Human fetal liver as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation

The CFU-GM and T cell contents of human fetal livers were studied at various times between 6-14 weeks of gestation. The number of CFU-GM increased parallel to gestational age, especially after week 10. Cells bearing mature T cell markers, however, were found only in one case out of 35 fetal liver samples. Cryopreservation of fetal liver cells hardly affected the viability and proliferative capacity of CFU-GM in the sample. According to these findings fetal liver is, at least up to the 14th gestational week, practically free of mature T cells but it does contain a considerable amount of CFU-GM (an accepted indicator of pluripotent stem cell content), consequently fetal liver can be considered as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation for children.     

Expression of megakaryocytic and myeloid markers in blasts of transient abnormal myelopoiesis in a stillbirth with Down syndrome: report of histopathological findings of an autopsy case

Transient abnormal myelopoiesis in neonates with Down syndrome is an unusual leukemia that spontaneously regresses within several months of life and is thought to arise in the fetal liver. It is largely unknown how the leukemic blasts proliferate and differentiate in fetal tissues. We report the histopathological findings of an autopsy case of a stillbirth with transient abnormal myelopoiesis. Blood vessels in almost all organs were filled with immature leukemic cells, most of which expressed megakaryocyte antigen CD42b. In contrast, leukemic cells infiltrating the tissues, including the pericardium, expressed myeloperoxidase. These findings indicate that leukemic progenitors in transient abnormal myelopoiesis can differentiate along both megakaryocytic and myeloid lineages, which may be influenced by microenvironmental factors. Numerous dysplastic mature/immature megakaryocytes and blasts were present in the liver, whereas the bone marrow contained predominantly myeloid cells at various stages of differentiation, suggesting that the fetal liver is the major organ for proliferation of blasts in transient abnormal myelopoiesis.     

Human follicular dendritic cells (FDC): a study with monoclonal antibodies (MoAb).

A collection of new and established FDC-reactive MoAb has been used in an immunohistological study designed to throw light on (a) the nature of FDC (which are of unknown lineage) as judged by their sharing of antigens with other cell types and (b) the reason for the strong expression of some B cell antigens on FDC. The MoAb were tested on: (1) sections of tonsil, (2) sections of lymph nodes from four cases of non-Hodgkin's lymphoma, (3) peripheral blood cells and (4) cells of cultured haemopoietic cell lines. Only one of the ten new MoAb bound to FDC and no other component of the tissues screened. It resembled R4/23, a MoAb known to be specific for FDC. The other nine antibodies showed a range of cross-reactivity patterns involving one or more of the following: monocytes, macrophages, platelets, epithelium, endothelium and connective tissue fibres. Some of the MoAb reacted with B lymphocytes and cells of B lymphoblastoid lines but none showed the restricted FDC-staining pattern associated with MoAb which detect the CD23, P45 antigen. The findings are discussed in terms of the intrinsic or extrinsic nature of the antigens detected.     

Characterization of human lymphocytes separated from fetal liver and spleen at different stages of ontogeny

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and B-lymphocytes (up to 16% and 45%, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells less than 10% and B cells less than 15%). The majority of early CD3+ spleen cells (21st-28th week) expressed TCR alpha beta but not TCR gamma delta, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity. Later spleen T cells (29th-36th week) expressed the phenotype as mature adult-type T cells (CD3+TCR alpha beta +CD4/8+CD5+). During ontogeny in fetal spleen, a growing number of B cells could be estimated without any changes in the proportion of subsets, expressing the different light and heavy chains. However, the proportion of CD5+ B cells decreased with gestational age. The results suggest that the functional immaturity of antenatal splenocytes could not be caused by dramatic phenotypical differences in comparison with adult-type splenic lymphocytes.     

T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver

The developmental potential of individual cells in the Lin-c-kit+CD45+IL-7R+ (IL-7R+) population from murine fetal liver was investigated using a clonal assay capable of determining the potential of a progenitor to give rise to myeloid, T, and B cells. Unipotent progenitors generating T cells (p-T) or B cells (p-B) but not other types of progenitors were found in the IL-7R+ population. A large proportion of progenitors at day 12 of gestation are p-T, whereas the frequency of p-T dramatically decreases with gestational age. In marked contrast, p-B are very rare by day 12, but they rapidly increase thereafter. These findings strongly suggest that the commitment of multipotent progenitors to T and B cell lineages occurs independently.     

Ontogeny of human T and B lymphocytes during stressed and normal gestation: phenotypic analysis of umbilical cord lymphocytes from term and preterm infants

Cord blood lymphocytes from premature and stressed term infants were phenotyped and contrasted with T and B lymphocytes from healthy newborns at term. Cytofluorometric analysis shows that in the early third trimester, 80-85% of fetal T cells belong to the T4+ inducer population, and 10% to the T8+ suppressor/cytotoxic subset. As gestation progresses, the T4:T8 ratio shifts toward adult values and there is an increase in expression of the mature antigen, T12. The B-cell-differentiation antigens B1, B2, and B4 do not appear to change during gestation in healthy infants. Antenatal stress which threatens fetal survival, however, leads to circulating cells of both lineages which are phenotypically less mature than expected for gestational age. Most notably, in response to severe antenatal hypoxic stress, cells expressing the very early B-cell markers B2 and B4 increase and exceed the numbers of more mature B1+ cells in cord blood.     

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.     

A New Surface Antigen on Human Lymphocytes Defined by the Murine Monoclonal Antibody IVF7

The murine monoclonal antibody (MoAb) IVF7 was produced against tumour cells from a patient with a CD3+, CD4+, CD8- T-cell chronic lymphatic leukaemia (T-CLL). The MoAb IVF7 showed reactivity with subpopulations of normal peripheral blood lymphocytes (PBL), as well as with a few cell lines of haematopoietic origin. Thirty-six per cent of PBL were stained with IVF7. Analysing subpopulations, we found that 80% of NK cells, 25% of T cells, and 10-20% of B cells were positive. The myelomonocytic cell line KG-1 was also stained. The molecular weight of the molecule was 40 kDa under reducing conditions. The antigen was found to be trypsin-sensitive. MoAb IVF7 could modulate the antigen from the cell surface. The antibody did not stimulate PBL to DNA synthesis, nor did it significantly influence NK cell-mediated killing.     

Development of pre-B and B lymphocytes in the human fetus

Cell suspensions from human fetal liver, bone marrow and spleen were systematically studied at between the fetal ages of 8 and 20 weeks by the direct immunofluorescence technique for the presence of pre-B and B cells. Pre-B cells were characterized as lymphoid cells containing cytoplasmic mu heavy chains but lacking surface IgM. Based on their size and morphological appearance, these cells were subdivided into large and small pre-B cells. In the livers of 8 week old fetuses, more than 90% of the total pre-B plus B cell population consisted of pre-B cells; the relative number of liver pre-B cells gradually decreased with increasing gestational age and, after the 14th week, B cells outnumbered pre-B cells. At 20 weeks, the ratio of pre-B to B cells was only 0.25. In contrast, the number of pre-B cells in fetal bone marrow (12-20 weeks) was always greater than that of the B cells. Large and small pre-B cells were present in the liver and bone marrow. Small pre-B cells outnumbered the large ones in both organs and with increasing gestational age the ratio of small to large pre-B cells increased four-fold. In fetal spleen (12-20 weeks), no large pre-B cells were seen and the small ones comprised only a minor fraction of the total B-cell population. It can be concluded from these data that during early human fetal life the liver is an important site of pre-B cell production. From 12 weeks onwards, this function is gradually taken over by the bone marrow. During the second half of pregnancy, pre-B cell production in fetal liver becomes very much less as compared with the bone marrow. No generation of pre-B cells takes place in the fetal spleen, but a certain amount of maturation of cells of the B cell line may take place in this organ.     

Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3.

Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3- CD16- cells were sorted and cloned by limiting dilution. Two representative clones, FL121 and FL125, were expanded and characterized. They shared the phenotype of CD2+CD3-CD4-CD5-CD6+CD16-CD56+. FL121 did not express CD8 whereas FL125 expressed CD8 alpha but not CD8 beta. Both clones were found to express cytoplasmic CD3 delta and CD3 epsilon Ag while CD3- NK clones isolated from PBL were negative for them. These results indicate that FL121 and FL125 were committed to the T-cell lineage. Southern blot analysis showed that the TCR beta genes and the TCR gamma genes of these clones were in the germ-line configuration. The establishment of FL121 and FL125 may provide a novel insight into the earliest stage of T-cell development in man.     

Development of lymphocyte subpopulations identified by monoclonal antibodies in human fetuses

We have used a panel of monoclonal antibodies to examine the development of lymphoid and myeloid sub-populations of cells in thymus, bone marrow, and liver of 16 fetuses from 12 to 16 weeks of gestational age. Pre-B and IgM⁺ B cells were present at a ratio of approximately 2:1 in all of the fetal bone marrow and liver samples; cells of both phenotypes were HLA-DR⁺ but did not express the mature B-cell antigen, HB-2. Cells expressing the myelomonocytic antigen, MMA or Leu-M1, were more frequent in bone marrow (40%) than in fetal liver (10%), and cells expressing the HNK-1 or Leu-7 antigen were rare (<1%) in all of the fetal tissues examined. Each of the T-cell antigens, T1, 4, 5, 6, and 8, was expressed by a majority of thymocytes irrespective of the age of the fetal donor. In contrast, cells with the T1, 4, 5, and 8 antigens were not seen in bone marrow and liver before the 13th week of gestation, and T6⁺ cells were never seen in these hemopoietic tissues. These results suggest that fetal liver and bone marrow precursors in humans do not express these T-cell antigens prior to thymic entry and the onset of thymocyte differentiation.     

Immunocytochemical characterization of lymphocyte development in human embryonic and fetal livers

Lymphohemopoietic progenitor cells and the development of lymphocytes in human embryonic and fetal livers during the 4 to 11 weeks of gestation were examined immunocytochemically by using a panel of monoclonal antibodies. CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable from the 8th gestational week. CD2+ and CD3+ cells of T cell lineage were observed from the 10th gestational week. Tdt+ cells first appeared on the 43rd day of gestation. Both CD34+ and Ia+ cells were observed in all examined livers, and these cells appeared morphologically as small lymphoid cells from the 43rd day of gestation. These seemed to suggest that B lymphocytes developed in fetal liver from 8 weeks of gestation and lymphohemopoietic progenitor cells were comprised in Tdt+ cells in liver during the 43rd to 56th day of gestation.     

Differential expression of leukocyte common antigen in human fetal lymphoid organs.

To investigate the differential expression of various types of leukocyte common antigen (LCA) isoforms during development, we analyzed human fetal lymphoid organs, including the thymus, liver, spleen, and bone marrow from 14 weeks to 29 weeks of gestational age by immunohistochemical and flow cytometric methods. In fetal thymus, over 90% of thymocytes throughout the entire fetal life expressed CD45RO and CD45RB, while CD45RA was expressed only in less than 5% of thymocytes. This expression pattern of LCA isoforms was established by a gestational age of 14 weeks or earlier, and persisted throughout the fetal period. The tissue distribution was different from each isoform; CD45RO-positive thymocytes were found in both the cortex and medulla at the 14th week with low intensity, but was localized in the cortex with increasing fetal age. CD45RB-positive thymocytes distributed mainly in the medulla from early gestational age. Among extrathymic lymphoid organs, a small portion of lymphoid cells expressing leukocyte common antigens appeared first in the liver at 10-12 weeks of gestational age and was followed by a small number in the spleen and bone marrow by 13-15 weeks. All lymphoid cells in these extrathymic lymphoid organs at this stage were CD19+ B cells. The number of these CD19+ cells increased abruptly during the early period of mid-gestational age. The pattern of tissue distribution of each LCA isoform in the fetal liver and spleen correlated well with the patterns of quantitative analysis by flow cytometry. In summary we found that different LCA isoforms expressed in cell-type-specific pattern and showed different tissue distribution during the period of fetal development, and that LCA was the earliest antigen expressed by lymphocytes in the thymus and extrathymic lymphoid organs in our series.     

Immunohistological Analysis of Human Fetal Lymph Nodes

A panel of monoclonal antibodies directed against various lymphoid and non-lymphoid cell subsets was used to study the lymph nodes of human fetuses of 16-40 weeks. B cells were of intermediate size and were present at all ages in primitive follicles and in the outer cortex. The fetal B-cell immunophenotype is indicative of an intermediate stage of development, just preceding the differentiation to mature B cell. Forty to sixty per cent Leu1+ B cells were observed in the follicles until the end of the second trimester. At all stages, T cells showed an immunophenotype similar to type III thymocytes, different from adult peripheral T cells, with a marked predominance of CD4+ T cells. Leu7+ NK cells were generally absent. OKIa+ interdigitating reticulum cells were present in T-cell areas. Some axillary lymph nodes showed strongly CD1+ dendritic cells, probably Langerhans' cells. Macrophages and granulocytes were present in varying numbers. Altogether, our results indicate that fetal lymph nodes are quite well differentiated at an early fetal age, although T and B cells do not (yet) show adult immunophenotypes. The expression of the CD38 antigen may be a main marker related to the immaturity of fetal T and B cells.     

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.     

Reconstitution of SCID mice with haemopoietic precursors: a detailed analysis of gamma delta T-cell reconstitution.

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

A transgenic marker expressed on discrete populations during B-cell development

We describe a transgenic mouse strain that selectively express a surface marker (huCD25) on transitional B cells, pre-B cells and a lineage unidentified bone marrow (BM) population. We show that a subpopulation of B cells in Peyer's patches, spleen, blood and BM expressed the transgenic huCD25 marker on the cell surface. In the spleen, the huCD25 expression was found on transitional B cells, that had not yet been recruited into the recirculating pool. In the BM a fraction of the B220low surface immunoglobulin (Ig) negative PB493+ pre-B cells were huCD25+. HuCD25 expression was also seen on practically all immature B cells while the mature recirculating B cells did not express huCD25. A huCD25+B220- cell population was also found in the BM that had not rearranged the Ig heavy chain locus and did not express the lineage markers CD3, T-cell receptor (TCR), CD19 and Mac-1. A low expression of CD4 on these cells may indicate that they represent a noncommitted, hematopoetic progenitor cell population.     

The biological role and potential therapeutic application of interleukin 7

Interleukin (IL)-7 is a pleiotropic, non-redundant cytokine necessary for the development of B and T lymphocytes, in particular gammadelta T cell receptor-positive cell differentiation. The cytokine can function as a cofactor during myelopoiesis and the generation of cytotoxic T cells and natural killer cells, can activate monocytes/macrophages, and support the survival of mature T cells. A role for IL-7 in promoting the formation of Peyer's patch anlage has also been demonstrated. IL-7 is constitutively expressed in the thymus, bone marrow stromal cells, epithelial and dendritic cells, keratinocytes, as well as in fetal and adult liver. IL-7 acts on various cells through its receptor (IL-7R), a heterodimer consisting of an alpha chain (CD127) that specifically binds IL-7 and a common gamma(c) chain (CD132) shared by other cytokine receptors. The receptor is expressed on bone marrow progenitor cells, lymphoid T and B precursors, and mature T cells. IL-7 activity towards murine endothelial cells has been recently described. The presence of IL-7R on human endothelial cells has also been demonstrated. Several therapeutic applications of recombinant IL-7 have been proposed. These have focused on the enhancement of lymphopoiesis, promotion of stem cell engraftment, and the anti-tumor activity of the cytokine.     

A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA).

We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.