Residual erythroid progenitors in W/W mice respond to erythropoietin in the absence of steel factor signals.

Erythropoiesis is severely impaired in mice with inactivating mutations in the Steel factor (SF) gene (Sl/Sl mice) or in c-kit, in the SF receptor gene (W/W mice), and in mice with null mutations in the genes for either erythropoietin (EPO) or the erythropoietin receptor (EPO-R). Previous studies indicated that EPO is sufficient for colony development from colony-forming units-erythroid (CFU-E). However, recent studies have shown that there is a physical association between these 2 receptors and that c-kit can phosphorylate EPO-R. To examine the role SF and EPO play in regulating erythropoiesis, we examined the effect of SF and EPO on colony development from cells of the embryonic aorta-gonad-mesonephros (AGM) region, yolk sac, and liver of fetal wild-type and W/W mice. The maturation of CFU-E from these sites did not require the addition of SF to clonal cultures, whereas the efficient development of erythroid bursts required both EPO and SE The number of erythroid colony-forming cells was reduced in both the AGM region and liver of fetal W/W mice. The residual CFU-E present in W/W mice were dependent on EPO and independent of SF. These results indicate that EPO/EPO-R can function to support colony formation in the absence of an SF signal.

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

Erythropoietin receptors that signal through Stat5 or Stat3 support fetal liver and adult erythropoiesis: lack of specificity of stat signals during red blood cell development.

Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

Loss of flk-2/flt3 expression during commitment of multipotent mouse hematopoietic progenitor cells to the mast cell lineage.

The expression of c-kit and flk-2/flt3 was analyzed in various stages of mast cell differentiation using reverse transcriptase polymerase chain reaction (RT-PCR). Mouse fetal liver cells were sorted using antibodies for Sca-1 (Ly6A/E) and CD43 to obtain a population enriched for early progenitors; committed mast cell progenitors were absent from this population. Mouse fetal liver-derived, IL-3-dependent blast cell colonies provided a source of committed mast cell progenitors, and mast cell colonies provided mature mast cells. Comparison of these populations showed that some uncommitted cells express both c-kit and flk-2/flt3. At the time of commitment to the mast cell lineage, the expression of c-kit increases compared to that of uncommitted progenitors, and the expression of flk-2/flt3 becomes undetectable. Previous studies have shown that steel factor, the ligand for c-kit, supports mast cell differentiation in vivo and in vitro. In contrast, the ligand for flk-2/flt3 is inactive on mast cells. Thus, receptor gene expression appears to be an important determinant of the response or lack of response of mast cells to the ligands for these two homologous receptors.

Expression of an activated erythropoietin or a colony-stimulating factor 1 receptor by pluripotent progenitors enhances colony formation but does not induce differentiation.

Whether the presence of specific receptors on the surface of developing cells is the cause or consequence of lineage restriction is not known. If activation of specific receptors is the driving event in differentiation, the premature expression of specific receptors would promote differentiation along that pathway. In this study pluripotent progenitors, obtained from blast cell colonies (pooled or individual) of 5-fluorouracil-treated mice, were infected with retroviral vectors containing either an activated receptor for erythropoietin (EPO), an erythroid progenitor growth factor, or the receptor for colony-stimulating factor 1 (CSF-1), a macrophage growth factor. These receptors exhibit expression patterns restricted to committed progenitors. The developmental potential of infected pluripotent progenitors was not changed, although they expressed the exogenous genes, suggesting that in these cells activation of lineage-specific receptors does not induce differentiation. Acquisition of a constitutively activated EPO receptor allowed erythroid development in mixed colonies in the absence of EPO, as expected. Infection of progenitors with a virus containing the CSF-1 receptor promoted the development of granulocyte/macrophage (GM) colonies but did not alter the differentiation potential of either colony-forming unit (CFU)-GM or CFU-mix.

A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth.

We tested the ability of a constitutively activated erythropoietin receptor [EpoR(R129C)] to alter the growth requirements of primary hematopoietic precursors that terminally differentiate in culture. Two recombinant retroviruses expressing EpoR(R129C), spleen focus-forming virus (SFFVc-EpoR) and myeloproliferative sarcoma virus (MPSVcEpoR), were used to infect fetal liver cells that served as a source of hematopoietic progenitors. Methylcellulose cultures were incubated in the absence of any added growth factors or in combination with selected growth factors. EpoR(R129C) completely abrogated the Epo requirement of erythroid colony-forming units to form erythrocytes after 2-5 days in culture and did not interfere with the differentiation program of these cells. In the absence of added growth factors EpoR(R129C) did not enhance erythroid burst-forming unit development. In contrast to experiments in heterologous cell lines, EpoR(R129C) did not render progenitor cells independent of interleukin 3 or granulocyte/macrophage colony-stimulating factor (GM-CSF). However, when progenitors were cultured with added steel factor, but not with interleukin 3 or GM-CSF, EpoR(R129C) augmented the growth and differentiation of erythroid bursts, mixed erythroid/myeloid, and granulocyte/macrophage (GM) colonies. Furthermore, both viruses were capable of expressing EpoR(R129C) in erythroid, mixed erythroid/myeloid, and GM colonies. Thus an aberrantly expressed and constitutively activated EpoR can stimulate proliferation of some GM progenitors. The ability of EpoR(R129C) to abrogate the Epo requirement of primary hematopoietic cells, but not the requirement for other cytokines, is consistent with the induction of erythroblastosis in vivo.

Enhancement of the proliferation of murine fetal liver erythroid progenitors by infection with Harvey sarcoma virus.

We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.

Fetal liver blast cell colonies: a source of erythroid progenitors.

Isolated progenitors of defined type are useful for definitive studies of commitment, growth factor specificity, and identification of viral target cells. Blast cell colonies from cultures of spleen cells obtained from mice treated with 5-fluorouracil have proven to be a useful source of pluripotent progenitors. These colonies also contain committed progenitors of some lineages such as the macrophage lineage; however, the incidence of progenitors for pure erythroid colonies is low in this population. We report here the development of an alternative blast cell colony assay using fetal liver cells that is based on the greater sensitivity of early progenitors to interleukin 3. Replating studies demonstrated a variety of types of secondary colonies, including macrophage, neutrophil, neutrophil/macrophage, megakaryocyte, erythroid, mast cell, and mixed. The incidence of pure erythroid and mast cell colony-forming cells was substantially higher in fetal liver blast cell colonies than in colonies cultured from adult spleen.

In vitro retroviral transfer of ras genes to single hemopoietic progenitors.

Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.

A stochastic model for mast cell proliferation in culture.

A birth-death model was developed for the proliferation of mast cells. According to the model, each secondary mast cell colony starts with one proliferative cell. At each generation each cell chooses among three possibilities: 1) division into two proliferative cells; 2) division into two non-proliferative cells; or 3) disappearance. At each step, a non-proliferative cell either does nothing or disappears. A computer simulation of this model could be fitted reasonably well to our data for the size distributions of secondary mast cell colonies recorded after different culture periods. Our model predicts that proliferative cells comprise a larger fraction of the colony in large secondary colonies than in small ones. This prediction was successfully tested by examination of tertiary colony formation. This is a general model for cell proliferation that may be applicable to other types of cells.

Analysis of pure and mixed murine mast cell colonies.

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5-44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, that mast cells are derived from pluripotent hemopoietic stem cells, and that mast cells with metachromatic granules can have a high proliferating ability.