Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC.

Zygotic epigenetic reprogramming is the major initial event in embryo development to acquire a totipotent potential. However, the patterns of epigenetic modifications in bovine zygote were not well clarified, especially in the first cell cycle of bovine somatic cell nuclear transfer (SCNT) embryos. This study was conducted to examine the patterns of DNA methylation (5-methylcytosine [5mc] and 5-hydroxymethylcytosine [5hmc]) and histone H3 lysine 9 methylation (H3K9m2 and H3K9m3) in the first cell cycle of bovine in vitro fertilization (IVF) and SCNT embryos. In bovine zygotic development, the 5mc in the paternal pronucleus (pPN) undergoes partial demethylation from PN1 to PN3, and remethylation from PN4 to PN5, while 5hmc exhibits absolutely different patterns. The 5mc in SCNT embryos underwent much more dramatic demethylation and much earlier de novo methylation compared with their IVF counterparts, while 5hmc stayed stable from PN1 to PN4, and significantly increased at PN5, which made significantly higher level of 5mc and 5hmc at the end of the first cell cycle in SCNT embryos. Different H3K9m2 and H3K9m3 patterns were also observed between IVF and SCNT embryos. H3K9m2 and H3K9m3 asymmetrically distributed in parental genomes in IVF zygote, highly present in the maternal pronucleus, whereas faintly stained in the pPN. H3K9m2 and H3K9m3 in the somatic cell genome were gradually demethylated from PN1-PN4, and significantly increased at the end of the first cell cycle. TET3 dioxygenase was highly present in the first cell cycle of embryos compared with TET1 and TET2. Our results showed that SCNT embryos underwent aberrant epigenetic reprogramming in the first cell cycle; much more dramatic demethylation and significant higher remethylation were observed compared with IVF counterparts.

Oxidative stress can impede maturation of the nucleus and cytoplasm of oocytes during in vitro maturation (IVM). Rhodiola sachalinensis, an herb commonly used in traditional Chinese medicine, conveys antioxidative effects to cryopreserved bovine sperm. Therefore, the aims of this study were to evaluate the effects of different concentrations of R. sachalinensis aqueous extract (RSAE) on IVM and subsequent in vitro embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT). The results showed that RSAE supplementation (6 and 60 mg/L) significantly increased intracellular glutathione levels, but had no effect on maturation rates or reactive oxygen species. After in vitro culture, greater blastocyst formation was observed in PA embryos (6 mg/L RSAE), as well as in IVF and SCNT embryos (60 mg/L) matured in RSAE-supplemented IVM media. In conclusion, although there was no significant improvement in the maturation rate, RSAE supplementation conveyed an antioxidative effect during IVM, and improved subsequent embryonic development in vitro. Further studies are needed to explore gene expression pattern in oocytes and embryos treated with RSAE.

Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher (p < 0.05) than that of the SCNT method (96.6% ± 1.1% vs. 86.7% ± 6.0%, 29.5% ± 2.6% vs. 22.1% ± 3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7 ± 0.2 vs. 2.9 ± 0.3, p < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group (p < 0.05), and the relative acH3K56 level in the CICT group was significantly (p < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the in vitro developmental competence and quality of cloned embryos.

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

To evaluate age-related changes in T cells and stem cell-related genes in mice and the changes in T cells and stem cell-related genes after allogenic bone marrow mesenchymal stem cell (BMSC) transplantation, BALB/c mice were divided into young (2 months, n = 5) and old (20 months, n = 5) groups and 1 × 107 BMSCs from 3-week-old C57BL/6J mice were injected into the old mice (n = 5). T lymphocytes including CD3+, CD8+, CD8+CD28+, and CD8+CD44lowCD62LhighSca-1+ stem cell-like memory T cells from spleens were analyzed by flow cytometry. mRNA transcriptions of the tumor suppressor p16INK4A and the senescence inhibiting AUF 1 and stem cell-related ADAM12, GIL3, c-MYC, NANOG, Wnt, HOX11, Sox2, Oct3/4, and KLF4 genes were analyzed by quantitative reverse transcription-polymerase chain reaction for comparison between young and old mice and old mice after BMSC application. Stem cell-related genes were reduced transcribed in old mice, an action that could be partly or completely reversed for some genes by BMSC injections. The proportion of CD8+CD28+ T cells in the spleens of old mice was significantly reduced (p < 0.01), indicating advanced proliferative T cell senescence. The CD8+CD44lowCD62Lhigh cell fraction was significantly reduced and that of CD8+CD44lowCD62LhighSca-1+ increased in splenic CD8+ cells of old mice, both actions of which were reversed by BMSC injections. p16INK4A transcription was enhanced and AUF1 transcription was reduced in old mice, the latter effect partly reversed by BMSC injections. BMSC injections led to recovery of stem cell-related gene activation or BMSC stem cell-related gene expression tolerance in spleens of old mice.

DNA methylation is an essential epigenetic mark for embryo development and can be susceptible to environment factors such as in vitro conditions. The aim of this study was to verify the effect of in vitro culture until Day (D) 14 of the development on the embryo size and DNA methylation pattern of the insulin-like growth factor 2 (IGF2)-imprinted gene. To achieve this, we produced bovine embryos completely in vivo, completely in vitro, and in vitro until D7 and then in vivo up to D14. The embryos produced in in vitro were smaller than those in other two groups (p = 0.024); no differences in embryo size were observed between genders. The in vitro embryos showed a higher level of DNA methylation in the IGF2 as compared with that in the completely in vivo-produced (IVV) embryos (p = 0.009). Furthermore, totally in vitro-produced male embryos showed higher levels of DNA methylation as compared with those observed for the totally IVV male embryos (p = 0.034). No differences were observed among genders for IGF2 DNA methylation. These results showed that the window between D7 and D14 is critical for embryo development and alterations in the environmental conditions during this period can impair DNA methylation establishment of important developmental imprinted genes. This study brings unprecedented data for bovine embryos regarding the impact of the environmental conditions during the posthatching development.

Induced pluripotent stem cells (iPSCs) remain a promising approach to target diseases with a loss of functional parenchyma. This technology comes with a number of concerns for clinical applications, including teratogenic potential and genomic instability. Here we focused on evaluating the safety of cross-species Sendai viral reprogramming, as well as investigating the transcriptional dynamics during reprogramming and differentiation. We established that Sendai viral vectors carrying human Oct4, Sox2, Klf4, and c-Myc (OSKM) could produce mouse iPSCs free of transduced viral materials. Gene expression analysis revealed an efficient silencing of the virally-introduced human pluripotency factors and upregulation of the endogenous pluripotency network over time. In addition, single cell gene expression analysis of proof-of-principle-derived cardiomyocytes revealed distinct expression patterns indicative of subspecialized cardiac cell lineages. Moreover, our results demonstrate the importance of monitoring genomic aberrations before any clinical or preclinical applications, as we detected a high prevalence of chromosomal instability. Taken together, we demonstrated the successful use of a clinically germane method to reprogram terminally differentiated mouse cells and their potential to generate specialized cardiac cell types. Additionally, our results suggest a plasticity of OSKM to reprogram more divergent species and provide a new application of an established reprogramming approach.

Cellular ReprogrammingVol. 21, No. 1 Retraction NoticeFree AccessRetraction of: Bone Mesenchymal Stem Cell-Conditioned Medium Regulates the Differentiation of Neural Stem Cells Via Notch Pathway Activation, by Li H-M, Tong Y, Xia X, Huang J, Song P-W, Zhang R-J, Shen C-L. Cell Reprogram. [Epub ahead of print; Dec 27, 2018] DOI: 10.1089/cell.2018.0042This article retracts the following:Retracted: Bone Mesenchymal Stem Cell-Conditioned Medium Regulates the Differentiation of Neural Stem Cells Via Notch Pathway ActivationPublished Online:8 Feb 2019https://doi.org/10.1089/cell.2018.0042.retractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail The online-ahead-of print e-pub version of the article entitled, Bone Mesenchymal Stem Cell-Conditioned Medium Regulates the Differentiation of Neural Stem Cells Via Notch Pathway Activation, by Li H-M, Tong Y, Xia X, Huang J, Song P-W, Zhang R-J, Shen C-L, utilizing the DOI number 10.1089/cell.2018.0042 is being officially retracted from Cellular Reprogramming.The original version of the paper was submitted to the journal for peer review on July 29, 2018, with the revised version after peer review submitted on October 21, 2018. The paper was accepted for publication on November 20, 2018 and was subsequently published online ahead of print on December 27, 2018. After the e-publication of the article, the editor received an email from the corresponding author on January 14, 2019 requesting “to withdraw the above-mentioned manuscript for further consideration, due to a technical reason (we have done a further experiment and found this article need add more results).”Though it is unclear why the authors were not able to determine these faults with the paper within the six months the manuscript was in review, revision, and production, the editorial leadership of the Journal has determined that the paper requires a full retraction from the literature as Cellular Reprogramming is committed to upholding the strictest standards and best practices of scientific publishing.FiguresReferencesRelatedDetailsRelated articlesRetracted: Bone Mesenchymal Stem Cell-Conditioned Medium Regulates the Differentiation of Neural Stem Cells Via Notch Pathway Activation4 Dec 2019Cellular Reprogramming Volume 21Issue 1Feb 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Retraction of: Bone Mesenchymal Stem Cell-Conditioned Medium Regulates the Differentiation of Neural Stem Cells Via Notch Pathway Activation, by Li H-M, Tong Y, Xia X, Huang J, Song P-W, Zhang R-J, Shen C-L. Cell Reprogram. [Epub ahead of print; Dec 27, 2018] DOI: 10.1089/cell.2018.0042.Cellular Reprogramming.Feb 2019.61-61.http://doi.org/10.1089/cell.2018.0042.retractPublished in Volume: 21 Issue 1: February 8, 2019PDF download

One of the most important concerns cancer survivors face is fertility. Current treatment modalities often result in damage to the reproductive system. Different options have been proposed to preserve the fertility of affected women, and many attempts have been made to improve their chance of childbearing after therapy. Cryopreservation of ovarian tissue and follicles before the onset of cancer treatment and then either transplantation of ovarian tissue or culture of ovarian tissue and individual follicles in vitro is a commonly cited approach. Extensive research is being done to design an optimal condition for the culture of ovarian follicles. Improving follicle culture systems by understanding their actual growth needs might be a crucial step toward fertility preservation in cancer patients. This review article will try to provide a summary of the role of different factors and conditions on growth of human and bovine preantral follicles in vitro.