Nuclear Transfer Embryonic Stem Cells Research Articles

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

Healthy cloned offspring derived from freeze-dried somatic cells

Maintaining biodiversity is an essential task, but storing germ cells as genetic resources using liquid nitrogen is difficult, expensive, and easily disrupted during disasters. Our aim is to generate cloned mice from freeze-dried somatic cell nuclei, preserved at −30 °C for up to 9 months after freeze drying treatment. All somatic cells died after freeze drying, and nucleic DNA damage significantly increased. However, after nuclear transfer, we produced cloned blastocysts from freeze-dried somatic cells, and established nuclear transfer embryonic stem cell lines. Using these cells as nuclear donors for re-cloning, we obtained healthy cloned female and male mice with a success rate of 0.2–5.4%. Here, we show that freeze-dried somatic cells can produce healthy, fertile clones, suggesting that this technique may be important for the establishment of alternative, cheaper, and safer liquid nitrogen-free bio-banking solutions.

Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials

Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.

Induction of Bone Formation by 3D Biologically Active Scaffolds Containing RGD‐NPs, BMP2, and NtMPCs

AbstractIt is demonstrated that nuclear transfer embryonic stem cells (NT‐ESCs) can be transformed into mesenchymal progenitor cells (NtMPCs) via culture under appropriate conditions and it is proposed that NtMPCs can be used for bone regeneration instead of bone marrow‐derived mesenchymal stem cells (BMSCs). In a 2‐dimensional culture, NtMPCs undergo chondrogenic and adipogenic differentiation more readily than BMSCs upon culture in appropriate media. However, the osteogenic differentiation ability of NtMPCs is lower than that of BMSCs. 3‐dimensional (3D) biologically active scaffolds (BAs) comprising poly(lactic‐co‐glycolic acid) (PLGA) microspheres are constructed to improve osteogenic differentiation. To generate BAs, nanoparticles containing an l‐Arginyl‐Glycyl‐l‐Aspartic acid (RGD) sequence (RGD‐NPs) and dexamethasone (DEX), a drug used to control differentiation and inflammation, are simultaneously encapsulated by PLGA to form particles measuring 150–250 µm. After that, bone morphogenetic protein 2 (BMP2) is immobilized on the surface of polyethylenimine (PEI)‐coated microspheres via formation of ionic bonds between O‐sulfate and N‐sulfate in heparin with lysine/arginine residues of BMP2. Gene expression profiling is performed via QuantSeq 3′ mRNA‐sequencing in mice transplanted with BAs containing NtMPCs and BMSCs. Expression of the osteogenic differentiation‐related genes collagen Type I Alpha 2 (COL1A2), Homeobox protein MSX‐2 (MSX2), Runt‐related transcription factor 2 (RUNX2), and bone morphogenetic protein 2 (BMP2) is 2–3‐fold higher in mice transplanted with BAs containing NtMPCs than in mice transplanted with BAs containing BMSCs.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

Fasudil Increases the Establishment of Somatic Cell Nuclear Transfer Embryonic Stem Cells in Mouse

Fasudil Increases the Establishment of Somatic Cell Nuclear Transfer Embryonic Stem Cells in Mouse

Effects of reprogramming on genomic imprinting and the application of pluripotent stem cells.

Pluripotent stem cells are considered to be the ideal candidates for cell-based therapies in humans. In this regard, both nuclear transfer embryonic stem (ntES) cells and induced pluripotent stem (iPS) cells are particularly advantageous because patient-specific autologous ntES and iPS cells can avoid immunorejection and other side effects that may be present in the allogenic pluripotent stem cells derived from unrelated sources. However, they have been found to contain deleterious genetic and epigenetic changes that may hinder their therapeutic applications. Indeed, deregulation of genomic imprinting has been frequently observed in reprogrammed ntES and iPS cells. We will survey the recent studies on genomic imprinting in pluripotent stem cells, particularly in iPS cells. In a previous study published about six years ago, genomic imprinting was found to be variably lost in mouse iPS clones. Intriguingly, de novo DNA methylation also occurred at the previously unmethylated imprinting control regions (ICRs) in a high percentage of iPS clones. These unexpected results were confirmed by a recent independent study with a similar approach. Since dysregulation of genomic imprinting can cause many human diseases including cancer and neurological disorders, these recent findings on genomic imprinting in reprogramming may have some implications for therapeutic applications of pluripotent stem cells.

Deconstructing age reprogramming

It has been proposed that age reprogramming enables old cells to be rejuvenated without passage through an embryonic stage (Singh and Zacouto in J. Biosci. 35 315-319, 2010). As such, age reprogramming stands apart from the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell therapies where histo-compatible cells are produced only after passage through an embryonic stage. It avoids many of the disadvantages associated with iPS and NT-ES cell therapies. Experimental evidence in support of age reprogramming is burgeoning. Here, we discuss possible new approaches to enhance age reprogramming, which will have considerable benefits for regenerative therapies.

Stem cells and reproduction

Reproductive biotechnology has developed rapidly and is now able to overcome many birth difficulties due to infertility or the transmission of genetic diseases. Here we introduce the next generation of assisted reproductive technologies (ART), such as mitochondrial replacement technique (MRT) or genetic correction in eggs with micromanipulation. Further, we suggest that the transmission of genetic information from somatic cells to subsequent generations without gametes should be useful for people who suffer from infertility or genetic diseases. Pluripotent stem cells (PSCs) can be converted into germ cells such as sperm or oocytes in the laboratory. Notably, germ cells derived from nuclear transfer embryonic stem cells (NT-ESCs) or induced pluripotent stem cells (iPSCs) inherit the full parental genome. The most important issue in this technique is the generation of a haploid chromosome from diploid somatic cells. We hereby examine current science and limitations underpinning these important developments and provide recommendations for moving forward.

Effects of Embryo Aggregation and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

To improve the cloning efficiency of somatic cell nuclear transfer (SCNT) and to establish nuclear transfer embryonic stem cells (NT-ESCs) reliably, it is necessary to produce high-quality blastocysts derived from mice SCNT embryos. Therefore, the present study aims to investigate an optimal method for mouse SCNT embryo production and NT-ESCs derivation by comparing the effects of two methods: the treatment of histone deacetylase inhibitor PXD101 after SCNT, embryo aggregation and their combination treatment. The results suggest that embryo aggregation at four-cell stage and 50 nM PXD101 treated for 10 hours during and after activation could improve both mouse SCNT embryos' development (PXD101: 40.0% vs. 18.5%; p < 0.05; aggregation: 40.2% vs. 18.5%; p < 0.05) and also enhance the isolation rate of NT-ESCs (PXD101: 38.2% vs. 12.5%; p < 0.05; aggregation: 39.0% vs. 12.5%; p < 0.05). The combination of their treatments had a higher development rate (43.6%) and significantly higher NT-ESCs isolation rate (54.7%), therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 10 hours after SCNT and then aggregated at four-cell stage) is considered as the optimal way for the in vitro development of SCNT embryo and subsequent NT-ESCs isolation in mice, providing a new approach for the practical improvement of mouse cloning techniques and opening new opportunities to improve cloning efficiencies in other species.

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy.

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

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.

High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds

A combination of nanotopographical cues and surface modification of collagen and fibronectin is a potential platform in primordial germ cells (PGCs) differentiation. In the present study, the synergistic effect of nanotopography and surface modification on differentiation of nuclear transfer embryonic stem cells (nt-ESCs) toward PGC lineage was investigated. In order to achieve this goal, poly-anyline (PANi) was mix within poly-L-lactic acid (PLLA). Afterward, the random composite mats were fabricated using PLLA and PANi mix solution. The nanofiber topography notably upregulated the expressions of prdm14, mvh and c-kit compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and surface modification resulted in more enhancement of PGCs differentiation compared with non-modified nanofibrous scaffold. Additionally, gene expression results showed that mvh and c-kit were expressed at higher intensity in cells exposed to collagen and fibronectin rather than collagen or fibronectin solitary. These results demonstrated the importance of combined effect of collagen and fibronectin in order to develop a functional extracellular matrix (ECM) mimic in directing stem cell fate and the potential of such biofunctional scaffolds for treatment of infertility.

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

Lymphoid lineage differentiation potential of mouse nuclear transfer embryonic stem cells

Stem cells therapy is considered as an efficient strategy for the treatment of some diseases. Nevertheless, some obstacles such as probability of rejection by the immune system limit applications of this strategy. Therefore, several efforts have been made to overcome this among which using the induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cell (nt-ESCs) are the most efficient strategies. The objective of this study was to evaluate the differentiation potential of the nt-ESCs to lymphoid lineage in the presence of IL-7, IL-3, FLT3-ligand and TPO growth factors in vitro. To this end, the nt-ESCs cells were prepared and treated with aforementioned growth factors for 7 and 14 days. Then, the cells were examined for expression of lymphoid markers (CD3, CD25, CD127 and CD19) by quantitative PCR (q-PCR) and flow cytometry. An increased expression of CD19 and CD25 markers was observed in the treated cells compared with the negative control samples by day 7. After 14 days, the expression level of all the tested CD markers significantly increased in the treated groups in comparison with the control. The current study reveals the potential of the nt-ESCs in differentiation to lymphoid lineage in the presence of defined growth factors.

Generation of fertile offspring from Kit(w)/Kit(wv) mice through differentiation of gene corrected nuclear transfer embryonic stem cells.

Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit(w)/Kit(wv) mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit(w)/Kit(wv) somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit(w)/Kit(wv) infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.

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

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

Comparable Frequencies of Coding Mutations and Loss of Imprinting in Human Pluripotent Cells Derived by Nuclear Transfer and Defined Factors

The recent finding that reprogrammed human pluripotent stem cells can be derived by nuclear transfer into human oocytes as well as by induced expression of defined factors has revitalized the debate on whether one approach might be advantageous over the other. Here we compare the genetic and epigenetic integrity of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal, and adult origin. The two cell types showed similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs had comparable numbers of de novo coding mutations, but significantly more than parthenogenetic ESCs. As iPSCs, NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of derivation approach.