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Biomedical subjects

Alan Trounson

Publications and source records attributed to Alan Trounson.

At least 19 recordsLinked to original sources
PubMed2007

Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating.

The procedures of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are routinely used in modern medicine to overcome infertility and, in animal husbandry, to propagate lines with compromised fertility. However, there remains concern that manual selection and injection of whole sperm into oocytes could contribute to pre- and postnatal developmental defects. To address this, we have used gene expression profiling and immunophenotyping to characterize offspring generated by these procedures. We used gametes from glutathione peroxidase 1 knockout (Gpx1-/-) mice as a sensitized screen responsive to oxidative stress from artificial reproduction technologies (ART). There were no differences between IVF and ICSI derived offspring in gene expression patterns, and minor differences in hematopoietic parameters. Furthermore there were only minor differences between these IVF and ICSI pups and those derived from natural mating. These data demonstrate for the first time in that there is no significant phenotypic affects of ICSI when compared to IVF and we identified a relatively minor influence of the artificial fertilization methods on phenotype of offspring compared with natural mating. These observations would support the use of ICSI for derivation of mutant mouse lines and may be of some importance for the use of this technique in human ART.

Animals↗
PubMed2006

The production and directed differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

Biomarkers↗
PubMed2006

A role for neurotrophins in embryonic stem cell growth.

Neurotrophins act on embryonic cells through TRK receptors to inhibit apoptosis by phosphorylation of AKT. In a recent paper in Nature Biotechnology, show that the presence of selected neurotrophins enables cloning of trypsinized single embryonic stem cells and potentially increases the availability and usefulness of these stem cells.

Apoptosis↗
PubMed2006

Spindle abnormalities in oocytes.

Human oocytes have been found to exhibit spindle abnormalities that might lead to chromosomal errors when matured in vitro. However, the immature oocytes recovered from patients with polycystic ovary syndrome might include a high proportion of oocytes from degenerating and atretic follicles that are not selected for maturation in vivo.

Cell Differentiation↗
PubMed2006

Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts.

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured before in vitro fertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

Animals↗
PubMed2005

Neural stem cells express non-neural markers during embryoid body coculture.

The capacity of neural stem cells (NSC) to transdifferentiate into a wide range of non-neuronal lineages is the subject of debate. One approach to test NSC plasticity is to ectopically place NSCs in permissive or instructive microenvironments in which the signals driving differentiation of multiple cell types are being elicited. Here we produce embryoid body neurosphere aggregates by combining neurosphere derivatives from fetal mice constitutively expressing green fluorescent protein with embryonic stem (ES) cells isolated from Zin40 mice constitutively expressing nuclear beta-galacosidase. Under these conditions, we assess neurosphere-derivative-immunoreactivity to anti-neurofilament heavy chain, anti-pan-cytokeratin, anti-smooth muscle alpha-actinin and anti-alpha-fetoprotein-specific antibodies. Furthermore, we determine lineage-specific transgene expression and undertake fluorescence in situ hybridization to assess ES cell-neural stem cell-fusion indices. Our data demonstrate that following coculture in hanging drops with ES cells, neurosphere derivatives display immunoreactivity to non-neural markers, in particular smooth muscle, which is not dependent upon cell-cell fusion. These results suggest that given an appropriate environment, NSC may lose their in vivo restrictions and display non-neuronal phenotypes.

Animals↗
PubMed2005

Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Animals↗
PubMed2005

The early days of IVF outside the UK.

In this article the history of IVF in geographical regions outside the UK are traced by pioneers of that time. Following the birth of Louise Brown in 1978, live births after IVF occurred in Australia in 1980, in the USA in 1981 and in Sweden and France in 1982. Following the first IVF birth in Australia, the Government of Victoria established a review of IVF research and practice which led to the proclamation of the Infertility (Medical Procedures) Act 1984, the first legislation to regulate IVF and its associated human embryo research. Despite such restriction, IVF doctors and scientists from Victoria, especially those under the leadership of Carl Wood, Alan Trounson and Ian Johnston continued to initiate new treatments for infertility and new methods for delivering this treatment. In the USA IVF research began on animals as early as the 1930s, when Pincus and Enzmann at Harvard were involved in attempts at IVF in the rabbit. In the 1940s, John Rock attempted human IVF with 138 human oocytes without success. In 1965, Bob Edwards was with Georgeanna and Howard Jones at Johns Hopkins where attempts were made to fertilize oocytes in vitro. Clinical IVF began in earnest in the USA in 1980 with the first birth in 1981 achieved by the use of HMG--a first successful use with IVF. In France, two groups Frydman and Testart (Clamart) and Cohen, Mandelbaum and Plachot (Sevres) focused their research in particular directions. In 1981, the Clamart group developed a plasma assay for the initial rise in LH. The Sevres group developed a transport technique. Plachot produced a long series of cytogenetic analyses of oocytes and human embryos. Mandelbaum described the microstructures of the human oocyte. The start of IVF in France benefited from the help of animal researchers from the Institut National de la Recherche Agronomique. The first babies were born in Clamart in February 1982 and in Sèvres in June 1982. Important contributions to the development of IVF from the Nordic countries include techniques for ovarian stimulation, sonographic techniques for monitoring and vaginal oocyte retrieval and also unique possibilities for monitoring IVF safety. These developments, in combination with relatively permissive laws for the practice of reproductive medicine and relatively generous reimbursement policies, as well as a general public confidence in IVF, have led to an exceptionally high availability of IVF, within international comparison.

Australia↗
PubMed2005

Derivation characteristics and perspectives for mammalian pluripotential stem cells.

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.

Animals↗
PubMed2005

A novel method for somatic cell nuclear transfer to mouse embryonic stem cells.

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.

Animals↗
PubMed2005

Developments in infertility therapy--diagnosis of genetic disease in embryos.

BACKGROUND: The general practitioner plays a vital role in assisting couples through the difficult and emotional field of assisted reproductive techniques including in vitro fertilisation (IVF). OBJECTIVE: This article discusses the most recent developments in IVF so that GPs may better explain this ever changing field of medicine to their patients. DISCUSSION: This article begins by explaining how sperm can be brought together with oocytes for fertilisation outside the body, and then moves on to selecting the most genetically appropriate embryos for implantation. From this comes the issue of examining the fertilised oocytes to detect abnormalities, and the options available to couples with ethical concerns. The promise of regenerative medicine through the use of sperm cells is also discussed.

Cryopreservation↗
PubMed2004

Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin.

Human embryonic stem cells differentiate spontaneously in vitro into a range of cell types, and they frequently give rise to cells with the properties of extra-embryonic endoderm. We show here that endogenous signaling by bone morphogenetic protein-2 controls the differentiation of embryonic stem cells into this lineage. Treatment of embryonic stem cell cultures with the bone morphogenetic protein antagonist noggin blocks this form of differentiation and induces the appearance of a novel cell type that can give rise to neural precursors. These findings indicate that bone morphogenetic protein-2 controls a key early commitment step in human embryonic stem cell differentiation, and show that the conservation of developmental mechanisms at the cellular level can be exploited in this system--in this case, to provide a facile route for the generation of neural precursors from pluripotent cells.

Base Sequence↗
PubMed2004

Stem cells, plasticity and cancer - uncomfortable bed fellows.

Stem cell research is a vibrant and rapidly moving field of science that investigates self-renewing cells in the adult and embryo. Two debates currently exist in the stem cell field concerning the transcriptional redirection of stem cell differentiation and fate, and the reorganization of cell commitment through nuclear reprogramming caused by cell fusion and nuclear transfer. The recent Keystone Symposium in Colorado on stem cells, organised by Fiona Watt and Leonard Zon, brought together both leading and upcoming researchers in the field to explore stem cell biology and these issues.

Adult↗
PubMed2004

Keystone symposia - stem cells.

The Keystone Symposia stem cell conference focused on the biological nature of self-renewing cell populations that exist in special regulatory niches and form other cell types that enable maintenance of tissue function, repair and, in some cases, regeneration. Adult stem cells are present in most, if not all tissues, and their regulation as renewing populations, the nature of the niche and the factors driving decisions for differentiation in primary pathways remain the dominant interest in the field. The more vexed question concerning possible transdifferentiation of adult stem cells from one tissue type to another has become focused on the role of fusion and whether this is a recapitulation of normal processes in embryonic and adult development. The possible linkage of cancer phenotypes with stem cells and downstream progenitor cell types through loss of regulatory influences in renewal, expansion and lineage commitment is a rapidly growing interest. Pluripotent embryonic stem (ES) cells and their maintenance and directed differentiation into a variety of tissue types remains an optimistic field of research in which some debate exists about true pluripotency of adult stem cells. The role of epigenetic influences on differentiation and development has drawn on data from nuclear transfer studies in cloning the whole organism and in ES cell production. The formation of germ cells and their renewal and differentiation was also a major interest of the symposium.

Animals↗
PubMed2003

Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains.

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications.

Animals↗