self renewal. Both genes reverted back to normal expression levels after adding Cort to the drinking water. The signaling Notch1 intracellular domain (NICD), a product of Notch1 cleavage by γ-secratase, was also upregulated in R1KO BM hematopoietic and stromal precursors. Finally, inhibition of Notch1 activation by adding the γ-secratase inhibitor DAPT to BM cultures prevented the stimulating effect of low Cort on colony formation, suggesting that this effect is mediated by Notch1 signaling. Taken together, our study suggests that abolishment of circadian Cort production coupled with continuous low Cort levels increases HSPC proliferation directly and indirectly via Notch1 signaling, while impairing bone maintenance and structure. We propose Cort oscillations and levels to be critical for integrating balanced bone turnover and HSPC function. Disclosures: No relevant conflicts of interest to declare.