Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

396 Oct-4 EXPRESSION IN CAT INNER CELL MASS-DERIVED CELLS CULTURED IN MEDIUM WITH DIFFERENT PROTEIN SOURCES

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
T. S. Rascado ◽  
J. F. Lima-Neto ◽  
S. E. R. S. Lorena ◽  
B. W. Minto ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Domestic Cat ◽  
Santa Cruz

The domestic cat can be used as a biological model for humans because of similarities in some disease and genetically transmitted conditions. Embryonic stem cells might complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective culture system for inner cell mass (ICM)-derived cells in the domestic cat, testing the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies in the presence of fetal calf serum (FCS) and Knockout serum (KS). Moreover, knowing that the transcription factor Oct-4 is important for the maintenance of pluripotency in human and murine embryonic stem cells, the expression of this factor was evaluated in in vitro-produced blastocyst and in the attached ICM. Domestic cat oocytes were matured, fertilized, and cultured in vitro until the blastocyst stage. The ICM was mechanically isolated (n = 60) using a scalpel blade and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 μg mL-1 mitomicin C. The base culture media (BM) was DMEM/F12 supplemented with nonessential amino acids, glutamine, leukemia inhibitory factor, fibroblast growth factor-2, 2-mercaptoethanol, and antibiotics. Three groups were tested: G1 = BM with 20% FCS (20); G2 = BM with 20% KS (20); G3 = BM with 15% FSC and 5% KS (20). Culture was performed in a 5% CO2 in air incubator at 38.5°C. No statistical difference was observed among groups in relation to ICM attachment (chi-square, P > 0.05). Ninety percent of the ICM presented good adhesion after 3 days of culture and started to grow in all media tested. However, until now, no good colonies were formed. Fifteen blastocysts and 10 attached ICM were fixed in 3% paraformaldehyde and permeabilized in 0.2% triton X-100 in PBS. Subsequently, to block nonspecific binding of the primary antibody, the preadsorption for 2 h at room temperature with OCT4 blocking peptide (sc-8628P, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Samples were incubated with Oct4 antibody (N-19 : sc 8628, Santa Cruz Biotechnology) and with the appropriate secondary antibody (A21431, Invitrogen) and examined by fluorescence microscopy. Oct4 protein was detected both in the ICM and trophoderm cells, and it was distributed in cytoplasm and nuclei. These embryos were also stained with Hoechst 33342. Although further standardization of the culture media is needed, it seems that the KS can be replaced by FCS in cat embryonic stem cell culture. Furthermore, the immunostain of the trophoderm with Oct-4 indicates a difference in the expression of this factor when compared with its expression on human and murine blastocysts. This could be related to in vitro production, or Oct 4 is not a good pluripotency marker for cat embryos and cat embryonic stem cell, consequently. This fact has been noted in goat, bovine, and porcine embryos. Acknowledgment is given to FAPESP.

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.

Tracking the progression of the human inner cell mass during embryonic stem cell derivation

2012 ◽  
Vol 30 (3) ◽  
pp. 278-282 ◽  
Author(s):  
Thomas O'Leary ◽  
Björn Heindryckx ◽  
Sylvie Lierman ◽  
David van Bruggen ◽  
Jelle J Goeman ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

scholarly journals The Ethical Case against Stem Cell Research

2003 ◽  
Vol 12 (4) ◽  
pp. 372-383 ◽  
Author(s):  
SØREN HOLM
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Stem Cell Research ◽  
Human Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Ethical Analysis ◽  
Considerable Debate ◽  
Stem Cell Lines

The possibility of creating human embryonic stem cell lines from the inner cell mass of blastocysts has led to considerable debate about how these scientific developments should be regulated. Part of this debate has focused on the ethical analysis and part on how this analysis should influence policymaking.

scholarly journals Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines

2007 ◽  
Vol 22 (12) ◽  
pp. 3051-3058 ◽  
Author(s):  
S. Strom ◽  
J. Inzunza ◽  
K.-H. Grinnemo ◽  
K. Holmberg ◽  
E. Matilainen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Human Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Lines ◽  
Mechanical Isolation

279 COMPARISON OF DIFFERENT METHODS OF ISOLATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO-PRODUCED GOAT BLASTOCYSTS

2008 ◽  
Vol 20 (1) ◽  
pp. 219 ◽  
Author(s):  
A. K. De ◽  
D. Malakar ◽  
Y. S. Akshey
Keyword(s):  
Amino Acids ◽  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Colony Formation ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Es Cell

The present study was carried out to compare different isolation procedures for embryonic stem (ES) cell-like cells from in vitro-produced goat blastocysts. Goat oocytes were aspirated from slaughterhouse-derived ovaries; matured in vitro in maturation medium containing TCM-199 (HEPES modified) with 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, BSA, and 10% estrous goat serum; and fertilized by freshly collected buck semen. The fertilized oocytes were cultured in embryo development medium containing TCM-199 (HEPES modified), 50 µg mL–1 sodium pyruvate, 3.5 µg mL–1 L-glutamine, gentamicin, essential amino acids, nonessential amino acids, BSA, and 10% fetal calf serum, along with goat oviductal cells for further development. A total of 250 blastocysts and 50 hatched blastocysts were used to isolate and culture goat ES cell-like cells. Inner cell mass cells (ICMs) were isolated mechanically from 100 blastocysts and 20 hatched blastocysts and enzymatically from 50 blastocysts and 15 hatched blastocysts. The ICMs were cultured on 10 µg mL–1 mitomycin-C-inactivated goat fetal fibroblast feeder layer. Primary colony formation was significantly higher (P < 0.01) when hatched blastocysts were used for ICM isolation (28/35; 80%) than when blastocysts were used (77/150; 51.3%). However, when ICMs were isolated mechanically, the percentage of primary colony formation was significantly higher (P < 0.01) for blastocysts (66/100; 66%) as well as for hatched blastocysts (18/20; 90%) than when ICMs were isolated enzymatically (15/50; 30%, and 11/15; 73%), respectively, for blastocysts and hatched blastocysts. ES cell-like cells were also isolated from intact blastocysts and intact hatched blastocysts. But the primary colony formation (27/100; 27%), and (6/15; 40%), respectively, was significantly low (P < 0.01) compared to that for mechanically isolated ICMs. Statistical analysis was done by chi-squire test. When the putative ES cell-like cells were subcultured enzymatically (0.25% trypsin–EDTA), they differentiated after 3 passages. However, when the mechanical subculture procedure was followed, the ES cell-like cells maintained their pluripotent state up to the 9th passage, and three goat ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). The putative ES cell-like cells were stained with alkaline phosphatase substrate solution (Sigma, St. Louis, MO, USA). The RT-PCR mediated expression of Oct-4 was done by a special kit Cells-to-cDNA™ II (Ambion, Austin, TX, USA). The ES cell-like cells expressed both alkaline phosphatase and Oct-4. It may be concluded that mechanical isolation of hatched goat blastocysts is the best method for isolation of goat embryonic stem cell-like cells, and they are positive for alkaline phosphatase and Oct-4.

scholarly journals Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast

Cell Reports ◽  
2016 ◽  
Vol 15 (12) ◽  
pp. 2651-2664 ◽  
Author(s):  
Dario Acampora ◽  
Daniela Omodei ◽  
Giuseppe Petrosino ◽  
Arcomaria Garofalo ◽  
Marco Savarese ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Binding Site ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem
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