The ability of Steel Factor (SF) to stimulate colony formation and progenitor cell generation by hemopoietic stem cells (HSCs) in vitro in the absence of interleukin 3 (IL-3) was investigated. IL-3 was required for HSC proliferation, and no or restricted proliferation occurred in the presence of SF, IL-6, IL-11, or IL-12 as single factors or in combination. Neutralizing concentrations of anti-transforming growth factor (TGF)-beta 1 antibodies enhanced progenitor cell generation 2-3-fold in the presence of IL-3, but 75 to over 300-fold when cultures contained at least SF in the absence of IL-3. Exogenous TGF-beta 1 fully abrogated the antibody effects. In the presence of antibodies to TGF-beta 1, SF alone stimulated the delayed formation of small blast cell colonies and SF synergized with IL-6, IL-11, or IL-12 to greatly hasten colony formation, enhance colony number and size, and increase colony forming unit-culture (CFU-C) output from suspension cultures of enriched HSC populations. Secondary CFU-C colonies were significantly larger when IL-3 was absent during the suspension culture phase. Single cell and limiting dilution analysis using a homogenous colony forming unit-spleen (CFU-S) day-12 population and an 800-fold enriched long-term repopulating HSC fraction, respectively, indicated that TGF-beta 1 was an autocrine product of these HSC subsets. Addition of nucleosides, insulin, extra glucose, or serum could not replace the effects of the anti-TGF-beta 1 antibody. While these data offer one possible explanation for reports on the inability of SF to stimulate HSC proliferation, they present the basis for a novel model of the regulation of HSC activation wherein: 1) close-range interactions of HSCs with mesenchymal stromal cells do not exclusively determine maintenance of HSC quiescence; 2) competence acquisition by dormant HSCs may involve the down-regulation or inactivation of autocrine TGF-beta 1; and 3) SF may act as a primary growth factor rather than exclusively as a synergistic cytokine.

The radiation dose-survival of various hematopoietic cell subsets in murine bone marrow (BM) was determined in the cobblestone area forming cell (CAFC) assay under conditions of single-, split-, and multiple- dose irradiation. A greater recovery in cell survival with decreasing dose per fraction, or increasing fraction number, was observed for primitive CAFC day-28 and day-35 than for CAFC day-6 and day-12 (colony- forming unit (CFU)-granulocyte macrophage and CFU-spleen day-12 equivalents). Linear quadratic (LQ) model analysis of CAFC survival data provided an estimate of the alpha/beta ratio that is an inverse index of the fractionation effect and is known to be lower for late than for acutely responding tissues. This analysis gave decreasing alpha/beta ratios with increasing primitiveness of the CAFC subset. These values were found to be comparatively low (about 4 Gy) for CAFC day-28 and day-35 and are in general agreement with previous studies on long-term repopulation in vivo. In contrast, alpha/beta ratios of CAFC day-6 and day-12 were relatively high (above 6 Gy) and are consistent with values obtained from acute marrow failure. Delayed harvesting of BM after a single dose of 6 Gy showed little evidence of proliferative repopulation over 1 week and hence the differential dose-sparing effect of fractionation among the CAFC subsets appears to be mostly attributable to the influence of sublethal damage repair. These results require a reevaluation of previous notions of marrow stem cell radiosensitivity and repair based on acute marrow lethality (LD50/30) or spleen colony (CFU-S) data, especially when applied to fractionated total body irradiation effects on long-term repopulating stem cells in a BM transplant setting.

Summary:The polyfunctional alkylating agent thiotepa (N, NЈ, NЉ-Thiotepa (TT) has long been considered for inclusion triethylene thiophosphoramide) (TT) has now been in cliniin clinical bone marrow transplant (BMT) conditioning cal use for more than 40 years. Myelodepression is well regimens in an attempt to prevent allograft rejection recognized as the major dose-limiting toxicity and there is and leukemia relapse. These studies have been encourcurrent interest in applying escalating doses of TT in conaged by initial murine experiments showing a clear junction with autologous bone marrow transplantation improvement in allogeneic bone marrow engraftment (BMT) (eg Refs 1-3). This agent has also been considered with addition of TT to total body irradiation (TBI)for inclusion in the conditioning regimen prior to allogeneic where it was assumed that TT enhances donor-type BMT 4-7 and there is at least experimental evidence for chimerism via ablation of competing stem cells in the enhanced engraftment of donor stem cells when TT was recipient. The aim of the present study was to re-evaluadded to total body irradiation (TBI). 8 The latter finding in ate the hematological toxicity of TT among different an H-2-incompatible allogeneic BMT model in mice was stem cell subsets that included primitive cells capable attributed to the reduction of stem cell competition through of long-term repopulation and to assess how the combiadditional myeloablation and this effect has been compared nation of TT with TBI influences the development of with host stem cell toxicity afforded by another alkylating donor engraftment in both syngeneic (B6-Gpi-1 a → B6-agent, busulfan. However, it has since been appreciated that Gpi-1 b ) and H-2 compatible allogeneic (BALB.B10 → while the acute myelodepression caused by various cyto-B6) BMT models. At 24 h after TT (20 mg/kg) the femtoxic agents generally reflects depletion of committed prooral content of different stem cell subsets was detergenitor cell populations in the bone marrow, it may not mined from the frequency of transient repopulating, necessarily follow that primitive stem cells capable of longand the more primitive cobblestone area-forming, cells term repopulation are similarly affected. 9-11 The aim of the (CAFCs) growing in stroma-supported cultures. This present study was to re-address the hematological properassay showed a large TT-induced depletion (2% ties of TT with respect to survival of the different progenisurvival) of early clones developing at day 7 in culture tor and stem cell subsets in the mouse bone marrow by but survival recovered towards normal for later appearenumerating cobblestone area-forming cell (CAFC) subsets ing clones developing from more primitive CAFC subafter treatment. We then evaluated how the combination of sets. The sparing of these primitive stem cells was TT with TBI influences the magnitude of short-and longreflected as undetectable levels of donor marrow term engraftment of transplanted marrow cells using conrepopulation in recipients given TT ...

Peripheral blood stem cells (PBSC) are used for stem cell support in patients after intensive chemotherapy and generally permit faster hematopoietic recovery than bone marrow. The development of different protocols for chemotherapy conditioning, mobilization, and ex vivo manipulation of PBSC may potentially lead to loss of primitive hematopoietic stem cells or reduction of their quality. To characterize the frequency of different stem cell subsets and their quality per mobilized PBSC, we have studied 47 leukapheresis products (LPs) of 21 cancer patients using stroma-dependent long-term culture (LTC) and limiting dilution-type cobblestone area forming cell (CAFC) assays. A large variation in CAFC week-type frequencies between the LPs was observed. The frequencies of CAFC week 2 as a tentative indicator of progenitor cells and transiently repopulating hematopoietic stem cells ranged from 0.89 to 205 per 10(5) mobilized nucleated cells and the frequencies of more primitive CAFC week 6 varied between 0.37 and 48. The average total colony-forming cell (CFC) production per CAFC at week 6 varied between 1.2 and 730, as determined in parallel LTC. In contrast to LPs, bone marrow samples generated 4.2 to 48 CFC per CAFC at week 6. Notably, a poor stem cell quality was consistently found in LPs that contained less than 5,000 CAFC week 6 per kilogram of body weight. Frequency analyses of CFCs, CAFC subtypes, and immunophenotypic subsets showed a good level of mutual correlation, suggesting identical mobilization kinetics of different stem cell subsets. The premobilization chemotherapy intensity was directly correlated with both decreasing frequency and quality of the CAFC week 6 in LPs. The frequency of CFCs, immunophenotypic subsets, and CAFC subsets transplanted and the transplant quality as determined in LTC assays was related to the neutrophil and platelet recovery time after PBSC transplantation. Although the number of progenitor cells transplanted and the in vitro transplant quality showed the best correlation with early hematopoietic recovery, the data did not permit determination of which stem cell subsets are predominantly responsible for early posttransplantation recovery. As a result, frequency and quality analysis of stem cell subsets may be a useful tool to monitor and calibrate the efficacy of novel mobilization regimens and ex vivo manipulation of PBSC.

(LTBMCs). At this time a fixed complement of 10 congenicallv marked donor bone marrow cells (B6-Gpi-10-* B6-Gpi-PI) was infused in the drug-treated mice and erythroid engraftment was followed over 36 weeks. Diverse effects on early-and late-developing CAFC frequencies were found for the different drugs: these were generally related to the pattern of donor bone marrow engraftment in treated recipients. Melphalan was more toxic to earlydeveloping than to late-developing CAFC subsets. and the transplant only offered an earlv wave of blood chimerism followed bv return of host cells. CY and BCNU had minimal to moderate effects on CAFC content and engraftment with no apparent preference for any particular haemopoietic cell subset. IMS also had a relatively low toxic effect on host marrow CAFC frequencies but appeared exceptional in its ability to allow for more donor-type engraftment. The dimethane sulphonate compounds busulphan and DMB were especially potent at depleting late CAFC subsets and ensured high and lasting levels of donor bone marrow engraftment. These studies support the value of CAFC measurements for predicting the fate and growth of transplanted bone marrow cells in recipients pretreated with a variety of cytotoxic agents.