(1/83) Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from in vitro fertilization.

BACKGROUND: Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested. RESULTS AND DISCUSSION: We contrast experimental culture of single blastomeres obtained by 1) non-destructive biopsy of embryos destined for transfer, and 2) isolation of karyotypically normal blastomeres from disaggregated ("dead") embryos considered unsuitable for transfer, and evaluate these approaches with regard to production of ESCs. Pluripotency was confirmed by morphological criteria and by quantification of divergent homeodomain proteins specific to undifferentiated cell development. Following ESC isolation and identification, assessment was conducted according to a novel ESC grading system, also proposed here. CONCLUSION: The role of reproductive endocrinology in ESC research remains paramount. In this report, we hypothesize new and expand on existing strategies having the potential to enhance human ESC isolation, identification and in vitro maintenance.  (+info)

(2/83) Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organized into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organized neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here, we show that bovine inner cell masses from nuclear transfer and fertilized embryos, grown on feeders in serum-free medium, form polarized rosette structures expressing nestin, Pax6, Pax7, Sox1, and Otx2 and exhibiting interkinetic nuclear migration activity and cell junction distribution as in the developing neural tube. After in vitro expansion, neural rosettes give rise to p75-positive neural crest precursor cell lines capable of long-term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells, and chondrocytes, recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2, and Nkx6.1 and differentiation of mature astrocytes and neurons of central nervous system ventral identity, demonstrating appropriate response to inductive signals. All together, these findings indicate that neural rosettes directly derived from cloned and fertilized bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.  (+info)

(3/83) Aggregating embryonic but not somatic nuclear transfer embryos increases cloning efficiency in cattle.

Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.  (+info)

(4/83) Murine inner cell mass-derived lineages depend on Sall4 function.

Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages.  (+info)

(5/83) Enhanced development of porcine embryos cloned from bone marrow mesenchymal stem cells.

In the present study, we have characterized an isolated population of porcine bone marrow mesenchymal stem cells (MSCs) for multilineage commitment and compared the developmental potential of cloned embryos with porcine MSCs and fetal fibroblasts (FFs). MSCs exhibited robust alkaline phosphatase activity and later transformed into mineralized nodules following osteoinduction. Furthermore, MSCs underwent adipogenic and chondrogenic differentiation by producing lipid droplets and proteoglycans, respectively. Primary cultures of FFs from a female fetus at ~30 day of gestation were established. Donor cells at 3-4 passage were employed for nuclear transfer (NT). Cell cycle analysis showed that the majority of MSCs in confluence were in the G0/G1 stage. Cumulus-oocyte complexes were matured and fertilized in vitro (IVF) as control. The cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs and FFs (84.54.6% vs. 52.25.4% and 50.85.2%, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs (20.62.5% and 18.43.0%) did not differ, but were significantly (P<0.05) higher than NT derived from FFs (9.52.1%). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs (34.45.2 and 0.380.08, respectively) were significantly (P<0.05) higher than those from FFs (22.65.5 and 0.180.12, respectively). Proportions of TUNEL positive cells in NT embryos from FFs (7.31.8%) were significantly (P<0.05) higher than in MSCs (4.61.3%) and IVF (2.50.9%). The results clearly demonstrate that multipotent bone marrow MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned porcine embryos.  (+info)

(6/83) Histone arginine methylation regulates pluripotency in the early mouse embryo.

It has been generally accepted that the mammalian embryo starts its development with all cells identical, and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage. These differences depend on the orientation and order of the cleavage divisions that generated them. Because epigenetic marks are suggested to be involved in sustaining pluripotency, we considered that such developmental properties might be achieved through epigenetic mechanisms. Here we show that modification of histone H3, through the methylation of specific arginine residues, is correlated with cell fate and potency. Levels of H3 methylation at specific arginine residues are maximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. We confirm this prediction by overexpressing the H3-specific arginine methyltransferase CARM1 in individual blastomeres and show that this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, our results identify specific histone modifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination.  (+info)

(7/83) Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development.

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.  (+info)

(8/83) Blastocyst axis is specified independently of early cell lineage but aligns with the ZP shape.

The mechanisms controlling the establishment of the embryonic-abembryonic (E-Ab) axis of the mammalian blastocyst are controversial. We used in vitro time-lapse imaging and in vivo lineage labeling to provide evidence that the E-Ab axis of the mouse blastocyst is generated independently of early cell lineage. Rather, both the boundary between two-cell blastomeres and the E-Ab axis of the blastocyst align relative to the ellipsoidal shape of the zona pellucida (ZP), an extraembryonic structure. Lack of correlation between cell lineage and the E-Ab axis can be explained by the rotation of the embryo within the ZP.  (+info)