Human Fetal Spleen Research Articles

Expression of the myelin proteolipid protein gene in the human fetal thymus

We have analyzed human fetal thymus and spleen for expression of the proteolipid protein (PLP) gene. We demonstrate that the PLP gene is transcribed in both tissues, and that both the PLP and DM-20 mRNAs are produced. Western blot analyses revealed that both the PLP and DM-20 protein isoforms were present in the fetal thymus and spleen. Immunohistochemical analyses indicated that the PLP/ DM-20 proteins were detected in cells which have the distribution and morphology of thymic macrophages. These results provide further evidence that the PLP and DM-20 proteins are expressed in cell types other than myelin forming cells and possess function(s) unrelated to myelin structure. Furthermore, these data demonstrate that the PLP and DM-20 proteins are not shielded from the immune system behind the blood-brain barrier. These observations directly impinge upon the debate concerning acquisition of tolerance and the recognition that the encephalitogenic nature of PLP in diseases, such as Multiple Sclerosis, may not simply be related to its ‘sequestration’ from a ‘naive’ immune system.

The Gene for Human Glutaredoxin (GLRX) Is Localized to Human Chromosome 5q14

Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescencein situhybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human–hamster and a human–mouse hybrid panel and using a human glutaredoxin cDNA as a probe.

Anti‐B cell autoantibodies encoded by VH 4–21 genes in human fetal spleen do not require in vivo somatic selection

We isolated immunoglobulin (Ig) VH4 genes that were rearranged in the genomic DNA of 160 day human fetal spleen. Productively rearranged VH 4-21 genes were cloned into pRTM1, a human IgM expression vector. This allowed us to generate IgM kappa-expressing transfectomas by co-transfecting each of these constructs with pSVG-V kappa 3, an Ig kappa light-chain expression vector that has a variable region encoded Humkv325, a conserved V kappa gene that is frequently expressed early B cell ontogeny. We find that all transfectomas expressing IgM kappa encoded by VH 4-21 make IgM autoantibodies reactive with i, a linear poly-N-acetyllactosamine determinant present on neonatal red blood cells and a B cell-restricted isoform of the CD45 surface molecule. In contrast, a transfectoma expressing pSVG-V kappa 3 and pRTM1 containing a rearranged VH4-59 (V71-4) gene isolated from a chronic lymphocytic leukemia B cell population, designated WIL, produced IgM kappa antibodies that had no detectable anti-i binding activity. However, transfectomas expressing VH 4-21 fused onto the Ig heavy-chain third complementarity determining region (CDR3) of WIL are found to make anti-B cell autoantibodies with anti-i activity. These studies indicate that VH 4-21 genes rearranged in human fetal B cell ontogeny can encode anti-B cell autoantibodies with a binding specificity that does not require in vivo somatic selection.

In the human fetus, vascular endothelial growth factor is expressed in epithelial cells and myocytes, but not vascular endothelium: implications for mode of action.

Vascular endothelial growth factor (VEGF) is potentially an important regulator of angiogenesis, particularly during the extensive tissue growth and remodeling that occur in utero. In the present study, we have investigated the role of VEGF during human fetal development by analyzing the distribution of VEGF messenger RNA as well as the tissue- and cell-specific localization of VEGF peptide in the human midgestation (16-22 weeks) fetus. As a comparison, we conducted parallel studies on several human adult tissues. Messenger RNA encoding VEGF was detected in all fetal tissues studied and was most abundant in human fetal lung, kidney, and spleen; moderately abundant in heart, adrenal, pancreas, intestine, liver, testis, skin, muscle, and brain; and minimally detected in thymus and placenta. VEGF peptide, detected by immunohistochemistry, always was intracytoplasmic and localized principally in epithelial cells and myocytes, including the smooth muscle cells lining blood vessels. VEGF was not detected in vascular endothelial cells. As the cellular localization of VEGF in several human adult tissues was similar to that found in the cognate fetal tissues, VEGF is probably important not only in angiogenesis, but also in the maintenance of existing vessels. As VEGF was localized primarily in epithelial cells and myocytes and not in endothelial cells, these data are consistent with a paracrine mechanism of action whereby VEGF secreted by nonendothelial cells modulates activities in adjacent vascular endothelium.

Experimental study on interleukin 6 activity against tumor; Regulation of interleukin-2 induced lak cell activity

The ability of human recombinant interleukin-6 (IL-6) to regulate the induction and cytotoxic function of lymphokine activated killer (LAK) cells from human fetal spleen was studied. The results showed that IL-6 alone was unably to induce fetal splenic mononuclear cells (FSMC) to develop functional LAK cells, nor was it able to affect the number of IL- 2- induced LAK cells and to alter the lytic activity of LAK cells against tumor cells in effector phase. However, when IL- 6 was used in combination with IL- 2 during the induction phase, the resulting LAK cells displayed considerably greater lytic, activity than that with IL-2 alone (P<0.01), and this effect was IL-6 dose-dependent. The possible machanism of the synergistic effect of IL-2 and IL-6 in LAK cell induction from human fetal spleen is discussed.

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3 +, 76% CD4 +, 84% CD8 +, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR α β + (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of α/β T cells over γ/δ T cells. Propagated γ/δ T cells of liver and spleen were predominantly TCR γ δ 2 + . Whereas propagated cells of liver and spleen consisted of approximately 10% γ/δ + cells, thymus-derived cells expanded in culture were only an average of 2% TCR γ/δ +, demonstrating a rarity of IL2-responslve γ/δ T cells in middle gestation fetal thymus.

Development of multicomponent immunoassay and micro-localization analysis by laser-photoacoustic and microscopic image analyzer

Laser photoacoustic spectroscopy (PAS) has a potential to be developed as a sensitive and solid-phase analytical method and was applied to enzyme immunoassay. The test sample for immunoassay was prepared by adsorbing multi-component immunoglobulins on a nitrocellulose membrane filter. Human lambda- and kappa-chains, which are used as a principal indication of malignant lymphoreticular disease, and immunoglobulin G were used as model proteins, and PAS immunoassay was applied to the individual detection of these three proteins in the urine. Furthermore, in order to develop a sensitive analysis for particular biological components in tissues or cells, laser photoacoustic microscopy (PAM) and video intensified microscopy (VIM) were developed. PAM was shown to be applicable to the detection and quantification of human lambda-chain in a micro-region of the tissue sections of the human fetal spleen and pancreas. VIM was applied to the detection of stimulation-response processes in a cell. By using neutrophils which are stimulated by many substances and produce active oxidants as the results, dynamic changes in the stimulation-response process in a living cell were visualized as fluorescence or chemiluminescence images by the VIM system.

Growth of primary T-cell non-Hodgkin's lymphomata in SCID-hu mice: requirement for a human lymphoid microenvironment

We reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T- cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to V beta 5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.

Growth of Primary T-Cell Non-Hodgkin's Lymphomata in SCID-hu Mice: Requirement for a Human Lymphoid Microenvironment

AbstractWe reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T-cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to Vβ5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.

Sequence Analysis and Idiotypic Relationships of BEG-2, a Human Fetal Antibody Reactive with DNA

Monoclonal antibody (mAb) BEG-2 is a dsDNA binding IgM lambda derived from a 12-week human fetus. Two binding site idiotypes (BEG-2 Id alpha and BEG-2 Id beta) have been defined with the use of polyclonal rabbit anti-idiotypic anti-serum. BEG-2 Id alpha is located on the lambda light chain and has been described previously. The BEG-2 Id beta is present on the mu heavy chain. By means of a direct binding ELISA, BEG-2 Id beta has been identified on EBV-derived mAbs from human fetal liver or spleen (5%), human cord blood (2.7%) and adult peripheral blood (1%). In addition, the Id is present on 8.5% of adult spleen-derived hybridoma antibodies and 6% of RA synovium-derived hybridoma antibodies. In all populations the presence of the Id is strongly associated with binding to DNA and other polyanions. Competition assays indicated that the Id was located at or near the antigen-binding site on these molecules. To explore the structural basis of this binding, a major part of the BEG-2 heavy chain was sequenced and found to be encoded by a member of the VH4 family joined to a variant of JH5 by a very short Diversity or N region. Of the BEG-2 Id beta positive mAbs for which the VH family has been determined, five are encoded by VH4 and two are encoded by VH6, but none is encoded by other families. Thus, the BEG-2 Id beta identifies a set of polyreactive antibodies that are common in fetal life, persist into adulthood and are encoded by VH6 and, a subset of VH4 genes.

Regulatory effects of human recombinant IL-6 on natural killer cell activity of human fetal spleens

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrIL-6 was dose-dependent, and significnatly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%,P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

Immunochemical detection of sodium channel in human tissue and cell lines.

Mouse monoclonal antibodies have been raised to the C-terminal region of rat type II voltage-dependent sodium channel. One of these, designated 13A4-G4, recognizes a 260 Kd putative sodium channel protein in human fetal brain, heart and skeletal muscle, at 14-18 weeks of gestation. Faint immunoreactivity is also present in human fetal kidney but none has been detected in human fetal liver, lung or spleen. The antibody reacts in both western blots and immunocytochemical preparations with the human neuroblastoma cell lines SK-N-SH and SK-N-MC, the rhabdomyoblastoma cell line TE671, Y79 retinoblastoma cells and IPSB-18 astrocytoma cells. No 13A4-G4 immunoreactivity has been detected in several human cell lines derived from tissues that do not normally express sodium channels.

Uniform high frequency expression of autoantibody-associated crossreactive idiotypes in the primary B cell follicles of human fetal spleen.

At 23 wk of gestation, the fetal spleen contains follicles of lymphocytes that coexpress B cell differentiation antigens, surface Ig, and the 67-kD pan-T lymphocyte antigen, CD5 (Leu-1). Such cells are thought to represent the normal equivalent cells of B chronic lymphocytic leukemia (CLL). This B cell leukemia is distinctive in that high proportions of patients have leukemic cells that express sIg bearing one or more crossreactive idiotypes (CRIs) that commonly are found on IgM autoantibodies. We performed immunohistochemical studies on fetal spleen at 23 wk of gestation using a panel of mAbs specific for autoantibody-associated CRIs. We find that high proportions (5-17%) of the lymphocytes within each follicle react with any one of the anti-CRI mAbs. Furthermore, there is little variation between primary follicles in the proportions of cells that express a particular CRI. Using a cocktail of four anti-CRI mAbs, we detect autoantibody-associated CRIs on approximately one-third of the lymphocytes within each of the primary B cell follicles. These data indicate that the many of the Igs produced during early B cell development may be structurally related to IgM autoantibodies and to Ig expressed in CLL and related CD5 B cell malignancies. Furthermore, these studies suggest that the repertoire of Ig V genes expressed in each primary B cell follicle may be representative of the total restricted Ig V gene repertoire expressed during early B cell ontogeny.

Immunomodulation of human fetal cells by the fungal metabolite gliotoxin

Gliotoxin (GT) is a fungal metabolite that reduces the ability of murine macrophages to react immunologically in vitro. It is also capable of modulating the immunogenicity of murine bone marrow cells, so that the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras is delayed. The present study examines the effect of GT on human fetal cells, both in terms of reduction of immunogenicity and toxicity. GT (10 micrograms/ml) significantly decreased the responsiveness in mixed lymphocyte cultures of cells derived from human fetal pancreas, spleen, liver and bone marrow. This concentration of GT was, however, mildly toxic to explants of the pancreas, with a significant reduction in insulin secretion from this tissue during the first day of its organ culture, but not thereafter. GT-treated pancreatic explants were lighter and contained less insulin than the untreated controls 3 months after the tissue had been implanted beneath the renal capsule of nude mice. This difference was not apparent 3 weeks after transplantation into these animals. It is hypothesized that the immunomodulating effect of GT (at a concentration less than 10 micrograms/ml) may be of benefit in treating allografted human fetal pancreas before it is transplanted, as it has for murine adult bone marrow cells.

Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells.

Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5'-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3'-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.

Immuno-architecture of human fetal lymphoid tissues

Spleen, thymus and lymph node of human fetuses from the 12th to the 38rd week (spleen from 9 weeks) were investigated in an immunohistological study on B5-fixed paraffin embedded tissues, employing a panel of recently developed monoclonal antibodies, reactive with antigens resistant against fixation and paraffin embedment. The monoclonal antibodies included were MT1, MT2, MB1, MB2, MB3, LN1, LN2, LN3, LeuM1, Leu7, VIE-G4, together with polyclonal antibodies reactive with immunoglobulin heavy and light chains, and with lysozyme and S100-protein. The preservation of morphological detail together with immunoperoxidase staining of cellular subsets, allowed an accurate determination of the ontogenic development of the different cell types in situ, in relation to their micro-environment. The use of paraffin tissue reactive (monoclonal) antibodies gives an extra dimension to the study of fetal lymphoid tissues. This is of particular advantage in studies on very fragile tissues as in early embryonal and fetal ontogeny.

Differential expression of HLA class II antigens in fetal human spleen: relationship of HLA-DP, DQ, and DR to immunoglobulin expression.

Frozen sections of human fetal spleen from 12 to 20 wk gestation were examined by using polyclonal antibodies to Ig isotypes, monoclonal antibodies to HLA class II subregion locus products, B and T cells, and follicular dendritic cells. Scattered lymphoid cells in spleen sections from fetuses of 12 to 13 wk gestational age expressed IgM but not IgD. The appearance of lymphoid cells expressing IgD occurred at 14 to 15 wk before the formation of loose clusters of B cells at 16 wk. IgD expression was associated mainly with cells in these clusters, which by 17 wk had become definite follicles. Follicular dendritic cells were not detectable until 20 wk. OKT3-positive T cells were not detected until 17 wk, and at 20 wk constituted 5% of the nucleated cell population. HLA-DR- and DP-positive lymphocytes and macrophages were detectable in fetal spleen from 12 wk onward; DR was expressed on more cells than DP, and the numbers of cells stained by HLA-DR-specific monoclonal antibodies exceeded the number of Ig-positive cells in all spleens examined. HLA-DQ was expressed by consistently fewer cells than HLA-DR and -DP in all spleens tested. The small number of DQ-positive cells in spleens from 12- to 13-wk fetuses had the morphology of macrophages; HLA-DQ expression by lymphoid cells followed a similar pattern to IgD expression and was associated mainly with follicular lymphocytes. It could be demonstrated by double-labeling experiments that all follicular IgM-positive cells in 17- to 20-wk spleens expressed HLA-DP, DQ, and DR antigens: IgM-positive cells in 12- to 16-wk spleens and interfollicular IgM-positive cells in 17- to 20-wk spleens all expressed HLA-DR, but only 59% and 43% expressed DP and DQ, respectively. Ninety-one to 100% of IgD-positive cells in all spleens examined expressed HLA-DQ in addition to DR and DP. In these experiments IgD-negative, DQ-positive cells had the morphologic appearance characteristic of macrophages. These data suggest that class II antigens are differentially expressed on developing lymphoid cells; DR and DP expression occurring in the earliest spleens examined, with expression of DP on a subpopulation of DR-positive cells; IgD and DQ expression appears to be coincident on maturing B cells as they begin to form follicles. An immunoregulatory role for HLA-DQ in B cell development is implicated and remains to be fully investigated.

Leu-1+ (CD5+) B cells. A major lymphoid subpopulation in human fetal spleen: phenotypic and functional studies.

Examination of the cell surface phenotype of fetal splenic lymphocytes demonstrated a major, novel subpopulation of B cells that co-express Leu-1 (CD5) in addition to B cell differentiation antigens (Leu-1+ B cells). These cells are similar to some conventional B cells in that they express HLA-DR, Leu-12, and B1, as well as both immunoglobulin (Ig) M and IgD. They comprise 40 to 60% of total splenic B cells in the fetus but are infrequent in fetal liver and adult spleen. Fetal Leu-1+ B cells do not respond to pokeweed mitogen with either proliferation or Ig secretion, and in contrast to the murine counterpart, Ly-1 B cells, they do not constitutively produce Ig. Leu-1+ B cells were incapable of augmenting Ig production of Leu-1- B cells when suboptimal numbers of T cells were present; however, they did require the presence of T cells to secrete antibody. They do not cap either the CD5 protein or surface Ig. These cells are a unique subpopulation of fetal splenic B cells that do not function as conventional B cells. Their role in the humoral immune response is unknown. They may represent the normal stage of B cell development, which is reflected in the phenotype of B cell CLL cells.

Transformation of human fetal thymus and spleen lymphocytes by human T-cell leukemia virus type I.

Co-cultivation of human thymus and spleen lymphocytes, which were obtained from 26-week and 27-week fetuses, with a lethally-irradiated human cord T-cell line harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of T-cell lines positive for adult T-cell leukemia-associated antigens and producing HTLV-I. These cell lines had the phenotype of a helper/inducer subset of peripheral T-cells as evidenced by the reactivity with monoclonal antibodies to human T-cells.

Differentiation of red pulp and evaluation of hemopoietic role of human prenatal spleen.

Both the development of red pulp and hemopoietic activity in spleen obtained from 62 human embryos and fetuses between 30 days and 20 weeks after ovulation were investigated light and electron microscopically. The spleen develops in the left-posterior portion of the dorsal mesogastrium at 35-40 days after ovulation. At the 8th week after ovulation, reticular cells formed a three-dimensional meshwork. Two types of reticular cells (dark and clear reticular cells) were observed in the splenic cord in the 12-13th week after ovulation. Mature hemopoietic cells, mostly of the erythroblastic series, increased in number in the extravascular spaces with the development of the fetus. However, presumptive hemopoietic stem cells or "undifferentiated mononuclear cells" (Fukuda, 1973a) did not appear in the spleen. Moreover, immature hemopoietic cells such as proerythroblasts, myeloblasts and megakaryoblasts could not be detected. Therefore, despite the occurrence of a well-developed reticular cell network, hemopoiesis was judged to have not taken place in human fetal spleen. Macrophages appeared in the spleen at the 8th week after ovulation and increased in number with the the development of the fetus. Phagocytosis of decrepit blood cells proved to be an essential function of the spleen.