Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony- stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.
Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.
There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors. Leukemia (2000) 14, 662-670.
blythi during a part of the annual cycle. All species examined except Miniopterus schreibersi reach their southern limits of distribution in Algeria. The duration of hibernation was shorter in Algeria than in Europe, but the reproductive cycle was similar in that insemination took place in autumn and fertilization in spring. Parturition occurred earlier than in European populations of the same species. Observations were made on the choice of roosts, formation of intraand interspecific associations, as well as individual and geographic variation in dates of parturition. The tendency of M. blythi to form harems during the mating season was noted.
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