Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk+ cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk+ cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk+ cells contained both albumin+ and cytokeratin 19+ cells, indicating that a majority of colony-forming Dlk+ cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition,numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk+ cells under this condition. Furthermore, 7% of the colony-forming Dlk+cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk+ cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk+cells differentiated into hepatocytes in vivo. These results indicate that Dlk+ cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.

The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a Gata2 promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following Gata2-directed GFP fluorescence, and that they can also be monitored in vivo. Each individual GFP-positive cell lay in a G 0͞G1 cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered.GATA-2 ͉ GFP knock-in mouse ͉ imaging

Mouse long-term hematopoietic reconstituting cells exist in the c-Kit ؉ Sca-1 ؉ Lin ؊ (KSL) cell population; among them, CD34 low/؊ cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34 low/؊ c-Kit ؉ Sca-1 ؉ Lin ؊ (34 ؊ KSL) cells with the HES-1 gene, which encodes a basic helix-loophelix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34 ؊ KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34 low/؊ profile in the bone marrow KSL population in each recipient mouse at ratios 3.5-and 7.8-fold those produced by nontransduced 34 ؊ KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34 ؊ KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34 ؊ KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells. IntroductionHematopoietic stem cells (HSCs) are generated during ontogeny and supply all mature hematopoietic lineages throughout life with their self-renewal and multilineage differentiation capacity. 1 Efforts have been made to expand HSCs ex vivo without loss of their original potency. Long-term reconstitution capacity of mouse and human HSCs is maintained for up to 2 to 3 weeks by coculture with certain stromal cells. [2][3][4] For expansion of HSCs without stromal cells, various combinations of cytokines that are active for immature hematopoietic progenitors have been surveyed. [5][6][7][8][9] Of interest are approaches using Notch signaling, since it has been shown to inhibit differentiation of diverse types of cells in vertebrates. [10][11][12][13][14] Notch signals are mediated by interactions between Notch receptors and their membrane-anchored ligands expressed in adjacent cells. 15 In the hematopoietic compartment, Notch receptors and ligands are expressed in hematopoietic progenitors and certain stromal cells, respectively. [16][17][18][19] It was recently reported that the Notch ligand Jagged-1 maintained the severe combined immunodeficiency (scid)-repopulating activity of human cord blood-derived CD34 ϩ CD38 Ϫ cells in vitro significantly longer than the control. 20 Further evidence implying the potential usefulness of Notch signaling in HSC expansion comes from the establishment of a line of cytokinedependent cells which differentiate into myeloid and lymphoid lineages in vivo when transplanted into syngeneic mice, by retroviral transduction of stem cell-enriched bone marrow cells with an activated form of Notch-1. 21 In these previous investigations, however, it was not certain whether HSC expansion was...