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Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. EH domain-binding protein 1 like 1 (EHBP1L1) is an adaptor protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in non-epithelial cells remains unknown. In this study, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization prior to enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and non-epithelial cells.

Hematopoietic stem cells (HSCs) reside in and are maintained by special microenvironments, termed niches. It is assumed that the HSC niche space remains occupied by endogenous cells and that myelosuppressive conditioning is required to achieve high levels of HSC engraftment. We herein demonstrate that upon the transplantation of very large numbers of purified HSCs into normal mice not exposed to myeloablation, donor HSCs engrafted in niches distant from filled HSC niches without replacing host HSCs and subsequently proliferated and generated hematopoietic progenitors, leading to marked increases in the overall HSC numbers in bone marrow. Additionally, stem cell factor that is produced by CXC chemokine ligand 12-abundant reticular cells is involved in HSC engraftment. In contrast, host granulocyte/macrophage progenitors (GMPs) were replaced by the progeny of transplanted donor HSCs, and overall GMP numbers remained unchanged. Thus, inconsistent with the classical concept, numerous empty HSC niches are available for engraftment and proliferation in bone marrow.

Myelodysplastic syndromes (MDS) is a clonal disorder of hematopoietic stem cells (HSCs) characterized by clonal hematopoietic stem cells (HSCs) with cytopenia, morphological abnormalities, genetic alteration, ineffective normal hematopoiesis, and frequent progression to AML. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually come to dominate the bone marrow space. Despite several studies of mesenchymal stem cells (MSCs), one of the principal components of HSC niche supporting normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. In this study, we examined the mechanism by which less-proliferative MDS cells outcompete normal hematopoiesis through the effects on MSCs using serially transplantable Abcg2-induced MDS/AML model we recently generated. The recipient-derived normal BM cells displayed a considerably lower colony output with markedly decreased numbers of the hematopoietic stem progenitor cells (HSPCs) . However, there were no direct effects on the colony-forming ability of the recipient HSPCs co-cultured with MDS/AML cells, indicating that MDS/AML cells inhibited hematopoiesis through alteration of bone marrow microenvironment, such as MSCs, rather than direct interaction between normal and malignant HSCs. We next analyzed histological features of BM specimens. Interestingly, bone sections from the MDS/AML mice showed a reduced trabecular bone and narrowed growth plates. Moreover, micro computed tomography (micro-CT) analysis of the femora showed a significant reduction of the trabecular bone volume in the recipient mice transplanted with the MDS/AML BM cells. We detected decreased bone formation based on the calcein double labeling, but unchanged numbers of the TRAP-positive mononuclear or multinucleated (osteoclastic) cells in the MDS/AML samples, suggesting that the reduced bone volume was caused by suppressed bone formation. The impaired bone formation was also observed in the human MDS patients in terms of lower bone volume and decreased expression of BGLAP, one of osteogenic markers. In line with the above findings, single cell qRT-PCR analyses of mouse MSCs displayed downregulation of a line of osteolineage markers, indicating that MDS/AML cells suppress bone formation through inhibiting osteolineage differentiation of MSCs. Based on the findings, we next examined if re-induction of osteolineage differentiation of the MDS/AML-derived MSCs could rescue the potential of MSCs to support normal hematopoiesis. Importantly, the number of colony-forming cells (CFCs) was significantly restored by inducing differentiation of MDS/AML-derived MSCs toward osteogenic lineage both in vitro and in vivo. These results indicate that the impairment of osteolineage differentiation is the principal cause for an impaired normal hematopoiesis in MDS/AML, and that restoring the supportive niche will be a potential therapeutic option. Since extracellular vesicles (EVs) derived from MDS/AML cells are critical mediators of intercellular communication, we examined the molecular mechanism underlying the dysfunction of MSCs via EVs. As expected, EVs from MDS/AML cells were incorporated into the normal MSCs where osteolineage marker genes were clearly downregulated, and the number of CFCs significantly decreased in the HSPCs co-cultured with MSCs treated by the MDS/AML-derived EVs. Moreover, by comprehensively analyzing microRNAs (miRNAs) enriched in EVs derived from MDS/AML cells, we identified several miRNAs that impaired the differentiation of normal MSCs. These results suggested that miRNAs in EVs derived from MDS/AML cells disrupted the hematopoietic supporting niche through suppressing an osteolineage differentiation of MSCs. Here we uncover a heretofore unrecognized mechanism of bone marrow failure in MDS via the impairment of osteolineage differentiation in MSCs. EVs from MDS cells will be an attractive therapeutic target to restore the supportive niches, such as MSCs, for the remaining normal HSCs. Figure Disclosures No relevant conflicts of interest to declare.

Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)–producingcells, are thought to play central roles in antiviral immunity and the pathogenesis of some autoimmune diseases. pDCs are produced from hematopoietic stem cells in bone marrow. However, the environmental regulation of the development of pDCs is not fully understood. Here, we show that the numbers of pDCs and their earliest progenitors are severely reduced in the absence of CXCR4, the primary physiologic receptor for CXC chemokine ligand 12 (CXCL12), also known as stromal cell–derived factor-1 (SDF-1) in vivo. In vitro, CXCL12 induces a significant increase in pDC numbers generated from primitive hematopoietic cells, and pDCs and their progenitors migrate to CXCL12. In addition, most pDCs are in contact with CXCL12-abundant reticular (CAR) cells in the intersinal space of bone marrow, although many primitive hematopoietic cells adjoin CAR cells surrounding sinusoidal endothelial cells or residing near the bone surface. Thus we identified CXCL12 as a key regulator of pDC development produced by cellular niches, providing new targets for pDC therapeutic control.

Abstract 2337 Although hematopoietic stem cells (HSC) are characterized with self-renewal and pluri-potential, their cell-cycle status and differentiating behavior do fluctuate according to the physiological requirement. In the homeostatic state of adult bone marrow (BM), HSC are likely to be quiescent so that they can evade exhaustion or mutation. However, when BM is injured by irradiation and/or anti-cancer drugs, HSC need to proliferate to restore normal hematopoiesis. Then, after re-establishment of homeostasis, activated HSC return to be quiescent. Molecular crosstalk between HSC and BM microenvironment is thought to elaborately control the status of HSC, but precise mechanisms remain unknown. If the conversion of HSC between dormancy and self-renewal could be accurately monitored, the method should be useful to understand how the HSC status is regulated. Our previous study demonstrated that endothelial cell-selective adhesion molecule (ESAM) is a useful marker for murine HSC throughout life. In the present study, we examined if the ESAM level reflects the HSC status between dormancy and activation. Firstly we monitored ESAM levels of the Lin− Sca1+ c-kit+ (LSK) HSC-enriched fraction in BM after a single 5-FU injection (150 mg/kg) by flow cytometry. From 2 to 9 days after the 5-FU injection, ESAM levels on the LSK fraction remarkably increased. Indeed, the mean fluorescence intensity of ESAM expression on HSC increased by 9.6-fold in 5 days after 5-FU injection. The increase of ESAM expression was more drastic than that of other endothelial-related markers such as CD34 (1.6-fold). After reaching to the maximum peak around day 5–6, ESAM level gradually decreased and returned to the homeostatic level by 12 days after 5-FU. Interestingly, the ESAM up-regulation on HSC was abrogated when inhibitory drugs for NF-kB and topoisomerase-II were given after 5-FU injection. Furthermore, short-term BrdU exposure proved that the ESAMhi cells after 5-FU treatment are actually active in the cell cycle status. Then, the immuno-histochemical analyses were performed to locate the activated HSC in 5-FU treated BM. Since more than 80% of the Lin− ESAMhi Sca1+ cells were found within 20 μm from vascular endothelium, the activated HSC seemed to be intimate with endothelial cells and/or vascular-related cells. Next, we performed functional assessments of the ESAMlow LSK and ESAMhi LSK fractions sorted from 5-FU-treated BM. In methylcellulose cultures, while both fractions contained a number of hematopoietic progenitors, CFU-Mix, primitive multipotent progenitors, were significantly enriched in the ESAMhi fraction (10±0 vs. 48.5±2.1 per 200 ESAMlow or ESAMhi LSK cells, respectively). In the in vivo long-term reconstitution assays, we transplanted 2,000 CD45.1+ ESAMlow or ESAMhi LSK cells with 2 × 105 CD45.2+ competitor BM cells into lethally irradiated CD45.2+ mice. Sixteen weeks after transplantation, the mice transplanted with ESAMhi LSK cells showed significantly higher chimerisms of CD45.1+ cells than those transplanted with ESAMlow LSK, suggesting that long-term HSC are enriched in the ESAMhi fraction. It is noteworthy that the ESAMhi CD45.1+ LSK fraction re-constituted a CD45.1+ LSK population in the CD45.2+ recipient BM, whose ESAM expression levels lowered to the homeostatic level. The results above suggested that ESAM expression level mirrors the activation status of HSC after BM injury. However, it remains unclear if ESAM plays an important role in the hematopoietic recovery. Although we did not observe significant phenotypes except slight anemia in homeostatic ESAM KO mice, we presumed that substantial BM stress might reveal physiological importance of the ESAM expression. At day 5 after injecting 200mg/kg 5-FU, we found that leukocytes and platelet were remarkably decreased in KO mice. Furthermore, the KO mice showed severe anemia (Hb; WT 10.4±1.1 g/dl vs. KO 6.0±1.7 g/dl at day 10), and two of five mice died at day 12. In addition, we observed LSK Flt3− HSC as well as total mononuclear cells more significantly decreased in the KO mice. In summary, our data have shown that ESAM serves as a strong tool to monitor the conversion between dormancy and proliferation of adult BM HSC. In addition, the data from ESAM KO mice have suggested that ESAM is indispensable for normal hematopoietic recovery after BM injury. Further studies should address physiological meanings of the high ESAM level on active HSC. Disclosures: No relevant conflicts of interest to declare.