Parthenogenetic Embryonic Stem Cells Research Articles

Generation of Parthenogenetic Induced Pluripotent Stem Cells from Parthenogenetic Neural Stem Cells

Somatic cells can achieve a pluripotent cell state in a process called pluripotential reprogramming. Multipotent stem cells can differentiate into cells of only one lineage, but pluripotent stem cells can give rise to cells of all three germ layers of an organism. In this study, we generated induced pluripotent stem (iPS) cells from bimaternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with either four (4F: Oct4, Klf4, Sox2, and c-Myc) or two (2F: Oct4 and Klf4) transcription factors. The resultant maternal iPS cells, which were reprogrammed directly from pNSCs, were capable of generating germ line-competent chimeras. Interestingly, analysis of global gene expression and imprinting status revealed that parthenogenetic iPS cells clustered closer to parthenogenetic ESCs than to female ESCs, with patterns that were clearly distinct from those of pNSCs.

Parthenogenetic babies from IVM eggs and tetraploid complementation

OBJECTIVE: Produce offspring from parthegenotes to demonstrate their pluripotency, and test whether activated eggs can contribute to all tissues, and therefore provide a valuable source of cells for regenerative medicine. Women facing cancer therapy or combat could benefit from pluripotent cells for regenerative medicine. Parthenogenetic embryonic stem (pES) cells produced from activated eggs avoid ethical constraints, but their digyneic origin raises concerns about abnormal imprinting, including hypomorphic placenta and limited contributions to some fetal tissues. In vitro maturation (IVM) disrupts imprinting, so paradoxically may help erase digyneic imprinting of parthenogenetic embryos. Also, stem cells undergo extensive epigenetic reprogramming, and tetraploid complementation can furnish a placenta to support development and differentiation of pES cells to pups. DESIGN: Laboratory-based mouse study. MATERIALS AND METHODS: IVM-produced B6C3F1 mouse eggs were activated with strontium, cultured to blastocyts, pES cells produced and characterized, injected into KM tetraploid embryos, transferred to ICR surrogates, and the contribution of pES cells to tissues in resulting pups studied. PCR analysis of DNA microsatellites and allele heterozygosity determined the pups' origin. RESULTS: pES cells were Oct 4, SSEA-1, AP, TERT, Nanog postive, karyotypically normal and produced teratomas in nude mice. We produced for the first time term pups from parthenogenesis by using IVM eggs and tetraploid complementation. Microsatellite and heterozygoisity analyses confirmed the pES cell origin of all pups. CONCLUSIONS: Epigenetic reprogramming from IVM and passage through the stem cell state overcome digyneic imprinting, and tetraploid complementation furnishes placentas to produce term pups from pES cells. This does not justify fatherless reproduction, but does suggest that pES cells may provide a valuable and ethically justifiable source of private pluripotent cells.

Hepatic differentiation of pluripotent stem cells

In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

Human parthenogenetic embryonic stem cells: one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem (iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies. However, the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells. Embryonic stem cells (ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy. Recent studies on human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics, but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions. To generate various pluripotent stem cells, diverse reprogramming techniques and approaches will be developed and integrated. This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology, and ultimately benefit cell therapy and regenerative medicine.

Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells

Mammalian parthenogenetic embryos are not viable and die because of defects in placental development and genomic imprinting. Parthenogenetic ESCs (pESCs) derived from parthenogenetic embryos might advance regenerative medicine by avoiding immuno-rejection. However, previous reports suggest that pESCs may fail to differentiate and contribute to some organs in chimeras, including muscle and pancreas, and it remains unclear whether pESCs themselves can form all tissue types in the body. We found that derivation of pESCs is more efficient than of ESCs derived from fertilized embryos, in association with reduced mitogen-activated protein kinase signaling in parthenogenetic embryos and their inner cell mass outgrowth. Furthermore, in vitro culture modifies the expression of imprinted genes in pESCs, and these cells, being functionally indistinguishable from fertilized embryo-derived ESCs, can contribute to all organs in chimeras. Even more surprisingly, our study shows that live parthenote pups were produced from pESCs through tetraploid embryo complementation, which contributes to placenta development. This is the first demonstration that pESCs are capable of full-term development and can differentiate into all cell types and functional organs in the body.

Correlation of expression and methylation of imprinted genes with pluripotency of parthenogenetic embryonic stem cells

Mammalian parthenogenetic embryos (pE) are not viable due to placental deficiency, presumably resulting from lack of paternally expressed imprinted genes. Pluripotent parthenogenetic embryonic stem (pES) cells derived from pE could advance regenerative medicine by avoiding immuno-rejection and ethical roadblocks. We attempted to explore the epigenetic status of imprinted genes in the generation of pES cells from parthenogenetic blastocysts, and its relationship to pluripotency of pES cells. Pluripotency was evaluated for developmental and differentiation potential in vivo, based on contributions of pES cells to chimeras and development to day 9.5 of pES fetuses complemented by tetraploid embryos (TEC). Consistently, pE and fetuses failed to express paternally expressed imprinted genes, but pES cells expressed those genes in a pattern resembling that of fertilized embryos (fE) and fertilized embryonic stem (fES) cells derived from fE. Like fE and fES cells, but unlike pE or fetuses, pES cells and pES cell-fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1 and U2af1-rs1. Coincidently, global methylation increased in pE but decreased in pES cells, further suggesting dramatic epigenetic reprogramming occurred during isolation and culture of pES cells. Moreover, we identified decreased methylation of Igf2r, Snrpn, and especially U2af1-rs1, in association with increased contributions of pES cells to chimeras. Our data show that in vitro culture changes epigenetic status of imprinted genes during isolation of pES cells from their progenitor embryos and that increased expression of U2af1-rs1 and Snrpn and decreased expression of Igf2r correlate with pluripotency of pES cells.

Change in gene expression of mouse embryonic stem cells derived from parthenogenetic activation

We previously established parthenogenetic mouse embryonic stem cells (ESCs) and this study was subsequently conducted for elucidating the influence of oocyte parthenogenesis on gene expression profile of ESCs. Gene expression of parthenogenetic ESC (pESC)-1 or pESC-2 was separately compared with that of two normally fertilized ESC (nfESC) lines (B6D2F1 and R1 strains), and quantification of mRNA expression was conducted for validating microarray data. In two sets of comparison, reaction of 11 347 and 15 454 gene probes were altered by parthenogenesis, while strain difference changed the expression of 15 750 and 14 944 probes. Level of correlation coefficient was higher in the comparisons between normal fertilization and parthenogenesis (0.974-0.985) than in the comparisons between strains of nfESCs (0.97-0.971). Overall, the expression of 3276-3329 genes was changed after parthenogenesis, and 88% (96/109) of major functional genes differentially (P < 0.01) expressed in one comparison set showed the same change in the other. When we monitored imprinted genes, expression of nine paternal and eight maternal genes were altered after parthenogenesis and 88% (14/16) of these was confirmed by mRNA quantification. The change in gene expression after parthenogenesis was similar to, or less than, the change induced by a strain difference under a certain genetic background. These results may suggest the clinical feasibility of parthenogenesis-derived, pluripotent cells.

Improved Establishment of Autologous Stem Cells Derived from Preantral Follicle Culture and Oocyte Parthenogenesis

This study was conducted to improve establishing autologous embryonic stem cells (ESCs) by culture of preantral follicles and parthenogenetic activation of oocytes. First, paternal inheritance of the follicle donor was changed without altering maternal heredity by employing B6CBAF1 instead of B6D2F1 mice. A significant increase in the establishment of parthenogenetic ESCs was detected after the change, and a different gene expression pro-file was detected in the ESCs established. Among 62 stemness-related genes showing different expression level between two strains, 35 (56.5%) were lower in the rarely established ESCs (B6D2F1) than in the easily established ESCs (B6CBAF1). Several paternally expressed genes were aberrantly expressed in the rarely established ESCs. Second, the establishment of parthenogenetic ESCs in B6D2F1 was significantly improved when preantral follicles were cultured in glutathione (GSH)-containing medium. In the ESCs derived from GSH-treated follicles, 77% of the 62 genes showing the difference increased their expression. Translation of several proteins related to stemness (Wnt-1, beta-catenin, p-p44/42, and smad) was similar between the parthenogenetic ESCs established after GSH treatment and the control E14 ESCs. We concluded that change in genetic inheritance and exposure of in vitro-growing ovarian follicles to GSH contributes to improving establishment of parthenogenetic ESCs, which may help increase the feasibility of the established lines for patient-specific, stem cell therapy.

Human parthenogenetic stem cells

Human parthenogenetic embryonic stem cells (pESCs) were previously thought to be better cell sources for ESC banking because the homozygous Human Leukocyte Antigen (HLA) would strongly increase the matching degree for certain population with relatively smaller cohorts of cell lines. While recent studies have shown that upon currently used strategy to artificial activate oocyte without second polar body extrusion, most pESCs derived would be predominantly heterozygous due to early recombination events in oocyte. Here we reported successful derivation of human homozygous and heterozygous pESC lines from one pronuclear zygote following routine in vitro fertilization treatment and artificial parthenoactivation. The hESC characters, SNPs genotyping, gene expression profiles were conducted and compared to other normal hESCs. The in vitro differentiation ability and the epigenetic stability of imprinted genes of these two cell lines before and post differentiation were further evaluated.

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

Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy

To evaluate whether cryopreserved newborn mouse ovaries can generate sufficient numbers of parthenogenetic mouse embryonic stem (pmES) cells for autologous stem cell therapy. Prospective study. Reproductive clinic of Xinhua Hospital in Shanghai. Kunming, C57BL/6J, BALB/c, and NOD-SCID mice. Cryopreserved newborn mouse ovaries were thawed, grafted into immunodeficient mice, treated with pregnant mare serum gonadotropin to promote follicular maturation, and collected oocytes activated in vitro to generate parthenogenetic embryonic stem cells. Preimplantation development and stem cell characterization. This new protocol yielded a large number of oocytes from cryopreserved ovaries over a long period. These oocytes were used to derive pmES cell lines, which expressed embryonic stem cell-specific markers and differentiated into embryoid bodies in vitro and teratomas in vivo. The pmES cell line was propagated in an undifferentiated state for more than 30 passages and maintained a diploid karyotype. The pmES cells lines established by our protocol exhibited the same degree of pluripotency as standard embryonic stem cell lines. This approach may be used for exploring autologous stem cell therapies.

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.

ASCR-005 Inducing human parthenogenetic embryonic stem cells into insulin-producing cells

ASCR-005 Inducing human parthenogenetic embryonic stem cells into insulin-producing cells

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure

Homozygous human embryonic stem cells (hESCs) are thought to be better cell sources for hESC banking because their human leukocyte antigen (HLA) haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line (chHES-32) from a one-pronuclear oocyte following routine in vitro fertilization treatment. chHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year (>P50) and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, chHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of chHES-32 cells was further confirmed. The results indicated that 'unwanted' one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategy for obtaining homozygous hESC lines from parthenogenetic haploid oocytes.

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.

Generation and characterization of mouse parthenogenetic embryonic stem cells containing genomes from non‐growing and fully grown oocytes

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem cells. Unfortunately, differentiation capacity of the parthenogenetic embryonic stem cells was rather lower than fertilized embryos derived ES cells, which might be the result of the absence of male genome. It had been found that some maternally expressed genes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, maternal genome from non-growing oocytes can partially act as "sperm genome". In the present study, parthenogenetic blastocysts containing genome from non-growing and fully grown oocytes (named as NF-pBlastocysts) were produced by germinal vesicle transfer, and three newly established parthenogenetic embryonic stem (named as NF-pES) cell lines were derived from the resulting parthenogenetic blastocysts. All three NF-pES cell lines were positive for ES cell markers, such as alkaline phosphatase (AKP), stage-specific embryonic antigen 1 (SSEA-1) and octamer-binding transcription factor (Oct-4). They have a normal chromosome karyotype (40) and can be maintained in an undifferentiated state for extended periods of time. When NF-pES cells were injected into severe combined immunodeficient mice, teratomas with all three embryonic germ layers were obtained. The in vitro differentiation potential of NF-pES cells was analyzed by embryonic bodies (EB) formation. The expression of germ layer markers, such as nestin (ectoderm), desmin (mesoderm), and alpha-fetoprotein (endoderm) demonstrated that the NF-pES cells can differentiate into all three germ layers.

Loss of Genomic Imprinting in Mouse Parthenogenetic Embryonic Stem Cells

In mammals, complementary contributions of both the maternal and the paternal genomes are required for normal development because of the parental-allele-specific modification of the genome, called genomic imprinting. Therefore, parthenogenetic embryos (PG) with two maternal genomes cannot develop to term, and PG chimeras show a restricted cell contribution of donor cells and reduced weight, although they can develop to term. On the other hand, parthenogenetic embryonic stem cells (PGES) chimeras are more normal in their tissue contribution of donor cells and body weight compared with PG chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the imprint status in donor cells of PGES and PG chimeras. In somatic lineages, genomic imprinting was lost in some PGES chimeras, whereas those in PG chimeras were almost totally maintained. Moreover, loss of imprints correlated to the gene expression pattern of imprinted genes. Therefore, this loss of imprinting in PGES chimeras could improve the tissue contribution and body weight to a normal level. On the other hand, in germ lineages, both PGES and PG in chimeras showed normal erasure of imprints, indicating that the reprogramming in germ lineages is an inevitable event, regardless of the imprint status of primordial germ cells.

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.

Recombination Signatures Distinguish Embryonic Stem Cells Derived by Parthenogenesis and Somatic Cell Nuclear Transfer

Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.