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

2009 ◽  
Vol 21 (9) ◽  
pp. 20 ◽  
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.

387 DERIVATION OF PUTATIVE EMBRYONIC STEM CELLS FROM PORCINE PARTHENOGENETIC BLASTOCYSTS AND THE LOSS OF PARENTAL-SPECIFIC EXPRESSION PATTERNS IN Igf2/H19 GENES

2010 ◽  
Vol 22 (1) ◽  
pp. 350
Author(s):  
C. K. Lee ◽  
K. J. Uh ◽  
J. K. Park ◽  
H. S. Kim ◽  
H. M. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Polar Body ◽  
Expression Patterns ◽  
Severe Combined Immunodeficiency ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Specific Expression

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).

Isolation and In Vitro Characterization of Putative Porcine Embryonic Stem Cells from Cloned Embryos Treated with Trichostatin A

2011 ◽  
Vol 13 (3) ◽  
pp. 205-213 ◽  
Author(s):  
Ivan Vassiliev ◽  
Svetlana Vassilieva ◽  
Kam P. Truong ◽  
Luke F.S. Beebe ◽  
Stephen M. McIlfatrick ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Trichostatin A ◽  
Embryonic Stem ◽  
In Vitro Characterization

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

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.

Establishment of mouse androgenetic embryonic stem cells by double sperm injection and differentiation into beating embryoid body

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 405-412
Author(s):  
Lei Lei ◽  
Lili Hu ◽  
Tong Li ◽  
Xinghui Shen ◽  
Xiao Liang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Patient Specific ◽  
Imprinted Genes ◽  
Significant Difference ◽  
Androgenetic Embryos

SummaryAndrogenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.

scholarly journals Blastocyst Development after Fertilization with in vitro Spermatids Derived from Non-Human Primate Embryonic Stem Cells

F&S Science ◽  
2021 ◽  
Author(s):  
Sujittra Khampang ◽  
In Ki Cho ◽  
Kanchana Punyawai ◽  
Brittany Gill ◽  
Jacqueline N. Langmo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Blastocyst Development ◽  
Human Primate

scholarly journals Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

2021 ◽  
Author(s):  
Eun-Young Shin ◽  
Seah Park ◽  
Won Yun Choi ◽  
Dong Ryul Lee
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Male Hypogonadism ◽  
Testosterone Levels ◽  
Sequence Of Events ◽  
Short Period ◽  
Molecular Compounds

Abstract Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.

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