48 QUIESCENCE INDUCES LONG-TERM EPIGENETIC CHANGES IN BOVINE FIBROBLASTS THAT IMPROVE THEIR REPROGRAMMING INTO CLONED ANIMALS

2013 ◽  
Vol 25 (1) ◽  
pp. 171 ◽  
Author(s):  
P. K. Kallingappa ◽  
P. Turner ◽  
A. Green ◽  
J. Oliver ◽  
M. Eichenlaub ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Donor Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Starvation ◽  
Functional Assay ◽  
Epigenetic Changes ◽  
G1 Cells

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) bovine adult male fibroblasts versus non-starved, diploid G1 controls. Mechanically synchronized G1 cells were generated by manual selection or mitotic shake-off and processed within 3 h post-mitosis. Based on morphological assessment and 5-ethyl-2′-deoxyuridine (EdU) incorporation during continuous labelling, >93% of cells were captured in G1. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay (ELISA), we show that G0 fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3, and H3K27me3, but not H3K9me1. They were also significantly hypoacetylated at H3K9 and H4K5, hyperacetylated at H4K12, and unchanged at H4K16 positions. Furthermore, G0 cells significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as polycomb group proteins EED, SUZ12, PHC1, and RING2. Following NT into metaphase-arrested oocytes, G0 chromatin condensed slower than that of G1 cells, indicating a more relaxed configuration. After 7 days of in vitro culture, H3K9me3, but not H4K4me3, H3K27me3, SUZ12, and RING2, remained hypomethylated in G0- versus G1-derived NT blastocysts, both in the inner cell mass and trophectoderm (730 v. 550 nuclei from 55 v. 42 G0 v. G1 blastocysts, respectively; n = 7 NT runs). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. Expression of other pluripotency-related factors (NANOG, SOX2, STELLA, and IIFITM3), imprinted genes (SNRPN), and histone demethylases (KDM4A) was not affected in G0-derived blastocysts (32 G0 v. 55 G1 blastocysts; n = 4). Following NT, G0 donors developed significantly better into cloned blastocysts (175/382 = 46% v. 122/332 = 37% for G0 v. G1, respectively; n = 7, P < 0.05). Likewise, after transfer into surrogate mothers, G0-derived blastocysts developed significantly better into live calves (5/18 = 28% v. 1/25 = 4% for G0 v. G1, respectively; n = 2, P < 0.05). In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability. This research was supported by FRST C10X0303.

2 BOVINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2017 ◽  
Vol 29 (1) ◽  
pp. 108
Author(s):  
Y. S. Bogliotti ◽  
J. Wu ◽  
M. Vilariño ◽  
K. Suzuki ◽  
J. C. Belmonte ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basal Medium ◽  
Primitive Endoderm ◽  
Culture Dish ◽  
Rock Inhibitor ◽  
Immunodeficient Mice

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date, it has been challenging to establish pluripotent ESC lines for domestic animals, which could be important for biotechnological applications, such as genetic engineering and SCNT, and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech) with 4 mg mL−1 BSA for 7 days until they reached the blastocyst stage (Ross et al., 2009 Reproduction 137, 427–437). At that point, the zona pellucida (ZP) was removed using 1 mg mL−1 Pronase (Sigma, St. Louis, MO), and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts, mechanically isolated ICM, and immunosurgery-derived ICM. In each case, individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20 ng mL−1 FGF2 and 2.5 μM IWR1 (CTFR) (Wu et al. 2015 Nature 521, 316–321). After 48 h, blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter, the media was changed daily. Outgrowths (after 6–7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632, 10 μM) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF), RNA-seq, and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16, 56.3%), mechanical ICM isolation (7/12, 58.3%), and immunosurgical ICM isolation (7/16, 43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency), but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1, NANOG, SOX2, LIN28B, DNAMT3B, UTF1, SALL4) were expressed (RPKM > 0.4), while trophectoderm markers (CDX2, GATA2, GATA3, FGF4, TFAP2A) and primitive endoderm markers (GATA6, HNF4A) were not expressed (RPKM < 0.4). Finally, bESC lines (n = 2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1, endoderm: FOXA2, and mesoderm: ASM). In conclusion, the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC.

scholarly journals β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1593
Author(s):  
Meng Zhang ◽  
Yiwei Lai ◽  
Vladislav Krupalnik ◽  
Pengcheng Guo ◽  
Xiangpeng Guo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Ground State ◽  
Transcription Initiation ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Cell Cycle Gene

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Chiaki Sano ◽  
Asako Matsumoto ◽  
Eimei Sato ◽  
Emiko Fukui ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Long Term Culture ◽  
Oct4 Expression ◽  
Feeder Layers

SummaryEmbryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

scholarly journals Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

2021 ◽  
Author(s):  
Hongling Zhang ◽  
Yuanyuan Li ◽  
Yongjian Ma ◽  
Chongping Lai ◽  
Qian Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Early Passage ◽  
Developmental Potential ◽  
Passage Cells ◽  
Androgenetic Haploid

AbstractThe use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.

scholarly journals MED20 is essential for early embryogenesis and regulates NANOG expression

Reproduction ◽  
2019 ◽  
Vol 157 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Wei Cui ◽  
Chelsea Marcho ◽  
Yongsheng Wang ◽  
Rinat Degani ◽  
Morgane Golan ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Blastocyst Stage ◽  
Mediator Complex ◽  
Mammalian Development ◽  
Lineage Specification ◽  
Mammalian Embryo ◽  
Pol Ii

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.

52 THE EFFECTS OF DONOR CELL CYCLE AND THE TIMING OF OOCYTE ACTIVATION ON DEVELOPMENT OF BOVINE NUCLEAR TRANSFERRED EMBRYOS IN VIVO

2011 ◽  
Vol 23 (1) ◽  
pp. 132 ◽  
Author(s):  
K. Matsukawa ◽  
S. Akagi ◽  
K. Fukunari ◽  
Y. Hosokawa ◽  
C. Yonezawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Embryo Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Serum Starvation ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
G0 Phase ◽  
G1 Cells

The cell cycle of donor cells and recipient cytoplasts are important factors affecting development of nuclear transferred (NT) embryos. We previously showed that bovine NT embryos using pre-activated cytoplasts and early G1 cells had a high in vitro developmental rate (SSR, 2008, 41st Annual Meeting). The objective of the present study was to evaluate the effects of donor cell cycle (early G1 or G0 phase) and the timing of oocyte activation on fetal development of bovine NT embryos. Adult fibroblasts from ear skin tissue of Japanese black cattle were used as donor cells. The G0 phase cells were synchronized by serum-starvation, and the G1 phase cells were prepared from actively dividing M phase cells. NT embryo production was performed by 2 kinds of protocols as follows: 1) recipient oocytes were activated by Ca ionophore (CaI), followed with cycloheximide (CH) for 2 h, and fused with synchronized donor cells followed with cytochalasin D (CD) and CH for 1 h, then CH for 4 h (pre-activated), 2) unactivated oocytes were fused with synchronized donor cells and activation was performed by CaI 1 h after fusion, followed by with CD and CH 1 h, then CH for 4 h (post-activated). After activation treatments, NT embryos were cultured in IVD101 medium for 7 days. Then, blastocysts were transferred to recipient cows. Diagnosis of pregnancy was made by ultrasonography at days 30, 60, and 90 (Day 0 = the day of embryo transfer). As shown in Table 1, the blastocyst formation rate of the NT embryos derived from early G1 cells in the pre-activated group was higher than that from G0 cells in the post-activated group (36% v. 23%, P < 0.05). After embryo transfer, 29, 67, and 50% of recipient cows were pregnant at Day 30 in G0 post-, G1 post-, and G1 pre-activated groups, respectively. However, only 1 embryo (14%) of G0 post-activated group developed to term. In conclusion, bovine NT embryos using early G1 cells and pre-activated cytoplasts showed a high blastocyst formation rate, but the full-term development of bovine NT embryos could not be improved by using early G1 cells and pre-activated cytoplasts. Table 1.Effect of the timing of oocyte activation on developmental ability of bovine NT embryos derived from early G1 or G0 phase cells

Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro

2017 ◽  
Vol 29 (6) ◽  
pp. 1064 ◽  
Author(s):  
A. Murillo-Ríos ◽  
V. Maillo ◽  
M. Muñoz ◽  
A. Gutiérrez-Adán ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gestation Length ◽  
Individual Culture ◽  
Cell Counts ◽  
Activating Transcription Factor 4 ◽  
Short And Long Term

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24 h in culture (P = 0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P < 0.06) mostly as a result of a higher number of miscarriages (P < 0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P < 0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P = 0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P < 0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.

001. ISOLATION AND CHARACTERISATION OF PORCINE ES CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 1
Author(s):  
M. B. Nottle ◽  
I. M. Vassiliev ◽  
S. Vassilieva ◽  
L. F. S. Beebe ◽  
S. J. Harrison ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
The Body ◽  
Domestic Species ◽  
Serum Replacement

Embryonic stem (ES) cellshave the capacity for self renewal, can remain undifferentiated in long term culture and can contribute to all the cells in the body including the germ cells. EScells have been isolated in mice and have also been described for humans. However despite considerable effort for more than two decades ES cellswhich can contribute to the germline are yet to be isolated for the pig or any domestic species for that matter. We have developed a new method for isolating porcine ES cells which uses whole embryos cultured in alpha MEM with 10% serum replacement plus additives under 5% O2. Unlike methods employed previously this method results in homogenous outgrowths whose cells resemble ES cells and which express Oct 4 and Nanog and SSEA-1 [1]. These cells can be passaged and cryopreserved repeatedly resulting in the establishment of cell lines at similar efficiencies to that reported previously for 129Sv mice [2]. These cells can form embryoid bodies and can be differentiated to various cell types representative of all three germ layers [3]. Following their injection into blastocysts these cells localise /become incorporated in the inner cell mass and can be used to produce chimaeras when these embryos are transferred to recipient animals [2]. To date we have produced chimaeric pigs from one male ES cell line [2]. These are currently being mated to demonstrate germline transmission. Future studies will examine the applicability of our method to other species commencing with mice and cattle before extending these to humans.

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