Osteolineage Cells Research Articles

Osteocyte-Mediated Parathyroid Hormone (PTH) Signaling Regulates Hematopoietic Stem Cells Under Physiologic and Continuous PTH Exposure

The bone marrow microenvironment, including osteolineage cells, regulates hematopoietic stem cell (HSC) fate choices. Intermittent pharmacologic treatment of mice with parathyroid hormone, PTH (1-34), indirectly increases HSCs through their niche, as HSCs do not express the PTH receptor (PTH1R). Osteocytes, the most abundant osteolineage cells in bone, are a critical target of the skeletal actions of PTH and coordinate multiple cell types that are components of the HSC niche including osteoblasts, osteoclasts and resident macrophages. While osteocytes express the PTH1R, the role of osteocytes in HSC regulation is unclear. Therefore, we studied the role of osteocyte-mediated PTH regulation of HSCs, using cre recombinase driven by the 8kb-DMP1 promoter to conditionally delete PTH1R in osteocytes (OCyPTHRko mice).OCyPTHRko mice were viable, fertile, did not exhibit any significant skeletal defect as juveniles or at 6 months of age, had no significant difference in serum PTH levels, and had no significant difference in osteoblastic or mesenchymal stem cell numbers compared to WT mice. In juvenile OCyPTH1Rko mice there was a decrease in long-term HSCs as measured by flow cytometric analysis (0.0029 ± 0.00028 vs. 0.0021 ± 0.00021 % of cells, WT vs. OCyPTH1Rko p≤0.05 N≥19 mice/group). OCyPTH1Rko mice had 4 fold lower long-term engraftment capacity as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥10 mice/group) that was evident in all hematopoietic lineages. Short-term engraftment however was increased in OCyPTH1Rko mice as measured by primary competitive transplantation (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.01, N≥9 mice/group). These data demonstrate that physiologic PTH signaling in osteocytes regulates the balance of long-term and short-term HSC potential in juvenile, growing mice. Adult OCyPTH1Rko mice also had 5 fold lower long-term engraftment as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥15 mice/group). These findings demonstrate a previously unrecognized physiologic role of PTH signaling in HSC regulation. Having demonstrated a role for PTH signaling in HSC homeostasis, we investigated if sustained PTH elevations (as are found in vitamin D deficiency and in hyperparathyroidism) alter HSC function. Therefore, we utilized a murine model of secondary hyperparathyroidism caused by a low calcium (LCa) diet. In juvenile mice placed on the LCa diet immediately upon weaning, serum PTH levels were significantly elevated. Fourteen days on the LCa diet caused a significant reduction in long-term engraftment potential as measured by secondary competitive transplants over 22 weeks (Normal vs. LCa diet donors, 2-way ANOVA p≤0.001, N≥20 mice/group), while there was no decrease in HSCs when adult mice were placed on the LCa diet. These data suggest that sustained PTH signaling decreases microenvironmental support for HSCs in juvenile mice. We utilized the OCyPTHRko mice to study the role of osteocytes in hyperparathyroidism-induced loss of functional HSCs. In juvenile mice the lack of PTH signaling in osteocytes rescued the long-term engraftment defects, suggesting that PTH signaling in osteocytes mediates the loss of long-term HSC support caused by the LCa diet. In further support of a deleterious effect mediated by the PTH1R in osteocytes in the setting of continuous PTH, adult OCyPTH1Rko mice placed on LCa diet had superior long term HSC function.Our findings demonstrate a physiologic role for PTH in HSC regulation and identify osteocytes as a critical constituent of the HSC niche that, either directly or indirectly, contribute to maintenance of the long-term repopulating HSC pool. In addition, we show that continuous exposure to elevated levels of PTH in a model of secondary hyperparathyroidism leads to osteocyte-mediated loss of long-term engraftment potential of HSCs in juvenile mice. We speculate that removing the effect of continuous PTH from osteocytes uncovers additional HSC-supportive effects of continuous PTH, mediated by non-osteocyte HSC niche cellular populations. Together these data establish PTH as a critical regulatory signal in the HSC niche, and show that the relative contributions of niche populations to HSC regulation are modulated by age. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Restoration of the Bone Marrow Microenvironment Improves Hematopoietic Function in a Murine Model of Myelodysplastic Syndrome

While data suggest that, in the myelodysplastic syndromes (MDS), the bone marrow microenvironment (BMME) is abnormal, the lack of examination of the BMME in a robust in vivo model has limited progress in the understanding of reciprocal MDS-BMME interactions. If microenvironmental defects contribute to disease progression, targeting the BM niche may offer an alternative approach for therapeutic benefit. We sought to define the MDS BMME in a well-established transgenic murine model that recapitulates hallmark features of human MDS. In this model, hematopoietic tissue-specific expression of the NUP98-HOXD13 (NHD13) fusion gene is driven by Vav regulatory elements, resulting in peripheral cytopenias by 16 weeks of age and mortality from transformation to leukemia at a median time of 11 months of age. Mice were analyzed at 15-36 weeks of age, when the MDS phenotype is prominent in the absence of leukemia. Flow cytometric quantification of BM stroma in 23-week old NHD13 mice showed a 6.5-fold increase in frequency of CD51+/Sca1- osteoblastic cells (OBC) compared to WT (p<0.05). CD51+/Sca1+ multipotent stromal cells (MSC) and CD31+/Sca1+ endothelial cells were also significantly increased in NHD13 compared to WT mice. This was not due to loss of hematopoietic cells in the marrow of NHD13 mice. While an expansion of functional MSCs and osteoblastic cells could result in skeletal changes, micro CT imaging of the femora and tibiae of 20-week old NHD13 mice revealed no differences in skeletal parameters compared to WT mice. These data suggest that the expanded osteolineage cells in NHD13 mice are not functional bone-forming cells. While stromal populations were not altered in bone-associated cells of 23-week old NHD13 compared to WT mice, 36-week old NHD13 mice also showed increased bone-associated OBCs, MSCs, and endothelial cells. Therefore, there are significant time-dependent shifts in critical stromal populations in this in vivo model of MDS, which may contribute to an abnormal BMME. To determine if the MDS BMME contributes to hematopoietic failure, NHD13 BM (CD45.2) was transplanted with WT competitor BM (CD45.1) in a 1:1 ratio into lethally irradiated NHD13 or WT (CD45.2) recipients, thus exposing the same MDS hematopoietic cells to either MDS or WT microenvironments. Using this transplantation paradigm, we previously reported improvement of hematopoiesis when NHD13 BM is exposed to a WT BMME. Surprisingly, CD45.1+ WT competitor-derived cells exhibited myeloid skewing when transplanted into NHD13 recipients compared to WT recipients, suggesting that interaction of WT BM with an MDS BMME can induce myeloid skewing, a feature of the NHD13 model. NHD13 BM was next transplanted non-competitively into lethally irradiated NHD13 and WT mice. At 10 weeks post-transplant, WT recipients had a 2.5-fold increase in peripheral leukocytes (p<0.05), significant improvement of anemia, and significant mitigation of BM long term-HSC loss compared to NHD13 recipients, suggesting that WT BMME components can rescue hematopoietic function in MDS. Together, our studies strongly suggest that a murine model recapitulates MDS microenvironmental abnormalities, and that exposure of MDS hematopoietic cells to a non-malignant microenvironment is sufficient to improve hematopoietic function. Thus, improvement of the BM microenvironment represents a novel therapeutic strategy to ameliorate hematopoietic function in MDS. DisclosuresBecker:Millenium: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties.

Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice. Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells. There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes. Disclosures No relevant conflicts of interest to declare.

Heterogeneity in the Making of Blood

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches. Disclosures No relevant conflicts of interest to declare.

Loss of TGF-β Signaling in Bone Marrow Mesenchymal Progenitors Promotes Adipocyte over Osteoblast Differentiation but Does Not Disrupt the HSC Niche

Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. Several mesenchymal stromal cells have been implicated in hematopoietic niches, including osteoblasts, pericytes, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem cells (MSCs). Members of the transforming growth factor (TGF) superfamily, in particular TGF-β, have a well-documented role in regulating osteoblast development. However, the contribution of TGF family member signaling to the establishment and maintenance of hematopoietic niches is largely unknown. Here, we characterize the role of transforming growth factor-β (TGF-β) signaling in mesenchymal stromal cells on the HSC niche.TGF-β receptor 2 (encoded by Tgfbr2) is required for all TGF-β signaling. To selectively disrupt TGF-β signaling in bone marrow mesenchymal stromal cells, we generated Osx-C re Tgfbr2fl/fl mice. Osx-Cre targets most bone marrow mesenchymal stromal cells (including osteoblasts, CAR cells, MSCs, pericytes, and adipocytes) but not endothelial cells or hematopoietic cells. Osx-C re Tgfbr2fl/fl mice are severely runted and most die by 4 weeks of age. We analyzed mice at 3 weeks, when the mice appeared healthy. Osteoblast number was severely reduced in Osx-C re Tgfbr2fl/fl mice, as assessed by histomorphometry and immunostaining for osteocalcin. Accordingly, microCT analysis demonstrated reduced tissue mineral density and cortical thickness of long bone and marked trabecularization of long bones in diaphyseal regions. Surprisingly, marrow adiposity, as measured by osmium tetroxide staining with microCT, was strikingly increased in Osx-C re Tgfbr2fl/fl mice. CAR cells are mesenchymal progenitors with osteogenic and adipogenic potential in vitro. To assess CAR cells, we generated Osx-Cre Tgfrb2fl/fl x Cxcl12gfp mice. Surprisingly, CAR cell number was significantly increased. However, despite the increase in CAR cells, the number of CFU-osteoblast (CFU-OB) in Osx-C re Tgfbr2fl/fl mice is nearly undetectable. Together, these data suggest that TGF-b signaling contributes to lineage commitment of mesenchymal progenitors. Specifically, our data suggest that TGF-β signaling suppresses commitment to the osteoblast lineage, while increasing adipogenic differentiation.We next asked whether alterations in bone marrow stromal cells present in Osx-C re Tgfbr2fl/fl mice affect HSC number or function. The increase in marrow adipocytes and loss of osteolineage cells is predicted to impair HSC maintenance, while the increase in CAR cells might augment HSCs. Osx-Cre Tgfrb2fl/fl mice have modest leukopenia, but normal red blood cell and platelet counts. Bone marrow and spleen cellularity are reduced, even after normalizing for body weight. The frequency of phenotypic HSCs (defined as Kit+ lineage- Sca+ CD34- Flk2- cells) is comparable to control mice. To assess HSC function, we performed competitive repopulation assays with bone marrow from Osx-Cre Tgfrb2fl/fl or control mice. Surprisingly, these data show that the long-term multi-lineage repopulating activity of HSCs from Osx-Cre Tgfrb2fl/fl mice is normal. Moreover, serial transplantation studies suggest that the self-renewal capacity of HSCs is normal. Thus, despite major alterations in mesenchymal stromal cell populations, the HSC niche is intact in Osx-Cre Tgfrb2fl/fl mice.Collectively, these data show that TGF-b signaling in mesenchymal progenitors is required for the proper development of multiple stromal cell populations that contribute to hematopoietic niches. Studies are underway to assess the impact of post-natal deletion of Tgfbr2 in mesenchymal stromal cell on hematopoietic niches. Since drugs that modulate the activity of TGF-b are in development, this research may suggest novel approaches to modulate hematopoietic niches for therapeutic benefit. DisclosuresNo relevant conflicts of interest to declare.

Communication of bone cells with hematopoiesis, immunity and energy metabolism.

The bone contains the bone marrow. The functional communication between bone cells and hematopoiesis has been extensively studied in the past decade or so. Osteolineage cells and their modulators, such as the sympathetic nervous system, macrophages and osteoclasts, form a complex unit to maintain the homeostasis of hematopoiesis, called the 'microenvironment'. Recently, bone-embedded osteocytes, the sensors of gravity and mechanical stress, have joined the microenvironment, and they are demonstrated to contribute to whole body homeostasis through the control of immunity and energy metabolism. The inter-organ communication orchestrated by the bone is summarized in this article.

Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model.

The bone marrow niche is recognized as a central player in maintaining and regulating the behavior of hematopoietic stem and progenitor cells. Specific gain-of and loss-of function experiments perturbing a range of osteolineage cells or their secreted proteins had been shown to affect stem cell maintenance (Calvi et al, 2003 [1]; Stier et al., 2005 [2]; Zhang et al., 2003 [3]; Nilsson et al., 2005 [4]; Greenbaum et al., 2013 [5]) and engraftment (Adam et al., 2006, 2009 [6], [7]). We used specific in vivo cell deletion approaches to dissect the niche cell-parenchymal cell dependency in a complex bone marrow microenvironment. Endogenous deletion of osteocalcin-expressing (Ocn+) cells led to a loss of T immune cells (Yu et al., 2015 [8]. Ocn+ cells express the Notch ligand DLL4 to communicate with T-competent progenitors, and thereby ensuring T precursor production and expression of chemotactic molecules on their cell surface for subsequent thymic seeding. In contrast, depletion of osterix-expressing (Osx+) osteoprogenitors led to reduced B immune cells. These distinct hematopoietic phenotypes suggest specific pairing of mesenchymal niche cells and parenchymal hematopoietic cells in the bone marrow to create unique functional units to support hematopoiesis. Here, we present the global gene expression profiles of these osteolineage subtypes utilizing a triple fluorescent transgenic mouse model (OsxCre+;Rosa-mCh+;Ocn:Topaz+) that labels Osx+ cells red, Ocn+ cells green, and Osx+ Ocn+ cells yellow. This system allows isolation of distinct osteolineage subsets within the same animal by flow cytometry. Array data that have been described in our study [8] are also publically available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE66042. Differences in gene expression may correlate with functional difference in supporting hematopoiesis.

Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development.

Vascular endothelial growth factor A (Vegfa) has important roles in endochondral bone formation. Osteoblast precursors, endothelial cells and osteoclasts migrate from perichondrium into primary ossification centers of cartilage templates of future bones in response to Vegfa secreted by (pre)hypertrophic chondrocytes. Perichondrial osteolineage cells also produce Vegfa, but its function is not well understood. By deleting Vegfa in osteolineage cells in vivo, we demonstrate that progenitor-derived Vegfa is required for blood vessel recruitment in perichondrium and the differentiation of osteoblast precursors in mice. Conditional deletion of Vegfa receptors indicates that Vegfa-dependent effects on osteoblast differentiation are mediated by Vegf receptor 2 (Vegfr2). In addition, Vegfa/Vegfr2 signaling stimulates the expression and activity of Indian hedgehog, increases the expression of β-catenin and inhibits Notch2. Our findings identify Vegfa as a regulator of perichondrial vascularity and osteoblast differentiation at early stages of bone development.

Modulation of Interaction of Human Osteoprogenitor Cells with Hematopoietic Stem and Progenitor Cells

Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system.Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions.To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p < 0.05).Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors >50 years old compared to <50 years old donors (2.240e-005 ± 3.300e-006 N=2 vs. 0.0001146 ± 4.163e-005 N=9). We identified non-statistically significant decrease in the frequency of CFU-OBs in bone marrow SACs from MDS patients compared to normal donors (1.090e-005 ± 1.400e-006 N=2 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.179); and similar decrease in frequency of osteoprogenitor cells in the bone marrow aspirates from AML patients compared to normal donors (2.303e-005 ± 9.371e-006 N=3 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.251). These data support our hypothesis that OPCs in patients with MDS and AML are negatively impacted compared to normal bone marrow.These data demonstrate that human SACs contain HSPC-supportive cells which can be stimulated to improve HSPC function. Human SACs comprise MSCs and osteolineage cells including osteoprogenitor cells. Aging decreases OPC pools in SACs. Our data in our small sample also suggest that dysplastic bone marrow microenvironment may negatively impact OPCs, which may in turn decrease OPC support of HPSCs. PTH treatment in our in-vitro model shows the potential to improve the interaction between the OPCs and HSPCs, resulting in amelioration of HSC function. Together these data suggest a strategy where targeting the MDS microenvironment may add to the currently available treatment modalities. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Proximity-Based Single Cell Analysis of the Bone Marrow Niche Identifies Interleukin-18 As a Quiescence Regulator of Early Hematopoietic Progenitors

Background. Niche-derived signals are essential for maintenance and expansion of stem/progenitor cells in the bone marrow. Recently published studies used elegant genetic tools to dissect predominant cellular sources of known HSPC regulators, such as CXCL12 and kit-ligand (Ding et al, Nature 2012, Greenbaum et al, Nature 2013). However, strategies enabling identification of novel niche-derived factors are lacking. Here, we describe an approach which utilizes spatial proximity between osteolineage cells (OLC) and HSPC in the post-transplant bone marrow niche as a guide to niche factor discovery and reveals the role of Interleukin-18 (IL18) as a quiescence regulator of early hematopoietic progenitors.Results. We established a neonatal bone marrow (BM) transplantation model, in which adult BM-derived LKS CD34-Flk2- HSPCs fluorescently labeled with DiI were transplanted into irradiated newborn col2.3GFP recipients (in this mouse strain, the majority of OLCs are labeled with GFP). Forty-eight hours later, we obtained sections of femoral trabecular bone from transplanted mice and located rare OLCs harboring DiI-positive HSPCs in their immediate proximity. We harvested individual OLCs located within two cell diameters (proximal OLCs) and greater than five cell diameters (distal OLCs) from DiI-labeled HSPCs and performed comparative transcriptome analysis by single cell RNA-Seq. Remarkably, we found that proximal OLCs were distinct from their distal counterparts and showed a higher expression levels of known niche-associated molecules, as well as secreted factors not previously linked to regulatory niche function, including a pro-inflammatory cytokine IL18.Quantification of primitive subsets in the BM of IL18 knock-out (IL18KO) mice showed no abnormalities. However, cell cycle studies revealed that while long-term HSCs (which did not express the IL18 receptor) were unaffected by IL18 deletion, early progenitors - short-term HSC, multi-potent progenitor and common lymphoid progenitor – were more actively cycling. Accordingly, following sublethal exposure to 5-fluorouracil (5FU), IL18KO animals displayed a 2-fold increase in frequency of lin-kit+Sca1+ (LKS) cells, lin-kit+ myeloid progenitors and CLPs, as compared wild-type (WT) controls (Figure 1). On the other hand, exogenous administration of recombinant IL18 to 5FU-treated WT animals was associated with 25% mortality, while no deaths were observed in the vehicle-treated group. Taken together, these data illustrate that IL18 constrains the ability of the progenitor pool to respond to a genotoxic stress.Next, we tested if IL18 acts in a non cell-autonomous fashion. We transplanted progenitor-enriched bone marrow fraction (LKS cells) into IL18KO or WT hosts and observed an approximately 2-fold increase in both myeloid and lymphoid cells in peripheral blood of IL18KO animals. This finding was recapitulated in a reciprocal experiment, when LKS cells from IL18 receptor knock-out animals were transplanted into WT hosts, supporting the possibility that the effect of IL18 on progenitor proliferation is direct.Finally, we investigated if the loss of IL18-mediated quiescence can be therapeutically exploited in the transplant setting. To this end, we assessed survival of lethally irradiated WT and IL18KO mice following transplantation with a limiting BM dose, i.e. predicted to confer survival to 50% or less of WT animals. Notably, we observed reduced 30-day post-transplant mortality (33% versus 72%, p value = 0.05) in IL18KO animals, which likely resulted from accelerated progenitor expansion in the absence of IL18 in the host microenvironment (Figure 2).Conclusions.Our study demonstrates the capability of proximity-based single cell analysis of the post-transplant bone marrow microenvironment to identify a novel niche factor – IL 18. We show that IL18 specifically regulates quiescence of early hematopoietic progenitors, particularly under the conditions of stress hematopoiesis, such as exposure to 5FU or post-transplant hematopoietic expansion. The results presented here provide an insight into poorly understood molecular mechanisms of progenitor regulation by the niche. Moreover, improved post-transplant survival of animals in the absence of IL18 suggests that pharmacological inhibition of IL18 has the potential of improving clinical outcomes in transplant recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Osteoblastic VEGF Coordinates Remodeling of the Hematopoietic Stem Cell Niche

Osteoblasts (OBs) and the bone marrow (BM) vasculature constitute hematopoietic stem and progenitor cell (HSC/HSPC) niches. Immature perivascular osteolineage cells enforce HSC quiescence while signals from mature OBs are capable of bone and vascular remodeling. We previously demonstrated that bone anabolic parathyroid hormone (PTH) stimulation increases HSPCs with short and long-term repopulating activity. Based upon the coupled nature of osteogenesis and angiogenesis as well as the multifaceted role blood vessels play in HSC regulation, we investigated effects of PTH on the BM vasculature. Both genetic PTH receptor activation in maturing OBs (Col1caPTH1R) and systemic PTH treatment increased functional blood vessel branching imaged intravitally in the calvaria (14±2 vs 34±3 and 14±2 vs 27±4 branchpoints, n=3-4 mice/group, 3-7 regions analyzed per mouse). These changes were confirmed histologically in long bones, where PTH-induced tortuosity was accompanied by the emergence of α smooth muscle actin (αSMA)+ bone-associated vascular networks, likely small-caliber arterioles, containing red blood cell rouleaux, while control marrow displayed sparse, non-bone-associated αSMA+ vessels. Further, PTH increased morphologically-heterogeneous BM microvessels (167±18 vs 348±39 per section, n=5 mice/group) and endothelial cell (EC) abundance (0.09±0.02% vs 0.2±0.02%, n=4-5 mice/group). VEGF-A and FGF2, known to be important proangiogenic signals in bone, were increased after PTH treatment of osteoblastic cell lines and primary osteolineage cells in vitro, and in bone-associated cells of mice treated in vivo. Because the perivascular milieu is heterogeneous and can support HSC quiescence or activation depending on vessel type and stimuli, we tested whether tuning BM angiogenic responses modulated effects of PTH on HSCs. Treatment with the anti-VEGF-A monoclonal antibody bevacizumab (αVEGF) precluded PTH-induced vascular branching in calvarial BM and reduced pre-established vascular branching in Col1caPTH1R mice (34±3 vs 21±1 branchpoints, n=4 mice/group, 3-5 regions analyzed per mouse), while PTH-induced bone anabolism, EC abundance and bone-associated αSMA+ small-caliber vessels were sustained. Moreover, αVEGF did not change the frequency of CD51+ PDGFRα+ or PDGFRα+ Sca1+ immature mesenchymal cells reported to regulate HSCs. The altered balance of marrow sinusoidal vs arteriolar structures we quantified in PTH + αVEGF-stimulated BM would be expected to improve HSC support based on niche remodeling, therefore we tested the hematopoietic consequences. αVEGF did not alter frequencies of phenotypically-defined HSCs in the BM or mature hematopoietic cells and platelets in the peripheral blood (PB). Remarkably, αVEGF augmented PTH-induced repopulation of the PB in primary BM transplantation (p = 0.0013, n=10 recipients/group) and sustained the repopulating ability (p < 0.0001, n=10 recipients/group) and BM engraftment (~70 fold) of cells in secondary transplantation. Competitive transplantation of HSPCs sorted from PTH + αVEGF-treated mice showed enhanced repopulating ability, confirming niche-mediated improvement of HSPC function. Unbiased analysis of sorted stem and progenitor cells demonstrated that PTH, alone or in combination with αVEGF, broadly reduced multipotent progenitor (MPP) gene expression, including markers of cell proliferation. Notably, microenvironmental activation with PTH alone uniquely decreased a cluster of transcripts associated with hematopoietic differentiation in MPPs, suggesting progenitor cell reeducation by PTH activation versus niche reapportioning by combined PTH and VEGF antagonism. Because PTH also increases FGF2, reported to expand HSCs and stabilize blood vessels, we tested whether FGF signaling is necessary for PTH to establish long-term HSC niches. In vivo FGF receptor 1 inhibition significantly reduced long-term hematopoietic repopulating ability of PTH + αVEGF-treated BM cells in secondary transplantation (p = 0.0046, n = 16-17 mice/group), suggesting VEGF-A and FGF2 have opposing HSC effects in the PTH-activated microenvironment. These data define the HSC niche as a regulatory network, and identify mature OBs as the cellular source of signals that serve to coordinate HSC-supportive niches, demonstrating functional cooperation of the different constituents of the bone marrow microenvironment. DisclosuresOff Label Use: Bevacizumab (trade name Avastin, Genentech/Roche) is used by the authors as a strategy to block VEGF-A in the bone marrow microenvironment.. Calvi:Fate Therapeutics: Patents & Royalties.

Expansion of the Endosteal Hematopoietic Stem Cell Niche Following Myeloablative and Reduced Intensity Conditioning Is Triggered By Hematopoietic Cell Loss

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 < 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 >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. DisclosuresNo relevant conflicts of interest to declare.

Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation.

Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated--to our knowledge for the first time--that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.

Development of the Fetal Bone Marrow Niche and Regulation of HSC Quiescence and Homing Ability by Emerging Osteolineage Cells

Hematopoietic stem cells (HSCs) reside within a specialized niche where interactions with vasculature, osteoblasts, and stromal components regulate their self-renewal and differentiation. Little is known about bone marrow niche formation or the role of its cellular components in HSC development; therefore, we established the timing of murine fetal long bone vascularization and ossification relative to the onset of HSC activity. Adult-repopulating HSCs emerged at embryonic day 16.5 (E16.5), coincident with marrow vascularization, and were contained within the c-Kit(+)Sca-1(+)Lin(-) (KSL) population. We used Osterix-null (Osx(-/-)) mice that form vascularized marrow but lack osteolineage cells to dissect the role(s) of these cellular components in HSC development. Osx(-/-) fetal bone marrow cells formed multilineage colonies invitro but were hyperproliferative and failed to home to and/or engraft transplant recipients. Thus, in developing bone marrow, the vasculature can sustain multilineage progenitors, but interactions with osteolineage cells are needed to regulate long-term HSC proliferation and potential.

The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.

Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.

Osteoblasts as leukemia-initiating cells.

Osteoblasts as leukemia-initiating cells.

A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.

Regulation of hematopoiesis in endosteal microenvironments.

After birth, the hematopoietic system develops along with bone formation in mammals. Osteolineage cells are derived from mesenchymal progenitor cells, and differentiate into several types of bone-forming cells. Of the various types of cell constituents in bone marrow, osteolineage cells have been shown to play important roles in hematopoiesis. Early studies have identified osteoblasts as a hematopoietic stem cell niche component. Since that time, the role of endosteal microenvironment as a critical regulator of hematopoietic stem/progenitor cell (HSC/HPC) behavior has been appreciated particularly under stress conditions, such as cytokine-induced HSC/HPC mobilization, homing/engraftment after bone marrow transplantation, and disease models of leukemia/myelodysplasia. Recent studies revealed that the most differentiated osteolineage cells, i.e., osteocytes, play important roles in the regulation of hematopoiesis. In this review, we provide an overview of recent advances in knowledge of regulatory hematopoietic mechanisms in the endosteal area.

Bone's dark side: mutated osteoblasts implicated in leukemia

Bone-lining osteolineage cells were previously implicated as contributors to hematological disorders and malignancies. A recent report in Nature now demonstrates that a specific mutation in mouse collagen-expressing osteoblastic cells leads to MDS and AML with 100% penetrance and is associated with strikingly similar findings in human patients.

Enrichment and Characterization of Two Subgroups of Committed Osteogenic Cells in the Mouse Endosteal Bone Marrow with Expression Levels of CD24

Primary osteogenic cells have been known to reside within the CD45-CD31-Ter119-Sca-1- cell fraction, particularly in the CD51+ subpopulation. However, detailed determination of the frequency of osteogenic cells within this Sca-1- cell population remains yet to be determined. In addition, it is not clear that other cell surface markers can be used to further sub-fractionate this Sca-1-CD51+ osteogenic cell population and to define their developmental stages. In this report, both Sca-1-CD24med and Sca-1- CD24-/lo cells have been shown to be two small subsets of the Sca-1-CD51+ cell fraction. These two cell fractions show subtle difference in the expression level of osteogenic marker genes such as Osx and Opn, and in vitro proliferate rate. All these observations suggest that they may be at different developmental stages of osteogenesis. The Sca-1-CD24med cell fraction is enriched for the more mature osteolineage cells than the Sca-1-CD24-/lo counterpart. In contrast, most of the Sca-1-CD24hi and Sca-1+CD24-/lo cells do not contain CFU-ALP nor express osteogenic gene markers. The high proliferation ability and osteo-adipogenic differentiation potentials confirm that the Sca-1+CD24-/lo cells are the multipotential mesenchymal stromal cells. The determination of individual stromal cell subpopulations will lead to a better understanding in the hierarchical organization of these osteolineage cells.