Parthenogenetic Cells Research Articles

Accumulation of instability in serial differentiation and reprogramming of parthenogenetic human cells

Human leukocyte antigen-homozygous parthenogenetic stem cells (pSC) could provide a source of progenitors for regenerative medicine, lowering the need for immune suppression in patients. However, the high level of homozygosis and the lack of a paternal genome might pose a safety challenge for their therapeutic use, and no study so far has evaluated the spread and significance of gene expression changes across serial potency changes in these cells. We performed serial rounds of differentiation and reprogramming to assess pSC gene expression stability, likely of epigenetic source. We first derived pSC from activated MII oocytes, and differentiated them to parthenogenetic mesenchymal stem cells (pMSC). We then proceeded to induce pluripotency in pMSC by over expression of the four transcription factors Oct4, Sox2, Klf4 and c-Myc. pMSC-derived iPS (piPS) were further differentiated into secondary pMSC (pMSC-II). At every potency change, we characterized the obtained lines both molecularly and by functional differentiation, and performed an extensive genome-wide expression study by microarray analysis. Although overall gene expression of parthenogenetic cells resembled that of potency-matched biparental lines, significantly broader changes were brought about upon secondary differentiation of piPS to pMSC-II compared with matched biparental controls; our results highlight the effect of the interplay of epigenetic reprogramming on a monoparental background, as well as the importance of heterozygosis and biparental imprinting for stable epigenetic reprogramming.

5 PARTHENOGENETIC EMBRYONIC STEM CELLS ARE CONNECTED BY FUNCTIONAL INTERCELLULAR BRIDGES

We previously reported that parthenogenetic stem cells display abnormal centrosome and spindle formation that results in severe chromosome missegregation, with a high incidence of hypoploid karyotypes. Unexpectedly, this is not accompanied by a correspondingly high rate of apoptosis and, by contrast, parthenogenetic cells share the pluripotency, self-renewal and in vitro differentiation properties of their bi-parental counterparts. We hypothesise that this is possible through a series of adaptive mechanisms that include the presence of intercellular bridges similar to those that connect germ cells during spermatogenesis. This would provide a way for mutual exchange of missing cell products, thus alleviating the unbalanced chromosome distribution that would otherwise hamper normal cell functions. The presence of intercellular bridges was investigated in pig parthenogenetic embryonic stem cells (PESC) by transmission electron microscopy (TEM). Cultured cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration in ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica). Thin sections were stained and observed with a Jeol 1010 electron microscope. Pig PESC were also subjected to scanning electron microscopy (SEM). To this purpose, they were fixed and dehydrated as described above, covered with a 9-nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech) and examined with an SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluorescent 10-kDa dextran was injected into the cytoplasm of a single cell with FemtoJet Microinjector (Eppendorf). Movement of the molecule from the injected cell to others was observed with a Nikon Eclipse TE200 microscope. Ultra-structural analysis of PESC demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterised by the presence of stabilising actin patches. Furthermore, extensive movement of 10-kDa dextran among cells demonstrated functional intercellular trafficking through these communication canals, suggesting their use for transfer of mRNA, proteins and ribosomes among cells. Our results demonstrate that PESC present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions. This process is commonly observed in transformed cells and gives further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines. Supported by AIRC IG 10376. PG was supported by INGM.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

Parthenogenesis in mammals: pros and cons in pluripotent cell derivation

Abstract Embryonic stem cells (ESCs) represent a useful tool for cell therapy studies, however the use of embryos for their derivation give rise to ethical, religious and legal problems when applied to the human. During the last years parthenogenesis has been proposed as an alternative source to obtain ESCs. Based on the fact that parthenotes avoid many concerns surrounding the “ad hoc” in vitro production and following destruction of viable human embryos. Unfortunately many aspects related to parthenogenetic cell biology are not fully understood and still need to be elucidated. In this review we describe advantages and limits of these cells. We discuss their typical ESC morphology and high telomerase activity, which disappears after differentiation. We examine the pluripotency signature that they share with bi-parental ESCs. We review their high differentiation plasticity that allow for the derivation of several mature cell type populations when we expose these cells to adequate conditions. On the other hand, in-depth analysis demonstrated chromosome mal-segregation and altered mechanisms controlling centriole arrangement and mitotic spindle formation in these cells. We hypothesize their monoparental origin as one of the possible cause of these anomalies and suggest a great caution if a therapeutic use is considered.

Global analysis of parental imprinting in human parthenogenetic induced pluripotent stem cells

To study the role of parental imprinting in human embryogenesis, we generated parthenogenetic human induced pluripotent stem cells (iPSCs) with a homozygote diploid karyotype. Global gene expression and DNA methylation analyses of the parthenogenetic cells enabled the identification of the entire repertoire of paternally expressed genes. We thus demonstrated that the gene U5D, encoding a variant of the U5 small RNA component of the spliceosome, is an imprinted gene. Introduction of the U5D gene into parthenogenetic cells partially corrected their molecular phenotype. Our analysis also uncovered multiple miRNAs existing as imprinted clustered transcripts, whose putative targets we then studied further. Examination of the consequences of parthenogenesis on human development identified marked effects on the differentiation of extraembryonic trophectoderm and embryonic liver and muscle tissues. This analysis suggests that distinct regulatory imprinted small RNAs and their targets have substantial roles in human development.

Parthenogenetic Cell Lines: An Unstable Equilibrium Between Pluripotency and Malignant Transformation

Human parthenogenetic embryos have been recently proposed as an alternative, less controversial source of embryonic stem cells. However many aspects related to the biology of parthenogenetic cell lines are not fully understood and still need to be elucidated. These cells have great potentials; they possess most of the main features of bi-parental stem cells, show the typical morphology and express most of the pluripotency markers distinctive of ESC. They also have high telomerase activity, that disappears upon differentiation, and display great plasticity. When cultured in appropriate conditions, they are able to give rise to high specification tissues and to differentiate into mature cell types of the neural and hematopoietic lineages. However, their injection in immune deficient mice has been reported to result in tumor formations. Aberrant levels of molecules related to spindle formation, cell cycle check points and chromosome segregation have also been detected in these cells, that are characterized by the presence of an abnormal number of centrioles and massive autophagy. All these observations indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation versus differentiation in parthenogenetic stem cells. In this manuscript we summarize data related to these aberrant controls and describe experimental evidence indicating their uniparental origin as one of the possible cause. Finally we propose their use as an intriguing experimental tool where the pathways controlling potency, self renewal and cell plasticity are deeply interconnected with cell transformation, in an unstable, although highly controlled, equilibrium between pluripotency and malignancy.

120 SUPPLEMENTATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR DURING IN VITRO MATURATION OF PORCINE IMMATURE CUMULUS - OOCYTE COMPLEXES AND SUBSEQUENT DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

In the present study, pig cumulus–oocyte complexes were cultured in medium supplemented with different concentrations (0, 5, 50, and 500 ng mL–1) of vascular endothelial growth factor (VEGF), and then the maturation and intracellular glutathione (GSH) concentration of oocytes were examined. In addition, the development of oocytes matured with different concentrations of VEGF after parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT) was observed. Although the maturation rate of oocytes was not affected by VEGF concentrations (81.13 ± 2.61%, 83.93 ± 1.97%, 82.14 ± 4.03%, 75.24 ± 2.68%, respectively), the intracellular GSH concentrations of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (12.68 ± 0.08, 12.33 ± 0.53 pMol/oocyte, respectively) than those of oocytes matured with 0 or 500 ng mL–1 VEGF (10.19 ± 0.66, 10.54 ± 0.54 pMol/oocyte, respectively). The blastocyst formation rates after PA of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (58.99 ± 4.70% and 54.00 ± 1.09%, respectively) than that of oocytes matured with 0 or 500 ng mL–1 VEGF (30.15 ± 4.52%, 34.79 ± 4.01%, respectively). Total cell number of PA blastocyst after oocytes matured with 5 and 50 ng mL–1 VEGF was significantly higher (83.21 ± 4.89, 78.16 ± 6.15, respectively) than that of control and 500 ng mL–1 VEGF (56.91 ± 4.78, 55.93 ± 3.89, respectively). Similarly, the blastocyst formation rate after SCNT of oocytes matured with 5 ng mL–1 VEGF was significantly higher (14.54 ± 1.42%) than that of oocytes matured without VEGF (7.95 ± 1.44%). Total cell number of SCNT blastocyst after oocytes matured with 5 ng mL–1 VEGF was significantly higher (67.83 ± 6.56) than control (48.09 ± 5.36). Fully cumulus cell expansion was significantly higher in the 5 ng mL–1 VEGF treated group (85.37 ± 0.73%) compared with the control (58.89 ± 0.88%). In conclusion, adding 5 ng mL–1 VEGF during IVM improved the developmental potential of PA and SCNT in porcine embryos by increasing the intracellular GSH level. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

67 PIG OOCYTES WITH A LARGE PERIVITELLINE SPACE MATURED IN VITRO HAVE GREATER DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER

The size of perivitelline space (PVS) is closely related with the frequency of polyspermic fertilization in pig oocytes. It has been reported that enlargement of PVS is attributed to accumulation of glycoproteins synthesised and secreted from cumulus cells and that culture of immature oocytes in low-salt medium enlarges PVS in pigs. This study examined the developmental competence of pig oocytes after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in relation to the size of the PVS of oocytes matured in vitro (IVM). Cumulus–oocyte complexes were matured in medium 199 (Experiment 1) or porcine zygote medium (PZM)-3 (Experiment 2) supplemented with pig follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then cultured in hormone-free medium for an additional 22 h. IVM oocytes were activated electrically for PA or used as recipient cytoplasts for SCNT. PA and SCNT embryos were cultured for 7 days in PZM-3 medium supplemented with bovine serum albumin. The intracellular glutathione (GSH) level in IVM oocytes was determined by analysing the fluorescence intensity of oocytes after staining with CellTracker Blue CMF2HC. The expression of CDK1, PCNA, and ERK2 mRNA in IVM oocytes was analysed by RT-PCR. Data were analysed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P < 0.05. In Experiment 1, oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.0 pixels/oocyte v. 0.6 pixels/oocyte) and blastocyst formation (54% v. 37%) after PA than oocytes with smaller PVS. In Experiment 2, maturation culture of oocytes in PZM-3 with reduced (61.6 mM) NaCl concentration significantly increased (P < 0.05) the size of the PVS (5.2 μM v. 3.3 μM) compared with control oocytes that were matured in PZM-3 containing 108 mM NaCl, although the treatment did not alter the nuclear maturation. Moreover, oocytes with increased PVS expressed more CDK1, PCNA, and ERK2 mRNA and had higher (P < 0.05) intracellular GSH levels (1.6 pixels/oocyte v. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52% v. 41%) and SCNT (32% v. 18%) compared with control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation resulting from the enhanced GSH level and transcription factor expression and that enlargement of PVS by the culture in low-NaCl medium also improves developmental competence of pig oocytes. This work was supported by grants (#20070301034040 and #20080401034072) from the BioGreen 21 Program (Rural Development Administration, Republic of Korea).

Functional full-term placentas formed from parthenogenetic embryos using serial nuclear transfer

Mammalian parthenogenetic embryos invariably die in mid-gestation from imprinted gene defects and placental hypoplasia. Based on chimera experiments, trophoblastic proliferation is supposed to be inhibited in the absence of a male genome. Here, we show that parthenogenetic mouse embryonic cell nuclei can be reprogrammed by serial rounds of nuclear transfer without using any genetic modification. The durations of survival in uteri of cloned foetuses derived from green fluorescent protein (GFP)-labelled parthenogenetic cell nuclei were extended with repeated nuclear transfers. After five repeats, live cloned foetuses were obtained up to day 14.5 of gestation; however, they did not survive longer even when we repeated nuclear transfer up to nine times. All foetuses showed intestinal herniation and possessed well-expanded large placentas. When embryonic stem (ES) cells derived from fertilised embryos were aggregated with the cloned embryos, full-term offspring with large placentas were obtained from the chimeric embryos. Those placentas were derived from parthenogenetic cell nuclei, judging from GFP expression. The patterns of imprinted gene expression and methylation status were similar to their parthenogenetic origin, except for Peg10, which showed the same level as in the normal placenta. These results suggest that there is a limitation for foetal development in the ability to reprogramme imprinted genes by repeated rounds of nuclear transfer. However, the placentas of parthenogenetic embryos can escape epigenetic regulation when developed using nuclear transfer techniques and can support foetal development to full gestation.

324 CELL LINES DERIVED FROM MAMMALIAN PARTHENOGENETIC EMBRYOS DISPLAY ABNORMAL CHROMOSOME COMPLEMENTS AND ABERRANT CENTRIOLE NUMBER

Mature oocytes can be activated in vitro, leading to the generation of parthenotes that will develop in culture forming blastocysts morphologically indistinguishable from those derived from fertilized eggs. Parthenotes have been used as a source of pluripotent cells that show the traditional features associated with their biparental counterpart: expression of totipotency markers, telomerase activity, embryoid body formation, in vitro differentiation and, in most cases, teratoma formation. However, many aspects still need to be elucidated and, in particular, little attention has been paid to the inci- dence of aneuploidy in these cells. Limited data available for parthenotes derived from different mammalian species indicate a high rate of aneuploidy, whichis consideredtobecaused by the lackofthe paternal contribution, because alterations of the centrosome are knowntolead to multipolar spindles that, in turn, cause aneuploid cells. In this study, we analyzed the rate of aneuploidy and centriole distribution (as a marker of centrosome anomalies) in pluripotent cell lines (pSC) previously derived in our laboratory from pig parthenogenetic embryos and in primary fibroblast cultures and sections obtained from sheep parthenogenetic fetuses (n = 3) that reached 24 days of development in vivo. This protocol was chosen to separate the effect related tooocyte activation from those of the procedures used to derive pSC lines. Centriole number and distribution were assessed both by immunocy- tochemical analysis using an anti-centrin-1 antibody (1 : 200, Abcam, Cambridge, UK) and an appropriate secondary antibody, and by ultrastructural evaluation of thin sections, using a Jeol 1010 EX electron microscope (Jeol, Tokyo, Japan). Karyotyping was performed on mitotically active cells. Metaphases were fully karyotyped under a Leica HC microscope (Wetzlar, Germany). Images were then captured with a Leica DC250 digital camera and cells karyotyped using the Leica CW4000 Karyo software. The results obtained indicate that cell lines of parthenogenetic origin have, in all examined cases, an incidence of aneuploidy significantly higher than that of their respective controls. In particular, although the diploid configuration represented the modal value, the majority of the cells displayed a consistently lower number of chromosomes, between <1N (hypohaploid) and >1N to <2N (hypodiploid).This resultis possibly related toa lossofchromosomes during the mitotic process.Ahigher incidence ofmultiple centrioles was also detected, suggesting that aneuploidy may be related to the lack of paternal contribution that results in abnormal centrosome formation, incorrect control of the process of spindle rearrangement, and consequent chromosomal malsegregation.Abnormal segregation and multicentriolar distribution were not limited to parthenogenetic cell lines but was observed in parthenotes as well, indicating that culture artifacts are unlikely to be the cause. PUR 2007, PUR 2008.

104. PROLIFERATION ABILITY, TELOMERASE ACTIVITY AND MOLECULAR CHARACTERIZATION OF PLURIPOTENT CELL LINES FROM IVF AND PARTHENOGENTIC PIG EMBRYOS

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.

237 SHORT-TERM TREATMENT OF 6-DMAP AND DEMECOLCINE IMPROVE DEVELOPMENTAL COMPETENCE OF PORCINE PARTHENOGENETIC EMBRYOS

Artificial activation is one of the essential factors for successful development of embryos derived from parthenogenetic activation or somatic cell nuclear transfer (SCNT). This study was carried out to confirm whether the short-term treatment of 6-dimethylaminopurine (6-DMAP) or demecolcine would improve developmental competence of porcine parthenogenetic activated embryos and find out the optimal activation protocol. Ovaries were collected from gilts at a local slaughterhouse and cumulus–oocyte complexes were cultured for 44 h in the tissue culture medium-199 with 10% of porcine follicular fluid. Matured oocytes were chemically activated with 10 min treatment of 0.2 mm thimerosal (Thi) followed by 30 min treatment of 8 mm dithiothrethiol (DTT). Additional treatments of 6-DMAP (2 mm) and/or demecolcine (0.4 μg mL–1) for a short term (40 min) or a long term (3 h) were conducted. Activated embryos were cultured in PZM-3. Cleavage rate, blastocyst (BL) formation rate and total cell number of BL were assessed at Day 2, 7 of culture, respectively. All data were subjected to one-way ANOVA followed by Tukey’s test using Prism version 4.0 (Graphpad Software, San Diego, CA, USA) to determine differences among experimental groups. To begin with, we evaluated that effect of short-term treatment of 6-DMAP or demecolcine. Short-term treatment of 6-DMAP improved developmental competence of activated embryos compared to long-term treatment (47.0 ± 1.0% v. 50.5 ± 1.5% for cleavage rate, 5 ± 3% v. 3 ± 1% for BL formation rate, 70 ± 13 v. 37 ± 6 for total cell numbers). Moreover, short-tem treatment of demecolcine also inhibited second polar body extrusion effectively compared with nontreated control (14 ± 2.1 v. 8.3 ± 4.1%). We confirmed that short-term treatment of 6-DMAP or demecolcine is valid. To optimize activation procedure with short-term treatment, development of embryos derived from each treatment were compared among nontreated control (C), 6-DMAP only treatment (6O), demecolcine only (DO) treatment, and 6-DMAP + demecolcine treatment (6 + D) groups. Cleavage rates of 4 groups were similar (53.5 ± 3.0, 62.1 ± 2.9, 53.6 ± 3.0 and 57.0 ± 3.0%, respectively). However, 6 + D group shows significantly greater BL formation rate (18.0 ± 2.5%) compared with C, 6O, or DO groups (9.4 ± 1.9, 14.0 ± 2.3 and 4.3 ± 1.3%, respectively). Total cell number of BL also increased in 6 + D group (52.8 ± 13.0) compared with C, 6O, or DO groups (47 ± 21.5, 31.5 ± 4.22 and 45.2 ± 13.7, respectively). In conclusion, we elucidated that short-term treatment of 6-DMAP or demecolcine improves developmental competence of embryos compared with long-term treatment. We recommend a combination of Thi/DTT with short-term treatment of both 6-DMAP and demecolcine for optimal activation procedure of porcine embryos. This study was supported by BK21, KOSEF (#M10625030005-07N250300510), HANWHA L&C Corp, and Gyeonggido Veterinary Service.

272 LEUKEMIA INHIBITORY FACTOR SIGNALING PATHWAY IN PIG PARTHENOGENETIC PLURIPOTENT CELLS

Leukemia inhibitory factor (LIF), its receptor heterodimer (LRβ-gp130), and the related signal transducer and activator of transcription-3 (STAT3) constitute a system controlling self-renewal and pluripotency of embryonic stem cells (ESC) in the mouse, where LIF withdrawal or direct inhibition of STAT3 causes ESC differentiation. By contrast, several studies have demonstrated that LIF is not required to maintain human ESC pluripotency. Scattered information is available in other species, and data on the role of LIF in pig ESC are scanty. The aims of the present study were (a) to characterize the expression profile of gp130, LRβ, and STAT3 in pig parthenogenetic cell lines (ppC), previously derived in our laboratory and shown to be positive for the main pluripotency related markers; (b) to evaluate the role of LIF pathway in maintaining the pluripotency of these cells. To this purpose, ppC were cultured as previously described (Brevini et al. 2007 Theriogenology 68, 206–214) and screened by RT-PCR for the two LIF receptor subunits (LRβ and gp130) and STAT3. Pig granulosa cells were used as positive controls. To better investigate the possible role of LIF in maintenance of pluripotency in ppC, the formation of embryoid bodies (EB) was induced in the presence or in the absence of the cytokine. Undifferentiated cells were cultured in hanging drops either with or without LIF for 12 days. The EB formation and the expression of molecular markers specific for the three germ layers was evaluated at the end of the differentiation period. Molecular analysis allowed us to detect transcription of STAT3, whereas no signal for LRβ and gp130 was detected in ppC. These results seem to indicate that LIF does not play a role in the maintenance of pluripotency in the pig. However, after removal of LIF, ppC routinely formed EB that expressed molecular markers specific for the three germ layers. On the other hand, when LIF was added to the differentiation medium, pig cells were unable to form EB. They kept proliferating in an undifferentiated state, and no expression of molecular markers specific for the three germ layers was detected. Moreover, when re-plated on inactivated feeder-layers, they formed distinct colonies that maintained expression of pluripotency markers. Our results show that a role of LIF in pluripotency maintenance through a classical LRβ-gp130 and STAT3 activation pathway is unlikely. However, interaction with an alternative nonclassical activation signaling pathway cannot be ruled out. Indeed, the presence of the cytokine in the medium used for differentiation experiments actively inhibited EB formation, indicating a possible role in preventing differentiation in the porcine species. Further studies are needed to elucidate these aspects. Supported by: PRIN2005; PRIN2006; First 2006; First2007.

Production of aneuploids of the cotton hybrid G. barbadense × G. hirsutum L. via intergeneric pollination with Abelmoschus esculentus

The possibility to induce embryo development after pollination of F1 interspecific cotton hybrids (Gossypium barbadense × Gossypium. hirsutum) with pollen from Abelmoschus esculentus was investigated to determine if wide-cross hybrid, haploid or other types of progeny might result. Small numbers of progeny (Pa) were indeed recovered after numerous alien pollinations and in-planta development or in-vitro culture. The Pa plants were characterized phenotypically and studied cytogenetically and microscopically to help establish their origin documenting their reproductive basis. Root-tip chromosome counts and meiotic Metaphase-I analyses revealed that chromosome numbers among cells of the Pa plants ranged from 33 to 44 and that the differences in chromosome number among cells of the same plant ranged from 1 to 3, indicating somatic instability. Flow cytometric analysis also indicated the aneuploid nature of Pa plants. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that alien pollinations may have resulted in parthenogenetic (Pa) egg cell development, or some other unusual reproductive events. The production of wide-crosses and high degrees of aneuploidy could be of use for several types of genomic studies, e.g., functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.

278 DIRECTED NEURONAL DIFFERENTIATION OF PLURIPOTENT CELL LINES DERIVED FROM PIG PARTHENOGENETIC EMBRYOS

Embryonic stem cells (ESCs) can be differentiated into many cell types in vitro, thus providing a potentially unlimited supply of cells for cognitive in vitro studies and cell-based therapy. However, suitable animal models are crucial prerequisites for the development of safe preclinical protocols in biomedical research. Pig has been proposed as an alternative to the mouse model, due to the morphological and functional affinity of the porcine species with the human. We have previously obtained pluripotent cell lines from pig parthenogenetic blastocysts (Brevini et al. 2007 Theriogenology 67, 54–63). These cells expressed pluripotency-related molecules, such as Oct-4, Nanog, SSEA-4, etc., and were negative for markers specific for ectodermal, endodermal, and mesodermal differentiation (Brevini et al. 2007 Theriogenology 68, 206–213). In this study, we investigated their ability to differentiate into the neural lineage. Since no information was available in the literature, we tested and compared two differentiation protocols. Neurospheres were obtained by culturing, for 2 days, undifferentiated parthenogenetic pluripotent cells in hanging drops of DMEM:Ham F10 medium (1:1), supplemented with knockout (KO) serum replacement (10%), fetal calf serum (5%), retinoic acid (10 µm), and Sonic Hedgehog (100 ng mL–1). In protocol A, neurospheres were disaggregated and plated in adherent cultures in neural progenitor basal medium supplemented with neural survival factor-1 (NSF-1, 2%) and brain-derived neurotrophic factor (BDNF, 20 ng mL–1) for 3 weeks. In protocol B, cells were cultured in hanging drops for a further 7 days. Neurospheres were then plated in monolayer and cultured for 3 weeks in DMEM:Ham F12 medium (70:30), supplemented with epidermal growth factor (EGF; 200 ng mL–1), basic fibroblast growth factor (bFGF; 200 ng mL–1), B27 (2%), and N2 (1%). At the end of the culture period, cells were either harvested and screened by RT-PCR for neural differentiation-related markers or fixed and subjected to immunocytochemical fluorescent staining. Protocol A was not suitable for neural differentiation of pig pluripotent cell lines and did not seem to induce any differentiation. In contrast, protocol B could efficiently drive cells toward ectodermal differentiation and neural lineage. Neurospheres showed expression of neurofilament H after culture in hanging drops. At the end of the 21-day culture in monolayer, approximately 18% of the cells originally plated differentiated into more mature cell types of the neural lineage, as shown both by morphology and by staining with nestin and β-tubulin III antibodies. Our results indicate that pig pluripotent parthenogenetic cells can be induced to differentiate into both early and more mature neural subpopulations. These experiments, however, also indicate that the direct use of culture conditions developed for mouse and human cells is not always appropriate for pig cells, and the identification of specifically targeted protocols and media formulations for pig cells are required. This work was supported by PRIN 2005 and FIRST 2006.

CPEB controls oocyte growth and follicle development in the mouse

CPEB is a sequence-specific RNA-binding protein that regulates polyadenylation-induced translation. In Cpeb knockout mice, meiotic progression is disrupted at pachytene due to inhibited translation of synaptonemal complex protein mRNAs. To assess the function of CPEB after pachytene, we used the zona pellucida 3 (Zp3) promoter to generate transgenic mice expressing siRNA that induce the destruction of Cpeb mRNA. Oocytes from these animals do not develop normally; they undergo parthenogenetic cell division in the ovary, exhibit abnormal polar bodies, are detached from the cumulus granulosa cell layer, and display spindle and nuclear anomalies. In addition, many follicles contain apoptotic granulosa cells. CPEB binds several oocyte mRNAs, including Smad1, Smad5, spindlin, Bub1b, Mos, H1foo, Obox1, Dnmt1o, TiParp, Trim61 and Gdf9, a well described oocyte-expressed growth factor that is necessary for follicle development. In Cpeb knockdown oocytes, Gdf9 RNA has a shortened poly(A) tail and reduced expression. These data indicate that CPEB controls the expression of Gdf9 mRNA, which in turn is necessary for oocyte-follicle development. Finally, several phenotypes, i.e. progressive oocyte loss and infertility, elicited by the knockdown of CPEB in oocytes resemble those of the human premature ovarian failure syndrome.

222 LOSS OF IMPRINTS OF PARTHENOGENETIC EMBRYONIC STEM CELLS IN MURINE CHIMERAS

Mammalian parthenotes with the 2 maternal genomes cannot develop to term. By contrast, chimeras produced by parthenogenetic and normal embryos can develop to term. However, parthenogenetic cells contribute to restricted cells and body weights of the chimeras are reduced. These effects are due to aberrant expressions of imprinted genes, with complete methylation of the maternally methylated genes and complete loss of the paternally methylated genes. On the other hand, parthenogenetic ES (PGES) chimeras show more normal tissue contribution of donor cells and body weight compared to parthenogenetic embryo (PG) chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the epigenetic status of maternally methylated genes in murine PG and PGES chimeras. To make parthenogenetic chimeras, PG and PGES cells which express green fluorescent protein (GFP) were introduced into normal host embryos. Mouse embryonic fibroblasts (MEFs) from E13.5 chimeric fetuses were sorted by the fluorescence-activated cell sorter (FACS). Methylation status of parthenogenetic cells was analyzed by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Methylation of maternally methylated genes, Peg1/Mest, Snrpn, and Igf 2r, was almost totally maintained in PG chimeras. Average methylatation percentages of PG-derived MEFs were 80% in Peg1/Mest, 84% in Snrpn, and 81% in Igf 2r (n = 6). In contrast, methylation in some PGES chimeras was partially reduced to normal level in all 3 genes (10–45%, n = 7). To clarify whether demethylation is correlated with expression of the imprinted genes, gene expression was analyzed by quantitative real-time RT-PCR. Among maternally imprinted genes, Peg1/Mest and Snrpn are expressed from the paternal allele, whereas Igf 2r is expressed from the maternal allele. Therefore, in parthenogenetic cells, loss of imprints is expected to up-regulate Peg1/Mest and Snrpn expression, and down-regulate Igf 2r expression. In fact, PGES-derived MEFs were up-regulated in Peg1/Mest and Snrpn expression, and down-regulated in Igf 2r expression. This study revealed that variations of imprint status were observed frequently in somatic cells of PGES cell origin. Demethylation could have occurred during establishment and/or maintenance of PGES cells. This demethylation that occurred in PGES cells could reprogram the maternally methylated imprinted genes and improve tissue contribution and body weight to normal level. The PGES cells with reprogramming ability might be utilized for fertility treatment and regenerative medicine.

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.

Chapter 20 Genomic imprinting and the maternal brain

Those parts of the genome that contain imprinted genes are relatively small (between 100 and 150 genes predicted) but their impact on mammalian development and evolution is substantial. Most of the imprinted genes that have been studied are regulatory: transcription factors, alternative splicers, oncogenes, tumor suppressors, growth factors, or are involved in complex signalling pathways such as the tumor necrosis factor (TNF) and ubiquitin pathways. This review considers the effects of imprinted genes on brain development by examining the distribution of androgenetic and parthenogenetic cells in the brains of chimeric mice using in situ markers. At birth, cells that are disomic for the paternal genome (androgenetic) contribute substantially to the hypothalamus, septum, preoptic area and bed nuclei of the stria terminalis and fail to survive in the developing neocortex and striatum. In contrast, cells that are disomic for the maternal genome (parthenogenetic) proliferate in the cortex and striatum but are excluded from the diencephalic structures. Growth of the brain is enhanced by the presence of parthenogenetic cells and hence increased maternal gene dosage, whereas the brains of androgenetic chimeras are smaller. Mest and Peg3, two imprinted genes that are paternally expressed, have been disrupted by gene targeting and show high levels of expression in regions where androgenetic cells accumulated, namely the hypothalamus, preoptic area and septum. Although of different structural classes and located on different chromosomes, both of these paternally expressed genes influence placental growth and maternal behavior. The implications of these findings for brain evolution and maternal behavior are discussed.

Different influences of genomic imprinting on the development of parthenogenetic cell clones in C57BL/6 and CBA mice

Clonal analysis of parthenogenetic chimeric mouse embryos C57B1/6(PG)<-->BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG)<-->BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.