Evaluation of in vivo and in vitro repopulation assays for murine haemopoietic stem cells

Three assays to determine the repopulation potential of stem cells in murine bone marrow grafts were evaluated on their reliability with regard to the ranges in graft size. In vivo, marrow repopulating ability, as calculated from the number of in vitro clonable progenitors (colony-forming units in culture, CFU-C) generated by the graft in the femur of an irradiated recipient, appeared to be independent of the input over a wide range of cell numbers grafted. A second assay, erythroid repopulating ability is a measure of the number of new reticulocytes or erythrocytes in the blood generated by the graft, and significantly underestimates stem cell activity of the graft. The third assay measures the long-term repopulating ability of bone marrow cells in vitro on pre-established stromal cell layers by determination of the number of CFU-C produced in these cultures. Calculations of short-term in vitro repopulating ability, done from measurements of the production of non-adherent CFU-C in the first week of culture, appeared to be independent of cell input. Long-term in vitro repopulating potential, measured by the CFU-C content of the adherent layer at 4 weeks, is also independent of the numbers of cell input.     

Murine haemopoietic stem cells with long-term engraftment and marrow repopulating ability are more resistant to gamma-radiation than are spleen colony forming cells.

The radiation sensitivity of various subsets in the haemopoietic stem cell hierarchy was defined using a limiting dilution type long-term bone marrow culture technique that was previously shown to allow quantification of cells with spleen colony-forming potential (day-12 CFU-S) and in vivo marrow repopulating ability (MRA). Primitive stem cells that generate new in vitro clonable colony-forming cells (CFU-C) in the irradiated marrow (MRA) and have long-term repopulation ability (LTRA) in vitro (cobblestone area forming cell, CAFC day-28) had D0 values of 1.25 and 1.38 Gy, respectively. A lower D0 was found for the less primitive CFU-S day-12, CAFC day-12 and cells with erythroid repopulating ability (0.91, 1.08 and 0.97 Gy, respectively). CFU-S day-7 were the most radiosensitive (D0 equalling 0.79 Gy), while CFU-C and CAFC day-5 were relatively resistant to irradiation (D0 1.33 and 1.77 Gy). Split-dose irradiation with a 6 h interval gave dose sparing for stem cells with MRA and even more with in vitro LTRA, less for CFU-S day-12 and CAFC day-10 and none for CFU-S day-7. The cell survival data of the specified stem cell populations were compared with the ability of a fixed number of B6-Gpi-1a donor bone marrow cells to provide for short- and long-term engraftment in single- and split-dose irradiated congenic B6-Gpi-1b mice. Serial blood glucose phosphate isomerase (Gpi) phenotyping showed less chimerism in the split as compared to the single radiation dose groups beyond 4 weeks after transplant. Radiation dose-response curves corresponding to stable chimerism at 12 weeks for single and fractionated doses revealed appreciable split-dose recovery (D2-D1) in the order of 2 Gy. This was comparable to D2-D1 estimates for MRA and late-developing CAFC (1.27 and 1.43 Gy, respectively), but differed from the poor dose recovery in cells corresponding to the committed CFU-S day-7/12 and CAFC day-10 population (0.14-0.33 Gy). These data are together consistent with differential radiosensitivity and repair in the haemopoietic stem cell hierarchy, and provide a cellular basis for explaining the dose-sparing effect of fractionated total-body irradiation conditioning on long-term host marrow repopulation.     

In myelodysplastic syndromes progression to leukemia is directly related to PHA dependency for colony formation and independent of in vitro maturation capacity

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.     

Studies of the Haemopoietic Microenvironments

The possible relation between haemopoietic activity and glycosaminoglycan levels in the haemopoietic microenvironment was examined in ectopic erythropoiesis, induced by phenylhydrazine treatment, in the liver of adult mice. The hepatic glycosaminoglycan content in the liver was determined biochemically over a period of 9 d following induction of haemolytic anaemia. After induction sulphated glycosaminoglycan levels increased up to d 4, then decreased to subnormal levels on d 5 and returned to normal values on the following days. The pattern of changes in GAG content coincided with changes in the number of CFU-S and with the number of erythroblasts in the liver. This observation fits in with the hypothesis of McCuskey et al (1972) stating that high sulphated glycosaminoglycan levels are favourable for stem cell proliferation, whereas low sulphated glycosaminoglycan levels favour erythropoiesis.     

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.     

Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.     

Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light- density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild- type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240- fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.