294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance. The authors acknowledge N.D.R.I for the financial and infrastructural assistance.

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Nuclear transfer and oocyte cryopreservation

Reproduction Fertility and Development ◽

10.1071/rd08218 ◽

2009 ◽

Vol 21 (1) ◽

pp. 37 ◽

Cited By ~ 6

Author(s):

Ching-Chien Chang ◽

Li-Ying Sung ◽

Tomokazu Amano ◽

X. Cindy Tian ◽

Xiangzhong Yang ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Regenerative Medicine ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Present Review ◽

Oocyte Cryopreservation ◽

Patient Specific ◽

Therapeutic Cloning ◽

Human Oocytes

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.

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Patient-Specific Pluripotent Stem Cells for Hematologic Disease.

Blood ◽

10.1182/blood.v114.22.sci-40.sci-40 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-40-SCI-40

Author(s):

George Q. Daley

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Nuclear Transfer ◽

Es Cells ◽

Somatic Cells ◽

Embryonic Stem ◽

Ips Cells ◽

Patient Specific ◽

Direct Reprogramming ◽

Disease Specific

Abstract Abstract SCI-40 Pluripotent stem cells can be isolated from embryos (embryonic stem cells; ES cells) or generated by direct reprogramming of somatic cells (induced pluripotent stem cells; iPS cells). Both types can be differentiated into a multitude of cell lineages to serve disease research and cell replacement therapies. Additionally, genetically matched pluripotent stem cells generated via nuclear transfer (ntES cells), parthenogenesis (pES cells), or direct reprogramming (iPS cells) are a possible source of histocompatible cells and tissues for transplantation. We have used customized ntES cells to repair genetic immunodeficiency in mice (Rideout et al., Cell 2002); however, generation of ES cells by nuclear transfer remains inefficient, and to date has not been achieved with human cells. We have also generated ES cells with defined histocompatibility loci by direct parthenogenetic activation of the unfertilized oocyte (Kim et al., Science 2007). Compared to ES cell lines from fertilized embryos, pES cells display comparable in vitro hematopoietic activity, but appear compromised in repopulating hematopoiesis in irradiated adult mouse recipients. We are currently comparing the performance of ntES, pES, and iPS cells in murine models of thalassemia. We have generated human iPS cells by direct reprogramming of human somatic cells with OCT4, SOX2, MYC, and KLF4 (Park et al., Nature 2008), and have generated disease-specific iPS cells from patients with a number of hematologic conditions (Park et al., Cell 2008; Agarwal et al., submitted). Applications of disease-specific cells for investigating the mechanisms of reprogramming and for probing aspects of human bone marrow disorders will be discussed. Disclosures Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.

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

Cloning and Stem Cells ◽

10.1089/clo.2005.7.265 ◽

2005 ◽

Vol 7 (4) ◽

pp. 265-271 ◽

Cited By ~ 27

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

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Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6755-6758.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6755-6758

Author(s):

B R Stanton ◽

S W Reid ◽

L F Parada

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Embryonic Development ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽

10.1182/blood-2005-09-3621 ◽

2006 ◽

Vol 107 (4) ◽

pp. 1265-1275 ◽

Cited By ~ 51

Author(s):

Abby L. Olsen ◽

David L. Stachura ◽

Mitchell J. Weiss

Keyword(s):

Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Basic Research ◽

Cell Types ◽

Patient Specific ◽

Es Cell ◽

Blood Disorders ◽

Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

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Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

10.1101/2019.12.30.891168 ◽

2019 ◽

Author(s):

Aseda Tena ◽

Yuxiang Zhang ◽

Nia Kyritsis ◽

Anne Devorak ◽

Jeffrey Zurita ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Es Cells ◽

Embryonic Stem ◽

Neural Progenitors ◽

Neural Genes ◽

Genome Wide ◽

Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

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Techniques for Nuclear Transfer to Mouse Embryonic Stem Cells

Nuclear Transfer Protocols ◽

10.1385/1-59745-154-1:269 ◽

2006 ◽

pp. 269-284

Author(s):

Danièle Pralong ◽

Paul J. Verma

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

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2. Embryonic stem cells

10.1093/actrade/9780198869290.003.0002 ◽

2021 ◽

pp. 21-37

Author(s):

Jonathan Slack

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Inner Cell Mass ◽

Es Cells ◽

Practical Importance ◽

Cell Mass ◽

Embryonic Stem ◽

Human Embryos ◽

Mouse Es Cells ◽

Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

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Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice

Journal of Diabetes Research ◽

10.1155/2015/257230 ◽

2015 ◽

Vol 2015 ◽

pp. 1-17 ◽

Cited By ~ 8

Author(s):

Tatsuhito Himeno ◽

Hideki Kamiya ◽

Keiko Naruse ◽

Zhao Cheng ◽

Sachiko Ito ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Blood Vessels ◽

Progenitor Cell ◽

Therapeutic Strategy ◽

Es Cells ◽

Embryonic Stem ◽

Diabetic Polyneuropathy ◽

Mouse Embryonic Stem Cells ◽

Intraepidermal Nerve Fiber

Background. Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted, a universally effective treatment of diabetic polyneuropathy has not yet been established. Recently, regenerative medicine studies in diabetic polyneuropathy using somatic stem/progenitor cell have been reported. However, the effectiveness of these cell transplantations was restricted because of their functional and numerical impairment in diabetic objects. Here, we investigated the efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived from mouse embryonic stem cells.Methods and Results. Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improved several physiological impairments in diabetic polyneuropathy: hypoalgesia, delayed nerve conduction velocities, and reduced blood flow in sciatic nerve and plantar skin. Furthermore, pathologically, the capillary number to muscle fiber ratios were increased in skeletal muscles of transplanted hindlimbs, and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skin. Transplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injection sites. Moreover, several transplanted cells constructed chimeric blood vessels with recipient cells.Conclusions. These results suggest that transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabetic polyneuropathy.

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