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Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+progenitor cells when incubated for 40 hours. However, after prolonged (>40 hours) preincubation, the viability-promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum-supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca-1+bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.

Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly active in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.

A large variability in the stem cell content of the spleens of female mice by comparison with that of the bone marrow, or with that of the spleen and bone marrow of male mice, is demonstrated. This variability is shown to be associated with the estrous cycle. Stem cells are assayed by the spleen colony-forming test, either exogenously by transplantation to irradiated recipient mice, or endogenously by colony development in sublethally irradiated female mice. It is shown that, for mice in pre-estrus, the spleens contain up to three times as many hemopoietic stem cells (CFU) as during the estrus and post-estrus stages of the cycle. Although the association may be indirect, it is suggested that the changes in splenic stem cell numbers may be related to the hormonal milieu at the time of observation.

The scanning electron microscope was used to examine the effects vitamin A has on the surface of human erythrocytes. The hemolytic properties of vitamin A, both time- and dose-dependent, are confirmed, and the observations are extended to the establishment of a sequence of cellular events that leads to the destruction of the red cell. The biochemical means whereby vitamin A induces these changes are discussed. In addition, the present study indicates that only selective areas of the erythrocyte membranes are responsive to the action of vitamin A, thereby supporting the proposal that the erythrocytic membrane may not be a homogeneous mass.

This paper, the first of a series on the regulation of the hematopoietic stem cell, examines the effects of stem cell kinetics during an acute exposure to hypoxic-hypoxia. This physiologic perturbation was chosen because of the well-established role it plays in influencing erythropoiesis. BDF1male mice were exposed to a simulated altitude of 22,000 ft for periods of time ranging from 3 to 48 hr. The following parameters were assessed: Packed red cell volume, number of nucleated cells per femur and spleen, relative and total number of colony-forming units (CFU, i.e., stem cells) in the femoral shaft and whole spleen, as well as the turnover of this population of cells using the “3H-thymidine-killing” technique. The spleen responds by releasing more than one-half (i.e., approximately 108cells) of its prehypoxic complement of nucleated cells. On the other hand, the femoral cellularity did not decrease but showed a moderate increase. Within 12 hr of hypoxia exposure the total femoral CFU had markedly risen, in some cases between 50% and 75% above control levels. There were, nevertheless, great fluctuations over a short period of time. The population turnover kinetics revealed that after 3 hr of hypoxia an average of 25% of the CFU in both marrow and spleen were in the DNA synthetic phase. It is clear that stem cells are capable of responding rapidly to hypoxia. The possible regulatory mechanisms are discussed in terms of what is known about the kinetics of erythropoietin generation.

Hypobaric-hypoxia, an established stimulator of erythropoiesis, was used to perturb normal steady-state hematopoiesis and thereby to facilitate an analysis of stem cell kinetics. BDF1male mice were exposed to a simulated altitude of 22,000 ft for 1–15 days. The following parameters were assessed at daily intervals: packed red cell volumes (PCV), femoral and splenic nucleated cellularity, the relative concentration and total number of colony-forming units (CFU, i.e., stem cells) in the femoral shaft and whole spleen, as well as the turnover of this population of cells using the “3H-thymidine-killing” technique. By 10 days of hypoxia the PCV had risen to 65% and by a fortnight was elevated to, and stabilized at, 70%–75%. After an evanescent increase, the femoral cellularity became slightly hypocellular but returned to normal by 3 days, thence becoming hypercellular from days 5 to 15. The spleen was markedly depleted of cells for 48 hr and recovered to hypercellular levels between 4 and 6 days. It then oscillated from hypercellular to normal levels with a period interval of 5 days. Both femoral and splenic CFU demonstrated oscillations, as did the estimations made on the state of CFU turnover. The femoral and splenic CFU did not decrease in number during the 15 days of hypoxia, indeed, they both revealed cyclic increases. These results are discussed in terms of CFU kinetics and humoral mediation.