Fetal Liver Progenitor Research Articles

HLA expression in hemopoietic development. Class I and II antigens are induced in the definitive erythroid lineage and differentially modulated by fetal liver cytokines.

Abstract We investigated the expression of HLA Ag on hemopoietic progenitors (burst-forming unit E, CFU-E, and CFU-granulocyte-macrophage) and precursors from human embryonic fetal liver (FL) and peripheral blood at 5 to 9 wk postconception. The expression on progenitors was evaluated by complement-mediated cytotoxicity followed by assay of residual progenitors in clonogenic culture; immunofluorescence and RIA were used for differentiated precursors. HLA Class I and II Ag are not expressed on the primitive erythroid lineage, i.e., on yolk sac-derived megaloblasts circulating in 5- to 6-wk peripheral blood. However, they are gradually induced on the definitive erythroid lineage in FL. Their expression on progenitors is first detected at 6 wk and shows a progressive increase through 9 wk, up to greater than or equal to 50% of adult values. A similar expression pattern is observed for FL erythroblasts. Incubation of 6-wk FL erythroid cells with IFN-alpha, -beta, or -gamma, or TNF-alpha induces a sharp rise of the expression of HLA class I, but not HLA class II Ag on both progenitors and precursors. In contrast, incubation with PHA-stimulated adult leukocyte-conditioned medium causes a marked increase of both HLA-ABC and HLA class II Ag expression. We hence investigated the effect of cytokines present in leukocyte-conditioned medium on the expression of HLA class II Ag: although IL-1 alpha, IL-2, and granulocyte-macrophage-CSF do not exert a significant action, IL-1 beta and IL-4 induce a marked increase of HLA class II, but not class I, Ag expression on 6-wk FL progenitors and precursors. Low amounts of IFN-gamma, TNF-alpha, and IL-1 beta were detected in the supernatants and extracts of 6-wk FL cells. The concentrations of these cytokines in both supernatants and extracts sharply increase in the 7- to 8-wk period, particularly for TNF-alpha and IL-1 beta, thus indicating a direct correlation with the rise of HLA Ag expression on FL erythroid cells in the same period. In conclusion, the expression of HLA class I and II Ag is not detected on primitive megaloblasts, but is gradually induced on definitive FL progenitors and precursors, possibly via production of specific cytokines in the FL microenvironment, i.e., IFN-gamma and TNF-alpha for class I Ag and IL-1 beta for class II Ag.

Developmental Regulation of Erythropoiesis by Hematopoietic Growth Factors: Analysis on Populations of BFU-E From Bone Marrow, Peripheral Blood, and Fetal Liver

AbstractFetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.

Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver

Fetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.

Anti-HIV drugs: Comparative toxicities in murine fetal liver and bone marrow erythroid progenitor cells

The toxicity of anti-HIV drugs, 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine (d4T) and ribavirin was studied in vitro in murine fetal liver cells (FLC) and in bone marrow cells. These studies indicate that d4T is the least toxic drug and ribavirin is the most toxic agent in both models. However, the murine FLC system was found to be a more sensitive model for the assessment of toxicity of anti-HIV agents towards erythroid progenitor cells as indicated by the IC50 values.

Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose- dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma- interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma- interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL- 2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.

γ-Interferon Alters Globin Gene Expression in Neonatal and Adult Erythroid Cells

Interferons have the ability to enhance or diminish the expression of specific genes and have been shown to affect the proliferation of certain cells. Here, the effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes. This supports the concept that developmental globin gene switching can be regulated by environmental factors.

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.

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 marrow 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 granulocyte-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.

T cell progenitors in the mouse fetal liver.

Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process.

Identification and characterization of three classes of erythroid progenitors in human fetal liver

Identification and characterization of three classes of erythroid progenitors in human fetal liver

Identification and Characterization of Three Classes of Erythroid Progenitors in Human Fetal Liver

AbstractErythroid progenitors from 18 fetal livers (7–15-wk gestational period) and 13 normal adult marrows were grown in methylcellulose cultures containing erythropoietin (Ep) and burst-enhancing factor(s) (BEF). Additional experiments were carried out on 1 fetal, 15 neonatal, and 26 adult blood specimens. Three classes of progenitors (primitive or mature erythroid burst-forming unit(s), P-BFU-E, M-BFU-E respectively; erythroid colony-forming unit(s), CFU-E) have been identified in fetal liver. Identification was based on their differential clonogenic characteristics (i.e., colony morphology and number, time/growth curve, Ep and BEF sensitivity, in vitro 3H-thymidine suicide index). The multistep differentiation of fetal erythroid progenitors is associated with: (1) gradual amplification of their pool size, (2) progressive decline of their BEF response, (3) gradual enhancement of their Ep sensitivity. Marked differences are observed, however, between the characteristics of corresponding fetal and adult erythroid progenitors. Thus: (1) Differentiation of adult precursors entails a gradual increase of their proliferative rate, while all fetal progenitors are characterized by maximal cycling activity. The doubling time of cells in fetal bursts is distinctly lower than in adult ones, while intermediate values are observed in cord blood bursts. These observations suggest that ontogenic development is associated with a progressive decline of both (A) proliferative activity of early and intermediate erythroid progenitors, and (B) cell doubling time in erythropoietic differentiation. (2) Adherent cells apparently play a key role in BEF production in adult marrow cultures, but not in fetal liver ones. Finally, (3) the sensitivity to added Ep of fetal CFU-E and M-BFU-E is apparently more elevated than that of corresponding adult progenitors.

HbF and HbA Production in Erythroid Cultures From Human Fetuses and Neonates

The synthesis of γ- and β-globin chains was investigated in erythroid cultures from first and second trimester fetuses as well as neonates in order to examine whether the differences in production of HbA and F in the fetal and newborn ontogenetic stages reflect in vivo predetermined differences in commitment among erythroid progenitors. Cultures of erythroid progenitors from first and second trimester fetuses (12 experiments) produced amounts of HbF that were similar to those synthesized in vivo by fetal liver erythro-blasts and reticulocytes (range of HbF production: in vivo, 84%-93%; in culture, 80%-94%). There was no difference in HbF synthesis in cultures of fetal liver or peripheral blood-origin progenitors from the same fetus. In cultures of circulating progenitors from full-term newborns (nine experiments), amounts of fetal hemoglobin that are expected for the newborn ontogenetic stage (range of HbF in culture: 55%-80% of total Hb) and that are close to the levels of HbF production in cord-blood reticulocytes of the same fetuses, were observed. Varying the concentration of erythropoietin by 40-fold produced only minor changes in the proportion of γ and β chain synthesis in culture. The findings show that the fetal or newborn erythroid stem cells are programmed in vivo for expressing the characteristic, for each ontogenetic stage, pattern of non-α-globin synthesis. This is consistent with the proposition that the regulation of γ- and β-chain synthesis during development takes place at the level of erythroid progenitor cells rather than in the terminally mature erythron.