Comparison of hepatic properties and transplantation of Thy-1(+) and Thy-1(-) cells isolated from embryonic day 14 rat fetal liver

Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1(+) cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1(+) fetal liver cells. Therefore, we isolated Thy-1(+) and Thy-1(-) cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1(+). The vast majority of alpha fetoprotein(+), albumin(+), cytokine-19(+), and E-cadherin(+) cells were found in cultured Thy-1(-) cells, whereas nearly all CD45(+) cells were in the Thy-1(+) fraction. In normal rat liver, transplanted Thy-1(+) cells produced only rare, small DPPIV(+) cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1(+) fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1(-) fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell-derived factor (SDF)-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1(+) and Thy-1(-) fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1alpha expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy-treated liver, and this correlated with liver repopulation by Thy-1(+) cells. CONCLUSION: Highly enriched Thy-1(+) ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1(-) stem/progenitor cells, which repopulate the normal adult liver.     

Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34(+) stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34(+)CD38(-)CXCR4(-) and CXCR4(+) cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34(+)CXCR4(+) (R4(+)) and CD34(+)CXCR4(-) (R4(-)) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34(+) cells. Coinjection of purified cells with 10 microg anti-CXCR4 mAb significantly reduced engraftment of all CD34(+) subsets, and 50 microg completely abrogated engraftment by R4(-) and CD34(+) cells. Importantly, R4(-) cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4(-) cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34(+) cells but yet CXCR4 dependent. In conclusion, R4(-) cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1-dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34(+) stem and progenitor cells, regulating their motility and repopulation capacities.     

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

The SH2 domain-containing inositol 5'-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP(-/-) mice. Despite expansion of the compartment, SHIP(-/-) HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP(-/-) stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing.     

CXCL12 and CXCR4 in bone marrow physiology

This article discusses the multiple roles of CXCL12 and its receptor, CXCR4, in bone marrow (BM) hematopoietic stem cell (HSC) development and regulation. CXCL12 interaction with CXCR4 results in effects as varied as cell migration, proliferation and survival or apoptosis. The selective signaling pathways that mediate these varied outcomes are summarized briefly. The CXCL12/CXCR4 pair is crucially involved in homing and repopulation of HSCs in the specific BM niches. Mechanisms of HSC mobilization to the peripheral circulation in response to physiological requests and therapeutic stimulations, as well as recent data on the novel receptor for CXCL12, CXCR7, are reviewed.     

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice

Fanconi anemia (FA) is a heterogeneous inherited disorder characterized by a progressive bone marrow (BM) failure and susceptibility to myeloid leukemia. Genetic correction using gene-transfer technology is one potential therapy. A major hurdle in applying this technology in FA patients is the inability of granulocyte colony-stimulating factor (G-CSF) to mobilize sufficient numbers of hematopoietic stem (HSC)/progenitor cells (HPC) from the BM to the peripheral blood. Whether the low number of CD34(+) cells is a result of BM hypoplasia or an inability of G-CSF to adequately mobilize FA HSC/HPC remains incompletely understood. Here we use competitive repopulation of lethally irradiated primary and secondary recipients to show that in two murine models of FA, AMD3100 synergizes with G-CSF resulting in a mobilization of HSC, whereas G-CSF alone fails to mobilize stem cells even in the absence of hypoplasia.     

Changes in integrin expression are associated with altered homing properties of Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor

OBJECTIVE: Although migration of hematopoietic stem cells (HSC) is essential for normal hematopoiesis and successful hematopoietic cell transplantation, little is known about the mechanisms that underlie this movement. We have sought to characterize the factors that regulate HSC migration by analyzing changes in expression of particular adhesion receptors associated with cyclophosphamide/granulocyte colony-stimulating factor (Cy/G-CSF)-induced HSC mobilization. METHODS: Expression by Lineage(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) HSC of members of the beta1 integrin family of adhesion molecules was assessed in untreated or Cy/G-CSF-treated mice by multiparameter flow cytometry. In parallel, the in vivo homing properties of normal and mobilized HSC were compared following intravenous transfer of fluorescently marked HSC. RESULTS: Normal adult HSC express high levels of several beta1 integrin family members. Following Cy/G treatment, bone marrow HSC selectively downregulate alpha 2 integrin expression and upregulate alpha 5 expression. HSC found in the blood following Cy/G-CSF treatment express significantly lower levels of multiple integrins than their bone marrow and/or splenic counterparts. Changes in integrin expression by blood-borne HSC correlate with a 50% decrease in their ability to home to the bone marrow in short-term assays, and with previously observed defects in competitive engraftment by these HSC. Similar reductions in bone marrow (BM) homing are observed for BM HSC treated with alpha 4 integrin function blocking mAb prior to injection. Modulation of integrin expression induced by mobilization was not associated with cell-cycle progression. CONCLUSION: Changes in integrin expression and function are associated with HSC mobilization and likely significantly affect the engraftment potential of hematopoietic stem cells.     

Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice, with a corresponding retention of progenitors in blood. Remarkably, the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells, studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin, studied in parallel. Furthermore, the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies, in contrast to antibodies against alpha4 integrin, did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells.     

ANGPTL7 regulates the expansion and repopulation of human hematopoietic stem and progenitor cells

Successful expansion of hematopoietic stem cells would benefit the use of hematopoietic stem cell transplants in the clinic. Several angiopoietin-like proteins, including angiopoietin-like 7, can support the activity of hematopoietic stem cells. However, effects of ANGPTL7 on human hematopoietic stem cells and the downstream signaling cascade activated by ANGPTL7 are poorly understood. Here, we established a human hematopoietic stem and progenitor cell-supportive mouse fetal liver cell line that specifically expressed the Angptl7 protein. Furthermore, we found ANGPTL7 is capable of stimulating human hematopoietic stem and progenitor cell expansion and increasing the repopulation activities of human hematopoietic progenitors in xenografts. RNA-sequencing analysis showed that ANGPTL7 activated the expression of CXCR4, HOXB4 and Wnt downstream targets in human hematopoietic progenitors. In addition, chemical manipulation of Wnt signaling diminished the effects of ANGPTL7 on human hematopoietic stem and progenitor cells in culture. In summary, we identify the secreted growth factor ANGPTL7 as a regulator of both human hematopoietic stem and progenitor cell expansion and regeneration.     

Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo

OBJECTIVE: Hematopoietic stem cells are important clinically, both as targets of disease and as reagents for cellular therapy. Studies in hematopoietic stem cell biology have been hampered by difficulties in purifying and manipulating these cells. To facilitate these studies, we sought to develop a system for targeting genes of interest to the hematopoietic stem cell compartment in transgenic mice. MATERIALS AND METHODS: We used Sca-1, a glycosyl phosphatidylinositol-anchored protein expressed on the surface of all hematopoietic stem cells in commonly used inbred mouse strains. We created a mutant Sca-1 allele in which the enhanced green fluorescent protein (EGFP) cDNA is integrated into the Sca-1 locus by homologous recombination in embryonic stem cells. RESULTS: EGFP protein is detectable in all hematopoietic tissues of mice heterozygous for the mutant Sca-1 allele. Growth and development of these mice are normal. No adverse effects of long-term, high-level EGFP expression were noted. Sca-1 positive cells coexpress EGFP in all tissues and lineages examined, as predicted by the targeting strategy. Sca-1 and EGFP expression are coordinately up-regulated in splenocytes from mutant mice. The Lin(-)EGFP(+) bone marrow population contains all progenitor activity in Sca-1(+)(/EGFP) mice. The Lin(-)EGFP(+) bone marrow cells are equivalent to Lin(-)Sca-1(+) cells in long-term repopulation and serial transplantation assays. CONCLUSION: The hematopoietic stem cell compartment appears to be targeted in Sca-1(+)(/EGFP) mutant mice. This system should be useful for studying the normal biology of hematopoietic stem cells and for targeting other genes to this cellular compartment.     

Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34 cells after ex vivo expansion

OBJECTIVE: The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. METHODS: G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. RESULTS: We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. CONCLUSIONS: Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.     

Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4-/- chimeric mice

The physiologic role of CXCR4 on hematopoietic stem/progenitor cells (HSPCs) is not fully understood. Here, we show that radioprotection of lethally irradiated mice by embryonic day 14.5 (E14.5) CXCR4-/- fetal liver (FL) cells was markedly impaired when compared with CXCR4+/+ counterparts, but this defect was rescued when hosts were engrafted with high cell numbers. This quantitative defect contrasted with a similar content in hematopoietic colony-forming cells (CFCs), splenic colony-forming units (CFUs-S), and Lin- Sca-1+ c-kit+ cells in E14.5 CXCR4-/- and CXCR4+/+ livers. In addition, the homing of HSPCs in the bone marrow was not altered as detected with a CFSE-staining assay. In contrast, a 30-fold increase in CFCs was seen in the circulation of mice stably reconstituted with CXCR4-/- FL cells and this increment was already observed before hematopoiesis had reached a steady-state level. Together, the data strongly suggest that impaired retention may, at least in short-term hematopoietic reconstitution, lead to a diminution in the number of available progenitors required for radioprotection.     

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

OBJECTIVE: The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. MATERIALS AND METHODS: A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. RESULTS: We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1(+) HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PY(lo) and PY(hi) subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the PY(lo)Rh(lo)Ho(lo/sp) PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PY(hi)). We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. CONCLUSION: Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the Rh(lo)Ho(lo/sp) PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population.     

Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells

OBJECTIVE: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34(+) cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. MATERIALS AND METHODS: We used a LV to transduce human CB CD34(+) cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. RESULTS: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34(+) cells when CB CD34(+) cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34(+) cells were noted under these conditions. CONCLUSION: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.     

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in?vitro and their recovery in transplanted recipients in?vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.     

Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells

Hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC) homing to specific microenvironmental niches involves interactions between multiple receptor ligand pairs. Although CXCL12/CXCR4 plays a central role in these events, CXCR4 regulators that provide the specificity for such cells to lodge and be retained in particular niches are poorly defined. Here, we provide evidence that the sialomucin endolyn (CD164), an adhesion receptor that regulates the adhesion of CD34+ cells to bone marrow stroma and the recruitment of CD34+CD38(lo/-) cells into cycle, associates with CXCR4. The class II 103B2 monoclonal antibody, which binds the CD164 N-linked glycan-dependent epitope or CD164 knockdown by RNA interference, significantly inhibits the migration of CD133+ HPCs toward CXCL12 in vitro. On presentation of CXCL12 on fibronectin, CD164 associates with CXCR4, an interaction that temporally follows the association of CXCR4 with the integrins VLA-4 and VLA-5. This coincides with PKC-zeta and Akt signaling through the CXCR4 receptor, which was disrupted on the loss of CD164 though MAPK signaling was unaffected. We therefore demonstrate a novel association among 3 distinct families of cell-surface receptors that regulate cell migratory responses and identify a new role for CD164. We propose that this lends specificity to the homing and lodgment of these cells within the bone marrow niche.     

Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34(+)CD38(-/low)CXCR4(+) cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34(-)CD38(-/low)Lin(-) cells were not detected. Moreover, whereas freshly isolated CD34(+)CD38(+/high) cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34(+)CD38(+) cells. Homing of enriched human CD34(+) cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34(+)CD38(-/low)CXCR4(+) cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34(+) cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Galpha(i) proteins, inhibited SDF-1-mediated in vitro transwell migration but not adhesion or in vivo homing of CD34(+) cells. Homing of human CD34(+) cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34(+)CD38(-/low)CXCR4(+) cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.     

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal

OBJECTIVE: Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. METHODS: Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. RESULTS: Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. CONCLUSION: These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.     

SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection

Precise analysis of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin(-)) CD34(-) cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4- mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34(+) progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34(+) cells in vivo. These in vivo-generated CD34(+) cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34(-) SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34(-) SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.     

Peripheral blood stem cell mobilization. A role for CXC chemokines

Chemokines induce rapid hematopoietic stem and progenitor cell mobilization and synergize with hematopoietic cytokines in mobilizing stem and progenitor cells. These proteins alone and in combination offer new paradigms for autologous and allogeneic peripheral blood stem cell transplantation (PBSCT). The mechanisms responsible for hematopoietic stem cell (HSC) mobilization either with growth factors or chemokines are largely unknown, but a better understanding of these mechanisms will permit the development of novel, more rapid and efficacious regimens. Studies presented herein indicate that the CXCR2 chemokine receptor that interacts with selective chemokine ligands, particularly GRObeta/CXCL2 and GRObeta-T, may be the dominant receptor mediating hematopoietic cell mobilization, and that polymorphonuclear neutrophils may be the primary CXCR2 expressing target cell for stem and progenitor cell mobilization.