Parthenogenetic Embryonic Stem Cell Lines Research Articles

Meiotic Instability Generates a Pathological Condition in Mammalian Ovum.

Maintenance of metaphase-II (M-II) arrest in ovum is required to present itself as a right gamete for successful fertilization in mammals. Surprisingly, instability of meiotic cell cycle results in spontaneous exit from M-II arrest, chromosomal scattering and incomplete extrusion of second polar body (PB-II) without forming pronuclei so called abortive spontaneous ovum activation (SOA). It remains unclear what causes meiotic instability in freshly ovulated ovum that results in abortive SOA. We propose the involvement of various signal molecules such as reactive oxygen species (ROS), cyclic 3',5' adenosine monophosphate (cAMP) and calcium (Ca2+)in the induction of meiotic instability and thereby abortive SOA. These signal molecules through their downstream pathways modulate phosphorylation status and activity of cyclin dependent kinase (cdk1) as well as cyclin B1 level. Changes in phosphorylation status of cdk1 and its activity, dissociation and degradation of cyclin B1 destabilize maturation promoting factor (MPF). The premature MPF destabilization and defects in other cell cycle regulators possibly cause meiotic instability in ovum soon after ovulation. The meiotic instability results in a pathological condition of abortive SOA and deteriorates ovum quality. These ova are unfit for fertilization and limit reproductive outcome in several mammalian species including human. Therefore, global attention is required to identify the underlying causes in greater details in order to address the problem of meiotic instability in ova of several mammalian species icluding human. Moreover, these activated ova may be used to create parthenogenetic embryonic stem cell lines in vitro for the use in regenerative medicine.Graphical abstract.

Imprinting Status in Two Human Parthenogenetic Embryonic Stem Cell Lines: Analysis of 63 Imprinted Gene Expression Levels in Undifferentiated and Early Differentiated Stages.

Human parthenogenetic embryonic stem cells (hPESCs) represent a source of histocompatible tissues for transplantation and carry two copies of the maternal genome, but lack the paternal genome. In this study, we selected 63 known human imprinted genes to investigate the imprinting status of hPESC. The expression level of these genes, including 27 maternally and 36 paternally imprinted were illustrated in hPESC and human embryonic stem cells (hESCs) derived from fertilized embryo lines. The expression activity changes of these genes were analyzed in undifferentiated and early differentiated hPESC lines. In addition, the methylation status of four differentially methylated regions (DMRs) of the imprinted genes was analyzed in undifferentiated and early differentiated hPESC and hESC lines. As a result, we found that all the maternally imprinted genes were expressed at similar levels in the undifferentiated hPESC lines and the hESC lines, except ZNF264 and ATP10A. Twenty-one analyzed paternal imprinted genes were expressed at the same level in two separated hPESC lines as well as compared with the hESC lines, whereas 15 other paternal imprinted genes were significantly downregulated or inactivated in hPESC lines as compared with the hESC line. During prolonged passage, the expression levels of the majority of imprinted genes remained stable in two hPESC lines. The four DMRs, including PEG3/ZIM2 (DMRs), SNURF/SNRPN DMRs, and KVDMR1 DMRs are highly methylated in the genes of two undifferentiated hPESCs and its embryonic bodies (EBs), whereas the genes of the undifferentiated hESCs and its EBs are half methylated. During the early differentiation stage, the imprinted genes showed the same expression trend and the expression levels of H19, IGF2, SLC22A2, SLC22A3/SLC22A18, and CPA4 were significantly upregulated in both hPESC lines. As conclusion, hPESCs show a substantial degree of epigenetic stability with respect to some imprinted genes.

Generation of mouse parthenogenetic embryonic stem cells and preliminary study of the differentiation ability to motor neurons

In this study, we generated embryonic stem cells from parthenogenetic embryos (PESCs), and induced them to differentiate to motor neurons, which could be an alternative source of histocompatible cells for replacement of therapy and theoretical foundation for studying the relationship of genome imprint and neural differentiation. The parthenogenetic activation rate of B6D2F1 mouse oocytes was 93.26%. We established eight parthenogenetic embryonic stem cell lines and the establishment rate from parthenogenetic embryos was 23.53%. The pluripotency marker Oct4, the cell surface marker SSEA-1, and alkaline phosphatase exhibited in PESCs. Karyotype analysis showed normal 40 chromosomes when examined at passages 10 and 30, which was in accordance with their oocyte origins. Three germinal layers were differentiated in vivo and in vitro. Mouse PESCs, which were treated by tretinoin and sonic hedgehog with extracellular matrix, could generate motor neurons that expressed the specific markers such as HB9 and Olig2.

Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders

Feeder cells from animals raise considerable concern for contamination because they are directly in contact with embryonic stem cells. To address this issue we collected discarded foreskin tissue and prepared a fibroblast cell line. We transferred one parthenogenetic blastocyst on to these feeder cells, and later observed outgrowth. By this approach, we were able to derive a human parthenogenetic embryonic stem cell line successfully. The embryonic stem cells had normal morphology, expressed all expected cell surface markers, could differentiate to embryonic bodies upon culture in vitro, and differentiated further to derivatives of all three germ layers. This study indicates that homologous human fibroblasts can be used as feeder cells to support not only the propagation, but also the derivation of ES cells, and this should facilitate studies of therapeutic cloning for research and clinical applications.

Two active X Chromosomes and methylation of the imprinted gene in human parthenogenetic embryonic stem cell line

Two active X Chromosomes and methylation of the imprinted gene in human parthenogenetic embryonic stem cell line

Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem (pES) cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2af1-rs1, Peg3, Impact, Zfp127, Dlk1 and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activation of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.

171 NORMAL REPROGRAMMING OF IMPRINTING IN PARTHENOGENETIC FEMALE GERM CELLS

Mammalian parthenotes cannot develop normally to term. Mouse parthenogenetic embryos die by Day 10 of gestation. On the other hand, viable parthenogenetic chimeras were produced by normal host embryos, although parthenogenetic cells were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. This can be explained by the aberrant expressions of imprinted genes in parthenogenetic cells. In female mice, erasure of imprints occurs around the time that primordial germ cells enter the gonad, and establishment of imprints occurs in the postnatal growth phase of oogenesis. In this study, we investigated whether aberrant imprints in parthenogenetic embryonic stem (PgES) cells can be erased through the germline. Diploid parthenogenetic embryos were produced by activation of (CBA × C57BL/6-EGFP) F1 mouse superovulated unfertilized oocytes by exposure to Sr2+ and cytochalasin B. Ten parthenogenetic blastocysts were plated and three PgES cell lines were isolated. Chimeras were made by injecting 10–15 PgES cells into ICR(CD-1) mouse blastocysts. Chimeras and chimeric tissues were detected by fluorescent microscopy. In all, 173 chimeric blastocysts were transferred to 9 recipient females, and 101 live pups containing 9 female and 21 male chimeras were born. No significant growth retardation was apparent in PgES chimeras, irrespective of their degree of chimerism. In 5 male chimeras killed at 1 day postpartum (dpp), PgES cells showed a restricted tissue contribution. The contribution to lung, liver, and intestine was considerably lower than in the other tissues such as brain, heart, spleen, and kidney. PgES derived or host embryo derived non-growing oocytes were isolated from dissociated ovaries of female chimeras at 1 dpp under fluorescent microscopy. Methylation imprints in non-growing oocytes were analyzed for maternally methylated imprinted genes Peg1, Snrpn, and Igf2r by the combined bisulfite restriction analysis (COBRA). In normal oocytes, imprints are expected to be erased and these genes are unmethylated at this stage. We observed that these genes were unmethylated in both PgES derived and host embryo derived non-growing oocytes. These results suggest that aberrant imprints in PgES cells can also be erased normally through the germline.