Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo

We investigated roles of alpha4 integrins during hematopoiesis using mutant and chimeric mice. Yolk sac erythropoiesis and migration of hematopoietic progenitors to fetal liver, spleen, and bone marrow can occur without alpha4 integrins. Although terminal differentiation of these progenitors is possible without alpha4 integrins, these receptors are essential to maintain normal hematopoiesis in fetal liver, spleen, and bone marrow microenvironments. Moreover, alpha4-deficient erythroid progenitors and pre-B cells neither transmigrate beneath the stroma nor expand-properly in vitro. In contrast, alpha4-null cells migrate and differentiate efficiently into T lymphocytes within the thymus. In summary, alpha4 integrins are essential for normal development of all hematopoietic lineages in fetal liver, bone marrow, and spleen, likely by regulating the proliferation/differentiation balance of hematopoietic progenitors.     

Early fetal liver readily repopulates B lymphopoiesis in adult bone marrow

Fetal liver (FL) becomes a major organ of hematopoiesis at mouse embryonic day (E) 11 and E12, when definitive hematopoietic stem cells, originating from the aorta-gonads-mesonephros region, colonize the hepatic tissue. Unipotent B-cell progenitors are very rare in FL by day 12, whereas erythropoiesis prevails. We have studied hematopoiesis in FL from different gestational ages, with special emphasis on B lymphopoiesis. The mRNA levels of selected liver-specific genes, hematopoietic lineage-specific genes, and genes for selected cytokines/hormones as well as for their receptors were evaluated by real-time polymerase chain reaction in FL from E12.5, E14.5, and E17.5, adult liver and adult bone marrow (BM). The level of B lineage-related gene expression in FL was very low at E12.5. There was also a significantly lower fraction of B220+ and CD19+ B cells in E12.5 FL compared with E17.5 FL. To analyze whether these differences reflect different stem cell potentials occurring during FL development, 10(6) or 5 x 10(6) of FL cells collected from embryos at E12.5 or E17.5 and those from adult BM were transplanted into sublethally irradiated (3- or 6-Gy) congenic mice. Short-term and long-term repopulation of B and T cells and granulocyte/macrophage lineages from donor FL or adult BM cells were evaluated in competition to adult hematopoiesis of sublethally irradiated recipients. In short-term repopulation, the transplantation of E12.5 FL cells resulted in a lower blood chimerism compared with that of E17.5 FL cells. However, the proportion of B lymphopoiesis exerted by E12.5 FL cells was not different from that of E17.5 FL or adult BM. This study demonstrates that E12.5 FL contains hematopoietic stem cells with fully developed B-cell repopulating capacity and that the developmental period of fetal hematopoiesis between E12.5 and E17.5 is not an obligatory phase for the adult B lymphopoiesis.     

Protein phosphatase 2A catalytic subunit α (PP2Acα) maintains survival of committed erythroid cells in fetal liver erythropoiesis through the STAT5 pathway

Suppression of programmed cell death is critical for the final maturation of red blood cells and depends largely on the anti-apoptotic effects of EpoR-STAT5-Bcl-x(L) signaling. As the major eukaryotic serine/threonine phosphatase, protein phosphatase 2A (PP2A) regulates multiple cellular processes, including apoptosis. However, whether PP2A plays a role in preventing erythroid cells from undergoing apoptosis remains to be elucidated. We conditionally inactivated the catalytic subunit α of PP2A (PP2Acα), which is the predominant form of PP2Ac, during early embryonic hematopoiesis. Loss of PP2Acα in hematopoietic cells perturbed definitive erythropoiesis characterized by fetal liver atrophy, reduced Ter119(+) cell number, abnormal expression patterns of molecular markers, less colony formation, and a reduction in definitive globin expression. Levels of erythropoiesis-promoting cytokines and initial seeding with hematopoietic progenitors remained unchanged in PP2Acα(TKO) fetal livers. We noted impaired expansion of the fetal erythroid compartment, which was associated with increased apoptosis of committed erythroid cells. Mechanistically, PP2Acα depletion markedly reduced Tyr(694) phosphorylation of STAT5 and expression of Bcl-x(L). Unexpectedly, PP2Acα-deficient embryos did not manifest any early embryonic vascular defects. Collectively, these data provide direct loss-of-function evidence demonstrating the importance of PP2Acα for the survival of committed erythroid cells during fetal liver erythropoiesis.     

APOA-1 is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.     

Analysis of the human fetal liver hematopoietic microenvironment

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.     

Microenvironmental influences on human B-cell development

Summary: Mammalian B‐cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement – culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B‐cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints.     

小鼠胚胎早期血细胞发生的研究进展

根据时空上的不同,小鼠胚胎发育时期的血细胞发生目前被分为原始造血、红系/髓系祖细胞（EMPs）的产生和造血干细胞（HSCs）成熟并分化为各种血细胞3个阶段。最新的观点也把原始造血和EMPs的产生归结为非HSCs依赖性的血细胞谱系分化阶段,将第3阶段称为HSCs依赖性的血细胞谱系分化阶段。尽管胚胎时期的血细胞发生涉及多个造血器官,但本文中我们主要对近年在细胞和分子水平进行的造血细胞和HSCs在卵黄囊和腹主动脉-性腺-中肾（AGM）区发育的研究进行归纳总结,以此展示新近发现的胚胎发育早期血细胞发生的特点及其潜在机制。     

DL4‐mediated Notch signaling is required for the development of fetal αβ and γδ T cells

T‐cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta‐like 4 (DL4) on TECs by Notch1 expressed by blood‐borne BM‐derived precursors is essential for T‐cell commitment in the adult thymus. In contrast to the adult, the earliest T‐cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T‐cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T‐cell lymphopoiesis, we show here that DL4‐mediated Notch signaling is essential for the development of both αβ and γδ T‐cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T‐cell development and a dramatic reduction of all γδ T‐cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T‐cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4‐mediated Notch signaling in fetal and adult thymopoiesis.     

B-lymphopoiesis gains sensitivity to subsequent inhibition by estrogens during final phase of fetal development

Adult B-lymphopoiesis is suppressed by the inhibitory effects of elevated estrogens during pregnancy. At the same time, hematopoietic cells in the fetal liver are resistant to this suppression by estrogens and ensure active production of B-cells. We investigated whether this unresponsiveness to estrogens of fetal cells also applies to cells obtained from a newborn liver and projects into the adult hematopoiesis when fetal liver cells are transplanted to adult mice. Mixtures of fetal liver (E14.5), neonatal liver (P0.5) and adult bone marrow (BM) cells were co-transplanted into adult primary and secondary recipients treated with high doses of estrogen in the Ly5.1/Ly5.2 congenic mouse model. Total chimerism as a proportion of all nucleated blood cells, chimerism as a proportion of B220+ B-cells, and of other blood cell lineages as well, were determined by flow cytometry. B-lymphopoiesis derived from fetal liver (E14.5) stem cells remained resistant to estrogen after transplantation into both primary and secondary adult recipients, for up to 280 days. In contrast, B-lymphopoiesis derived from neonatal liver (P0.5) stem cells was resistant to estrogen only for approximately 50 days after the primary transplantation to the adult BM microenvironment. These results provide further evidence for a critical developmental period of B-lymphopoiesis during its fetal liver stage. In the mouse, critical developmental events that allow for the subsequent expressed sensitivity of B-lymphopoiesis for suppression by estrogens after sexual maturation appear to occur during the period of late-stage fetal liver hematopoiesis before its migration to the bone marrow.     

Loss of SDF-1 receptor expression during positive selection in the thymus

SDF-1 is a member of the CXC chemokines. In contrast to other chemokines that are induced by inflammation, SDF-1 is constitutively produced by stromal cells. In order to investigate the physiological roles of SDF-1, we constructed a fusion protein, SDF-1-Cgamma1, composed from murine SDF-1alpha and the constant region of human IgG. SDF-1-Cgamma1 stained EL-4 T lymphoma cells and the staining was blocked by rhSDF-1beta. The expression levels of SDF-1R altered along with the T cell maturation. Most c-kit+ hematopoietic precursors in fetal liver in gestational day (GD) 14.5 embryo were SDF-1R-, while c- kit+ double-negative (DN) thymocytes in the embryo were positive for SDF-1R. The receptor expression increased along with T cell maturation up to double-positive (DP) cell stage. Interestingly, SDF-1R expression was down-modulated after positive selection; CD69+CD3hi DP and CD3hi single-positive thymocytes were SDF-1R-/lo. Northern blot analysis demonstrated that SDF-1 and CXCR4 mRNAs were abundantly expressed in the thymuses of embryo and adult mice. These results demonstrate that SDF-1R expression is involved in T cell development in the thymus, particularly in positive selection.      

The role of Notch receptor expression in bile duct development and disease

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT-PCR and immunohistochemistry. RT-PCR showed Notch1-4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and alpha1-anti-trypsin deficiency (alpha1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and alpha1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases.     

Notch/Delta4 interaction in human embryonic liver CD34+ CD38- cells: positive influence on BFU-E production and LTC-IC potential maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.     

Hematopoiesis/erythropoiesis in myocardial infarcts.

Extramedullary hematopoiesis occurring in the myocardium has previously only been reported in a single case of a neonate with cyanotic congenital heart disease. Herein we report the incidental discovery of extramedullary hematopoiesis or pure erythropoiesis in four failing adult hearts with myocardial infarction. In two cases, extramedullary hematopoiesis or erythropoiesis was identified in cardiectomy specimens removed at orthotopic heart transplantation; in two other cases, erythropoiesis was found in left ventricular tissue removed at the time of implantation of left ventricular assist devices. Myocardial hematopoiesis/erythropoiesis was identified based on characteristic light-microscopic findings in routinely processed tissue and was confirmed by immunhistochemistry using monoclonal antibodies to the erythroid cell marker glycophorin A (positive in all cases), the megakaryocyte marker CD61, and the granulocyte marker neutrophil elastase (the latter two markers positive in one case only). None of the four patients had a myeloproliferative disorder or evidence of a myelophthisic process. No hematopoietic elements were identified in 109 cardiectomy specimens without acute or recent infarcts. Myocardial hematopoiesis or erythropoiesis could represent heretofore-unrecognized manifestations of altered cytokine expression in patients with heart failure due to myocardial infarction.