Differential Sensitivity of Mouse Hematopoietic Stem Cells to Merocyanine 540

In vivo and in vitro clonal assays of immature mouse blood cells showed that diffferent populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%–4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.     

Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody,YBM/42

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS-II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25-fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU-E). In the most enriched fraction, 20% of the cells are day 2 CFU-E. Day 7 CFC can therefore be well separated from day 2 CFU-E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G-CFC), macrophage (M-CFC), mixed granulocyte/macrophage (GM-CFC), pure erythroid (E), or mixed erythroid (E_mix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM-CFC and M-CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM-CFC and M-CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Mononuclear macrophage (M-CFC) colony formation in semisolid media in the absence of exogenous GM-CSF

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony-stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar-MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M-CFC) were formed in a combined culture of agar and MC. Aggregates of 5-40 cells were observed on day 7. Gradually, large compact colonies which survived for 10-12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M-CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell-dose-dependent. Depletion of monocytes increased the number of colonies in agar-MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar-MC cultures. CSF increased M-CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M-CFC growing for 2-5 weeks in CSF-deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, an increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Comparative studies of the granulopoietic enhancing effects of biosynthetic human insulin-like growth factors I and II

The effect of biosynthetic human insulin-like growth factor I (IGF-I) and IGF-II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG-CSF), granulocyte-macrophage CSF (rhGM-CSF), or interleukin-3 (rhIL-3), was investigated. IGF-I and IGF-II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF-I induced an increase of 62 ± 16%, 84 ± 13%, and 107 ± 18% in granulocyte colony numbers in the presence of G-CSF, GM-CSF, or IL-3, respectively. The values for IGF-II were 66 ± 13%, 96 ± 12%, and 91 ± 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G-CSF and GM-CSF-stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL-3 in such cultures. The growth-promoting effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against the IGF-I (Type I) membrane receptor. IGF-I and IGF-II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor. © 1993 Wiley-Liss, Inc.     

Plasma membrane lipid order of leukemic and normal immature avian erythroid cells

Mammalian erythroblasts and their leukemic counterparts contain characteristic disordered regions of plasma membrane identified as putative membrane protein collection sites. In order to determine whether erythroid cells which do not enucleate contain homologous membrane domains, immature avian erythroid precursor cells and avian erythroleukemic cells were examined using merocyanine 540 (MC540), a fluorescent dye whose binding is sensitive to the packing of membrane lipids. Results were found to contrast with previous studies of the murine equivalents of these cells. In birds, normal erythroid precursors, including basophilic erythroblasts from the bone marrow and spleen of anemic animals, contained no detectable (less than 0.1%) cells which were stained by the dye. But cells from chicks infected with avian erythroblastosis virus (AEV) did stain. Considering the pattern of staining observed on AEV-erythroblasts relative to other leukemic and normal phenotypes, however, we conclude that neither normal nor leukemic avian erythroid cells contain a functional equivalent to the membrane protein collection sites found on their mammalian counterparts.     

Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

The effect of natural killer cells on the development of syngeneic hematopoietic progenitors

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

Merocyanine 540 as a fluorescent probe of membranes: staining of electrically excitable cells.

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.     

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs. © 1995 Wiley-Liss, Inc.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.     

A self-renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi-solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self-renewal potential. Forty-seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty-one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor-dependent cell lines are maintained by self-renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.     

Cryopreservation of bovine hemopoietic progenitor cells in liquid nitrogen

Bovine marrow granulocyte/macrophage and erythroid progenitor cells maintained viability after storage in liquid nitrogen for 2 to 4 weeks. The granulocyte/macrophage progenitor cells maintained 100% viability for 4 weeks, while the erythroid progenitor cells maintained 100% viability for at least 2 weeks. The optimum concentration of either DMSO or glycerol was found to be 5–10%. DMSO was superior to glycerol as a cryopreservative of bovine granulocyte/ macrophage progenitor cells. Glycerol was found to be unable to cryopreserve bovine erythroid progenitor cells.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.     

ERYTHROPOIETIN-INDEPENDENT REGENERATION OF ERYTHROID PROGENITOR CELLS FOLLOWING MULTIPLE INJECTIONS OF HYDROXYUREA

It was shown previously that colony formation in vitro by early erythroid progenitor cells (BFUe) requires sequential stimulation with a specific glycoprotein termed BFA and erythropoietin (EP). The action exerted by BFA was characterized as induction of proliferation in BFUe resulting after several cell divisions in EP-responsive progeny. The present study is directed at detection of EP-independent regulation of erythroid progenitor cells in vivo. Haemopoietic regeneration was induced by multiple administrations of hydroxyurea (HU). The femoral regeneration patterns of haemopoietic stem cells (CFUs), granulocyte/macrophage progenitor cells (CFUgm) and erythroid progenitor cells (BFUe, day 3 BFUe and CFUe) were studied in hypertransfused mice in comparison to nontransfused controls. The results show that (1) the phase of exponential regeneration of none of the cell populations studied is affected by hypertransfusion; (2) each of these cell populations exhibit a distinct regeneration pattern, indicating that they behave as separate functional entities; and (3) the three erythroid cell populations are suppressed by hypertransfusion in the post-exponential phase of regeneration in contrast to CFUs and CFUgm. The results support a two-regulator model of erythropoiesis.