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The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.

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.

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.

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.

Treosulfan is a water-soluble structural analog of busulfan, acting as a prodrug of alkylating epoxide species. It does not induce severe hepatotoxicity or veno-occlusive disease at or above the maximum tolerated dose, lacks significant nonhematological toxicity and has a limited organ toxicity. It is mainly indicated for the treatment of patients with ovarian cancer. In the present study, we report that permanent donor-specific tolerance and stable mixed multilineage chimerism can successfully be achieved across haploidentical MHC barriers when Treosulfan is administered in combination with anti-T-cell mAb and T-cell-depleted donor bone marrow cells. Furthermore, we show that less T-cell suppression is required when Treosulfan is included in the conditioning regimen. In conclusion, Treosulfan is a well-tolerated myeloablative agent with a low toxicity, and is a promising candidate drug for conditioning prior to bone marrow transplantation.

To investigate whether we could create a radiation-free conditioning regimen to induce permanent mixed and multilineage chimerism and donor-specific tolerance, we treated recipient mice with anti-T-cell antibodies, varying and fractionated doses of Treosulfan and fully MHC disparate bone marrow cells. Treosulfan is mainly used in the treatment of ovarian cancer. It is a structural analog of busulfan, but it does not induce severe hepatotoxicity or veno-occlusive disease at or above the maximum tolerated dose, lacks significant nonhematological toxicity and has limited organ toxicity. We report here the successful induction of permanent mixed multilineage chimerism and donor-specific tolerance as was proven by skin transplantation and IFN-gamma ELISPOT. In conclusion, because of its lower nonhematological toxicity, compared with other myeloablative regimens (eg irradiation or busulfan admin- istration), Treosulfan could be a better candidate for conditioning to induce donor-specific allograft tolerance.

Recent studies describe beneficial effects of bone marrow-derived mesenchymal stem cell infusion in animal models as well as in patients. However, data on the homing abilities of primary and culture-expanded MSC are lacking. In order to systematically investigate MSC homing we compared the fate of both primary and cultured MSC in a syngeneic mouse model. Twenty-four hours after transplantation of uncultured EGFP-transgenic MSC into sublethally irradiated mice, as many as 55-65% of injected CFU-F were recovered from the BM and 3.5-7% from the spleen. In the subsequent 4 weeks these donor CFU-F expanded 100-fold, which resulted in a normalization of femoral and splenic CFU-F numbers. This highly efficient homing of primary CFU-F contrasted with the defective homing of MSC following culture. Following their infusion immortalized multipotent syngeneic stromal cells were undetectable in BM, spleen, lymph nodes or thymus. Remarkably, following transplantation of primary MSC that had been cultured for only 24 h the seeding fraction in the BM was reduced to 10%, while after transplantation of 48 h cultured primary MSC no CFU-F were detected in the lymphohematopoietic organs. These data suggest that in vitro propagation of BM-derived MSC dramatically decreases their homing to BM and spleen.

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.

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.

Objective: Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen., Methods: Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability., Results: Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells., Conclusion: These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.

To analyse the involvement of a possible numerical or qualitative stem cell defect in the development of sustained graft failure after autologous transplantation, we have determined the graft content of CD34+ nucleated cells, colony-forming cells and cobblestone area-forming cell subsets, as well as transplant ability to produce progenitors using the long-term culture colony-forming cell (LTC-CFC) assay. We evaluated material from the graft reference ampoules of 13 graft failure patients after bone marrow transplantation (BMT), four graft failure patients and four isolated thrombocytopenia patients after peripheral blood stem cell transplantation (PBSCT). We compared these data with those from six successfully engrafted BMT patients and 20 engrafted PBSCT patients respectively. In the BMT setting, the LTC-CFC 6-week assay represented a highly significant graft failure predictor. In the PBSCT setting, the total number of 2-week and 6-week LTC-CFCs transplanted per kg bodyweight (BW) showed the highest significant difference between the engrafted and the graft failure patients, as well as between the engrafted patients and the patients suffering from isolated thrombocytopenia after transplantation. These data show that the ability of a graft to generate progenitors in vitro rather than the number of primitive progenitors transplanted can have prognostic value for post-transplant haematological reconstitution.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Fetal cells present in the maternal circulation are a potential source of fetal DNA that can be used for the development of a prenatal diagnostic test. Since their numbers are very low, amplification of fetal cells has been discussed for a long time. So far, most studies have focused on culturing fetal erythroid cells. In this study, we evaluated whether limiting numbers of fetal haemopoietic progenitor cells present in an excess of maternal cells were able to overgrow the maternal component. Therefore, we used a model system in which limiting numbers of male CD34+ umbilical cord blood cells were diluted in 400 000 female CD34+ peripheral blood cells. The number of XY positive cells derived from umbilical cord blood was determined using two-colour in situ hybridization with X and Y chromosomal probes. We demonstrated a 1500-fold relative expansion of male umbilical cord blood cells over the peripheral blood component after three weeks of liquid culture, which also corresponded to the extent of expansion of CD34+ cells derived from 20-week fetal blood. However, application of the same culture protocol to maternal blood samples obtained at 7-16 weeks of gestation showed no preferential growth of fetal haemopoietic progenitor cells. This study, therefore, suggests that fetal primitive haemopoietic progenitor cells do either not circulate in maternal blood before 16 weeks of gestation, or require different combinations/concentrations of cytokines for their in vitro expansion., (Copyright 2000 John Wiley & Sons, Ltd.)

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area-forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells. (Blood. 2000;96:498-505)

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.

Nonobese diabetic/severe combined immune deficiency (NOD/SCID) mouse repopulating cells (SRC) have been proposed to represent a more primitive human stem cell subset than the cobblestone area-forming cell (CAFC) week (wk) 6 or the long-term culture-initiating cell (LTC-IC) wk 5 on the basis of their difference in frequency, phenotype, transfectibility, and multilineage outgrowth potential in immunodeficient recipients. We have assessed the percentage of various progenitor cell populations (colony-forming cell [CFC] and CAFC subsets) contained in unsorted NOD/SCID BM nucleated cells (nc), human umbilical cord blood (UCB) nc, bone marrow (BM) nc, peripheral blood stem cells (PBSC), and CD34(+) selected UCB nc, seeding in the BM and spleen of NOD/SCID mice within 24 hours after transplantation. The seeding efficiency of NOD/SCID BM CAFC wk 5 was median (range) in the spleen 2.9% (0.7% to 4.0%) and in the total BM 8.7% (2.0% to 9.2%). For human unsorted UCB nc, BM nc, PBSC, and CD34(+) UCB cells, the seeding efficiency for CAFC wk 6 in the BM of NOD/SCID mice was 4.4% (3.5% to 6.3%), 0.8% (0.3% to 1.7%), 5.3% (1. 4% to 13.6%), and 4.4% (3.5% to 6.3%), respectively. Using flow cytometry, the percentage CD34(+) UCB cells retrieved from the BM of sublethally or supralethally irradiated NOD/SCID mice was 2.3 (1.4 to 2.8) and 2.5 (1.6 to 2.7), respectively. Because we did not observe any significant differences in the seeding efficiencies of the various stem cell subsets, it may be assumed that the SRC seeding efficiency in NOD/SCID mice is similarly low. Our data indicate that the seeding efficiency of a graft can be of great influence when assessing stem cell frequencies in in vivo repopulation assays.