160 Derivation and evaluation of bovine embryonic stem cells from early and full blastocyst-stage embryos

Porcine embryonic stem cells (ESC) can be a useful tool for the production of a transgenic animal and the study of developmental gene regulation. The study of porcine parthenogenetic ESC might also provide advantages in the understanding of changes in human parthenogenetic embryonic stem cells in the culture environment. Because human embryonic stem cells must be maintained stably for therapeutic uses, parthenogenetic porcine embryonic stem cells can give us precious information to help understand human parthenogenetic embryonic stem cells. Three putative porcine embryonic stem cell lines were derived from 99 parthenogenetic embryos. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Diploid parthenogenetic zygotes were produced by electrical activation followed by cytochalasin B treatment to suppress second polar body extrusion. Embryos were cultured to the blastocyst stage. Hatched blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Primary colonies were formed after 7 days of culture, and the colonies were transferred to new culture dishes 7 days after. They were passsaged every 5 days by physical dissociation, with one colony divided into small clumps and maintained for over 30 passages. These cells morphologically resembled human embryonic stem cells and consistently expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, OCT-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. They could be maintained holding the previous characteristics after cryopreservation. Furthermore, we conducted experiments to confirm the expression patterns of the imprinted genes Igf2 and H19 in these ESC and IVF/parthenogenetic blastocysts using quantitative real-time PCR. At the blastocyst stage, the 2 genes were expressed in a parental-specific manner according to their origins in normal fertilized embryos and uniparental embryos. The putative parthenogenetic ESC, on the other hand, showed a high expression of Igf2, the paternally expressed gene, when compared with their blastocyst counterparts. Current work aims to confirm the authenticity of these ESC via teratoma formation in severe combined immunodeficiency mice following injection with these putative parthenogenetic ESC. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

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Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Development ◽

10.1242/dev.128.12.2333 ◽

2001 ◽

Vol 128 (12) ◽

pp. 2333-2339 ◽

Cited By ~ 12

Author(s):

Jennifer Nichols ◽

Ian Chambers ◽

Tetsuya Taga ◽

Austin Smith

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Mouse Embryos ◽

Preimplantation Mouse ◽

In The Wild ◽

Lactating Females ◽

Hoechst Staining

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130−/− embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130−/− delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.

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101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

Reproduction Fertility and Development ◽

10.1071/srb09abs101 ◽

2009 ◽

Vol 21 (9) ◽

pp. 20 ◽

Cited By ~ 1

Author(s):

K. P. Truong ◽

I. Vassiliev ◽

L. F.S. Beebe ◽

S. M. McIlfatrick ◽

S. J. Harrison ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Trichostatin A ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Electrical Pulse ◽

Blastocyst Development ◽

Significant Difference ◽

Cloned Pig

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P < 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.

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229 DERIVATION OF PRESUMPTIVE GONOCYTES IN VITRO FROM PRIMATE EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab229 ◽

2007 ◽

Vol 19 (1) ◽

pp. 231

Author(s):

T. Teramura ◽

N. Kawata ◽

T. Takehara ◽

N. Fujinami ◽

M. Takenoshita ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Lines ◽

Germ Cells ◽

Nonhuman Primates ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Embryoid Bodies

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis, because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES (cyES) cell lines and attempted to induce their differentiation into germ cells in order to obtain further information on the development of primate germ cells by observing the transcripts of some markers reported as specific for germ cells. CyES cell lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing superovulation, females were treated with 25 IU kg-1 pregnant mare serum gonadotropin once a day for 9 days, followed by 400 IU kg-1 hCG. Oocytes were collected at 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For cyES cell establishment, inner cell masses (ICMs) were isolated by immunosurgery. The ESC colonies developed at about 10 days after ICM plating, and 3 cell lines were successfully established (3/11; 27.3%). All cell lines expressed Oct3/4, SSEA-4, and ALP activity. These ESCs formed teratomas containing 3 different embryonic layers when injected into SCID mice. And the cells could be passaged over 50 times without losing their original properties. To observe in vitro gametogenesis, we attempted to induce differentiation by non-adherent conditions. When cyES cells differentiated spontaneously, the aggregated structures (i.e. embryoid bodies; EBs) accumulated vasa, the expression of which is restricted to germ cells, and some meiotic markers such as dmc1 and sycp1 that exist only in synaptonemal complexes in meiosis. The existence of these markers was also confirmed by immunocytochemistry on cryosections. Interestingly, these products expressed oct4 and nanog again at Day 16, though the expression of both genes diminished at once with onset of differentiation. In vivo, it is reported that vasa, oct4, and nanog are expressed in migrating PGCs, posibly throughout the development of germ cells into spermatocytes/oocytes. Given the results obtained with the meiotic markers, it is possible that developing germ cells such as PGCs or gonocytes could be formed in cynomolgus EBs as in previous cases with mouse or human EBs. These results demonstrate that cyES cells might contribute to putative germ cells in vitro by differentiating into EBs and could be used as a model for studying mechanisms of germ cell development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

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194 ESTABLISHMENT OF GOAT EMBRYONIC STEM CELL-LIKE LINES DERIVED FROM IN VIVO-PRODUCED BLASTOCYST-STAGE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab194 ◽

2006 ◽

Vol 18 (2) ◽

pp. 205

Author(s):

E. Behboodi ◽

I. Begin ◽

K. Rao ◽

N. Neveu ◽

J. Pierson ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Feeder Layer ◽

Feeder Cells ◽

Fetal Fibroblast ◽

Embryonic Disc

Pluripotent embryonic stem cells (ESC) derived from the inner cell mass (ICM) of mammalian blastocysts provide AN unlimited number of cells that can be used in gene targeting and be of great value to agriculture and medicine. Embryonic stem cells with capacity for germ line transmission have been verified only in mouse despite many efforts to derive ESC from other mammalian species. The methods for the derivation, propagation and differentiation of ESC from domestic animals have not been fully established. The objective of this study was the generation and initial characterization of goat embryonic stem cells (GESC) derived from in vivo-produced blastocyst-stage embryos. Goat compact morulae and blastocysts were collected from superovulated adult Saanen crossbreed donors 7 days after insemination with a fertile male. Embryos were collected by uterine flushing using a 12Fr. Foley catheter by means of a laparoscopically assisted mid-ventral laparotomy under general anesthesia. Twenty eight in vivo-derived blastosyst-stage embryos were cultured on a goat fetal fibroblast feeder layer (inactivated by Mitomycin C) in a medium of DMEM containing 0.1 mm �-mercaptoethanol, 0.1 mm MEM nonessential amino acids, 200 mm l-glutamine, and 10% FCS. Following three days in culture 25 of 28 embryos hatched. Ten embryos attached to the feeder layer, nine degenerated, and nine embryos were floating in the medium and expanding in size. After 5-7 days in culture four of the tehn attached embryos appeared with a prominent an ICM outgrowth and 3 of the nine floating embryos formed structures resembling ICM disc surrounded by trophectoderm cells. The ICM and the embryonic discs were isolated mechanically and cultured on goat feeder cells in DMEM medium containing 10 ng/mL horse leukemia inhibitory factor (hLIF) and 10% FCS. The ICM and embryonic disc outgrew into colonies on Day 4 post-culture. Compact colonies of cells from these outgrowths were isolated mechanically and passed onto fresh goat feeder cells every 4-5 days with the addition of 10 ng/mL hLIF to the culture media. Established colonies at passage 6 were tested for immunoreactivity against alkaline phosphatase (AP) and Oct-1 using standard protocols. The result was the estabilshment of embryonic stem cell-like colonies (57%) from both ICM and embryonic discs cultured on goat fetal fibroblast feeder cells. Colonies forming from these outgrowths (50%) of either ICM or embryonic disc stained positive for both AP and Oct-4. Embryoid bodies formed from colonies of either ICM or embryonic disc in suspension (DMEM containing 10% FCS with no hLIF). Two cell lines (one from ICM and one from embryonic disc) have been maintained so far through passage 8 and have been cryopreserved. These GESC-like lines will be used in further characterization and ultimately in transgenic animal production studies.

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