International Journal of Molecular Sciences

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11 pages, 2158 KiB

Open AccessArticle

Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study

by Hyun Ju Oh, Eugene Chung, Jaehwan Kim, Min Jung Kim, Geon A. Kim, Seok Hee Lee, Kihae Ra, Kidong Eom, Soojin Park, Jong-Hee Chae, Jin-Soo Kim and Byeong Chun Lee

Int. J. Mol. Sci. 2022, 23(5), 2898; https://doi.org/10.3390/ijms23052898 - 7 Mar 2022

Cited by 4 | Viewed by 2396

Abstract

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in [...] Read more.

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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19 pages, 25087 KiB

Open AccessArticle

Establishment of Bovine-Induced Pluripotent Stem Cells

by Yue Su, Ling Wang, Zhiqiang Fan, Ying Liu, Jiaqi Zhu, Deborah Kaback, Julia Oudiz, Tayler Patrick, Siu Pok Yee, Xiuchun (Cindy) Tian, Irina Polejaeva and Young Tang

Int. J. Mol. Sci. 2021, 22(19), 10489; https://doi.org/10.3390/ijms221910489 - 28 Sep 2021

Cited by 14 | Viewed by 7511

Abstract

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) [...] Read more.

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (K_dOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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16 pages, 30330 KiB

Open AccessArticle

Rapid Production and Genetic Stability of Human Mesenchymal Progenitor Cells Derived from Human Somatic Cell Nuclear Transfer-Derived Pluripotent Stem Cells

by Soo Kyung Jung, Jeoung Eun Lee, Chang Woo Lee, Sung Han Shim and Dong Ryul Lee

Int. J. Mol. Sci. 2021, 22(17), 9238; https://doi.org/10.3390/ijms22179238 - 26 Aug 2021

Cited by 4 | Viewed by 1732

Abstract

Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, [...] Read more.

Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, we produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. The recovery and purification of SCNT-PSC-Direct-MPCs were significantly accelerated compared to those of the SCNT-PSC-EB-MPCs, but both types of MPCs expressed typical surface markers and exhibited similar proliferation and differentiation potentials. Additionally, the analysis of gene expression patterns using microarrays showed very similar patterns. Moreover, array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Therefore, we suggest that the direct method of differentiating MPCs from SCNT-PSCs can improve the efficacy of SCNT-PSCs applied to allogeneic transplantation. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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12 pages, 1763 KiB

Open AccessArticle

Nuclear Transfer Arrest Embryos Show Massive Dysregulation of Genes Involved in Transcription Pathways

by Chunshen Long, Hanshuang Li, Xinru Li, Wuritu Yang and Yongchun Zuo

Int. J. Mol. Sci. 2021, 22(15), 8187; https://doi.org/10.3390/ijms22158187 - 30 Jul 2021

Cited by 3 | Viewed by 2004

Abstract

Somatic cell nuclear transfer (SCNT) technology can reprogram terminally differentiated cell nuclei into a totipotent state. However, the underlying molecular barriers of SCNT embryo development remain incompletely elucidated. Here, we observed that transcription-related pathways were incompletely activated in nuclear transfer arrest (NTA) embryos [...] Read more.

Somatic cell nuclear transfer (SCNT) technology can reprogram terminally differentiated cell nuclei into a totipotent state. However, the underlying molecular barriers of SCNT embryo development remain incompletely elucidated. Here, we observed that transcription-related pathways were incompletely activated in nuclear transfer arrest (NTA) embryos compared to normal SCNT embryos and in vivo fertilized (WT) embryos, which hinders the development of SCNT embryos. We further revealed the transcription pathway associated gene regulatory networks (GRNs) and found the aberrant transcription pathways can lead to the massive dysregulation of genes in NTA embryos. The predicted target genes of transcription pathways contain a series of crucial factors in WT embryos, which play an important role in catabolic process, pluripotency regulation, epigenetic modification and signal transduction. In NTA embryos, however, these genes were varying degrees of inhibition and show a defect in synergy. Overall, our research found that the incomplete activation of transcription pathways is another potential molecular barrier for SCNT embryos besides the incomplete reprogramming of epigenetic modifications, broadening the understanding of molecular mechanism of SCNT embryonic development. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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9 pages, 1593 KiB

Open AccessArticle

Optimal Treatment of 6-Dimethylaminopurine Enhances the In Vivo Development of Canine Embryos by Rapid Initiation of DNA Synthesis

by Hyun Ju Oh, Byeong Chun Lee and Min Kyu Kim

Int. J. Mol. Sci. 2021, 22(14), 7757; https://doi.org/10.3390/ijms22147757 - 20 Jul 2021

Cited by 3 | Viewed by 2004

Abstract

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine [...] Read more.

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 μM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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13 pages, 1830 KiB

Open AccessArticle

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

by Lianguang Xu, Seok-Hwan Song, Muhammad Idrees, Ayman Mesalam, Myeong-Don Joo, Tabinda Sidrat, Yiran Wei, Kyeong-Lim Lee, Wenfa Lu and Il-Keun Kong

Int. J. Mol. Sci. 2021, 22(11), 5841; https://doi.org/10.3390/ijms22115841 - 29 May 2021

Cited by 4 | Viewed by 2991

Abstract

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic [...] Read more.

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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16 pages, 9637 KiB

Open AccessArticle

Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells

by Seon-Ung Hwang, Kiyoung Eun, Mirae Kim, Junchul David Yoon, Lian Cai, Hyerin Choi, Dongjin Oh, Gabsang Lee, Hyunggee Kim, Eunhye Kim and Sang-Hwan Hyun

Int. J. Mol. Sci. 2021, 22(5), 2600; https://doi.org/10.3390/ijms22052600 - 5 Mar 2021

Cited by 1 | Viewed by 2925

Abstract

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible [...] Read more.

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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Review

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26 pages, 8229 KiB

Open AccessReview

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

by Kanokwan Srirattana, Masahiro Kaneda and Rangsun Parnpai

Int. J. Mol. Sci. 2022, 23(4), 1969; https://doi.org/10.3390/ijms23041969 - 10 Feb 2022

Cited by 15 | Viewed by 5351

Abstract

Mammalian oocytes can reprogram differentiated somatic cells into a totipotent state through somatic cell nuclear transfer (SCNT), which is known as cloning. Although many mammalian species have been successfully cloned, the majority of cloned embryos failed to develop to term, resulting in the [...] Read more.

Mammalian oocytes can reprogram differentiated somatic cells into a totipotent state through somatic cell nuclear transfer (SCNT), which is known as cloning. Although many mammalian species have been successfully cloned, the majority of cloned embryos failed to develop to term, resulting in the overall cloning efficiency being still low. There are many factors contributing to the cloning success. Aberrant epigenetic reprogramming is a major cause for the developmental failure of cloned embryos and abnormalities in the cloned offspring. Numerous research groups attempted multiple strategies to technically improve each step of the SCNT procedure and rescue abnormal epigenetic reprogramming by modulating DNA methylation and histone modifications, overexpression or repression of embryonic-related genes, etc. Here, we review the recent approaches for technical SCNT improvement and ameliorating epigenetic modifications in donor cells, oocytes, and cloned embryos in order to enhance cloning efficiency. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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16 pages, 975 KiB

Open AccessReview

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

by Daehwan Kim and Sangho Roh

Int. J. Mol. Sci. 2021, 22(9), 5011; https://doi.org/10.3390/ijms22095011 - 9 May 2021

Cited by 3 | Viewed by 3616

Abstract

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs [...] Read more.

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs. Full article

(This article belongs to the Special Issue Somatic Cell Nuclear Transfer in Embryonic Development and Stem Cell Research)

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