scholarly journals ID3018 Direct reprogramming of mouse somatic cells into pluripotent stem sells by pig germinal vesicle oocytes extract

2017 ◽  
Vol 4 (S) ◽  
pp. 30
Author(s):  
Thuan Van Nguyen
Keyword(s):  
Regenerative Medicine ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Ips Cells ◽  
Mouse Fibroblast ◽  
Fibroblast Cells ◽  
Cytoplasmic Extract ◽  
Reprogramming Factors ◽  
Genomic Reprogramming

Genomic reprogramming factors in the cytoplasm of mature oocytes could be reprogrammed somatic cell cells to totipotency cells and full-term development (cloned animals). Since then, this technique has been considered an important toot not only for animal reproduction but also for regenerative medicine, concervation of endangered species, and for study of genes function and cell biology. Moreover, in order to produce nuclear transfer embryonic stem (ntES) cells using somatic cell nuclear transfer (SCNT), the SCNT technique requires donated fresh oocytes, which raises ethical problems for production in human cloned embryo. For this reason, the use of induced pluripotent stem (iPS) cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, the use of iPS cells for human therapy by the technique of retroviruses used to insert the pluripotent genes into somatic cells could cause tumors in tissues grown from the host iPS cells. Recently, we found that genomic reprogramming factors in the cytoplasm of pig germinal vesicle (GV)-stage oocytes has been shown to improve the efficiency of producing cloned mouse offspring and could reprogram pig fibroblasts to stem-like cells. In this talk, we will discuss whether pig GV cytoplasmic extract could induce pluripotent stem cells from mouse fibroblast cells (interspecies reprogramming). We first established stem-like cells from mouse fibroblast cells treated with GV oocytes extracted (gviPS cells). We demonstrated that reactivation of Oct4 promoter in mouse Oct4-GFP fibroblast cells at day 10 after treated with pig GV oocyte cytoplasmic extract and the formation of colonies is observed at 3 weeks after treatment. In addition, mouse gviPS cells reprogrammed with pig GV cytoplasmic extract can in vitro re-differentiate into neuron-like cells. Interestingly, mouse gviPS cells injected into embryos of different mouse strain could be produced chimeric mice.

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

2008 ◽  
Vol 14 (5) ◽  
pp. 418-432 ◽  
Author(s):  
Zhisheng Zhong ◽  
Yanhong Hao ◽  
Rongfeng Li ◽  
Lee Spate ◽  
David Wax ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Mouse Fibroblast ◽  
Mitochondrial Morphology ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Donor Cells

AbstractWe previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

scholarly journals Stem cell therapies and benefaction of somatic cell nuclear transfer cloning in COVID-19 era

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Birbal Singh ◽  
Gorakh Mal ◽  
Vinod Verma ◽  
Ruchi Tiwari ◽  
Muhammad Imran Khan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Drug Testing ◽  
Human Interaction ◽  
Patient Specific ◽  
Wide Range

Abstract Background The global health emergency of COVID-19 has necessitated the development of multiple therapeutic modalities including vaccinations, antivirals, anti-inflammatory, and cytoimmunotherapies, etc. COVID-19 patients suffer from damage to various organs and vascular structures, so they present multiple health crises. Mesenchymal stem cells (MSCs) are of interest to treat acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection. Main body Stem cell-based therapies have been verified for prospective benefits in copious preclinical and clinical studies. MSCs confer potential benefits to develop various cell types and organoids for studying virus-human interaction, drug testing, regenerative medicine, and immunomodulatory effects in COVID-19 patients. Apart from paving the ways to augment stem cell research and therapies, somatic cell nuclear transfer (SCNT) holds unique ability for a wide range of health applications such as patient-specific or isogenic cells for regenerative medicine and breeding transgenic animals for biomedical applications. Being a potent cell genome-reprogramming tool, the SCNT has increased prominence of recombinant therapeutics and cellular medicine in the current era of COVID-19. As SCNT is used to generate patient-specific stem cells, it avoids dependence on embryos to obtain stem cells. Conclusions The nuclear transfer cloning, being an ideal tool to generate cloned embryos, and the embryonic stem cells will boost drug testing and cellular medicine in COVID-19.

scholarly journals Human therapeutic cloning, pitfalls and lack luster because of rapid developments in induced pluripotent stem cell technology

2014 ◽  
Vol 8 (1) ◽  
pp. 5-10
Author(s):  
Song Hua ◽  
Henry Chung ◽  
Kuldip Sidhu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Therapeutic Cloning ◽  
Induced Pluripotent

AbstractBackground: Therapeutic cloning is the combination of somatic cell nuclear transfer (SCNT) and embryonic stem cell (ES) techniques to create specific ES cells that match those of a patient. Because ES cells derived by nuclear transfer (SCNT ES cells) are genetically identical to the donor, it will not generate rejection by the host’s immune system and thus therapeutically may be more acceptable. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cell artificially derived from an adult somatic cell by inducing a forced expression of a set of specific pluripotent genes. In the past few years, rapid progress in reprogramming and iPS technology has been made, and it seems to shadow any progress made in SCNT programs.Objective: This review compares the application perspective of SCNT with that of iPS in regenerative medicine.Methods:We conducted a literature search using the MEDLINE (PubMed), Wiley InterScience, Springer, EBSCO, and Annual Reviews databases using the keywords “iPS”, “ES”, “SCNT” “induced pluripotent stem cells”, “embryonic stem cells”, “therapeutic cloning”, “regenerative medicine”, and “somatic cell nuclear transfer”. Only articles published in English were included in this review.Results: These two methods both have advantages and disadvantages. Nevertheless, by using SCNT to generate patient-specific cell lines, it eliminates complications by avoiding the use of viral vectors during iPS generation. Success in in vitro matured eggs from aged women and even differentiation of oocytes from germ stem cells will further enhance the application of SCNT in regenerative medicine.Conclusion: Human SCNT may be an appropriate mean of generating patient stem cell lines for clinical therapy in the near future.

scholarly journals Somatic cell nuclear transfer: failures, successes and the challenges ahead

2019 ◽  
Vol 63 (3-4-5) ◽  
pp. 123-130 ◽  
Author(s):  
Marta Czernik ◽  
Debora A. Anzalone ◽  
Luca Palazzese ◽  
Mami Oikawa ◽  
Pasqualino Loi
Keyword(s):  
Endangered Species ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Factors Affecting ◽  
Potential Applications ◽  
Mitochondrial Defects ◽  
Species Production

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

2012 ◽  
Vol 15 (3) ◽  
pp. 509-516 ◽  
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
D. Lipiński
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electrical Activation ◽  
Developmental Outcome ◽  
Fibroblast Cells ◽  
Cleavage Activity ◽  
New Strategy ◽  
Cytoplasmic Hybrids ◽  
Stimulation Of

Abstract The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory.

scholarly journals Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

2007 ◽  
Vol 22 (7) ◽  
pp. 1982-1990 ◽  
Author(s):  
B. Heindryckx ◽  
P. De Sutter ◽  
J. Gerris ◽  
M. Dhont ◽  
J. Van der Elst
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle

scholarly journals Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors

PLoS ONE ◽  
2011 ◽  
Vol 6 (4) ◽  
pp. e14805 ◽  
Author(s):  
Yan Jiang ◽  
Richard Kelly ◽  
Amy Peters ◽  
Helena Fulka ◽  
Adam Dickinson ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Reprogramming Factors

185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 251
Author(s):  
J.-G. Yoo ◽  
M.-R. Park ◽  
H.-N. Kim ◽  
Y.-G. Ko ◽  
J.-Y. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Transfer ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Vitro Fertilization ◽  
Miniature Pigs ◽  
Donor Cells

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

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