scholarly journals 24 TRANSGENESIS AND NUCLEAR TRANSFER USING STEM CELLS FROM CULTURED PORCINE PRIMORDIAL GERM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
K.S. Ahn ◽  
H.S. Yang ◽  
S.Y. Heo ◽  
H. Shim
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Donor Cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including their morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of both in vitro and in vivo differentiation have been established (Shim H et al. 1997 Biol. Reprod. 57, 1089–1095). Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide an inexhaustible source of karyoplasts in nuclear transfer (NT). This would be particularly advantageous in maintaining nuclear donor cells carrying a transgene. In addition, genome-wide demethylation of DNA occurs in pre-implantation embryos as well as PGC. Nuclear transfer embryos using EG cells rather than somatic cells may be close to embryos from normal fertilization in their DNA methylation status. If combined with NT technique, EG cells may potentially be useful for genetic manipulation in pigs. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblast and EG cells were compared. Two different techniques were used to perform NT. When conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes was used, the rates of development to the blastocyst stage were 16.8% (59/351) and 14.1% (50/354) in EG and somatic cell NT, respectively. In piezo-driven micromanipulation (Honolulu method) involving direct injection of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 7.5% (12/160), respectively. Although the differences between EG and somatic cell NT were statistically insignificant, the rates of blastocyst development in EG cell NT were comparable to the somatic cell counterpart regardless of NT methods used in the present study. To investigate if EG cells can be used for transgenesis in pigs, GFP gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29/137, 21.2%), and all embryos that developed to the blastocyst stage expressed GFP, based on observation under fluorescence microscope. In this study, the possibility of using EG cells as karyoplast donors in NT procedure was tested. The results suggest that EG cell NT may be used as an alternative to somatic cell NT, and transgenic pig embryos may be produced using EG cells. This research was supported by a grant (SC14033) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Susa Shin ◽  
Kwang Sung Ahn ◽  
Seong-Jun Choi ◽  
Soon Young Heo ◽  
Hosup Shim
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Neural Precursor ◽  
Blastocyst Development ◽  
Donor Cells

SummaryUndifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

scholarly journals The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

2015 ◽  
Vol 38 (2) ◽  
pp. 220-226 ◽  
Author(s):  
Vahid Mansouri ◽  
Mohammad Salehi ◽  
Mohsen Nourozian ◽  
Fatemeh Fadaei ◽  
Reza Mastery Farahani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Embryonic Stem

200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 198
Author(s):  
T. Xiang ◽  
S. Walker ◽  
K. Gregg ◽  
W. Zhou ◽  
V. Farrar ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Donor Cells

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

scholarly journals Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art

2015 ◽  
Vol 27 (1) ◽  
pp. 89 ◽  
Author(s):  
Charles A. Easley ◽  
Calvin R. Simerly ◽  
Gerald Schatten
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Induced Pluripotent ◽  
Normal Offspring

Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.

scholarly journals A Germ Cell-specific Gene, Prmt5, Works in Somatic Cell Reprogramming

2011 ◽  
Vol 286 (12) ◽  
pp. 10641-10648 ◽  
Author(s):  
Go Nagamatsu ◽  
Takeo Kosaka ◽  
Miyuri Kawasumi ◽  
Taisuke Kinoshita ◽  
Keiyo Takubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Cell Reprogramming ◽  
Specific Gene ◽  
Somatic Cell Reprogramming

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

scholarly journals Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Large Set ◽  
Mouse Escs ◽  
Epiblast Stem Cells ◽  
Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

A Novel Method for Somatic Cell Nuclear Transfer to Mouse Embryonic Stem Cells

2005 ◽  
Vol 7 (4) ◽  
pp. 265-271 ◽  
Author(s):  
Danièle Pralong ◽  
Krzysztof Mrozik ◽  
Filomena Occhiodoro ◽  
Nishanthi Wijesundara ◽  
Huseyin Sumer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Novel Method

Collagen-alginate microspheres as a 3D culture system for mouse embryonic stem cells differentiation to primordial germ cells

Biologicals ◽  
2017 ◽  
Vol 48 ◽  
pp. 114-120 ◽  
Author(s):  
Vahid Mansouri ◽  
Mohammad Salehi ◽  
Mir davood Omrani ◽  
Zahra Niknam ◽  
Abdolreza Ardeshirylajimi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cells ◽  
Culture System ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
3D Culture ◽  
Mouse Embryonic Stem Cells ◽  
3D Culture System

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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