scholarly journals 175 ESTABLISHMENT OF PORCINE EMBRYONIC STEM CELL LINE DERIVED FROM IN VIVO BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 238 ◽  
Author(s):  
S.-A. Ock ◽  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
L.-Y. Shi ◽  
S.-L. Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Feeder Layer ◽  
Es Cell ◽  
Stem Cell Line

A porcine embryonic stem (ES) cell line was established from an in vivo-flushed blastocyst. The present study evaluated the effectiveness of IVP, parthenotes and in vivo-produced embryos on establishment of an ES cell line. IVP blastocysts were produced from slaughterhouse ovaries based on the previously reported protocols (2000 Theriogenology 54, 787–797) with minor modifications. Parthenote blastocysts were produced by activation of oocytes matured in vitro with electric stimulation of 2 DC pulses at 2.0 kV/cm for 30 μsec in 0.3 M mannitol solution containing 100 μM CaCl2 and 100 μM MgCl2 in vivo blastocysts were recovered on Day 7 after AI (Day = 0) by flushing the uterus with D-PBS containing 10% FBS from three females. After removal of zona pellucida with 0.2% pronase, the blastocysts were subjected to immunosurgical treatment with 10% rabbit anti-pig serum to isolate the inner cell mass (ICM) as previously reported (1975 PNAS 72, 5099–5102). The ICM was seeded onto the feeder layer of STO which was inactivated by treatment with 10 μg/mL mitomycin for 2.5 h and cultured in DMEM with 0.1 mM β-mercaptoethanol, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 0.1 mM MEM non-essential amino-acid, 20 ng/mL rh-bFGF, 40 ng/mL rh-LIF, 0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, 0.01 mM thymidine, and 15% FBS. The culture was maintained by changing the medium every day after initiation of ICM attachment onto the feeder layer. Any ES-like colonies were individually picked off the feed layer, dissected with 0.25% trypsin-0.02% EDTA for 3–5 min and reseeded on to new STO feed layer. Out of 140 blastocysts (25, in vivo; 55, IVF; 60, parthenotes) used, attaching rates of the ICMs onto the feeder layer were 88% (22/25, in vivo), 56.4% (31/55, IVF), and 58.3% (35/60, parthenotes). A total of 15 primary ES-like colonies was formed in in vivo (3, 12%), IVF (5, 9.1%), and parthenote (7, 11.7%). However, only one ES cell line from in vivo blastocyst was established, which was confirmed as positive by AP activity (Promega, Madison, WI, USA), and was maintained through four passages. In conclusion, for establishment of an ES cell line in pig, the in vivo blastocyst method is superior to currently available methods utilizing IVF or parthenotes. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

197 PRODUCTION OF IN VITRO- AND IN VIVO-DERIVED CAT BLASTOCYSTS FOR THE ESTABLISHMENT OF FELINE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 207 ◽  
Author(s):  
J. Kehler ◽  
M. Roelke-Parker ◽  
B. Pukazhenthi ◽  
W. Swanson ◽  
C. Ware ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Feeder Layers

Identification and characterization of spontaneously occurring genetic diseases in cats has permitted the development of valuable models for testing potential treatments of similar human diseases. With the near completion of the feline genome project, establishment of pluripotential feline embryonic stem (ES) cells would facilitate the targeting of specific genetic loci to produce new feline medical models. Two approaches were used to produce feline blastocysts in an attempt to establish feline ES cells in culture. Naive queens were superovulated with an intramuscular (i.m.) injection of 150 IU of equine chorionic gonadotropin (eCG) followed by an i.m. injection of 100 IU of human chorionic gonadotropin (hCG) 80 h later; follicles were aspirated laparoscopically 24-26 h later for subsequent in vitro fertilization (IVF). On average, 29 mature cumulus oocyte cell complexes (COCs) were recovered from each queen. IVF was performed in 50 microliter drops of complete Hams F-10 medium containing 30 000 fresh, motile sperm. COCs were cultured overnight in 5% carbon dioxide at 38�C, and residual adherent cumulus cells were removed 12 to 16 h later by trituration in 0.1% hyaluronidase. Embryos were cultured in fresh drops of Hams F-10, and on average 25% developed to the early blastocyst stage after 7 days. Alternatively, estrus was induced in queens with a single i.m. injection of 100 IU of eCG, and then 72 h later queens were permitted six supervised matings with a fertile tom over the next two days. Queens underwent ovariohysterectomy 7 days after their first copulation, and compacted morulae and early blastocysts were flushed from the oviducts and uterine horns. On average, eight embryos were recovered from the reproductive tract of each queen. Both in vivo- and in vitro-matured blastocysts were subsequently cultured in standard mouse ES cell medium on inactivated mouse embryonic fibroblasts. When they failed to hatch in culture after 3 days, a 0.5% pronase solution was used to dissolve the zonae pellucidae under microscopic visualization. Denuded expanded blastocysts adhered to the heterotypic feeder layer and primary inner cell mass (ICM) outgrowths formed within 4 days. Outgrowths were mechanically disaggregated into small clusters of 15 to 20 cells and re-plated on fresh feeders. These colonies grew slowly and were transferred after one week onto new feeder layers. The addition of murine or human recombinant leukemia inhibitory factor had no effect on the survival and proliferation of primary outgrowths or subsequent colonies. After 3 weeks, all colonies derived from both in vivo- and in vitro-matured blastocysts had either differentiated or died. Additional experiments are ongoing to test the effects of homotypic feeder layers and alternative growth factors on promoting the establishment and survival of feline ES cell lines. Ultimately, germline transmission of any putative feline ES cell lines will need to be demonstrated in vivo for their utility in gene targeting experiments to be realized.

scholarly journals 173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 237 ◽  
Author(s):  
G. Lazzari ◽  
I. Lagutina ◽  
G. Crotti ◽  
P. Turini ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Es Cell ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

scholarly journals Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

2021 ◽  
Vol 22 (23) ◽  
pp. 12918
Author(s):  
Man-Ling Zhang ◽  
Yong Jin ◽  
Li-Hua Zhao ◽  
Jia Zhang ◽  
Meng Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Novel Approach ◽  
Porcine Embryo ◽  
Development And Differentiation ◽  
Distinct Signaling Pathway

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFβ signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

215 COMPARATIVE ANALYSIS OF PORCINE PLURIPOTENT CELL LINES DERIVED FROM SOMATIC CELLS AND VARIOUS EMBRYONIC ORIGINS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. K. Park ◽  
H. S. Kim ◽  
K. J. Uh ◽  
K. H. Choi ◽  
H. M. Kim ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Pluripotent Cells ◽  
Differentiation Potential

Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts, tremendous efforts have been made to establish embryonic stem cell (ESC) lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to derive pluripotent cells of naïve state that represents full pluripotency, due to the frequent occurrence of spontaneous differentiation into an EpiSC-like state during culture in pigs. We have been able to derive EpiSC-like porcine embryonic stem cell (pESC) lines of a differentiated non-ES cell state from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. In this study, we analysed characteristics such as marker expression, pluripotency and the X chromosome inactivation (XCI) status of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential; female XCI activity and a normal karyotype. Here we provide preliminary results that suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. This work was supported by the BioGreen 21 Program (#20070401034031, PJ0081382011), Rural Development Administration, Republic of Korea.

scholarly journals The birth of embryonic pluripotency

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130541 ◽  
Author(s):  
Thorsten Boroviak ◽  
Jennifer Nichols
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Positional Information ◽  
Eutherian Mammal ◽  
Primitive Endoderm ◽  
Early Cleavage ◽  
Fgf Signalling

Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is protected from ectopic and premature differentiation until it is required to respond to inductive cues to form the fetus. While positional information plays a major role in specifying the trophoblast lineage, segregation of primitive endoderm from epiblast depends upon gradual acquisition of transcriptional identity, directed but not initiated by fibroblast growth factor (FGF) signalling. Following early cleavage divisions and formation of the blastocyst, cells of the inner cell mass lose totipotency. Developing epiblast cells transiently attain the state of naive pluripotency and competence to self-renew in vitro as embryonic stem cells and in vivo by means of diapause. This property is lost after implantation as the epiblast epithelializes and becomes primed in preparation for gastrulation and subsequent organogenesis.

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