GATA-2 plays two functionally distinct roles during the ontogeny of hematopoietic stem cells.

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.

Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia.

Recently it was shown that, analogous to normal hematopoietic cells, the level of CXC chemokine receptor 4 (CXCR-4) expression on acute myeloid leukemia (AML) cells correlates with stromal cell derived factor-1 alpha (SDF-1)-induced chemotaxis. As we speculated that an anomalous organ distribution of AML cells could affect cell survival and thus result in an altered fraction surviving chemotherapy, we examined a possible correlation between patient prognosis and CXCR-4 expression in AML patients. We found that patients with a high CXCR-4 expression in the CD34(+) subset had a significantly reduced survival and a higher probability of relapse, resulting in a median relapse-free survival (RFS) of only 8.3 months. CXCR-4 expression was significantly higher in fetal liver tyrosine kinase-3 (Flt3)/internal tandem duplication (ITD) AML than in Flt3/wild-type (wt) AML. Covariate analysis indicated that the prognostic significance of Flt3/ITDs with respect to RFS was no more apparent when analyzed in conjunction with the expression of CXCR-4 in the CD34(+) subset, suggesting that the poor prognosis of Flt3/ITD AML might be subordinate to the increased CXCR-4 expression. Using a granulocyte colony-stimulating factor receptor (G-CSF-R)-expressing 32D cell line, we observed that SDF-1/CXCR-4 interaction is required for the survival of myeloid differentiating cells, and it also induces a block in G-CSF-induced myeloid differentiation. These data suggest that the SDF-1/CXCR-4 axis may influence therapy responsiveness and defines unfavorable prognosis in AML.

Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo.

The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling pathway is thought to play an important role in the induction of neutrophil mobilization from the bone marrow in response to granulocyte-colony stimulating factor (G-CSF) treatment. CXCR4 belongs to the family of G protein-coupled receptors. Multiple members of this receptor family are desensitized by agonist-induced G protein-coupled receptor kinase (GRK)-mediated phosphorylation. Here, we demonstrate that in vitro SDF-1-induced chemotaxis of bone marrow-derived neutrophils from GRK6-deficient mice is significantly enhanced and that desensitization of the calcium response to SDF-1 is impaired in GRK6-/- neutrophils. CXCR4 activation by SDF-1 provides a key retention signal for hematopoietic cells in the bone marrow. It is interesting that we observed that in the absence of GRK6, the G-CSF-induced increase in circulating neutrophils is profoundly impaired. Three days after injection of pegylated-G-CSF, significantly lower numbers of circulating neutrophils were observed in GRK6-/- as compared with wild-type (WT) mice. In addition, early/acute neutrophil mobilization in response to G-CSF (3 h after treatment) was also impaired in GRK6-/- mice. However, blood neutrophil levels in untreated GRK6-/- and WT mice were not different. Moreover, the percentage of neutrophils in the bone marrow after G-CSF treatment was increased to the same extent in WT and GRK6-/- mice, indicating that neutrophil production is normal in the absence of GRK6. However, the increased chemotactic sensitivity of GRK6-/- neutrophils to SDF-1 was retained after G-CSF treatment. In view of these data, we suggest that the impaired G-CSF-induced neutrophil mobilization in the absence of GRK6 may be a result of enhanced CXCR4-mediated retention of PMN in the bone marrow.

Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis.

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.

Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.

The cobblestone-area-forming cell assay.

Hematopoietic stem cell (HSC) subsets are defined by the capacity to which their offspring can contribute to the various mature blood-cell lineages. However, the proliferative potential of stem cells is highly dependent on the environment in which they reside, and it is only in retrospect that the characteristics of a stem cell can be identified. Partly because of these elusive properties of stem cells, various functional assays to measure their frequency have been developed, both in vivo and in vitro. Various chapters in this volume each describe one of these assays. All these assays have the ability to quantify stem cells with certain properties, but not others. Therefore, the determination of which stem-cell assay will be most appropriate to use is highly dependent on a particular experimental setting. This chapter describes the cobblestone-areaforming cell (CAFC) assay (1).

Hematopoiesis: Gap Junction Intercellular Communication is Likely to be Involved in Regulation of Stroma-dependent Proliferation of Hemopoietic Stem Cells.

The 80-100 fold increased immunohistological expression of the Gap Junction (GJ) protein Connexin-43 in murine bone marrow during the neonatal period and directly following cytoreductive treatment of adult mice suggests that the regulation of stem cell proliferation may involve GJ Intercellular Communication (GJIC). Using a series of stromal cell lines from foetal liver and neonatal bone marrow we observed that the percentage of cells with GJIC, as indicated by dye-coupling using microinjection of lucifer yellow, correlated with the stromal support for late appearing clones formed by primitive stem cells (CAFC week 3-5). In order to functionally block all GJIC between mutual stromal cells and stromal cells and hemopoietic cells, in long-term stroma-supported flask (LTC) and CAFC cultures, the lipophilic compounds amphotericin-B (AB), nystatin, alpha-glycyrrhetinic acid, tetraphenylboron, dipicrylamine and arachidonic acid were tested for their effect on GJIC and CAFC support. Only AB and nystatin, which induced complete and prolonged GJIC blockade, were able to dramatically inhibit cobblestone area (CA) formation and CFU-C generation in LTC. This inhibition could be fully abrogated by withdrawing AB within the first 2 weeks of culture. Low AB concentrations stimulated CA formation. The AB-mediated inhibition of hemopoiesis probably involved direct stromal contact with stem cells because a) AB did not inhibit CFU-C generation when stem cells were cultured in trans-well inserts above the stroma; b) conditioned media from AB-containing or normal LTC did not inhibit colony formation by normal cells in semi-solid, non-stromal cultures, and c) AB did not inhibit colony formation by bone marrow cells in semi-solid culture nor did it inhibit growth or maintenance of stromal cells. In addition, The inhibition of hemopoiesis by AB could also not be explained by changes in the amount of cytokine and chemokine transcripts, including TGF-b1, in AB-blocked stromal cells. Our findings support the involvement of GJIC in stroma-dependent regulation of hemopoietic stem cell proliferation.