Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.     

Transitional change of colony stimulating factor requirements for erythroid progenitors.

The course of the differentiation and proliferation of the human erythroid burst-forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL-3 requirement correlate with the erythroid differentiated state.     

Clonal origin of murine hemopoietic colonies with apparent restriction to granuclocyte-macrophage-megakaryocyte (GMM) differentiation

We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GEMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.     

Growth of human normal erythroid progenitors in liquid culture: a comparison with colony growth in semisolid culture

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.     

Characteristics of murine yolk sac erythroid progenitors and their population expansion in liquid culture

Remarkable differences were found between late erythroid progenitors (CFU-e) in cultures of murine yolk sac cells and those of fetal liver cells with respect to frequency, erythropoietin responsiveness and colony size. Cultures of yolk sac on day 11 of gestation showed a CFU-e population of lower frequency, less sensitivity to erythropoietin and smaller colony size than those from cultures of day 14 fetal liver cells. As the proportion of CFU-e to BFU-e was much lower in yolk sac than that in fetal liver, 48-96 h liquid culture experiments were done with these cells to examine the capacity of their precursors to generate a certain amount of CFU-e subpopulations. The cultures of yolk sac cells produced large numbers of CFU-e which formed some large-sized colonies but those of fetal liver cells generated only a small amount of CFU-e.     

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment

Background  

We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Target cells infected by avian erythroblastosis virus differentiate and become transformed

Transformation in vitro of bone marrow cells by avian erythroblastosis virus (AEV) gives rise to rapidly growing cells of erythroid nature. Target cells of neoplastic transformation by AEV are recruited among the early progenitors of the erythroid lineage, the burst-forming units-erythroid (BFU-E). They express a brain-related antigen at a high level and an immature antigen at a low level. We show that AEV-transformed cells express low levels of the brain antigen and high levels of the immature antigen. Their response to specific factors regulating the erythroid differentiation indicates that they are very sensitive to erythropoietin. Furthermore, cells transformed by a temperature-sensitive mutant of AEV differentiate into hemoglobin-synthesizing cells 4 days after being shifted to the nonpermissive temperature. All these properties are similar to those of late progenitors of the erythroid lineage, the colony-forming units-erythroid (CFU-E). These results indicate that the AEV-transformed cells are blocked in their differentiation at the CFU-E stage.     

In vitro analysis of cardiac progenitor cell differentiation

Cardiac myoblast commitment and differentiation were studied in the developing avian embryo. Single cell analysis of isolated cardiogenic cells grown in vitro established that stage 4 (newly gastrulated) mesodermal cells are capable of myocyte differentiation in the absence of intercellular contact or short range cellular interactions. While cardiac myocytes derived from single isolated progenitors expressed muscle-specific myosin heavy chains (MHC), atrial and ventricular MHCs characteristic of in vivo development were not detected. When the same progenitors were grown at high density or in organ cultures, cell-specific, expression of atrial and ventricular MHCs was observed, suggesting a role of cell density-dependent processes for differential MHC expression. Cardiogenic mesoderm (stages 4-8) was treated with the cocarcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA), maintained as organ cultures, and assayed for muscle differentiation in an attempt to identify possible stage-specific variations in cardiac progenitors. TPA irreversibly blocked the differentiation of early (stages 4-7) progenitors. When exposed to TPA, stages 4-7 cardiogenic cells failed to synthesize several muscle-specific proteins as determined by immunochemical analysis of myosin synthesis and two-dimensional gel electrophoresis of 35S-labeled proteins isolated from cardiogenic cultures. In addition, stages 4-7, TPA-treated cells did not differentiate after the withdrawal of TPA. In contrast, TPA had no effect on the expression of several muscle-specific proteins in late (stage 8) cells including the cell-specific expression of atrial and ventricular MHCs.     

Ia-positive nonlymphoid cells and T cell development in murine fetal thymus organ cultures: monoclonal anti-Ia antibodies inhibit the development of T cells

A fetal thymus organ culture system has been developed to study the differentiation of murine thymus-derived immunocompetent cells (T cells) such that cell yields can be easily monitored. This system has been used to study the effects of monoclonal anti-I-A antibodies on the growth of T cells. The addition of anti-I-A antibodies, but not anti-H2K monoclonal antibodies, to fetal thymus organ cultures resulted in a decreased yield of lymphoid cells. Anti-I-A-treated cultures did not produce cells that gave an immune response in MLC assays. Anti-I-A antibodies stained a small subpopulation of nonlymphoid cells in untreated cultures by indirect immunofluorescence that were no longer detectable in cultures that had been pretreated with anti-I-A antibody. Culture of fetal thymus lobes at low temperature (20 degrees C) for 1 wk resulted in a decrease in lymphocyte production, as well as a concomitant increase in the frequency of Ia-positive nonlymphoid cells. Co-culture of fetal liver or anti-thy-1 plus complement-treated adult bone marrow with such Ia-positive cell-enriched fetal thymus lobes at 37 degrees C resulted in the production of T cells. Anti-Thy-1.1 or -1.2 staining by indirect immunofluorescence of cells obtained from co-cultures that differed at the Thy-1 locus showed that the T cells produced were derived from the bone marrow or fetal liver. T cell production occurred in both syngeneic and allogeneic cocultures. However, if co-cultures were made by using 14-day gestation fetal thymus instead of fetal liver or bone marrow as donors of T cell precursors, T cell growth was observed only in syngeneic combinations. These results suggest that Ia-positive nonlymphoid cells play a role in the development of T cells in the fetal thymus, and that "thymus processed" T cell progenitors (but not the more immature progenitors in the fetal liver or bone marrow) are self-Ia restricted in their differentiation.     

Down-regulation of Myc is essential for terminal erythroid maturation

Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.     

Effect of colony-stimulating activity on hematopoiesis in the organ culture of mouse embryonal liver

The effect of the colony-stimulating activity (CSA) on hemopoiesis in a long-term culture (4.5 week) of mouse embryonal liver was studied. After addition of the spleen cell-conditioned medium containing CSA to the organ culture, there was a decrease in the number of CFUs and in the granulocyte and macrophage precursors. However, the production of granulocytes and macrophages in the test cultures either did not fall or increased with primary differentiation of neutrophils. Addition of the medium with CSA shifted the equilibrium in the culture towards more intense production of differentiated cells at a lower level of the maintenance of precursors.     

Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.     

Cell differentiation in the hippocampus of the fetal rat in cell culture

Cell cultures from hippocampus of 16 and 17 days old embryonal rats were cultivated up to 4 weeks. After 24 hours in vitro on 18.4 percent of cells and after 5 days in vitro on 70 percent of cells processes could be recognized. These are neuroblasts. The cells reaggregated. Nerve fibers after 4 weeks in vitro are 200 to 300 mum long. Small and big neurons with 12 mum to 26 mum diameters of perikarya, bi- and multipolar neurons after 4 weeks in vitro were observed. In cultures and meningothel-monolayer developed. Maintenance and differentiation of cultures are possible only by sowing in at least 60,000 cells/ml medium. The advantage of cell culture opposite to organ culture exists in experiments with immediate selective influence.