Parthenogenetic Cells Research Articles

Differentiation of uniparental human embryonic stem cells into granulosa cells reveals a paternal contribution to gonadal development.

Genomic imprinting underlies the mammalian requirement for sexual reproduction. Nonetheless, the relative contribution of the two parental genomes during human development is not fully understood. Specifically, a fascinating question is whether the formation of the gonad, which holds the ability to reproduce, depends on equal contribution from both parental genomes. Here, we differentiated androgenetic and parthenogenetic human pluripotent stem cells (hPSCs) into ovarian granulosa-like cells (GLCs). We show that in contrast to biparental and androgenetic cells, parthenogenetic hPSCs present a reduced capacity to differentiate into GLCs. We further identify the paternally expressed gene IGF2 as the most upregulated imprinted gene upon differentiation. Remarkably, while IGF2 knockout androgenetic cells fail to differentiate into GLCs, the differentiation of parthenogenetic cells supplemented with IGF2 is partly rescued. Thus, our findings unravel a surprising essentiality of genes that are only expressed from the paternal genome to the development of the female reproductive system.

Parthenogenetic mosaicism: generation via second polar body retention and unmasking of a likely causative PER2 variant for hypersomnia

BackgroundParthenogenetic mosaicism is an extremely rare condition identified only in five subjects to date. The previous studies indicate that this condition is mediated by parthenogenetic activation and is free from a specific phenotype ascribed to unmaking of a maternally inherited recessive variant in the parthenogenetic cell lineage.ResultsWe examined a 28-year-old Japanese 46,XX female with Silver-Russell syndrome and idiopathic hypersomnia. The results revealed (1) predominance of maternally derived alleles for all the differentially methylated regions examined; (2) no disease-related copy-number variant; (3) two types of regions for all chromosomes, i.e., four BAF (B-allele frequency) band regions with single major microsatellite peaks of maternal origin and single minor microsatellite peaks of non-maternal (paternal) origin, and six BAF band regions with single major microsatellite peaks of maternal origin and two minor microsatellite peaks of maternal and non-maternal (paternal) origin; (4) an unmasked extremely rare PER2 variant (c.1403G>A:p.(Arg468Gln)) with high predicted pathogenicity; (5) mildly affected local structure with altered hydrogen bonds of the p.Arg468Gln-PER2 protein; and (6) nucleus-dominant subcellular distribution of the p.Arg468Gln-PER2 protein.ConclusionsThe above findings imply that the second polar body retention occurred around fertilization, resulting in the generation of the parthenogenetic cell lineage by endoreplication of a female pronucleus and the normal cell lineage by fusion of male and female pronuclei, and that the homozygous PER2 variant in the parthenogenetic cells is the likely causative factor for idiopathic hypersomnia.

"Biomechanical Signaling in Oocytes and Parthenogenetic Cells".

Oocyte-specific competence remains one of the major targets of current research in the field of reproduction. Several mechanisms are involved in meiotic maturation and the molecular signature of an oocyte is considered to reflect its quality and to predict its subsequent developmental and functional capabilities. In the present minireview, we focus on the possible role of mechanotransduction and mechanosensor signaling pathways, namely the Hippo and the RhoGTPase, in the maturing oocyte. Due to the limited access to female gametes, we propose the use of cells isolated from parthenogenetic embryos as a promising model to characterize and dissect the oocyte distinctive molecular signatures, given their exclusive maternal origin. The brief overview here reported suggests a role of the mechanosensing related pathways in oocyte quality and developmental competence and supports the use of uniparental cells as a useful tool for oocyte molecular signature characterization.

Adiponectin Improves In Vitro Development of Cloned Porcine Embryos by Reducing Endoplasmic Reticulum Stress and Apoptosis.

Simple SummarySuccessful attenuation of endoplasmic reticulum (ER) stress signaling has a beneficial outcome in in vitro embryonal improvement. We evaluated the effect of adiponectin during in vitro culture in porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer (SCNT). We found that 15 and 30 μg/mL adiponectin treatment significantly improved cleavage rates, blastocyst formation rates, and total cell number (TCN) of blastocysts derived from parthenogenetic activation and reduced the expression levels of XBP1. In SCNT embryos, the cleavage rate, blastocyst formation rate, and TCN of blastocysts were significantly improved by 15 μg/mL adiponectin treatment compared to the control. In addition, the 15 μg/mL adiponectin treatment reduced the levels of XBP1 expression and ER stress-related genes, increased expression levels of pluripotency-related genes, and decreased apoptosis-related gene expression. Comprehensively, treatment with 15 μg/mL adiponectin enhanced the in vitro developmental capacity of early-stage SCNT porcine embryos by reducing ER stress and apoptosis.The main factor of embryonic demise is endoplasmic reticulum (ER) stress. Successful attenuation of ER stress results in an improvement in embryo development. We studied the impact of adiponectin in the in vitro culture (IVC) of porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer (SCNT). The first experiment revealed that 15 and 30 μg/mL adiponectin treatments improved cleavage, blastocyst rates, and total cell number (TCN) of parthenogenetic embryos and reduced the expression of XBP1 compared to the 5 μg/mL adiponectin treatment and control groups (p < 0.05). The second experiment showed that cleavage rate, blastocyst formation rate, and TCN of blastocysts were improved in the 15 μg/mL adiponectin treatment group compared with the control group, with significantly reduced XBP1 expression in ≥4-cell stage SCNT embryos and blastocysts (p < 0.05). Treatment with 15 μg/mL adiponectin significantly improved the expression of XBP1 and reduced the expression of ER stress-related genes (uXBP1, sXBP1, PTPN1, and ATF4), increased the expression levels of pluripotency-related genes (Nanog and SOX2), and decreased apoptosis-related gene expression (Caspase-3). These results suggest that 15 μg/mL adiponectin enhanced the in vitro developmental capacity of early-stage SCNT porcine embryos by reducing ER stress and apoptosis.

Regenerative medicine: could Parkinson's be the first neurodegenerative disease to be cured?

Parkinson's disease is one of the most insidious neurodegenerative diseases in developed countries. Today, human pluripotent stem cells are produced from embryonic or adult cells, multiplied, differentiated into neural cell lines and ultimately transplanted into disease animal models or patients. Nowadays, DOPAminergic neurons derived from human pluripotent stem cells and human parthenogenetic cells are being clinically tested in China and Australia, respectively. More importantly, good manufacturing practices have been developed and the neurons obtained have been successfully tested in nonhuman primates by teams in Europe, USA and Japan. However, there is a need for translational clinical studies with small molecules tested in vitro, as well as testing of the the efficacy of additional therapies.

Effects of Roscovitine on In Vitro Development of Porcine Oocyte Using Brilliant Cresyl Blue

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 75 μM of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

DNA methylation modulates H19 and IGF2 expression in porcine female eye

The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR) and bisulfite sequencing PCR (BSP). We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA) cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively). We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs). Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye.

101 TOWARDS OPTIMAL IN VITRO CULTURE CONDITIONS FOR PIG-MONKEY AGGREGATION CHIMERAS

Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in pure iPSC-medium (6.3% blastocyst rate), but grew in a mixture of PZM and iPSC medium. The best results were achieved with PZM and PZM with 10% or 25% iPSC medium (38.8% and 30% blastocyst rates, respectively). Next, we checked aggregation results of chimeric embryos produced with two different iPSC lines in the respective media (PZM, PZM+10% iPSC medium, PZM+25% iPSC medium). The porcine iPSC line (piPSC) and cynomolgus monkey iPSC line (ciPSC) carry fluorescent markers (piPSC: GFP, ciPSC: venus), thus facilitating detection of integration into the host embryos. After aggregation of iPS cells between two Day-3 parthenogenetic embryos (sandwich technique), blastocyst rates at Day 6 were determined (see Table 1). While culture in PZM allowed for the highest blastocyst rate (&gt;40%), the iPS cell participation was very low (0–11.1%). Cultivation in PZM+25% iPS medium was compatible with a high rate of embryo-iPSC chimeras (50–100%), but with a low blastocyst rate (7.7–16.7%). Possibly, the iPS cells proliferate more rapidly than blastomeres and the embryo is overgrown. The best results were obtained in medium mixture PZM+10% iPSC medium (25–40% blastocyst rate with 66.7–100% iPS cell participation). These results show that supplementation of the basic culture medium with 10% iPSC medium yields high blastocyst rates for chimeric embryos, and also ensures much higher iPS cell participation. This improves the use of 3-day-old embryos in aggregation chimeras. Table 1.Development of parthenogenetic porcine embryo aggregates with porcine (p) or cynomolgus monkey (c) iPS cells

Description of bovine major histocompatibility complex class IIa haplotypes using parthenogenetic embryo-derived cells.

In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.

Intercellular bridges are essential for human parthenogenetic cell survival

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition.

Brief report: Parthenogenetic embryonic stem cells are an effective cell source for therapeutic liver repopulation.

Parthenogenesis is the development of an oocyte without fertilization. Mammalian parthenogenetic (PG) embryos are not viable, but can develop into blastocysts from which embryonic stem cells (ESCs) have been derived in mouse and human. PG ESCs are frequently homozygous for alleles encoding major histocompatibility complex (MHC) molecules. MHC homozygosity permits much more efficient immune matching than MHC heterozygosity found in conventional ESCs, making PG ESCs a promising cell source for cell therapies requiring no or little immune suppression. However, findings of restricted differentiation and proliferation of PG cells in developmental chimeras have cast doubt on the potential of PG ESC derivatives for organ regeneration. To address this uncertainty, we determined whether PG ESC derivatives are effective in rescuing mice with lethal liver failure due to deficiency of fumarylacetoacetate hydrolase (Fah). In developmental chimeras generated by injecting wild-type PG ESCs into Fah-deficient blastocysts, PG ESCs differentiated into hepatocytes that could repopulate the liver, provide normal liver function, and facilitate long-term survival of adult mice. Moreover, after transplantation into adult Fah-deficient mice, PG ESC-derived hepatocytes efficiently engrafted and proliferated, leading to high-level liver repopulation. Our results show that--despite the absence of a paternal genome--PG ESCs can form therapeutically effective hepatocytes.

Genomic imprinting analysis of Igf2/H19 in porcine cloned fetuses using parthenogenetic somatic cells as nuclear donors

To gain insight into parthenogenesis in pigs, we report for the first time that using parthenogenetic somatic cells as nuclear donors (PSCNT), the porcine parthenogenetic fetus can develop to gestational day 39. Weight and morphological analysis revealed that PSCNT fetuses were smaller and developmentally retarded when compared to normally fertilized controls. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19 was over-expressed, whereas Igf2 was significantly reduced (p < 0.05) compared with their controls. In addition, bisulfite-sequencing PCR results demonstrated that H19 differentially DNA methylated regions (DMRs) were hypomethylated in PSCNT fetuses, while Igf2 DMRs were hypermethylated in both PSCNT and control fetuses. Our results suggest that extended development of the porcine parthenogenetic fetus can be accomplished using PSCNT and that abnormal DNA methylation of H19 DMRs might contribute to the critical barrier of parthenogenesis in pigs.

Intercellular bridges functionally connect parthenogenetic cells

Intercellular bridges functionally connect parthenogenetic cells

Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.

Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs

To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVF- and three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general “stemness” criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer.

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter ('small' ≤110µm; 'medium' >110µm; 'large' ≥120µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

147 AGGREGATION OF IN VIVO FERTILIZED AND PARTHENOGENETICALLY ACTIVATED MOUSE EMBRYOS

Embryonic chimerism – mixing of cells originated from at least two different fertilizations – has been used as a tool for stem cell pluripotency diagnosis, transgenic rodent production, and organogenesis studies. Additionally, parthenotes are used for studies related to gene imprinting and the ability of their cells to compose the placenta and/or an adult animal. The aim of this work was to validate the production and characterize developmental kinetic of parthenogenetic embryos, obtained from C57BL/6 EGFP mice (EGFP), to determine their potential to produce chimeras, and to localize parthenogenetic cells on the produced blastocyst. Embryos were harvested from superovulated females. For the aggregation, pre-compaction IVF (Swiss Webster/SW strain) or parthenogenetic activated (PGA; SW and EGFP) embryos were used. For PGA, strontium chloride hydrate (5 mM for 6 h) was used. Two experiments were outlined in order to: i) evaluate the development and kinetic from IVF (IVF and in vitro developed; 2-cell stage embryos, n = 53) and from PGA (parthenogenetically activated and in vitro developed; n = 409 oocytes) techniques, both from SW; ii) evaluate the aggregation between pairs of control embryos (C; n = 20, 4-cell in vivo produced embryos from SW) or pairs of 4-cell in vivo produced SW embryos and 4- to 8-cell PGA EGFP embryos (parthenogenetic embryo, PG; n = 40). After manipulation (removal of the zona pellucida and approximation of pairs, for C and PG groups), all the groups were kept in vitro culture (37°C, 5% CO2, and saturated humidity) for 48 to 60 h (C and PG), or up to blastocyst stage (IVF and PGA). Chimerism rate and parthenogenetic cells fluorescence (pre- and post-culture) were evaluated under an inverted microscope with epifluorescence source, and digital images were captured (Eclipse Ti and NIS-Elements, Nikon, Japan, respectively), by merging visible and UV light images. The rate of PGA (EGFP oocytes) was 54.5% (66/121), assessed 6 h post-activation by the presence of at least one pronucleus. The assessment was performed with 20× magnification and Hoffman modulation contrast in an inverted microscope. The rate of blastocyst production between IVF and PGA was significantly different (71.4 and 12.9%, respectively; P &lt; 0.001, Fisher’s exact test). When developmental kinetic was evaluated there a difference in the average time to the majority of embryos reach blastocyst stage for IVF (48 h) and PGA (120 h) was observed. The chimerism rate was assessed by the presence of a single and cohesive cell mass (C) or by the incorporation of EGFP cells in the SW embryo (PG). There was difference (P = 0.006; Fisher’s exact test) between C (55.0%; 11/20) and PG (17.5%; 7/40) chimerism rates, assessed from 48 to 60 h of culture. In PG group, the incorporation of EGFP cells in the obtained chimeras was mainly detected in the trophectoderm although one chimeric blastocyst contained EGFP cells only in the inner cell mass. It was concluded that parthenogenetic embryos have a slower developmental kinetic (until 120 h of culture) than embryos derived from IVF. Acknowledgment to FAPESP for fellowship and funding.

Functional Neuronal Cells Generated by Human Parthenogenetic Stem Cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.

Centrosome Amplification and Chromosomal Instability in Human and Animal Parthenogenetic Cell Lines

Parthenotes have been proposed as a source of embryonic stem cells but they lack the centriole which is inherited through the sperm in all mammalian species, except for rodents. We investigated the centrosome of parthenotes and parthenogenetic embryonic stem cells using parthenogenetic and biparental pig pre-implantation embryos, human and pig parthenogenetic and biparental embryonic stem cells, sheep fibroblasts derived from post implantation parthenogenetic and biparental embryos developed in vivo. We also determined the level of aneuploidy in parthenogenetic cells. Oocytes of all species were activated using ionomycin and 6-dimethylaminopurine (6-DMAP). Over 60% of parthenogenetic blastomeres were affected by an excessive number of centrioles. Centrosome amplification, was observed by microscopical and ultrastructural analysis also in parthenogenetic cell lines of all three species. Over expression of PLK2 and down regulation of CCNF, respectively involved in the stimulation and inhibition of centrosome duplication, were present in all species. We also detected down regulation of spindle assembly checkpoint components such as BUB1, CENPE and MAD2. Centrosome amplification was accompanied by multipolar mitotic spindles and all cell lines were affected by a high rate of aneuploidy. These observations indicate a link between centrosome amplification and the high incidence of aneuploidy and suggest that parthenogenetic stem cells may be a useful model to investigate how aneuploidy can be compatible with cell proliferation and differentiation.