Characterization of substantia nigra neurogenesis in homeostasis and dopaminergic degeneration: beneficial effects of the microneurotrophin BNN-20

Abstract Background Loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) underlines much of the pathology of Parkinson’s disease (PD), but the existence of an endogenous neurogenic system that could be targeted as a therapeutic strategy has been controversial. BNN-20 is a synthetic, BDNF-mimicking, microneurotrophin that we previously showed to exhibit a pleiotropic neuroprotective effect on the dopaminergic neurons of the SNpc in the “weaver” mouse model of PD. Here, we assessed its potential effects on neurogenesis. Methods We quantified total numbers of dopaminergic neurons in the SNpc of wild-type and “weaver” mice, with or without administration of BNN-20, and we employed BrdU labelling and intracerebroventricular injections of DiI to evaluate the existence of dopaminergic neurogenesis in the SNpc and to assess the origin of newborn dopaminergic neurons. The in vivo experiments were complemented by in vitro proliferation/differentiation assays of adult neural stem cells (NSCs) isolated from the substantia nigra and the subependymal zone (SEZ) stem cell niche to further characterize the effects of BNN-20. Results Our analysis revealed the existence of a low-rate turnover of dopaminergic neurons in the normal SNpc and showed, using three independent lines of experiments (stereologic cell counts, BrdU and DiI tracing), that the administration of BNN-20 leads to increased neurogenesis in the SNpc and to partial reversal of dopaminergic cell loss. The newly born dopaminergic neurons, that are partially originated from the SEZ, follow the typical nigral maturation pathway, expressing the transcription factor FoxA2. Importantly, the pro-cytogenic effects of BNN-20 were very strong in the SNpc, but were absent in other brain areas such as the cortex or the stem cell niche of the hippocampus. Moreover, although the in vitro assays showed that BNN-20 enhances the differentiation of NSCs towards glia and neurons, its in vivo administration stimulated only neurogenesis. Conclusions Our results demonstrate the existence of a neurogenic system in the SNpc that can be manipulated in order to regenerate the depleted dopaminergic cell population in the “weaver” PD mouse model. Microneurotrophin BNN-20 emerges as an excellent candidate for future PD cell replacement therapies, due to its area-specific, pro-neurogenic effects.

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MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.018 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S7-S8

Author(s):

Safina Gadeock ◽

Cambrian Liu ◽

Brent Polk

Keyword(s):

Stem Cell ◽

Stem Cell Niche ◽

Mesenchymal Cells ◽

Epithelial Stem Cells ◽

Epithelial Stem Cell ◽

Long Term Treatment ◽

Lgr5 Expression ◽

Cell Niche

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

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Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds Create An In Vivo Leukemia Niche in NOD/SCID Mice.

Blood ◽

10.1182/blood.v114.22.559.559 ◽

2009 ◽

Vol 114 (22) ◽

pp. 559-559

Author(s):

Sarah Rivkah Vaiselbuh ◽

Morris Edelman ◽

Jeffrey Michael Lipton ◽

Johnson M. Liu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Scid Mice ◽

Cxcr4 Antagonist ◽

Cell Niche

Abstract Abstract 559 Introduction: Different cellular components of the normal hematopoietic niche have been identified. However, the niche for malignant hematopoiesis remains to be elucidated. Recent work of other groups has suggested that hematopoietic stem cells (HSC) within the bone marrow anchor themselves in place by attaching to osteoblasts and/or vascular sinusoid endothelial cells. We have recently identified mesenchymal stem cells (MSC) as niche-maker cells and found a crucial role of the SDF-1/CXCR4 axis in this process. Stromal Derived Factor-1 (SDF-1/CXCL12) regulates stem cell trafficking and the cell cycle via its receptor CXCR4. Methods: Polyurethane scaffolds, coated in vitro with human bone marrow MSC, were implanted subcutaneously in non-irradiated NOD/SCID mice. CD34+ HSC or primary AML cells (from a leukapheresis product) were injected either in situ or retro-orbitally in the mice and analyzed for engraftment. The mice were treated twice per week with in situ injections of SDF-1, AMD3100 (a CXCR4 antagonist) or PBS (control). After 2 to 4 weeks, the scaffolds were processed and evaluated for cell survival in the mesenchymal niche by immunohistochemistry. Results: We created in vitro MSC-coated scaffolds that retained inoculated AML cells in the presence of SDF-1, while AML cells seeded on empty scaffolds were not retained. In vivo in NOD/SCID mice, the MSC-coated scaffolds, in the presence of SDF-1 enabled homing of both in situ injected normal CD34+ HSC and retroorbital- or in situ injected primary human AML cells. The scaffolds were vascularized and showed osteoclasts and adipocytes present, suggestive of an ectopic human bone marrow microenvironment in the murine host. Finally, the SDF-1-treated scaffolds showed proliferation of the MSC stromal layer with multiple adherent AML cells, while in the AMD3100-treated scaffolds the stromal lining was thin and disrupted at several points, leaving AML cells free floating in proximity. The PBS-treated control-scaffold showed a thin single cell MSC stromal layer without disruption, with few AML cells attached. Conclusion: The preliminary data of this functional ectopic human microenvironment in NOD/SCID mice suggest that AMD3100 (a CXCR4 antagonist) can disrupt the stem cell niche by modulation of the mesenchymal stromal. Further studies are needed to define the role of mesenchymal stem cells in maintaining the hematopoietic/leukemic stem cell niche in vivo. In Vivo Leukemia Stem Cell Niche: (A) Empty polyurethane scaffold. (B)Vascularization in SQ implanted MSC-coated scaffold (s) niche in NOD/SCID mice. (C) DAB Peroxidase (brown) human CD45 positive nests of AML cells (arrows) 1 week after direct in situ AML injection. (D) Human CD45 positive myeloid cells adhere to MSC in vivo (arrows). Disclosures: No relevant conflicts of interest to declare.

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Intestinal Stem Cell Niche Insights Gathered from Both In Vivo and Novel In Vitro Models

Stem Cells International ◽

10.1155/2017/8387297 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Nikolce Gjorevski ◽

Paloma Ordóñez-Morán

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Niche ◽

Cell Function ◽

Complex Structure ◽

Intestinal Stem Cell ◽

Intestinal Damage ◽

Cell Niche

Intestinal stem cells are located at the base of the crypts and are surrounded by a complex structure called niche. This environment is composed mainly of epithelial cells and stroma which provides signals that govern cell maintenance, proliferation, and differentiation. Understanding how the niche regulates stem cell fate by controlling developmental signaling pathways will help us to define how stem cells choose between self-renewal and differentiation and how they maintain their undifferentiated state. Tractable in vitro assay systems, which reflect the complexity of the in vivo situation but provide higher level of control, would likely be crucial in identifying new players and mechanisms controlling stem cell function. Knowledge of the intestinal stem cell niche gathered from both in vivo and novel in vitro models may help us improve therapies for tumorigenesis and intestinal damage and make autologous intestinal transplants a feasible clinical practice.

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Enhanced Hexokinase 2 Expression and Lactate Production Promote The Apoptosis of Dopaminergic Neurons in Parkinson’s Disease

10.21203/rs.3.rs-587226/v1 ◽

2021 ◽

Author(s):

Jingyi Li ◽

Longmin Chen ◽

Qixiong Qin ◽

Danlei Wang ◽

Jingwei Zhao ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mouse Model ◽

Dopaminergic Neurons ◽

Lactate Production ◽

Substantia Nigra Pars Compacta ◽

Hexokinase 2 ◽

Lactate Levels

Abstract Background: Parkinson’s disease (PD) is characterized by impaired mitochondrial function and decreased ATP levels. Glycolysis is upregulated and lactate production is enhanced in PD. Since lactate promotes apoptosis and α-synuclein accumulation in neurons, we hypothesized that the increased lactate resulted from upregulated glycolysis is involved in the apoptosis of dopaminergic neurons in PD.Methods: We examined the expression of hexokinase 2 (HK2) and lactate dehydrogenase (LDH), the key enzymes in glycolysis, and lactate levels in the substantia nigra pars compacta (SNpc) of MPTP-induced mouse model of PD and in MPP+-treated SH-SY5Y cells. We investigated the role of HK2, lactate and AMPK pathway in the apoptosis of dopaminergic neurons by intervened with 3-Brpa, the HK2 inhibitor, in in vivo and in vitro systems.Results: We found that the expression of HK2 and LDHA, and lactate levels were markedly increased in brain SNpc of MPTP-treated mouse and in MPP+-treated SH-SY5Y cells. Meanwhile, the apoptosis of dopaminergic neurons in the mouse model and the apoptosis of the SH-SY5Y in vitro system were increased. Intriguingly, using HK2 inhibitor or siRNA can decrease the lactate levels and suppressed the apoptosis of dopaminergic neurons both in vivo and in vitro. Mechanistically, lactate increased the activity of adenosine monophosphate activated protein kinase (AMPK), and suppressed the phosphorylation of serine/threonine kinase 1 (Akt) and mammalian target of rapamycin (mTOR). Conclusion：Inhibition of HK2 ameliorate the apoptosis of dopaminergic neurons through downregulating the lactate production and AMPK/ Akt/ mTOR pathway activation in PD.

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Runx1 Maintains Granulocytic-Monocytic Progenitor Numbers By Negatively Regulating Interactions with the Stem Cell Niche

Blood ◽

10.1182/blood.v124.21.2913.2913 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2913-2913

Author(s):

Carol Stocking ◽

Kira Behrens ◽

Birte Niebuhr ◽

Ursula Mueller

Keyword(s):

Stem Cell ◽

Dna Binding ◽

Stem Cell Niche ◽

Target Genes ◽

Self Renewal ◽

Cell Niche ◽

Progenitor Compartment ◽

Myeloid Progenitors

Abstract The Runx1 transcription factor is among the most frequently mutated genes in acute myeloid leukemia (AML). In addition to the generation of the RUNX1-RUNX1T1 fusion protein in patients with the translocation (8;21), circa 15% of patients with a normal karyotype harbor missense mutations or frame-shift mutations in the RUNX1 gene, in most cases resulting in either a highly compromised protein lacking DNA-binding activity or a null allele. Recent studies have demonstrated that AML transformation normally occurs at the level of the granulocyte-monocyte-progenitor (GMP), thus we sought to dissect the role of Runx1 at this critical stage of myeloid differentiation by examining the hematopoietic progenitor compartment in conditional Runx1 knockout (KO) mice. Earlier studies have demonstrated increased cell numbers in the stem cell compartment in these mice, but a rigorous assessment of myeloid progenitors has not been performed. Our analysis showed that loss of Runx1 resulted in increased levels of all myeloid progenitors, with a 2.2-fold increase in the absolute GMP numbers. Furthermore, Runx1-deficient GMPs gave rise to 40% more colonies than controls, demonstrating increased self-renewal activity within this population. Notably, in addition to being larger, Runx1-deficient colonies did not exhibit the typical structure of GM colonies, which reflects the differentiation status of the cells composing the colonies. Indeed, retarded or impaired differentiation of Runx1-defcient cells was demonstrated by analysis of cell morphology and cell surface markers, with little or no mature forms observed in colonies cultured for 7 to 10 days. Notably, no significant shift to either the G or M lineages was observed in vitro or in vivo. To identify Runx1 target genes that impact at the level of the GMP, expression analysis of Runx1 wild-type and KO GMPs was performed. In addition, the transcriptome of primary KO GMPs genetically engineered to conditionally re-express RUNX1 in vivo was determined before or after Runx1 induction. A total of 36 reciprocally regulated genes were identified using stringent criteria to assess expression levels and fold-induction. Notably, many of the Runx1 target genes encode adhesion factors important for retaining HSPC in the stem cell niche, and which are normally down-regulated during differentiation. These genes were up-regulated in Runx1-deficient GMPs and down-regulated after Runx1 induction. Genome wide DNA-binding analysis established Runx1 binding to regulatory regions of target genes and furthermore revealed cooperative binding of Runx1 with several other TFs implicated in complex networks regulating self-renewal and differentiation of early hematopoietic progenitors. In vitro adhesion assays confirmed that loss of Runx1 resulted in increased adhesion to hematopoietic stromal cells. In summary, we conclude that Runx1 has at least two critical functions in normal myeloid development. In the early stem cell and progenitor compartment, Runx1 represses genes that mediate adhesion to the stem cell niche, thereby facilitating the differentiation program at the expense of self-renewal. On the other hand, during myeloid maturation, Runx1 augments both G and M developmental programs, presumably by facilitating up-regulation of critical granulocytic and monocytic genes. We predict that this former function is of critical importance during leukemogenesis, in which reduced levels of functional Runx1 increases the number of myeloid progenitors with self-renewal potential, which are critical targets of secondary genetic mutations during the clonal evolution of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

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Nanotopography – potential relevance in the stem cell niche

Biomaterials Science ◽

10.1039/c4bm00155a ◽

2014 ◽

Vol 2 (11) ◽

pp. 1574-1594 ◽

Cited By ~ 39

Author(s):

Lesley-Anne Turner ◽

Matthew J. Dalby

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Osteogenic Differentiation ◽

Stem Cell Niche ◽

Potential Role ◽

Cell Niche

Nanotopographical cues observed in vivo (such as in the sinusoid and bone) closely resemble nanotopographies that in vitro have been shown to promote niche relevant stem cells behaviours; specifically, retention of multipotency and osteogenic differentiation on ordered and disordered nano-pits respectively. These and other observations highlight a potential role for nano topography in the stem cell niche.

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Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.682067 ◽

2021 ◽

Vol 9 ◽

Author(s):

Verónica Company ◽

Abraham Andreu-Cervera ◽

M. Pilar Madrigal ◽

Belén Andrés ◽

Francisca Almagro-García ◽

...

Keyword(s):

Substantia Nigra ◽

Ventral Tegmental Area ◽

Dopaminergic Neurons ◽

Functional Organization ◽

Substantia Nigra Pars Compacta ◽

Fasciculus Retroflexus ◽

Ventral Tegmental

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

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Phage-Based Artificial Niche: The Recent Progress and Future Opportunities in Stem Cell Therapy

Stem Cells International ◽

10.1155/2019/4038560 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Kshitiz Raj Shrestha ◽

So Young Yoo

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Niche ◽

Therapeutic Strategy ◽

Different Types ◽

Effective Performance ◽

Cell Niche ◽

Stem Cell Niches

Self-renewal and differentiation of stem cells can be the best option for treating intractable diseases in regenerative medicine, and they occur when these cells reside in a special microenvironment, called the “stem cell niche.” Thus, the niche is crucial for the effective performance of the stem cells in bothin vivoandin vitrosince the niche provides its functional cues by interacting with stem cells chemically, physically, or topologically. This review provides a perspective on the different types of artificial niches including engineered phage and how they could be used to recapitulate or manipulate stem cell niches. Phage-based artificial niche engineering as a promising therapeutic strategy for repair and regeneration of tissues is also discussed.

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Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche

Scientific Reports ◽

10.1038/s41598-020-79448-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Giada Bassi ◽

Silvia Panseri ◽

Samuele Maria Dozio ◽

Monica Sandri ◽

Elisabetta Campodoni ◽

...

Keyword(s):

Cell Culture ◽

Stem Cell ◽

Stem Cell Niche ◽

Drug Testing ◽

Tumour Microenvironment ◽

Osteosarcoma Cell ◽

Cellular Models ◽

Cell Niche

AbstractThe failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

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Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche

Blood ◽

10.1182/blood.v116.21.399.399 ◽

2010 ◽

Vol 116 (21) ◽

pp. 399-399

Author(s):

Ben S. Lam ◽

Cynthia Cunningham ◽

Gregor B. Adams

Keyword(s):

Stem Cell ◽

Stem Cell Niche ◽

Calcium Sensing Receptor ◽

Hematopoietic Stem ◽

Endosteal Surface ◽

Calcium Sensing ◽

Cd34 Cells ◽

Cell Niche

Abstract Abstract 399 The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. Disclosures: No relevant conflicts of interest to declare.

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