Regulation of hematopoietic cell clusters in the placental niche through SCF/Kit signaling in embryonic mouse.

Hematopoietic stem cells (HSCs) emerge from and expand in the mouse placenta at mid-gestation. To determine their compartment of origin and define extrinsic signals governing their commitment to this lineage, we identified hematopoietic cell (HC) clusters in mouse placenta, defined as cells expressing the embryonic HSC markers CD31, CD34 and Kit, by immunohistochemistry. HC clusters were first observed in the placenta at 9.5 days post coitum (dpc). To determine their origin, we tagged the allantoic region with CM-DiI at 8.25 dpc, prior to placenta formation, and cultured embryos in a whole embryo culture (WEC) system. CM-DiI-positive HC clusters were observed 42 hours later. To determine how clusters are extrinsically regulated, we isolated niche cells using laser capture micro-dissection and assayed them for expression of genes encoding hematopoietic cytokines. Among a panel of candidates assayed, only stem cell factor (SCF) was expressed in niche cells. To define niche cells, endothelial and mesenchymal cells were sorted by flow cytometry from dissociated placenta and hematopoietic cytokine gene expression was investigated. The endothelial cell compartment predominantly expressed SCF mRNA and protein. To determine whether SCF/Kit signaling regulates placental HC cluster proliferation, we injected anti-Kit neutralizing antibody into 10.25 dpc embryos and assayed cultured embryos for expression of hematopoietic transcription factors. Runx1, Myb and Gata2 were downregulated in the placental HC cluster fraction relative to controls. These observations demonstrate that placental HC clusters originate from the allantois and are regulated by endothelial niche cells through SCF/Kit signaling.

Cold exposure down-regulates zebrafish pigmentation.

Vertebrates use adaptive mechanisms when exposed to physiologic stresses. However, the mechanisms of pigmentation regulation in response to physiologic stresses largely remain unclear. To address this issue, we developed a novel pigmentation model in adult zebrafish using coldwater exposure (cold zebrafish). When zebrafish were maintained at 17 °C, the pigmentation of their pigment stripes was reduced compared with zebrafish at 26.5 °C (normal zebrafish). In cold zebrafish, gene expression levels of tyrosinase and dopachrome tautomerase, which encode enzymes involved in melanogenesis, were down-regulated, suggesting that either down-regulation of melanin synthesis occurred or the number of melanophores decreased. Both regular and electron microscopic observation of zebrafish skin showed that the number of melanophores decreased, whereas aggregation of melanosomes was not changed in cold zebrafish compared with normal zebrafish. Taken together, we here show that cold exposure down-regulated adult zebrafish pigmentation through decreasing the number of melanophores and propose that the cold zebrafish model is a powerful tool for pigmentation research.

Ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro.

Hmgn2 (high mobility group nucleosomal 2), a ubiquitous nucleosome-binding protein that unfolds chromatin fibres and enhances DNA replication, reportedly regulates differentiation of epithelial and mesenchymal cells. To investigate how Hmgn2 regulates HC (haemopoietic cell) differentiation, we quantified Hmgn2 expression in HCs of mouse FL (fetal liver) during erythroid differentiation. Hmgn2 expression levels were >10-fold higher in immature erythroid progenitors than in mature erythroid cells, suggesting that Hmgn2 antagonizes erythroid differentiation. To address this issue, Hmgn2 were transfected into both Friend erythroleukaemia cells and FL HCs. There was a 3.3-fold decrease in relatively mature c-Kit(+)/CD71(+) erythroid cells, a 2.9-fold increase in immature c-Kit(+)/CD71(-) erythroid cells in transfected Friend cells, a 1.1-fold decrease in relatively mature CD71(+)/Ter119(+) erythroid cells, and a 1.7-fold increase in relatively immature c-Kit(+)/CD71(+) erythroid cells in FL HCs accompanied by down-regulation of genes encoding the erythroid transcription factors, Gata1 and Klf1. Two days after Hmgn2 transfection of Friend erythroleukaemia cells, the number of S-phase cells increased, whereas the number of cells in G(1) decreased, while that of mitotic cells remained unchanged. We conclude that ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro, which may be due to enhancement of DNA replication and/or blocking entry of mitosis at S-phase.

Hepatoblasts comprise a niche for fetal liver erythropoiesis through cytokine production.

In mammals, definitive erythropoiesis first occurs in fetal liver (FL), although little is known about how the process is regulated. FL consists of hepatoblasts, sinusoid endothelial cells and hematopoietic cells. To determine niche cells for fetal liver erythropoiesis, we isolated each FL component by flow cytometry. mRNA analysis suggested that Dlk-1-expressing hepatoblasts primarily expressed EPO and SCF, genes encoding erythropoietic cytokines. EPO protein was detected predominantly in hepatoblasts, as assessed by ELISA and immunohistochemistry, and was not detected in sinusoid endothelial cells and hematopoietic cells. To characterize hepatoblast function in FL, we analyzed Map2k4(-/-) mouse embryos, which lack hepatoblasts, and observed down-regulation of EPO and SCF expression in FL relative to wild-type mice. Our observations demonstrate that hepatoblasts comprise a niche for erythropoiesis through cytokine secretion.

Embryonic regulation of the mouse hematopoietic niche.

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called "niche cells" surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis.

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts.

Fetal liver is the major embryonic hematopoietic organ and is extrinsically colonized by circulating hematopoietic stem cells (HSCs). Integrin beta-1 expression on HSCs is crucial for colonization, suggesting that interaction of Integrin beta-1 with extra-cellular matrix (ECM) factors promotes HSC adherence to fetal liver. However, little is known about how ECM production is regulated in fetal liver. Here we used flow cytometry to sort fetal liver compartments and detected ECM gene and protein expression predominantly in sorted hepatoblasts. mRNA and protein analysis suggested that TGF-beta-1 expressed by hepatoblasts, sinusoid endothelial cells and hematopoietic cells, binds to the TGF-beta receptor type-2 expressed on hepatoblasts to stimulate ECM production. Intra-cardiac injection of TGF-inhibitors into mouse embryos dramatically decreased fetal liver ECM gene expression. Taken together, our observations suggest that hepatoblasts predominantly produce ECM factors under control of TGF-beta-1 in fetal liver.

Intra-aortic clusters undergo endothelial to hematopoietic phenotypic transition during early embryogenesis.

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc.

Variation in hematopoietic potential of induced pluripotent stem cell lines.

Induced pluripotent stem (iPS) cells were originally generated from somatic cells by ectopic expression of four transcription factor genes: Oct3/4, Sox2, Klf4 and c-Myc. Currently, iPS cell lines differ in tissue origin, the combination of factors used to construct them, the method of gene delivery and expression of pluripotency markers. Thus to evaluate iPS cells for haematotherapy, the hematopoietic potential among iPS lines should be compared. Here, we compare differentiation capacity of six iPS lines into mesodermal cells and hematopoietic cells (HCs) through embryoid body (EB) formation. We show that the mouse embryonic fibroblast (MEF)-derived iPS lines 20D17 and 178B5 resemble CCE ES cells in terms of morphology in culture, number and size of EBs and differentiation capacity into mesodermal cells compared to iPS cells derived from adults, although all iPS lines could form EBs. The number of mesodermal cells differentiated from MEF-derived iPS cell lines showed a 3.9-407-fold increase compared to that from iPS lines derived from adults. Furthermore, 178B5 iPS cells generated Ter119(+) erythroid cells (3.35%) efficiently in culture. We conclude that hematopoietic potential differs among the six lines and that MEF-derived 20D17 and 178B5 iPS cells generate HCs more efficiently than adult-derived iPS cells.

Application of whole mouse embryo culture system on stem cell research.

The normal development of mouse embryo in vivo could be maintained in vitro up to 72 h in the presence of rat serum which is continuously supplied with the appropriate concentration of O(2) and CO(2). There are several applications of the whole mouse embryo culture model for study of cellular dynamics in hematopoiesis and its interaction with vasculogenesis. In this protocol, we have described details of manipulation techniques in combination with the whole embryo culture and also some advance techniques applied to the mouse embryo such as intra-cardiac inoculation of acetylated low density lipoprotein for cell-specific labeling and engraftment of donor yolk-sac from different genotype/phenotype mouse embryo onto the yolk-sac of host mouse for study of the dynamic distribution of hematopoietic cell.