Mature oocytes derived from purified mouse fetal germ cells.

BACKGROUND: Mouse fetal germ cells have been successfully purified from fetal gonads. However, there are no published reports describing a procedure for deriving mature oocytes from isolated fetal germ cells. The purpose of this present study is to explore whether purified fetal germ cells are able to differentiate into mature oocytes through an in vivo grafting procedure. METHODS AND RESULTS: First, intact 11.5 and 12.5 days post-coitum (dpc) female gonads with or without the attached mesonephros and the reaggregated female gonad cells were transplanted into the recipient mice. The results demonstrate both the gonad accompanied by mesonephroi and the innate gonad structure are not absolutely required for 11.5 dpc and 12.5 dpc oogonia to generate mature oocytes. Next, oogonia were purified from female gonads, aggregated with different ovarian somatic cells and transplanted into the recipient mice. Purified 12.5 dpc oogonia were able to generate mature oocytes by aggregating with 12.5 dpc ovarian somatic cells, but not with 16.5 dpc or 0 days postpartum ovarian somatic cells. We also tested 12.5 dpc male germ cells but they were unable to undergo oogenesis. CONCLUSIONS: Our study demonstrates that mature oocytes can be derived from purified fetal germ cells through an aggregation and transplantation procedure. It also suggests that the synchronized interactions between oogonia and gonadal somatic cells are important to ensure normal folliculogenesis.

Searching for a Cell-Based Therapeutic Tool for Haemophilia A within the Embryonic/Foetal Liver and the Aorta-Gonads-Mesonephros Region.

The development of new strategies based on cell therapy approaches to correct haemophilia A (HA) requires further insights into new cell populations capable of producing coagulation factor VIII (FVIII) and presenting stable engraftment potential. The major producers of FVIII in the adult are liver sinusoidal endothelial cells (LSECs) and in a lesser degree bone marrow-derived cells, both of which have been shown to ameliorate the bleeding phenotype in adult HA mice after transplantation. We have previously shown that cells from the foetal liver (FL) and the aorta-gonads-mesonephros (AGM) haematopoietic locations possess higher LSEC engraftment potential in newborn mice compared with adult-derived LSECs, constituting likely therapeutic targets for the treatment of HA in neonates. However, less is known about the production of FVIII in embryonic locations. Quantitative polymerase chain reaction and Western blot analysis were performed to assess the relative level of FVIII production in different embryonic tissues and at various developmental stages, identifying the FL and AGM region from day 12 (E12) as prominent sources of FVIII. Furthermore, FL-derived VE-cad+CD45-Lyve1+/- endothelial/endothelial progenitor cells, presenting vascular engraftment potential, produced high levels of F8 ribonucleic acid compared with CD45+ blood progenitors or Dlk1+ hepatoblasts. In addition, we show that the E11 AGM explant cultures expanded cells with LSEC repopulation activity, instrumental to further understand signals for in vitro generation of LSECs. Taking into account the capacity for FVIII expression, culture expansion and newborn engraftment potential, these results support the use of cells with foetal characteristics for correction of FVIII deficiency in young individuals.

Microvascular assembly and cell invasion in chick mesonephros grafted onto chorioallantoic membrane.

Embryonic tissues, in common with other tissues, including tumours, tend to develop a substantial vasculature when transplanted onto the chorioallantoic membrane (CAM). Studies conducted to date have not examined in any detail the identity of vessels that supply these grafts, although it is known that the survival of transplanted tissues depends on their ability to connect with CAM vessels supplying oxygen and nutrients. We grafted the mesonephros, a challenging model for studies in vascular development, when it was fully developed (HH35). We used reciprocal chick-quail transplantations in order to study the arterial and venous connections and to analyse the cell invasion from the CAM to the organ, whose degeneration in normal conditions is rapid. The revascularization of the grafted mesonephros was produced by the formation of peripheral anastomoses between the graft and previous host vasculatures. The assembly of graft and CAM blood vessels occurred between relatively large arteries or veins, resulting in chimeric vessels of varying morphology depending on their arterial or venous status. Grafts showed an increased angiogenesis from their original vasculature, suggesting that the normal vascular degeneration of the mesonephros was partially inhibited. Three types of isolated host haemangioblast were identified in the mesonephros: migrating angioblast-like cells, indicating vasculogenesis, undifferentiated haematopoietic cells and macrophages, which might have been involved in the angiogenesis. Tomato lectin was found to bind activated macrophages in avian embryos.

Cell contacts and sorting out in vivo: the behaviour of some embryonic tissues implanted into the developing chick wing.

The interaction of cells from embryonic liver, neural retina and mesonephros with cells from limb-bud mesenchyme has been investigated in vivo by grafting these tissues into the developing chick wing-bud. The implanted cells were in all cases from quail tissue which can be recognized histologically. As embryonic liver and neural tube are tissues that sort externally to limb-bud mesenchyme in mixed aggregates, it would be expected, from a differential adhesiveness hypothesis, that heterotypic adhesions along the borders of graft and host would be favoured over cell-cell adhesions in the graft. No morphological signs of this were evident: rather the grafted cells maximized like-like contacts. The cells of the grafts, including those from control mesenchyme, did not invade into the wing. The results were the same irrespective of whether the graft was a fragment of tissue or a pellet of reaggregated cells. This supports the idea that cells within tissues are not actively moving around and also provides controls for assaying the invasiveness of other cell types, such as malignant cells into the wing.

Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.

The AML1:CBFbeta transcription factor complex is essential for definitive hematopoiesis. Null mutations in mouse AML1 result in midgestational lethality with a complete lack of fetal liver hematopoiesis. While the cell autonomous nature and expression pattern of AML1 suggest an intrinsic role for this transcription factor in the developing hematopoietic system, no direct link to a functional cell type has been made. Here, we examine the consequences of AML1 loss in hematopoietic stem cells (HSC) of the mouse embryo. We demonstrate an absolute requirement for AML1 in functional HSCs. Moreover, haploinsufficiency results in a dramatic change in the temporal and spatial distribution of HSCs, leading to their early appearance in the normal position in the aorta-gonad-mesonephros region and also in the yolk sac.