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PubMed1999

Functional and molecular analysis of hematopoietic progenitors derived from the aorta-gonad-mesonephros region of the mouse embryo.

Herein, we show that CD34, c-kit double-positive (CD34(+)c-kit(+)) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34(+)c-kit(+) cells by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) shows coactivation of erythroid (beta-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and lambda5) genes. Additionally, most cells coexpress the stem cell-associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34(+)c-kit(+) population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.

Animals↗
PubMed1999

Emergence of T, B, and myeloid lineage-committed as well as multipotent hemopoietic progenitors in the aorta-gonad-mesonephros region of day 10 fetuses of the mouse.

We investigated the developmental potential of hemopoietic progenitors in the aorta-gonad-mesonephros (AGM) region, where the definitive type hemopoietic progenitors have been shown to emerge before the fetal liver develops. By using an assay system that is able to determine the developmental potential of individual progenitors toward the T, B, and myeloid lineages, we show that not only multipotent progenitors but also progenitors committed to the T, B, or myeloid lineage already exist in this region of day 10 fetuses. Bipotent progenitors generating myeloid and T cells or those generating myeloid and B cells were also detected, suggesting that the commitment to T and B cell lineages is in progress in the AGM region. The numbers of these progenitors, however, were only 1/200-1/1000 of those in fetal liver of day 12 fetuses. Such small numbers of progenitors suggest that hemopoiesis has just started in the AGM region of day 10 fetuses. Although most of T cell lineage-committed progenitors in the AGM region generated only a small number of immature T cells, some were able to generate a large number of mature T cells. The detection of various types of lineage-committed progenitors strongly suggests that the AGM region is not only the site of stem cell emergence, but also the site of hemopoiesis, including lineage commitment. The T cell progenitors found in the AGM region may represent the first immigrants to the thymus anlage.

Animals↗
PubMed2000

Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region.

In the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-beta1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-beta1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo. (Blood. 2000;96:1591-1593)

Bone Morphogenetic Protein 4↗
PubMed2000

CFU-S(11) activity does not localize solely with the aorta in the aorta-gonad-mesonephros region.

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site in the midgestation mouse conceptus and first contains colony-forming units-spleen day 11 (CFU-S(11)) at embryonic day 10 (E10). Because CFU-S(11) activity is present in the AGM region before the onset of hematopoietic stem cell (HSC) activity, CFU-S(11) activity in the complex developing vascular and urogenital regions of the AGM was localized. From E10 onward, CFU-S(11) activity is associated with the aortic vasculature, and is found also in the urogenital ridges (UGRs). Together with data obtained from organ explant cultures, in which up to a 16-fold increase in CFU-S(11) activity was observed, it was determined that CFU-S(11) can be increased autonomously both in vascular sites and in UGRs. Furthermore, CFU-S(11) activity is present in vitelline and umbilical vessels. This, together with the presence of CFU-S(11) in the UGRs 2 days before HSC activity, suggests both temporally and spatially distinct emergent sources of CFU-S(11). (Blood. 2000;96:2902-2904)

Animals↗
PubMed1975

[Development of the nephrons of the mesonephros of chicken embryo].

The number of nephron populations in the postinduction period was established in 6- and 8-days chicken embryos and the development of an individual nephron and its parts was studied. The investigation by microdissection method has shownand the number of nephrons is different along the length of the kidney. Only two layers ofthe nephrons were found in the cranial portion, while in the caudal direction their number increased up to 4-6 populations which distinguished from one another by the glomerule position, the length of the nephron and its segments. All the populations of the ventral nephrons enter immediately into the mesonephritic (Wolffian) duct, while the dorsal nephrons have a system ofcollecting tubes by which they are connected with the mesonephric duct. The development of mesonephros was accompained by the increase of the absolute length of the nephrons of all populationsand their segments. Laboratory of Individual Development, Institute of Physiology, Czechoslovakian Academy of Sciences, Prague, and Laboratory of the Evolution of the Kidney and Water-Salt Exchange, Sechenov Institue of Evolutionary Physiologyand Biochemistry, Leningrad.

Animals↗
PubMed2005

Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells.

BACKGROUND: Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice. METHODS: The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow (BM) of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+ cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo. RESULTS: AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC's expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times, but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs' homing to BM in vivo. CONCLUSIONS: AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation.

Animals↗
PubMed2006

[Supportive effects of human aorta-gonad-mesonephros-derived stromal cells on umbilical cord blood LTC-IC].

The objective of this study was to explore the supportive effects of human aorta-gonad-mesonephros (AGM)-derived stromal cells on human umbilical cord blood long-term culture-initiating cells (LTC-IC). A co-culture system was established with human AGM stromal cells and umbilical cord blood CD34(+) cells. Different stromal cells derived from human AGM region (hAGM S1-S5) were plated on 24-well plates as feeder cells. CD34(+) cells were positively selected from human umbilical cord blood through immunomagnetic bead selection method, seeded on the feeder cells, and co-cultured for 8 weeks. The hematopoietic cells were collected at 5, 6, 7 and 8 weeks for CFC analysis. Frequencies of LTC-IC in umbilical cord blood CD34(+) cells after co-culture with AGM stromal cells were detected through limiting dilute analysis (LDA). The results showed that there was no any hematopoietic CFC in the feeder cell-free culture system after 5 weeks of co-culture. However, in AGM feeder cells culture systems, there were still CFCs after 5 weeks of co-culture, which indicated that human AGM stromal cells could maintain LTC-IC in vitro. In groups of hAGM feeders, hAGMS3 and S4 had better supportive effects than other AGM groups (P < 0.05). The absolute number of LTC-IC in hAGM S3 and S4 culture systems got expansion up to (176 +/- 46)% and (187 +/- 52)% respectively without significant difference between hAGMS3 and S4 (P > 0.05). It is concluded that human AGM stromal cells S1-S5 support the maintenance of umbilical blood LTC-IC in vitro, while hAGMS3 and S4 cells have better effects on maintaining LTC-IC and expansion of LTC-IC.

Aorta↗
PubMed2006

Mesenchymal lineage potentials of aorta-gonad-mesonephros stromal clones.

BACKGROUND AND OBJECTIVES: The characterization of stem cell microenvironments throughout ontogeny is of fundamental interest in the field of stem cell biology. Within the adult blood system, hematopoietic stem cells (HSC) are supported in the osteoblastic and endothelial bone marrow microenvironments. During mouse mid-gestation, the first HSC emerge autonomously in the aorta-gonad-mesonephros (AGM) region. However, little is known about this microenvironment. To study the cellular complexity of the AGM hematopoietic microenvironment and its relationship to HSC, we examined the potential of AGM stromal clones to differentiate into several mesenchymal lineages. DESIGN AND METHODS: Stromal cell clones from the mid-gestation mouse were cultured in appropriate conditions known to support osteogenic, adipogenic, chondrogenic and endothelial differentiation. Potentials of the stromal cells were scored by morphological examination of the cultures, specific staining and gene expression profile. RESULTS: We show that most clones possess uni/bilineage osteogenic, adipogenic and/or endothelial potential. The differentiation potential of the stromal clones appears to relate to their site of origin but not to their ability to support hematopoiesis. Moreover, we show that AGM HSC activity is unaffected by the osteogenic differentiation of UG26.1B6 stromal cells. INTERPRETATION AND CONCLUSIONS: These results confirm the existence of mesenchymal stem/progenitor cells in the AGM region and suggest that the AGM hematopoietic microenvironment is highly complex, containing stromal cells with various mesenchymal lineage potentials.

Animals↗
PubMed2006

[Supportive effects of stromal cells from human embryonic aorta-gonad-mesonephros region on umbilical cord blood CD34+ cells].

OBJECTIVE: To explore the supportive effects of stromal cells from human aorta-gonad-mesonephros (AGM) region on umbilical cord blood CD34+ cells. METHODS: Stromal cells derived from human AGM region (hAGMS1-S5) and fetal trunk fibroblasts (hFf) were cultured on the bottom of 24-well plates as feeder cells. CD34+ cells positively selected from human umbilical cord blood through immunomagnetic beads selection method, were seeded into 24-well plates, and co-cultured for 28d. The number of total nucleated cells (TNC), CD34+ cells, CD34+ CD38- cells, and CFC were counted every week. RESULTS: Stromal cells from human AGM region significantly supported proliferation of the TNC, CD34+ cells, CD34+ CD38- cells and CFC, when compared with hFT and controls without feeder cells (P < 0.05). The TNC increased (25.13 +/- 4.83)-fold (peak value) at day 21 in group of co-culture with AGM stromal cells. CD34 and CD34+ CD38- cells increased (2.68 +/- 0.51)- and (2. 38 +/- 0.45)-fold respectively at day 14 of co-culture. In colony analysis, HPP-CFU increased (2.62 +/- 0.85)-fold at day 14 of co-culture. The supportive effects of human AGM S1-S5 were significantly different, hAGM S3 and S4 were better than hAGM SI, S2, and S5 (P < 0.05). CONCLUSIONS: Human AGM stromal cells S1-S5 could support the maintenance and expansion of umbilical cord blood CD34+ cells in vitro. hAGMS3, S4 cell had better effects on maintaining HSC activity, which would provide model cells and basic data for researches on hematopoiesis mechanism and hematopoietic differentiation of embryonic stem cells.

Antigens, CD34↗
PubMed1991

Development of the mesonephros, metanephros and gonads in the foetal dog.

We describe the chronology of the development of the renal system and gonads of 19 greyhound foetuses. The smallest specimens were embedded in paraffin wax and were sectioned serially at 10 microns; the rest were sectioned at 50 microns in an LKB 2250 PMV cryomicrotome. Selected sections were stained with haematoxylin and eosin. The mesonephros, metanephros and gonads were measured in drawings of the sections, using the area fraction programme of a Vids II image analyse.

Animals↗
PubMed1989

Detection of sex-specific proteins in chick embryo gonads and mesonephros: effects of estradiol benzoate or tamoxifen on their expression.

Gonadal and mesonephric protein patterns from 19 day old normal chick embryos were investigated by two-dimensional polyacrylamide gel electrophoresis. Under these conditions, several sex-specific polypeptides were detected. As concerns gonadal extracts, four sex-specific polypeptides, all restricted to the cytosol, were present in the testis, whereas three sex-specific polypeptides, two localized in the cytosol, the other being membrane-bound, were identified in the ovary. Among the ovary-specific polypeptides two proved to be estrogen-dependent. They appeared in the left testis of embryos after early estradiol benzoate treatment and their expression was reduced in the ovary after early exposure to the antiestrogen, tamoxifen. Mesonephros extracts of both sexes also differed in their protein composition since three additional polypeptides (one in both the cytosolic and membrane fractions, the others in the cytosol) not found in females were found to be present in males. None appeared to be affected after either estradiol or tamoxifen treatment.

Animals↗
PubMed1981

[Histochemical localization of carbonic anhydrase in flask cells of the clawfrog mesonephros (Xenopus laevis Daudin) (author's transl)].

Carbonic anhydrase activity was demonstrated in the flask cells of intermediate segments and collecting tubules of the clawfrog-mesonephros with the cobalt-bicarbonate method of Häusler (1958) and Hansson (1967). Additional the luminal surface of proximal and distal convoluted tubules reacted positively. By electron microscopy the enzyme activity in the flask cells was localized mainly on the surface of microvilli like cytoplasmic processes which lined the lumen of intracellular secretion capillaries. The existence of carbonic anhydrase in the flask cells is related to a possible physiological function of these cells.

Animals↗
PubMed1980

[Scanning electron microscopy of the chick embryo mesonephros].

The mesonephros of chick embryos from the 7th to the 15th day of incubation has been studied by scanning electron microscope. The material was submitted to the common techniques for scanning electron microscopy using a special fixing agent that does not damage the embryonic tissue (Litke e Low, 1977). At the 7th day of incubation, the mesonephric corpuscles show a capsule whose parietal lamina is formed by flattened cells, while the visceral lamina already shows podocytes provided with pedicels interdigitating around the capillary loops. In the same period the proximal tubules appear to be bounded by columnar cells with long and thick apical microvilli. The Henle's loop is lined by low cells with slightly dome-shaped apices provided with single cilium. The distal convoluted tubules are lined by a low columnar epithelium. The cell apices show microvilli shorter and more rare than those found in the proximal portion. This pattern is present till the 12th day of incubation, when regression phenomena occur. These are mainly represented by a progressive occlusion of the tubules and a gradual alteration of the podocytes.

Animals↗
PubMed1998

Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium.

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34(+) cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin-CD34(+)Sca-1(+)c-Kit+ phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.

Animals↗
PubMed1998

Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines.

We report here on a novel stromal cell line, AGM-S3, derived from the aorta-gonad-mesonephros (AGM) region of a 10.5 days postcoitum (dpc) mouse embryo. The AGM-S3 cells promoted production of hematopoietic progenitors and day-12 spleen colony-forming cells from Lin-c-Kit+Sca-1(+) murine primitive hematopoietic cells. They also supported for 6 weeks generation of human multipotential progenitors from cord blood CD34(+)CD38(-) primitive hematopoietic cells. Human long-term repopulating hematopoietic stem cells (LTR-HSC) with the potential to reconstitute hematopoiesis in NOD/SCID mice were maintained on AGM-S3 cells for at least 4 weeks. Flow cytometric analysis showed that CD13, vascular cellular adhesion molecule-1, and Sca-1 were expressed on AGM-S3 cells. Because stem cell factor, interleukin-6 (IL-6), and oncostatin M, but not IL-3, IL-11, leukemia- inhibitory factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, thrombopoietin, and Flk2 ligand were detected in reverse transcription-polymerase chain reaction analysis of AGM-S3 cells, the cells seem to express species-cross reactive molecule(s) other than the cytokines examined and which act on primitive hematopoietic progenitor/stem cells. This cell line is expected to elucidate molecular mechanisms regulating early hematopoiesis and pave the way for developing strategies for expansion of human transplantable HSC.

ADP-ribosyl Cyclase↗
PubMed2005

Generation of murine stromal cell lines: models for the microenvironment of the embryonic mouse aorta-gonads-mesonephros region.

We describe a method to derive cell lines and clones from cells of the murine midgestation aorta-gonads-mesonephros (AGM) microenvironment. We start from subdissected AGM regions in "explant-" or "single-cell suspension"-type cultures from embryos transgenic for tsA58, a temperature-sensitive mutant of the SV40 T antigen gene. The number of cells in such cultures initially expand, but in most cases, this expansion phase is followed by a stable or even decline in cell number. After this so-called crisis phase, cell proliferation is noticeable in more than 90% of the cultures. Stromal cell clones can be isolated from these cultures, some of which have been cultured for more than 50 population doublings. These stromal cell clones are valuable tools for the study of the regulation of hematopoietic stem and progenitor cells in the midgestation mouse embryo.

Animals↗
PubMed1977

[Involution of the mesonephros and differentiation of the epididymis in chickens: immunohistological study].

By means of purified rabbit anti-adult chicken kidney antibodies two types of antigens have been identified in the mesonephros: one, localised in the cells of the proximal segment of the secretory tubules, the other characteristic of the collecting segments derived from the Wolffian duct. During the transformation of certain mesonephric tubules into ductuli efferentes and conjugentes of the epididymis the collecting tubule antigen disappears whereas the proximal secretory tubule antigen not only persists but can also be detected in the parietal layer of Bowman's capsules as they transform before connecting with the rete testis.

Age Factors↗