De Cheng

Porcine Induced Pluripotent Stem Cells Require LIF and Maintain Their Developmental Potential in Early Stage of Embryos

Porcine induced pluripotent stem (piPS) cell lines have been generated recently by using a cocktail of defined transcription factors, however, the features of authentic piPS cells have not been agreed upon and most of published iPS clones did not meet the stringent requirements of pluripotency. Here, we report the generation of piPS cells from fibroblasts using retrovirus carrying four mouse transcription factors (mOct4, mSox2, mKlf4 and mc-Myc, 4F). Multiple LIF-dependent piPS cell lines were generated and these cells showed the morphology similar to mouse embryonic stem cells and other pluripotent stem cells. In addition to the routine characterization, piPS cells were injected into porcine pre-compacted embryos to generate chimera embryos and nuclear transfer (NT) embryos. The results showed that piPS cells retain the ability to integrate into inner and outer layers of the blastocysts, and support the NT embryos development to blastocysts. The generations of chimera embryos and NT embryos derived from piPS clones are a practical means to determine the quality of iPS cells ex vivo.

Vitamin C enhances the in vitro development of porcine pre-implantation embryos by reducing oxidative stress.

Vitamin C (Vc) is a natural compound supplemented to culture media to guarantee the appropriate reactive oxygen species (ROS) level, as well as protect cells from oxidative damage and apoptosis. The current study was conducted to determine the effects of Vc (0, 2.5, 5, 10, 20 and 40 μg/ml) on the ROS production, developmental ability and quality of in vitro produced porcine parthenotes. The results show that: (i) the ROS levels in the embryos significantly decrease in the Vc-treated groups compared with the control (p < 0.05), (ii) the rates of blastocyst formation and total cell numbers in each blastocyst are significantly higher in the Vc-treated groups than in the control (p < 0.05); the optimum concentration of Vc is 20 μg/ml, (iii) the relative expression of Bcl-xL significantly increases and that of Bax is downregulated after Vc treatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling analysis indicates that the ratio of apoptotic cells in the blastocyst is also significantly lower in Vc-treated groups (p < 0.05) and (iv) Vc treatment can also increase the expression of the Nanog gene in porcine embryos, with a fivefold increase in 20 μg/ml Vc treatment compared with the control (p < 0.05). Therefore, Vc improves the development of porcine embryos by reducing the ROS levels. Vc addition in PZM-3 medium can decrease the number of apoptotic cells and increase the cell numbers in blastocysts to produce high-quality porcine embryos in vitro.

Dual roles of c-Myc in the regulation of hTERT gene

Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at -165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.

Insulin–transferrin–selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.

Comparative gene expression signature of pig, human and mouse induced pluripotent stem cell lines reveals insight into pig pluripotency gene networks.

Reported pig induced pluripotent stem cells (piPSCs) have shown either a bFGF-dependent state with human embryonic stem cell (ESC) and mouse epiblast stem cell (EpiSC) morphology and molecular features or piPSCs exist in a LIF-dependent state and resemble fully reprogrammed mouse iPSCs. The features of authentic piPSCs and molecular events during the reprogramming are largely unknown. In this study, we assessed the transcriptome profile of multiple piPSC lines derived from different laboratories worldwide and compared to mouse and human iPSCs to determine the molecular signaling pathways that might play a central role in authentic piPSCs. The results demonstrated that the up-regulation of endogenous epithelial cells adhesion molecule (EpCAM) was correlated with the pluripotent state of pig pluripotent cells, which could be utilized as a marker for evaluating pig cell reprogramming. Comparison of key signaling pathways JAK-STAT, NOTCH, TGFB1, WNT and VEGF in pig, mouse and human iPSCs showed that the core transcriptional network to maintain pluripotency and self-renewal in pig were different from that in mouse, but had significant similarities to human. Pig iPSCs, which lacked expression of specific naïve state markers KLF2/4/5 and TBX3, but expressed the primed state markers of Otx2 and Fabp7, share defining features with human ESCs and mouse EpiSCs. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) were silenced in piPSCs as previously seen in mouse iPSCs that have limited ability to contribute to chimaeras. These key differences in naïve state gene and imprinting gene expression suggests that so far known piPSC lines may be more similar to primed state cells. The primed state of these cells may potentially explain the rare ability of piPSCS to generate chimeras and cloned offspring.

Retinoic acid induces myoblasts transdifferentiation into premeiotic Stra8-positive cells

Spermatogonia and sperm‐like cells can be derived in vitro via the addition of RA (retinoic acid) to pluripotent ES and EG cells. At present, however, these cells have not been derived from unipotent cells. Here, we have generated premeiotic Stra8‐positive cells from C2C12 myoblasts following treatment with 10 μM all‐trans‐RA for 8 days. The differentiated C2C12 cells exhibited spherical morphology similar to spermatogonia, and they expressed gene markers of premeiosis, meiosis and postmeiosis. In addition, some of the transdifferentiated Stra8‐positive cells had a tail‐like phenotype. Flow cytometry results indicated that up to 20% of RA‐induced C2C12 cells were Stra8‐positive. Mvh (mouse vasa homologue) protein, a germ cell‐specific ATP‐dependent RNA helicase and Prm1 (protamine 1) were detected in transdifferentiated cells. The DNA content in induced C2C12 cells showed that Stra8‐positive cells were diploid, suggesting that the myoblast transdifferentiation was in the premeiotic stage of spermatogenesis. The derivation of Stra8‐positive cells from C2C12 myoblasts has important implications for studying unipotent cell differentiation. Furthermore, C2C12 myoblasts may provide a useful in vitro cell model to study signal transduction and transdifferentiation during RA treatments.

The gene expression profiles of induced pluripotent stem cells (iPSCs) generated by a non-integrating method are more similar to embryonic stem cells than those of iPSCs generated by an integrating method

Induced pluripotent stem cells (iPSCs) obtained by the ectopic expression of defined transcription factors have tremendous promise and therapeutic potential for regenerative medicine. Many studies have highlighted important differences between iPSCs and embryonic stem cells (ESCs). In this work, we used meta-analysis to compare the global transcriptional profiles of human iPSCs from various cellular origins and induced by different methods. The induction strategy affected the quality of iPSCs in terms of transcriptional signatures. The iPSCs generated by non-integrating methods were closer to ESCs in terms of transcriptional distance than iPSCs generated by integrating methods. Several pathways that could be potentially useful for studying the molecular mechanisms underlying transcription factor-mediated reprogramming leading to pluripotency were also identified. These pathways were mostly associated with the maintenance of ESC pluripotency and cancer regulation. Numerous genes that are up-regulated during the induction of reprogramming also have an important role in the success of human preimplantation embryonic development. Our results indicate that hiPSCs maintain their pluripotency through mechanisms similar to those of hESCs.

Kinetic Analysis of Porcine Fibroblast Reprogramming Toward Pluripotency by Defined Factors

Induced pluripotent stem cells (iPSCs) are generated from somatic cells through ectopic expression of defined transcription factors. So far, many iPSC lines have been established in various species, including porcine. However, the molecular events during somatic cell reprogramming in pig are largely unknown. The aim of this study was to carry out porcine embryonic fibroblast (PEF) reprogramming by using mouse transcription factors and to monitor morphological and biological progress systematically at an early stage of cell reprogramming. The retrovirus-infected PEF cells retained the four transgenes used and showed morphological changes and alkaline phosphatase staining at day 5 after infection. The endogenous OCT4, NANOG, and TERT genes were activated, and their expression levels were increased significantly. BAX gene expression, a proapoptotic member of the BCL-2 family, was also increased at day 5, suggesting that c-Myc might trigger cell apoptosis. Omission of c-Myc from the cocktail of factors then greatly influenced the reprogramming efficiency and lowered the formation of iPSC colonies. The expression of paternally imprinted DLK1 and DIO3 was slightly downregulated after infection, but then recovered within 2 weeks. However, the expression of maternally imprinted GTL2 was silenced aberrantly at a very early stage of infection and did not recover. Together, these observations illustrated that upregulation of pluripotent-related gene expression, stabilizing the imprinted Dlk1-Dio3 domain, and inhibition of apoptotic events might be conductive to the promotion of the reprogramming process to produce complete porcine iPSCs.

Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes

Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo.

Human Specific Regulation of the Telomerase Reverse Transcriptase Gene

Telomerase, regulated primarily by the transcription of its catalytic subunit telomerase reverse transcriptase (TERT), is critical for controlling cell proliferation and tissue homeostasis by maintaining telomere length. Although there is a high conservation between human and mouse TERT genes, the regulation of their transcription is significantly different in these two species. Whereas mTERT expression is widely detected in adult mice, hTERT is expressed at extremely low levels in most adult human tissues and cells. As a result, mice do not exhibit telomere-mediated replicative aging, but telomere shortening is a critical factor of human aging and its stabilization is essential for cancer development in humans. The chromatin environment and epigenetic modifications of the hTERT locus, the binding of transcriptional factors to its promoter, and recruitment of nucleosome modifying complexes all play essential roles in restricting its transcription in different cell types. In this review, we will discuss recent progress in understanding the molecular mechanisms of TERT regulation in human and mouse tissues and cells, and during cancer development.