Background/Aims: The hematopoietic and hepatic systems are intertwined in the liver during fetal life. Cells expressing the hematopoietic stem cell marker CD34 and cytokeratin 7/8 (CK7/8) are hypothesized to be common stem cells for the hematopoietic and hepatic systems. Our aim was to determine if human fetal liver cells expressing CD34 and CK7/8 represent a common stem cell for both the hernatopoietic and hepatic systems. Methods: CD34(+)CK7/8(+) cells from midgestation livers were analyzed for the expression of various markers by flow cytometry and isolated based on their expression of CD34, nerve growth factor receptor (NGFR) and lack of CD45 expression. CD34(+)CD38(-) hematopoietic stem cells were also isolated and cultured in the presence of various hepatopoietins. Results: CD34(+)CK7/8(+) cells comprised 3.4-8.5% of the erythrocyte-depleted liver. CD34(+)CK7/8(+) cells had unique light-scatter properties compared to hematopoietic precursors and did not express most markers associated with hernatopoietic cells. They did stain with CD13, CD59, NGFR, desmin and alpha-smooth muscle actin. In culture, these cells had a stellate appearance. Cultured hernatopoietic stem cells failed to generate hepatocytes. Conclusions: CD34(+)CK7/8(+) cells are not common stem cells but rather appear to be hepatic stellate cells. A link between the hematopoietic and hepatic systems during fetal life requires further investigation. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Megakaryocyte growth and development factor (MGDF), or thrombopoietin, has received considerable attention as a therapeutic agent for treating thrombocytopenia or for its use in the ex vivo culture of hematopoietic stein cells. MGDF is known to support the growth of a broad spectrum of hematopoietic precursors obtained from adult or neonatal tissues, but its effects on the growth of fetal progenitors and stem cells has not been studied. Human CD38(+)CD34(2+) progenitors and CD38(-)CD34(2+) cells, a population that contains stem cells, were isolated from midgestation liver and grown under defined conditions with MGDF and various cytokines known to support the growth of primitive hematopoietic precursors. In clonal assays of colony-forming cells (CFCs), MGDF supported the growth of 15-25% of candidate stem cells when combined with granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), flk-2/flt3 ligand, or stem cell factor. MGDF was observed to strongly support the early stages of hematopoiesis and expansion of high proliferative potential CFCs. More mature progenitors were expanded nearly 78-fold in 1 wk of culture with MGDF+SCF+GM-CSF. MGDF alone was also found to support the short-term (2 d) survival of CD38(-)CD34(2+) high proliferative potential CFCs. The effects of MGDF were more modest on CD38(+)CD34(2+) progenitors with only additive increases in colony formation being observed. These findings suggest that MGDF administration in fetuses and neonates may strongly affect the growth and mobilization of primitive hematopoietic progenitors and that MGDF may find use in the ex vivo growth and expansion of fetal stem cells.

Alternatives to the use of radioisotopes to measure cell proliferation in mixed lymphocyte reactions (MLR) are desirable to avoid the hazards and costs associated with radioisotope use. The versatile fluorochrome 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to measure MLR and provides the opportunity to measure several different growth parameters. This study was aimed at determining which growth parameter is most practical and suitable for measuring murine MLR. The parameters measured were: the relative number of daughter T-cells, the relative number and frequency of reactive T-cell precursors and the relative number of mitotic events. Responder cells were CFSE-labeled unfractionated splenocytes from C57BL/6 mice. Stimulator cells included irradiated splenocytes from C57BL/6 (control), B6D2F(1) (haplo-allogeneic) or FVB/N (allogeneic) mice. Cultures were harvested daily for 1 week. Stimulator T-cells rapidly declined to less than 0.2-0.3% of the mixed population by day 2 of culture. Experimental groups had a significantly higher number of daughter T-cells and mitotic events after 2 days of culture with the number of daughter T-cells climbing exponentially after 5 days of culture. The number and frequency of reactive T-cell precursors were significantly higher in experimental groups on days 2-3, but this difference became insignificant by day 4. Among all the parameters, the relative number of daughter T-cells was the most practical for measuring MLR, after 5 days of culture, based upon the growth kinetics of responder T-cells and the survival of the stimulator cells. (C) 2003 Elsevier B.V. All rights reserved.

The ontogeny of the human immune system was studied by analyzing fetal and adult tissues for the presence of various lymphocyte populations and activation/maturation markers. CD95 (fas) was expressed in hematopoietic tissues during the final stages of development of monocytes, granulocytes, NK cells and T cells, but to a much lesser extent on B cells. In the periphery, CD95 expression declined on granulocytes and NK cells. CD95 was expressed at a higher level on CD45RA(+) peripheral T-cells in the fetus than in the adult. Contrary to the belief that most fetal T-cells are naive or resting, a notable number of CD45RO(+) T-cells were observed as well as an unique CD95(-)CD45RO(+) population. Activation markers CD25, CD122, CD69 and CD80 were also present on fetal T-cells. These findings indicate that in the initial weeks following thymic maturation, a high frequency of T-cells is activated in the periphery of the fetus. (C) 2003 Elsevier Science Ltd. All rights reserved.

Immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/ SzJ (NSG) mice are a valuable resource to study human hematopoietic stem cells. Prolonged multilineage hematopoiesis indi- cates stem cell engraftment and generally is measured by flow cytometry. In this study, we took advantage of the multi-parameter detec- tion afforded by modern flow cytometers to optimize detection of human hematopoiesis in NSG mice. Antigens widely expressed by mouse or human cells were evaluated as mark- ers to distinguish mixtures of these cells to optimize and test the limits of chimerism detection. The bone marrow, spleen, and liver of NSG mice transplanted with human hematopoietic cells were analyzed for evidence of engraftment.

Mouse bone marrow cells were best marked for exclusion by staining with a combination of CD45, TER-119, and anti-H-2Kd monoclonal antibodies, whereas live human cells were most accurately identified by elimination of cell doublets and positive staining for CD59. Human stem cells (CD34++CD133+CD38low) and progenitors were detected in the bone marrow and liver, but not in the spleen. An unusual pattern of myeloid antigen expression was detected in the bone marrow and CD3+CD4+CD8+ T-cells were detected in the spleen. We concluded that multicolor flow cyto- metric analysis that clearly distinguishes mouse and human cells offers accurate detec- tion of human chimerism in NSG mice. Human hematopoiesis can be detected in the bone marrow and liver of NSG mice with T- lymphopoiesis, possibly occurring in the spleen.

The effects of donor T cells, or their CD8(+) subset, on engraftment and tolerance induction in fetal transplantation were evaluated using an F-1-into-parent mouse-model that does not permit a graft-versus-host effect. Gestational day 13 C57BL/6 (H-2K(b)) fetuses were transplanted with B6D2F(1) (H-2K(b/d)) light density bone marrow cells (LDBMC) containing 1-2% T cells, T-cell depleted bone marrow cells (TDBMC, <0.1% T cells), or TDBMC with enriched CD8(+) T cells (CD8). Chimaerism levels in the peripheral blood, spleen and bone marrow were usually below 0.2% in all groups, indicating that T cells do not improve engraftment without a graft-versus-host effect. A significant, but transient, wave of donor cells was seen in the peripheral blood at 1 month of age in the CD8 and LDBMC groups. Relatively high levels of chimaerism (<17%) were sometimes detected in the peritoneal cavities of recipients. T-cell tolerance specific to donor cells was evaluated in mixed lymphocyte cultures. The CD8 and LDBMC groups had significantly lower T-cell responses than untransplanted controls. These findings indicate that in utero transplantation of haploidentical donor CD8(+) or CD3(+) cells can help to lessen the immune response of host T cells towards donor cells. The persistence of donor cells in the peritoneal cavity also correlated with tolerance induction.

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Results

Conclusions