Hematopoietic Cells In Fetal Liver Research Articles

Regulation of the embryonic erythropoietic niche: a future perspective.

The production of red blood cells, termed erythropoiesis, occurs in two waves in the developing mouse embryo: first primitive erythropoiesis followed by definitive erythropoiesis. In the mouse embryo, both primitive and definitive erythropoiesis originates in the extra-embryonic yolk sac. The definitive wave then migrates to the fetal liver, fetal spleen and fetal bone marrow as these organs form. The fetal liver serves as the major organ for hematopoietic cell expansion and erythroid maturation after mid-gestation. The erythropoietic niche, which expresses critical cytokines such as stem cell factor (SCF), thrombopoietin (TPO) and the insulin-like growth factors IGF1 and IGF2, supports hematopoietic expansion in the fetal liver. Previously, our group demonstrated that DLK1+ hepatoblasts support fetal liver hematopoiesis through erythropoietin and SCF release as well as extracellular matrix deposition. Loss of DLK1+ hepatoblasts in Map2k4−/− mouse embryos resulted in decreased numbers of hematopoietic cells in fetal liver. Genes encoding proteinases and peptidases were found to be highly expressed in DLK1+ hepatoblasts. Capitalizing on this knowledge, and working on the assumption that these proteinases and peptidases are generating small, potentially biologically active peptides, we assessed a range of peptides for their ability to support erythropoiesis in vitro. We identified KS-13 (PCT/JP2010/067011) as an erythropoietic peptide-a peptide which enhances the production of red blood cells from progenitor cells. Here, we discuss the elements regulating embryonic erythropoiesis with special attention to niche cells, and demonstrate how this knowledge can be applied in the identification of niche-derived peptides with potential therapeutic capability.

A model system to study Connexin 43 in the immune system

Connexin 43 (Cx43) is the predominant gap junction protein expressed in immune cells. Previous manuscripts have stated that gap junctions may play a role in antigen cross-presentation, dendritic cell maturation, T cell development, and regulatory T cell function. Many of these previous studies were performed in vitro. In vivo studies were not directly possible in adult mice because Cx43−/− mice die shortly after birth due to a cardiac malformation. To overcome these drawbacks, we have developed a mouse model that deletes Cx43 in the immune system while maintaining normal cardiac function. In our model, irradiated CD45.1+ wild-type mice were reconstituted with Cx43WT, Cx43±, or Cx43−/− hematopoietic fetal liver cells that were derived from CD45.2+ mice. The presence of CD45.2 allowed us to identify and track the donor cells following reconstitution. We determined that Cx43± and Cx43−/− hematopoietic cells were able to reconstitute irradiated mice as well as Cx43WT cells. Reconstitution was nearly 100% in the thymus and over 90% in the spleen. There appeared to be no difference in thymocyte development or in the ability of lymphocytes to transmigrate to peripheral lymphoid organs. However in response to inflammation, Cx43± radiation chimeras had increased peritoneal infiltration compared to Cx43WT and Cx43−/− groups. IgG responses were normal in all groups but the Cx43−/− reconstituted mice had an elevated IgM response. Our data suggests that Cx43 may not be involved in the normal development of the immune system but may regulate certain effector functions in vivo.

Angiotensin-converting enzyme (CD143) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues

AbstractPrevious studies revealed that mAb BB9 reacts with a subset of CD34+ human BM cells with hematopoietic stem cell (HSC) characteristics. Here we map BB9 expression throughout hematopoietic development and show that the earliest definitive HSCs that arise at the ventral wall of the aorta and surrounding endothelial cells are BB9+. Thereafter, BB9 is expressed by primitive hematopoietic cells in fetal liver and in umbilical cord blood (UCB). BB9+CD34+ UCB cells transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+BB9− counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population. Protein microsequencing of the 160-kDa band corresponding to the BB9 protein established its identity as that of somatic angiotensin-converting enzyme (ACE). Although the role of ACE on human HSCs remains to be determined, these studies designate ACE as a hitherto unrecognized marker of human HSCs throughout hematopoietic ontogeny and adulthood.

Kinetic of Iron Absorption and Expression of Iron Related Genes in Beta-Thalassemia.

We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (> 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.

Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene

Introduction: Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford ‘prope’ tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. Objectives: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain. Materials and methods: To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice. Results: Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9±1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6±1.2 days). Conclusions: IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant ( P<0.001) prolongation of the survival of donor skin allografts was observed.

Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver

During development, there are many factors to be considered in studying the efficacy of the hematopoietic system to provide the immune system with adequate numbers of functional cells within the immune repertoire. Hematopoietic cells must be able to develop, proliferate, and emigrate from the hematopoietic fetal liver to other tissues of the body, such as the thymus and bone marrow. There is evidence that ethanol consumption causes immune deficiencies in adults and that maternal ethanol consumption causes immune deficiencies in children, both with correlative effects on cells and cytokinetic regulators. Therefore, the ability of the hematopoietic system to seed the immune system may be jeopardized by maternal ethanol consumption. In this study, test groups included female rats (1) fed a Lieber–DeCarli ethanol liquid diet, (2) pair-fed a control liquid diet, or (3) fed standard laboratory chow. Livers were removed from fetuses 18 and 21 days postconception (dpc) and analyzed by immunophenotyping and flow cytometry. There was a 22% and 38% decrease in fetal body weight and a 25% and 30% decrease in fetal liver weights between ethanol-fed and pair-fed groups at 18 and 21 dpc, respectively. At 18 dpc, fetal body and liver weights of the pair-fed animals were also significantly reduced to those of the chow-fed group. However, by 21 dpc, both body and liver weights of the two control groups were not statistically different. The effects of maternal ethanol consumption on the distribution of hematopoietic cells were characterized by using monoclonal antibodies (mAbs) anti-CD43 (a progenitor hematopoietic cell marker) versus labeling with anti-Vβ8.2 (pre-T cells), anti-B220 (pre-B cells), and anti-NKR-P1A (pre-natural killer cells). With the use of flow cytometric analysis, at 18 dpc ethanol-exposed fetuses showed 40% and 62% decreases in B220 + and Vβ8.2 + cells, respectively, versus findings for pair-fed controls, with no significant change in NKR-P1A + cells. At 21 dpc, ethanol-exposed fetuses showed a 46% decrease in B220 + cells among the CD43 + cells, with a 50% increase in Vβ8.2 + cells. Observed alterations in gross fetal body and liver weights, together with modifications in the percentage of hematopoietic cells in the fetal liver after maternal ethanol consumption, strongly support the suggestion that the ability of the hematopoietic system to impart a repertoire of cells that are functional in the regulatory and effector mechanisms of the immune process may be compromised.

Role of Oncostatin M in hematopoiesis and liver development

Definitive hematopoietic stem cells (HSCs) first appear in the aorta/gonad/mesonephros (AGM) region and migrate to the fetal liver where they massively produce hematopoietic cells before establishing hematopoiesis in the bone marrow at a perinatal stage. In the AGM region, Oncostatin M (OSM) enhances the development of both hematopoietic and endothelial cells by possibly stimulating their common precursors, so-called hemangioblasts. During development of HSCs in the AGM region, the liver primodium is formed at the foregut and accepts HSCs. While fetal hepatic cells function as hematopoietic microenvironment for expansion of hematopoietic cells during mid to late gestation, they do not possess most of the metabolic functions of adult liver. Along with the expansion of hematopoietic cells in fetal liver, OSM is produced by hematopoietic cells and induces differentiation of fetal hepatic cells, conferring various metabolic activities of adult liver. Matured hepatic cells then lose the ability to support hematopoiesis. Thus, OSM appears to coordinate the development of liver and hematopoiesis in the fetus.

Up‐regulation of amphotrophic retroviral receptor expression in human peripheral blood CD34+ cells

Retroviral-mediated gene transfer into hematopoietic stem cells provides the only means of stable transduction of these cells and their progeny for use with a variety of potentially therapeutic genes. Expression of the Moloney amphotropic retroviral receptor—pit-2 or GLVR-2—is critical to the recognition and entry of Moloney leukemia virus-derived viruses into human target cells such as CD34+ hematopoietic cells. GLVR-2 functions as a sodium-dependent phosphate transporter as well as a receptor. We have previously shown that the expression of the murine homologue of the amphotropic receptor Ram 1, also a phosphate transporter, is developmentally regulated in murine hematopoietic fetal liver cells. We also demonstrated that culture of murine fetal liver cells in phosphate-free (PO4-free) medium increases levels of receptor mRNA and makes murine fetal liver cells susceptible to Moloney amphotropic viral gene transfer. We now examine the effect of culture conditions on the expression of GLVR-2 in human CD34+ cells. In this report, we demonstrate that there is a 2–3 fold increase in GLVR-2 mRNA levels in CD34+ cells after 3 days in culture with interleukin 3, interleukin 6, and stem-cell factor. In addition, the use of PO4-free medium increases expression of GLVR-2 an additional 2-fold in these cells during this time. These results indicate that GLVR-2 expression can be up-regulated on these cells, and may permit improved retroviral gene transfer efficiencies. Am. J. Hematol. 61:243–253, 1999. © 1999 Wiley-Liss, Inc.

Up-regulation of amphotrophic retroviral receptor expression in human peripheral blood CD34+ cells.

Retroviral-mediated gene transfer into hematopoietic stem cells provides the only means of stable transduction of these cells and their progeny for use with a variety of potentially therapeutic genes. Expression of the Moloney amphotropic retroviral receptor-pit-2 or GLVR-2-is critical to the recognition and entry of Moloney leukemia virus-derived viruses into human target cells such as CD34+ hematopoietic cells. GLVR-2 functions as a sodium-dependent phosphate transporter as well as a receptor. We have previously shown that the expression of the murine homologue of the amphotropic receptor Ram 1, also a phosphate transporter, is developmentally regulated in murine hematopoietic fetal liver cells. We also demonstrated that culture of murine fetal liver cells in phosphate-free (PO(4)-free) medium increases levels of receptor mRNA and makes murine fetal liver cells susceptible to Moloney amphotropic viral gene transfer. We now examine the effect of culture conditions on the expression of GLVR-2 in human CD34+ cells. In this report, we demonstrate that there is a 2-3 fold increase in GLVR-2 mRNA levels in CD34+ cells after 3 days in culture with interleukin 3, interleukin 6, and stem-cell factor. In addition, the use of PO(4)-free medium increases expression of GLVR-2 an additional 2-fold in these cells during this time. These results indicate that GLVR-2 expression can be up-regulated on these cells, and may permit improved retroviral gene transfer efficiencies.

Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells.

Expression of the transmembrane receptor protein Ram-1 may be critical to optimizing retroviral gene transfer. Ram-1 acts as both a sodium-dependent phosphate transporter and a receptor for amphotropic retroviruses. We previously reported detectable Ram-1 in murine hematopoietic fetal liver cells (FLC) despite resistance of these cells to amphotropic retroviral transduction (infection). We document here that Ram-1 expression is completely absent in murine yolk sac cells from days 9.5 through 13.5 of ontogeny and first appears at low levels in midgestational FLC between days 13.5 and 14.5. In addition, Ram-1 expression is detected only in more differentiated populations within FLC, day 14.5, and not in those highly enriched for stem cells, indicating developmental regulation of Ram-1 during murine hematopoiesis. Others have reported the in vitro use of phosphate-free medium as a stimulus to increase levels of Ram-1 mRNA in nonhematopoietic cells. We now demonstrate that Ram-1 poly(A)+ mRNA increases significantly following culture of FLC in phosphate-free medium. Further, transduction of FLC in phosphate-free medium with an amphotropic retrovirus containing the multiple drug resistance gene leads to gene transfer not observed previously. These data demonstrate that (i) the normal resistance of FLC to amphotropic transduction is most likely due to an insufficient number of Ram-1 molecules for efficient retroviral recognition and binding, and (ii) Ram-1 can be upregulated by increasing the need for phosphate transport across the cell membrane.

A Critical Role of VLA-4 in Erythropoiesis In Vivo

Hematopoiesis requires specific interactions with the microenvironments, and VLA-4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo.

Putative prethymic T cell precursors within the early human embryonic liver: a molecular and functional analysis.

Hematopoietic cells present in the liver in early human fetal life were characterized by phenotypic analysis using a broad panel of monoclonal antibodies. Expression of very late antigen 4 and leukocyte function-associated antigen 3 cell adhesion receptors and 4F2 cell activation molecules was found in all fetal liver hematopoietic cells before acquisition of T cell-, B cell-, or myeloid-specific surface markers, and before the time of intrathymic colonization. Molecular studies showed that expression of the interleukin 2 receptor beta (IL-2R beta) also occurred in the embryonic liver at this early ontogenic stage. In contrast, no expression of IL-2R alpha or IL-2 transcripts was found in fetal liver cells, whereas transcription of the IL-4 gene was detected in a small fetal liver cell subset. Putative T cell precursors were identified among the hematopoietic fetal liver cells by the expression of genes encoding the gamma, delta, epsilon, and zeta invariant chains of the CD3-T cell receptor (TCR) complex. However, no transcription of the polymorphic alpha and beta TCR genes was detected. Functional in vitro assays further demonstrated that fetal liver hematopoietic cells from those early embryos were capable of proliferating in response to T cell growth factors, including IL-4 and IL-2. However, whereas IL-4-induced proliferation paralleled the appearance in vitro of CD45+CD7-CD4dull cells expressing the CD14 myeloid antigen, as well as of CD34+ primitive hematopoietic progenitors, differentiation into CD45+CD7+CD8+CD3- immature T cells was observed when using IL-2. Moreover, coculture with thymic epithelial cell monolayers provided additional evidence that early fetal liver hematopoietic cells may include very primitive T cell precursors, which were able to differentiate in vitro into TCR alpha/beta+ mature T cells. Therefore, our results indicate that, after triggering of the T cell-specific maturation program in primitive fetal liver hematopoietic progenitors, specific signals provided intrathymically by epithelial cells may fulfill the requirements to drive terminal differentiation of prethymically committed T cell precursors.