Developmental ability of cloned embryos from neural stem cells

Mizutani, Eiji; Ohta, Hiroshi; Kishigami, Satoshi; Thuan, Nguyen Van; Hikichi, Takafusa; Wakayama, Sayaka; Kosaka, Mitsuko; Sato, Eimei; Wakayama, Teruhiko

doi:10.1530/rep.1.01010

Cited by 53 publications

(31 citation statements)

References 43 publications

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“…NS (Mizutani et al, 2006) and AFS cells may provide an alternative source of cells, without the need for ES cells, to generate cloned animals in porcine and other species. However, none of the studies on swine somatic cell cloning have compared the developmental ability between cloned embryos derived from NS, AFS cells and other donor cells such as differentiated cells from NS and AFS cells, FF cells, AF cells and MGE cells.…”

Section: R E T R a C T E Dmentioning

confidence: 99%

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells

Zhao

Zheng

2010

Animal

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The aim of this study was to determine the developmental ability of cloned embryos derived from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, differentiated cells from NS and AFS cells, fetal fibroblast (FF) cells, adult fibroblast (AF) cells and mammary gland epithelial (MGE) cells. NS, AFS and FF cells were isolated from embryonic day 30 porcine fetus, AF and MGE cells were isolated from adult pig. NS and AFS cells were induced to differentiate into different cell types, respectively. Stem cells and their differentiated cells were harvested for analysis of the markers using reverse transcription PCR. NS and AFS cells, their differentiated cells, FF, AF and MGE cells were used for nuclear transfer, respectively. A total of 100 two-cell stage cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that the neurospheres were positive for the undifferentiated neural cell marker, Nestin and NS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, Desmin, CD-90, Nanog and Sox2. AFS cells widely expressed NogoA, DCX, CyclinD2, CD133, Hes1, Nanog, Sox2, Oct4, Desmin and CD-90. Both NS and AFS cells were differentiated into astrocyte (GFAP 1 ), oligodendrocyte (GalC 1 ), neuron (NF

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Section: R E T R a C T E Dmentioning

confidence: 99%

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells

Zhao

Zheng

2010

Animal

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“…Unexpectedly, the best organ for source of donor nuclei was the brain, although the specific cell type (or types) of the donor cells has not been determined. Cloned mice have been obtained using the nuclei of embryonic or newborn neural cells (19)(20)(21). However, to our knowledge, no cloned mice have been generated from adult brain cells, and we have also failed to generate cloned mice from fresh adult brain tissues.…”

Section: Discussionmentioning

confidence: 99%

Production of healthy cloned mice from bodies frozen at −20°C for 16 years

Wakayama¹,

Ohta²,

Hikichi³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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112

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Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the ''resurrection'' of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at ؊20°C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to ''resurrect'' animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.brain ͉ cloning ͉ cryopreservation ͉ nuclear transfer ͉ reprogramming C urrent animal cloning techniques using nuclear transfer (1) can reconstruct animals from donor cells; clones so produced differ only in their mitochondrial DNA (2). This technology has produced a variety of cloned animals for scientific and commercial purposes. However, it is not known whether this method could be applied to the recovery of animals from frozen extinct species. Although most, if not all, mammalian cells lose their functional integrity if frozen without cryoprotective reagents, it is conceivable that some aspects of genomic information remain intact even in such dead bodies. By contrast, the genomes of spermatozoa are resistant to freezing (3), and even freeze-drying (4) without cryoprotectants. However, it has been difficult to demonstrate this in somatic cells, due to technical difficulties in assessing genomic integrity.One recent paper has reported on the nuclear integrity of dead cells after freeze-thawing by improved techniques (5). This study successfully produced cloned mouse embryos and generated germ line chimerae via nuclear transferred embryonic stem cell (ntES) cells. However, although all donor cells were dead after thawing, the donor cells were frozen in a medium including polyvinylpyrrolidone, which is known to exhibit cryoprotectant effect (6). Freeze-dried cells were also used for these nuclear transfer attempt, and all cells were dead after rehydration (7,8). Although cloned embryos were obtained from those dead cells following nuclear transfer, no cloned offspring were obtained. Moreover, so far all studies of nuclear transfer using ''dead'' material have used isolated single cells frozen in the laboratory using special media. In dead specimens frozen in natural conditions such as permafrost tundra, the cells of tissue will presumably bind strongly to each other and freeze gradually after death due to the large body size. It remains to be shown whether nuclei can be collected from whole bodies frozen without cryopro...

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“…These findings suggest that use of undifferentiated cells as donor cells in SCNT may improve cloning efficiency. However, the production efficiency of NT embryos for which pluripotent stem cells were used as nuclear donors has been reported to be comparable to, or lower than, that of NT embryos reconstructed with somatic cells [36,37,[51][52][53]. Thus, it remains uncertain whether use of undifferentiated cells as nuclear donors in NT is advantageous.…”

Section: Discussionmentioning

confidence: 99%

Production of Cloned Pigs by Nuclear Transfer of Preadipocytes Following Cell Cycle Synchronization by Differentiation Induction

Tomii

Kurome

Wako

et al. 2009

Journal of Reproduction and Development

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Abstract. Four methods of cell cycle synchronization of porcine preadipocytes for use as nuclear donors in somatic cell cloning were compared: serum starvation, differentiation induction, contact inhibition and roscovitine treatment. After three days of differentiation induction, the percentage of nuclear donor cells synchronized at the G0/G1 phase reached a peak value of 91.8%, which was significantly higher (P<0.05) than the percentage attained by serum starvation (84.9-89.8%), contact inhibition (78.3-83.7%) or roscovitine treatment (67.8-80.3%). Cell cycle synchronization by serum starvation, contact inhibition and roscovitine treatment all increased the percentage of apoptotic cells, while no increase was observed when the donor-cell cycle was synchronized by differentiation induction (Annexin V-positive: 15.7% to 19.3% vs. 7.7%, P<0.05; TUNEL-positive: 12.8% to 14.0% vs. 8.3%, P<0.05). Additionally, comparison of the in vitro development of nuclear transfer (NT) embryos formed from the nuclei of differentiation-induced or serum-starved preadipocytes revealed that, in both cases, a high proportion of embryos developed to the blastocyst stage (39.0 and 33.7%, respectively). In this study, NT embryos reconstructed with preadipocytes synchronized by differentiation induction were transferred to four recipient pigs, three of which gave birth to a total of 17 piglets (4.2%, 17/403). These results demonstrate that donor-cell cycle synchronization by differentiation induction enables effective production of cloned pigs. The findings also indicate that differentiation induction of multipotent cells is an excellent method of cell cycle synchronization that permits highly efficient synchronization of cells at the G0/G1 phase. Key words: Cell cycle synchronization, Multipotent cell, Nuclear transfer, Pig, Preadipocyte, Somatic cell cloning (J. Reprod. Dev. 55: [121][122][123][124][125][126][127] 2009) igs are not only raised as livestock for human consumption, but are also gaining recognition as valuable experimental animals. Expectations are particularly high for the use of cloned and genetically modified pigs produced by somatic cell nuclear transfer (SCNT) in cutting-edge medical research. Already, α1,3-galactosyltransferase gene knockout pigs have been produced as organ donors for xenotransplantation [1][2][3][4][5][6][7][8]. In addition, transgenic pigs serving as human disease models, such as those carrying the gene for endothelial cell nitric oxide synthase (eNOS), have been developed [9]. To further advance the use of cloned and genetically modified pigs in research, however, a SCNT system that enables more efficient generation of embryos is needed.In SCNT, the type and cell cycle phase of the donor cell are important factors that affect the efficiency of generating cloned animals [10][11][12][13][14]. In previous work, we demonstrated that preadipocytes have characteristics that render them excellent nuclear donor cells in porcine SCNT [15]. We have successfully produced cloned piglets by nuclear transfer ...

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Developmental ability of cloned embryos from neural stem cells

Cited by 53 publications

References 43 publications

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells

Production of healthy cloned mice from bodies frozen at −20°C for 16 years

Production of Cloned Pigs by Nuclear Transfer of Preadipocytes Following Cell Cycle Synchronization by Differentiation Induction

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