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Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn+ cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell–based adaptive immunity.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin−cKit+Sca1+ Flk2− (LKS Flk2−) and long-term culture–initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

The hematopoietic stem cell (HSC) niche is currently defined as the specific microenvironment in the bone marrow (BM) which anatomically harbors HSCs and governs their fate. It plays a pivotal role in regulating the survival and self-renewal ability of HSCs, protecting them from exhaustion while preventing their excessive proliferation. Many different stromal cell types have been proposed as putative constituents of the niche, but their integrated function is still unrevealed. Mechanisms by which stem/progenitor cell behavior is regulated in the niche include cell-to-cell interaction and the production of growth factors, cytokines, and extracellular matrix proteins. The HSC niche is a dynamic entity reflecting and responding to the needs of the organism. An understanding of how the niche participates in the maintenance of tissue homeostasis and repair offers new opportunities for the development of novel therapeutic tools.

Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, leading to reduced expression of frataxin protein. Evidence suggests that the mutation may induce epigenetic changes and heterochromatin formation, thereby impeding gene transcription. In particular, studies using FRDA patient blood and lymphoblastoid cell lines have detected increased DNA methylation of specific CpG sites upstream of the GAA repeat and histone modifications in regions flanking the GAA repeat. In this report we show that such epigenetic changes are also present in FRDA patient brain, cerebellum and heart tissues, the primary affected systems of the disorder. Bisulfite sequence analysis of the FXN flanking GAA regions reveals a shift in the FRDA DNA methylation profile, with upstream CpG sites becoming consistently hypermethylated and downstream CpG sites becoming consistently hypomethylated. We also identify differential DNA methylation at three specific CpG sites within the FXN promoter and one CpG site within exon 1. Furthermore, we show by chromatin immunoprecipitation analysis that there is overall decreased histone H3K9 acetylation together with increased H3K9 methylation of FRDA brain tissue. Further studies of brain, cerebellum and heart tissues from our GAA repeat expansion-containing FRDA YAC transgenic mice reveal comparable epigenetic changes to those detected in FRDA patient tissue. We have thus developed a mouse model that will be a valuable resource for future therapeutic studies targeting epigenetic modifications of the FXN gene to increase frataxin expression.

Summary Bone marrow niches for hematopoietic progenitor cells are not well defined despite their critical role in blood homeostasis. We previously found that cells expressing osteocalcin, a marker of mature osteolineage cells, regulate the production of thymic-seeding T lymphoid progenitors. Here, using a selective cell deletion strategy, we demonstrate that a subset of mesenchymal cells expressing osterix, a marker of bone precursors in the adult, serve to regulate the maturation of early B lymphoid precursors by promoting pro-B to pre-B cell transition through insulin-like growth factor 1 (IGF-1) production. Loss of Osx+ cells or Osx-specific deletion of IGF-1 led to a failure of B cell maturation and the impaired adaptive immune response. These data highlight the notion that bone marrow is a composite of specialized niches formed by pairings of specific mesenchymal cells with parenchymal stem or lineage committed progenitor cells, thereby providing distinctive functional units to regulate hematopoiesis., Graphical Abstract, Highlights • Loss of Osx+ osteolineage cells halted B cell maturation and caused immune failure • Mice with Osx+ cell-specific deletion of IGF-1 phenocopied Osx+ cell ablated mice • Osx+ cell promotes pro-B to pre-B cell transition through IGF-1 production • Specialized niche cell-hematopoietic progenitor pairings regulate hematopoiesis, In this article, Scadden, Ferraro, and colleagues show that cells expressing osterix, a marker of bone precursors, serves to regulate the maturation of early B lymphoid precursors through IGF-1 production. Together with the previous finding that mature osteolineage cells regulate the production of thymic-seeding T lymphoid precursors, these data highlight the notion that niche cells and parenchymal cells form distinct cell pairings to create specialized functional units to regulate different processes of hematopoiesis.

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.

Osteoblasts are a key component in the regulation of the hematopoietic stem cell (HSC) niche. Manipulating osteoblast numbers results in a parallel change in HSC numbers. We tested the activity of strontium (Sr), a bone anabolic agent that enhances osteoblast function and inhibits osteoclast activity, on hematopoiesis. In vitro treatment of primary murine osteoblasts with Sr increased their ability to form bone nodules, and in vivo it increased osteoblast number, bone volume, and trabecular thickness and decreased trabecular pattern factor. However, the administration of Sr had no influence on primitive HSCs, although the number of hematopoietic progenitors was higher than in control cells. When Sr-treated mice were used as donors for HSC transplantation, no difference in the engraftment ability was observed, whereas hematopoietic recovery was delayed when they were used as recipients. Despite the changes in osteoblast numbers, no increment in the number of N-cadherin+ osteoblasts and N-cadherin transcripts could be detected in Sr-treated mice. Therefore, increasing the overall number and function of osteoblasts without increasing N-cadherin+ cells is not sufficient to enhance HSC quantity and function. Our study further supports the notion that N-cadherin+ osteoblasts are fundamental in the hematopoietic niche.

Abstract 637 The hematopoietic stem cell niche is known to govern the function of hematopoietic and leukemic stem cells. One constituent of the niche is the extracellular matrix comprised of several elements including a group of molecules called heparan sulfate proteoglycans (HSPGs). These molecules are present in the microenvironment of all tissue stem cells and are critical in the dynamic interactions between stem cells and niche elements. Heparan sulfate proteoglycan synthesis depends upon the glycosyltransferase, Ext1, a known tumor suppressor with a skeletal phenotype (exostoses) when mutated in affected humans. In order to investigate the role of HSPGs in the hematopoietic stem cell niche we conditionally deleted the Ext1 gene in the recently described Mx1+ osteolineage stem/progenitor cell population (Park et al., 2012). Our studies show that Ext1 and its biological product, HSPGs, play a major role in the maintenance of hematopoietic stem cells (HSCs) by modulating the bone marrow microenvironment. Ext1 deletion in osteolineage cells results in a marked loss of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow and an increase in circulating colony forming units (CFU-Cs) and HSCs (designated as lineage -, c-Kit+, Sca1+, CD 150 + and CD48-) both in the spleen and peripheral blood, suggesting a key role of heparan sulfate proteoglycans in the retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. Importantly, histomorphometry analysis showed no defect in bone remodeling upon Ext1 deletion. In order to investigate whether the above phenotype could be therapeutically exploited in the context of bone marrow stem/progenitor cell mobilization, we administered the heparan sulfate proteoglycan inhibitors, heparin and protamine sulfate to mice in combination with conventional mobilization regimes such as G-CSF. Pharmacological inhibition of HSPG induced a significant improvement of hematopoietic stem/progenitor cell mobilization over G-CSF treatment alone, an effect that was completely abrogated in the absence of endogenous HSPGs. Furthermore, by combining HSPG inhibition with G-CSF, we could mobilize a population of HSPCs with superior in-vivo self-renewing ability. Mechanistically, we observed that Ext1 deletion in stromal cells leads to a constitutive activation of the AKT pathway through the PDK1-mediated phosphorylation of AKT-Thr-308, which in turn phosphorylates and inactivate FOXO1 transcription factor. Expression profiling of genes transcriptionally regulated by FOXO1 revealed that VCAM1, a key molecule driving HSCs adhesion to their niche, was highly down regulated in Ext1 deficient cells. VCAM1 protein levels were also decreased accompanied by a profound reduction in HSC adhesion to Ext1 deficient cells in vitro. Taken together our results suggest that Ext1/HSPGs control hematopoietic stem/progenitor cells retention in the bone marrow through an AKT/FOXO1/VCAM1 dependent pathway and that targeting heparan sulfate proteoglycans is a strategy for mobilizing stem cells with superior self renewing capability to the blood. We are grateful to Lexicon Genetics and the MMRRC for kindly providing the Ext1 mouse model. Disclosures: Wagers: BD Biosciences: Consultancy; iPierian, Inc.: Consultancy; MPM Capital: Consultancy; Novartis: Honoraria. Scadden:Fate Therapeutics: Consultancy, Equity Ownership; Genzyme: Consultancy.

Hematopoietic stem cell (HSC) function and number are tightly regulated in a specific microenvironment - the HSC niche - constituted of several cell types. Amongst them, osteoblasts have a prominent role since their increment is associated with an expansion of HSC. However, we demonstrated that an overall increase in osteoblasts does not necessarily promote hematopoiesis (Lymperi et al, Blood 2008 111:1173-81). We hypothesised that osteoclast mediated bone resorption, a component of the bone remodelling process which regulates osteoblast formation, takes active part in the HSC niche. To address this question we inhibited osteoclast function with the bone catabolic agents bisphosphonates. In vivo administration of bisphosphonates to C57BL/6 mice altered the architecture of the trabecular bone as defined by micro-CT and histomorphometric analysis. Treated mice exhibited increased bone volume and trabecular number and reduced trabecular separation and trabecular pattern factor compared to the untreated controls. This was accompanied to a decrease in the proportion and absolute number of HSC in the bone marrow (BM) as assessed by both the immunophenotypic enumeration of Lin-Sca1+c-kit+Flk2-HSC and the long-term culture- initiating cell (LTC-IC) assay. The number of the colony forming unit-cells (CFU-C) in the BM of bisphosphonate treated mice was increased, while more Lin-Sca1+c-kit+ enriched cells were found in the S/M phase of the cell cycle. These results indicate that, in osteoclast-impaired mice a proportion of HSC enters the cell cycle changing the balance in favour of hematopoietic progenitors. The ability of BM cells from bisphosphonate treated mice to engraft and reconstitute the hematopoietic system was tested in a competitive transplantation assay, whereby BM cells from treated and untreated mice were transplanted into syngeneic irradiated recipients. The long-term engraftment of treated BM cells was inferior to the one of controls, indicating a decrease in HSC numbers. Furthermore, recipient mice treated with bisphosphonates prior to conditioning and BM transplantation experienced a delayed hematopoietic recovery as compared to untreated recipients. Since it has been shown that parathyroid hormone (PTH) administration increases the number of HSC by stimulating osteoblast numbers, mice receiving micro-pump delivered PTH were also injected with bisphosphonates. We observed that the administration of bisphosphonates abolished the ability of PTH to increase the primitive HSC pool size as measured by FACS analysis of the Lin-Sca1+c-kit+Flk2- BM cells and their capacity to engraft and reconstitute irradiated recipients. Our findings demonstrate that the osteoclast function is fundamental for the HSC niche, possibly by promoting the recruitment of osteoblasts with HSC supporting capacity.