Search

The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist, Cinacalcet, we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro, including growth in stromal cell cocultures, adhesion to extracellular matrix molecules such as collagen I and fibronectin, and migration toward the chemotactic stimulus, stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing, CXCR4-mediated lodgment at the endosteal niche, and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. 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.

The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist, Cinacalcet, we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro, including growth in stromal cell cocultures, adhesion to extracellular matrix molecules such as collagen I and fibronectin, and migration toward the chemotactic stimulus, stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing, CXCR4-mediated lodgment at the endosteal niche, and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. 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.

Allogeneic hematopoietic cell transplant (HCT) improves survival in patients with relapsed or high risk acute myeloid leukemia (AML). Complete remission (CR) is typically a pre-requisite for transplantation, though many do not achieve a formal CR. The traditional AML treatment starts with induction chemotherapy, followed by assessment of response to guide next steps. Response criteria definitions differ between that of the National Comprehensive Cancer Network (NCCN), utilized by the majority of clinicians, and the Center for International Blood and Marrow Research (CIBMTR) data registry utilized by transplant centers, making interpretation of the impact of HCT difficult. Definitions for morphologic complete remission (CR) are the same, however complete remission with incomplete hematologic recovery (CRi) differs and the CIBMTR does not recognize the morphologic leukemia-free state (MLFS), thus mis-identifying such patients and preventing clear treatment guidelines for this population. We conducted a retrospective study, identifying a cohort of 35 AML patients at our center who underwent allogeneic HCT while in MLFS, to evaluate characteristics in patient demographics, disease status, treatment(s), and outcomes. From our cohort, the median overall survival (OS) was 14 months, however 37% were alive and in remission with median follow-up of survivors of five years. Twenty three percent had progression of disease following transplant. Non-relapse mortality (NRM) was 35% with leading cause of death being infection. Our study reveals that transplant can induce long-term survival in patients with acute leukemia who are in MLFS at the start of induction, similar to data for patients with high risk disease in early relapse or in later remissions. Early transplantation while in MLFS and not waiting for full count recovery may protect patients from toxicities of further chemotherapeutic agents or prevent unnecessary delays that may allow for infections or other barriers to arise, and requires further study.

Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic–severe combined immunodeficient (NOD/SCID)/β2m-/-mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.

Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic–severe combined immunodeficient (NOD/SCID)/β2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.

Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT. (Blood. 2003;102:4582-4593)

Bone marrow transplantation (BMT) is the only curative therapy for sickle cell disease (SCD). However, the morbidity and mortality related to pretransplantation myeloablative chemotherapy often outweighs the morbidity of SCD itself, thus severely limiting the number of patients eligible for transplantation. Although nonmyeloablative transplantation is expected to reduce the risk of BMT, it will likely result in mixed-chimerism rather than complete replacement with donor stem cells. Clinical application of nonmyeloablative transplantation thus requires knowledge of the effect of mixed chimerism on SCD pathophysiology. We have, therefore, created a panel of transplanted SCD mice that received transplants displaying an array of red blood cell (RBC) and white blood cell (WBC) chimerism. A significant enrichment of RBC over WBC chimerism occurred in these mice, because of the dramatic survival advantage of donor over sickle RBCs in the peripheral blood. Increasing levels of RBC chimerism provided progressive correction of hematologic and pathologic abnormalities. However, sickle bone marrow and splenic hematopoiesis was not corrected until peripheral blood sickle RBCs were fully replaced with donor RBCs. These results have important and unexpected implications for nonmyeloablative BMT for SCD. As the critical hematopoietic organs were not corrected without full RBC replacement, 100% peripheral blood RBC chimerism becomes the most important benchmark for cure after nonmyeloablative BMT. (Blood. 2003;102:4582-4593)

T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+but not CD4+T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/−NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+cells but not CD4+cells. This correlated with CD8+augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+cytoskeletal rearrangements abrogated CD34+transendothelial migration and impaired CD34+cell homing in vivo. CD8+cells did not migrate in tandem with CD34+cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+ but not CD4+ T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+ cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/− NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+ cells but not CD4+ cells. This correlated with CD8+ augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+ cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+ cytoskeletal rearrangements abrogated CD34+ transendothelial migration and impaired CD34+ cell homing in vivo. CD8+ cells did not migrate in tandem with CD34+ cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+ cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+ cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

The morbidity and mortality associated with sickle cell disease (SCD) is caused by hemolytic anemia, vaso-occlusion, and progressive multiorgan damage. Bone marrow transplantation (BMT) is currently the only curative therapy; however, toxic myeloablative preconditioning and barriers to allotransplantation limit this therapy to children with major SCD complications and HLA-matched donors. In trials of myeloablative BMT designed to yield total marrow replacement with donor stem cells, a subset of patients developed mixed chimerism. Importantly, these patients showed resolution of SCD complications. This implies that less toxic preparative regimens, purposefully yielding mixed chimerism after transplantation, may be sufficient to cure SCD without the risks of myeloablation. To rigorously test this hypothesis, we used a murine model for SCD to investigate whether nonmyeloablative preconditioning coupled with tolerance induction could intentionally create mixed chimerism and a clinical cure. We applied a well-tolerated, nonirradiation-based, allogeneic transplantation protocol using nonmyeloablative preconditioning (low-dose busulfan) and costimulation blockade (CTLA4-Ig and anti-CD40L) to produce mixed chimerism and transplantation tolerance to fully major histocompatibility complex–mismatched donor marrow. Chimeric mice were phenotypically cured of SCD and had normal RBC morphology and hematologic indices (hemoglobin, hematocrit, reticulocyte, and white blood cell counts) without evidence of graft versus host disease. Importantly, they also showed normalization of characteristic spleen and kidney pathology. These experiments demonstrate the ability to produce a phenotypic cure for murine SCD using a nonmyeloablative protocol with fully histocompatibility complex–mismatched donors. They suggest a future treatment strategy for human SCD patients that reduces the toxicity of conventional BMT and expands the use of allotransplantation to non–HLA-matched donors.

The morbidity and mortality associated with sickle cell disease (SCD) is caused by hemolytic anemia, vaso-occlusion, and progressive multiorgan damage. Bone marrow transplantation (BMT) is currently the only curative therapy; however, toxic myeloablative preconditioning and barriers to allotransplantation limit this therapy to children with major SCD complications and HLA-matched donors. In trials of myeloablative BMT designed to yield total marrow replacement with donor stem cells, a subset of patients developed mixed chimerism. Importantly, these patients showed resolution of SCD complications. This implies that less toxic preparative regimens, purposefully yielding mixed chimerism after transplantation, may be sufficient to cure SCD without the risks of myeloablation. To rigorously test this hypothesis, we used a murine model for SCD to investigate whether nonmyeloablative preconditioning coupled with tolerance induction could intentionally create mixed chimerism and a clinical cure. We applied a well-tolerated, nonirradiation-based, allogeneic transplantation protocol using nonmyeloablative preconditioning (low-dose busulfan) and costimulation blockade (CTLA4-Ig and anti-CD40L) to produce mixed chimerism and transplantation tolerance to fully major histocompatibility complex–mismatched donor marrow. Chimeric mice were phenotypically cured of SCD and had normal RBC morphology and hematologic indices (hemoglobin, hematocrit, reticulocyte, and white blood cell counts) without evidence of graft versus host disease. Importantly, they also showed normalization of characteristic spleen and kidney pathology. These experiments demonstrate the ability to produce a phenotypic cure for murine SCD using a nonmyeloablative protocol with fully histocompatibility complex–mismatched donors. They suggest a future treatment strategy for human SCD patients that reduces the toxicity of conventional BMT and expands the use of allotransplantation to non–HLA-matched donors.

Introduction: Outcomes of acute myeloid leukemia (AML) in older patients remain unsatisfactory with low rates of complete remission (CR), disease-free survival (DFS), and overall survival (OS). Microtransplantation - in which allogeneic cells are infused without substantial engraftment (<2% donor cell engraftment) - has the potential to improve anti-tumor immunity without the risks of allogeneic hematopoietic stem cell transplant and graft-vs-host disease (GVHD).

No relevant conflicts of interest to declare.

Abstract:Much of what is known about hemoglobin relates to its structure and function. However, there is a great deal to learn about its assembly. The application of new technologies has allowed us to investigate the process of hemoglobin synthesis extensively. It has been shown that heme, synthesized in the mitochondria, is exported into the cytoplasm where it rapidly binds with apoglobin synthesized by polyribosomes. In this study,subcellular localization of newly synthesized alpha and beta subunits of human hemoglobin were examined using fluorescence microscopic imaging of cultured human erythroleukemic cells (cell line HEL 92.1.7). Experiments were used to determine the location of newly synthesized protein subunits. This paper will highlight the current knowledge of hemoglobin assembly, function and cellular interactions; and illustrate how cellular labels can be used to investigate this protein. It is hoped that future experiments will help to determine more details of the mechanism of globin assembly, which will be useful in the investigation of hemoglobinopathies.

No relevant conflicts of interest to declare.

Abstract 2396

No relevant conflicts of interest to declare.

HMG CoA reductase inhibitors (statins) were recently reported to reduce the rate of venous thromboembolism (VTE) in healthy people. Statins reduce levels of inflammation biomarkers, however the mechanism for a reduction in VTE risk is unknown. We studied cross-sectional associations of statin use with hemostatic factors related to venous thrombosis risk in a large cohort of healthy people.Cross-sectional analyses were performed in the Multi-Ethnic Study of Atherosclerosis (MESA), a cohort study of 6814 healthy men and women age 45–84, free of clinical cardiovascular disease at baseline; 1001 were using statins. Twenty three warfarin users were excluded. Age, race, and sex-adjusted mean hemostatic factor levels were compared between statin users and nonusers, and multivariable linear regression models were used to assess associations of statin use with hemostasis factors, adjusted for age, race/ethnicity, education, income, hormone replacement therapy (in women) and major cardiovascular risk factors.The table shows that those using statins had significantly lower levels of D-dimer, C-reactive protein and factor VIII than non-users. Homocysteine and von Willebrand factor did not differ by statin use. Specific adjustment for LDL and triglycerides did not attenuate the observed differences in these factors by statin use.Findings of lower D-dimer, factor VIII and C-reactive protein levels with statin use suggest mechanisms whereby statins might lower VTE risk. These associations were not explained by lipid levels on treatment. A prospective study linking these biochemical differences to VTE outcomes is warranted.No relevant conflicts of interest to declare.

Abstract 399

Abstract 3178

Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitro culture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+ HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1− HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+ HSCs were found to be in the G0 phase of the cell cycle than VEGF-R1− cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+ HSCs or VEGFR1− HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+ HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+ HSCs in vitro. The functional relevance of this in vivo is currently being investigated.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow (BM), which remains the site of hematopoiesis throughout adulthood. In the BM the HSCs are located at the endosteal surface, where cells of the osteoblastic lineage comprise a key component of the stem cell niche. While hematopoiesis occurs in many bones, the process of bone formation can actually be split into those bones that develop through endochondral ossification (long bones) and those that form through membranous ossification (flat bones). We examined the role played by the microenvironment in these two distinct bones and whether these microenvironments have differing effects on the HSCs. In vitro analysis of the BM stromal cells isolated from long bones and flat bones has demonstrated that calvaria derived stromal layers can support cobblestone area-forming cells 10-fold greater than stromal layers derived from femurs and tibia. Real-time PCR analysis of gene expression has demonstrated that flat bone stromal cells have 5-fold greater expression of N-cadherin than long bone, while other cadherins such as VE-cadherin show no difference. Correlating with this, we found that calvarial derived HSCs demonstrated increased expression of N-cadherin and also increased expression of other genes associated with cadherin signaling, such as cyclinD1. However, no difference in the cell cycle status of the HSCs derived from long bone and flat bone was noted. Functional assays are being performed in order to assess the function of these distinct BM marrow niches in vivo. It is anticipated that we will be able to begin to define the molecular cues the govern HSC physiology in different locations within the mammalian skeleton and thus provide an understanding not only into the continual migration of HSCs between different HSC niches but also the regression of hematopoiesis that occurs from the appendicular skeleton to the axial skeleton during the adult human lifespan.

Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitroculture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1−HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+HSCs were found to be in the G0phase of the cell cycle than VEGF-R1−cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+HSCs or VEGFR1−HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+HSCs in vitro. The functional relevance of this in vivois currently being investigated.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow (BM), which remains the site of hematopoiesis throughout adulthood. In the BM the HSCs are located at the endosteal surface, where cells of the osteoblastic lineage comprise a key component of the stem cell niche. While hematopoiesis occurs in many bones, the process of bone formation can actually be split into those bones that develop through endochondral ossification (long bones) and those that form through membranous ossification (flat bones). We examined the role played by the microenvironment in these two distinct bones and whether these microenvironments have differing effects on the HSCs. In vitroanalysis of the BM stromal cells isolated from long bones and flat bones has demonstrated that calvaria derived stromal layers can support cobblestone area-forming cells 10-fold greater than stromal layers derived from femurs and tibia. Real-time PCR analysis of gene expression has demonstrated that flat bone stromal cells have 5-fold greater expression of N-cadherinthan long bone, while other cadherins such as VE-cadherin show no difference. Correlating with this, we found that calvarial derived HSCs demonstrated increased expression of N-cadherinand also increased expression of other genes associated with cadherin signaling, such as cyclinD1. However, no difference in the cell cycle status of the HSCs derived from long bone and flat bone was noted. Functional assays are being performed in order to assess the function of these distinct BM marrow niches in vivo. It is anticipated that we will be able to begin to define the molecular cues the govern HSC physiology in different locations within the mammalian skeleton and thus provide an understanding not only into the continual migration of HSCs between different HSC niches but also the regression of hematopoiesis that occurs from the appendicular skeleton to the axial skeleton during the adult human lifespan.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14-/- mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin-cKit+Sca1+ (LKS+) and CD34-/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD-, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD-, KO LKS+ 38.7% AnnexinV+7AAD-, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/−fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitroprimitive CFU-C or LTC-IC assay systems. However, Gsα−/−cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivoin a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivoas deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivoprior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.