[Show abstract][Hide abstract] ABSTRACT: Embryonic stem cells (ESCs) have the potential to reprogram somatic cells into ESC-like cells through cell fusion. In the present study, the potential of human (h)ESC cytoplasts and karyoplasts to reprogram human hepatocytes was evaluated. Green fluorescent protein (GFP) transfected hESCs (ENVY cells) were fused with SNARF-1 (CellTracker)-labeled human hepatocytes using polyethylene glycol (PEG) and fluorescence-activated cell sorting (FACS) to produce hESC-hepatocyte hybrids. Immunocytochemical analysis of ESC markers showed that the hybrids expressed OCT4, TRA-1-60, TRA-1-81, SSEA-4, and GCTM-2. However, SSEA-1, which is typically low or absent on hESCs, was detected on hESC–hepatocyte hybrids. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) showed that alpha-fetoprotein, which is highly expressed in hepatocytes, was erased in the hybrids. These results indicated that hESCs have the potential to reprogram hepatocyte phenotype to a relatively undifferentiated state, but such hybrid cells are not identical to hESCs. Although hESC–hepatocyte hybrids were aneuploid, they were able to differentiate into embryoid bodies and some types of somatic cells. Furthermore, cybrids of enucleated hESCs and hepatocytes were produced by cell fusion, but the cybrids were unable to self-renew in the same way as hESCs. Presumably, the reprogramming factors are associated with the karyoplast and not the cytoplast of hESCs.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by
their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs and the combination of hLIF and bFGF was unable to improve the culture condition.
Iranian Journal of Veterinary Research 01/2010; 11(3). · 0.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.
[Show abstract][Hide abstract] ABSTRACT: Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more divergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells.
[Show abstract][Hide abstract] ABSTRACT: Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.
Reproduction Fertility and Development 02/2008; 20(5):589-97. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reproductive technologies have made impressive advances since the 1950s owing to the development of new and innovative technologies. Most of these advances were driven largely by commercial opportunities and the potential improvement of farm livestock production and human health. Companion animals live long and healthy lives and the greatest expense for pet owners are services related to veterinary care and healthcare products. The recent development of embryonic stem cell and nuclear transfer technology in primates and mice has enabled the production of individual specific embryonic stem cell lines in a number of species for potential cell-replacement therapy. Stem cell technology is a fast-developing area in companion animals because many of the diseases and musculoskeletal injuries of cats, dogs and horses are similar to those in humans. Nuclear transfer-derived stem cells may also be selected and directed into differentiation pathways leading to the production of specific cell types, tissues and, eventually, even organs for research and transplantaton. Furthermore, investigations into the treatment of inherited or acquired pathologies have been performed mainly in mice. However, mouse models do not always faithfully represent the human disease. Naturally occurring diseases in companion animals can be more ideal as disease models of human genetic and acquired diseases and could help to define the potential therapeutic efficiency and safety of stem cell therapies. In the present review, we focus on the economic implications of companion animals in society, as well as recent biotechnological progress that has been made in horse, dog and cat embryonic stem cell derivation.
Reproduction Fertility and Development 02/2007; 19(6):740-7. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Preimplantation embryo development typically involves sequential morphological events connecting embryonic cleavage, morula compaction and blastocyst formation, and occurs in parallel with transcriptional regulation, specifically, the maternal to embryonic transition. The underlying homeostatic and metabolic mechanisms governing embryo development are influenced by both genetic and epigenetic factors that respond to environmental stimuli and may impact development during later gestational and fetal growth. There is a renewed interest in the identification and characterization of developmentally important genes during embryonic and fetal development. Perturbations in gene expression, resulting from environmental conditions, can have serious consequences on further embryonic development, homeostasis and disease pathogenesis. The bovine embryo is, however, capable of tolerating and adapting to a wide range of conditions, although little is known of the molecular fingerprint required for oocyte maturation, fertilization and development to term. The genomic revolution united with promising new technologies offer greater opportunity to elucidate the mechanisms behind this well-orchestrated biological process. This paper reviews the current literature on gene expression in the bovine embryo with reference to environmental interference and the development of new technologies to observe this biological process. Defining the difference in molecular signalling between in vivo and in vitro systems will undoubtedly improve the safety and efficiency of assisted reproductive technologies. The future challenge is to devise culture conditions that mimic the changing environment required by developing embryos to allow the correct temporal and spatial expression of a cohort of developmental genes in a manner similar to that seen in
Society of Reproduction and Fertility supplement 02/2007; 64:341-63.
[Show abstract][Hide abstract] ABSTRACT: In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.
[Show abstract][Hide abstract] ABSTRACT: Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.
[Show abstract][Hide abstract] ABSTRACT: The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.
[Show abstract][Hide abstract] ABSTRACT: Nuclear transfer (NT) experiments in mammals have demonstrated that adult cells are genetically equivalent to early embryonic cells and the reversal of the differentiated state of a cell to another that has characteristics of the undifferentiated embryonic state can be defined as nuclear reprogramming. The feasibility of interspecies somatic cell NT (iSCNT) has been demonstrated by blastocyst formation and the production of offspring in a number of studies. Embryo and oocyte availability is a major limiting factor in conducting NT to obtain, blastocysts for both reproductive NT studies in genetically endangered animals and in embryonic stem cell derivation for species such as the horse and human. One approach to generate new embryonic stem cells in human as disease models, or in species where embryos and oocytes are not widely available, is to use oocytes from another species. Utilization of oocytes for recipient cytoplasts from other species that are accessible and abundant, such as the cow and rabbit, would greatly benefit ongoing research on reprogramming and stem cell sciences. The use of iSCNT is an exciting possibility for species with limited availability of oocytes as well as for endangered or exotic species where assisted reproduction is needed. However, the mechanisms involved in nuclear reprogramming by the oocyte are still unknown and the extent of the "universality" of ooplasmic reprogramming of development remains under investigation.
[Show abstract][Hide abstract] ABSTRACT: Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.
Reproduction Fertility and Development 01/2006; 18(2). · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.
Molecular Reproduction and Development 01/2006; 72(4):471-82. · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to find an efficient and reliable chemically assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 microg mL(-1) demecolcine for 2 h. Subsequently, zonae pellucidae were digested with pronase, and one-third of the cytoplasm connected to an extrusion cone was removed by hand using a microblade. The remaining two-thirds were used as recipients for HMC, and reconstructed and activated embryos were cultured for 7 days. The time-dependent manner of the development of extrusion cones, the efficiency (oriented bisection per oocyte; 94%), reliability (success per attempted enucleation; 98%), and the blastocyst per reconstructed embryo rates (48%) were measured. Ultrastructural analyses demonstrated that demecolcine treatment resulted in disoriented and haphazardly orientated microtubules. The general ultrastructure of the oocyte organelles, however, appeared to be unaltered by the treatments. Considering that no oocyte selection based on polar body presence was performed, this system seems to be more efficient and reliable than any other enucleation method. Moreover, expensive equipment (inverted fluorescence microscope) and a potentially harmful step (staining and ultraviolet illumination) can be eliminated from the HMC procedure without compromising the high in vitro efficiency.
Reproduction Fertility and Development 02/2005; 17(8):791-7. · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.
Reproduction Fertility and Development 02/2005; 17(5):573-85. · 2.58 Impact Factor