Huayan Wang

The Na+/H+ Antiporter Potentiates Growth and Retinoic Acid-induced Differentiation of P19 Embryonal Carcinoma Cells

The Na+/H+exchanger is a ubiquitous plasma membrane protein that is responsible for pH regulation and is activated by growth factors. We examined the role of the Na+/H+ exchanger in cell growth and differentiation. Treatment of P19 cells with the Na+/H+ exchanger inhibitor Hoe 694 eliminated retinoic acid-induced differentiation in this cell line. We developed a P19 embryonal carcinoma cell line that was deficient in the Na+/H+ antiporter. Na+/H+ exchanger-deficient cells were reduced in the rate of cell growth and this effect was enhanced by the removal of added HCO3 − and by reducing extracellular pH. The antiporter-deficient cells were also markedly deficient in their ability to differentiate to neuronal-like cells and recovered this ability when the Na+/H+antiporter was reintroduced. The results show that the absence of Na+/H+ antiport as a pH regulatory mechanism can result in deficiencies in both cell growth and differentiation in embryonal carcinoma cells.

Cloning and analysis of the human myocardial Na+/H+ exchanger.

The Na+/H+ exchanger is an integral membrane protein that is universally distribute in mammalian tissues and is responsible for intracellular pH regulation. Several isoforms of the Na+/H+ exchanger exist (NHE-1-NHE-4). The first that was cloned is the amiloride sensitive isoform (NHE-1). Using a fragment of the rabbit cardiac Na+/H+ exchanger cDNA clone we isolated and sequenced Na+/H+ exchanger cDNA from a human heart coding for the complete human Na+/H+ exchanger (NHE-1 isoform). Two overlapping cDNA clones were obtained, giving a combined sequence that contained both 3′ and 5′ untranslated regions. The 5′ and 3′ untranslated regions proved to be highly homologous to human sequences described earlier but contained some variations that could affect the mRNA stability and/or the efficiency of translation of the Na+/H+ exchanger. Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the presence of the 5 kb NHE-1 message in primary cultures of isolated myocytes.

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.

Characterization of the Na+/H+ exchanger in human choriocarcinoma (BeWo) cells

Abstract We examined the expression and activity of the Na+/H+ exchanger in the human choriocarcinoma BeWo cell line. When treated with methotrexate, these cells differentiated from cytotrophoblast-like cells to enlarged multinucleate syncytiotrophoblast-like cells. There was no change in the apparent Km for Na+ between undifferentiated and differentiated cells. However, differentiated cells could transport more than five times the proton flux of undifferentiated cells. There was no difference in the Hill coefficient between undifferentiated and differentiated cells. However, the maximal flux (Jmax) for undifferentiated cells was higher than that for differentiated cells. Inhibition of Na+/H+ exchange activity by an amiloride analog and Hoe694 revealed a sensitive and a resistant component in both differentiated and undifferentiated cells. Northern blot analysis and immunocytochemistry suggested that the sensitive component was due to the NHE1 isoform of the protein while the resistant component was due to the NHE3 isoform. The NHE1 isoform was localized to the brush border membrane of BeWo cells and Western blot analysis showed that the NHE1 protein was more abundant in brush border membranes from differentiated BeWo cells compared to undifferentiated cells. The results show that BeWo cells contain the NHE1 and NHE3 isoforms of the Na+/H+ exchanger and that the NHE1 isoform is primarily localized to the brush border membrane.

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)‐like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self‐renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.—Mathew, R., Jia, W., Sharma, A., Zhao, Y., Clarke, L. E., Cheng, X., Wang, H., Salli, U., Vrana, K. E., Robertson, G. P., Zhu, J., Wang, S. Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J. 24, 2702–2715 (2010). www.fasebj.org

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.

Zidovudine and dideoxynucleosides deplete wild-type mitochondrial DNA levels and increase deleted mitochondrial DNA levels in cultured Kearns-Sayre syndrome fibroblasts

Kearns-Sayre syndrome is the most commonly diagnosed mitochondrial cytopathy and produces severe neuromuscular symptoms. The most frequent cause is a mitochondrial DNA deletion that removes a 4977-base pair segment of DNA that includes several genes encoding for respiratory chain subunits. Treatment of AIDS patients with nucleoside analogs has been reported to cause mtDNA depletion and myopathies. Here, we report that azidothymidine, dideoxyguanosine, and dideoxycytidine cause a depletion of wild-type mtDNA while increasing the levels of deleted mitochondria DNA in Kearns-Sayre syndrome fibroblasts. The result of these effects is a large increase in the relative amounts of delta mtDNA in comparison to wild type mtDNA. We found that Kearns-Sayre syndrome fibroblasts are a mixed population of cells with deleted mtDNA comprising from 0 to over 20% of the total mtDNA in individual cells. Treatment of cloned cell lines with dideoxycytidine did not result in increased levels of delta mtDNA. The results suggest that nucleoside analogs may act to increase the average delta mtDNA levels in a mixed population of cells by preferentially inhibiting the proliferation of cells with little or no delta mtDNA. This raises the possibility that modulation of deleted mtDNA levels may occur by similar mechanisms in vivo, in response to the influence of exogenous agents.

Regulation of Na+/H+ Exchanger Gene Expression ROLE OF A NOVEL POLY(dA·dT) ELEMENT IN REGULATION OF THE NHE1 PROMOTER

In this study we examine regulation of expression of the Na+/H+ exchanger promoter in L6 and NIH 3T3 cells. We have identified a highly conserved poly(dA·dT)-rich region that appears to be important in regulation of expression of the NHE1 gene. Deletion or mutation of this region results in dramatic decreases in promoter activity in both L6 and NIH 3T3 cells. In addition, DNase I footprinting experiments demonstrated that this region is protected by nuclear extracts from both cell types, and gel mobility shift assays showed that a protein or proteins specifically binds to the poly(dA·dT)-rich element. Using Southwestern blotting, we determined that a 33-kDa protein binds to the poly(dA·dT)-containing region. Mutations that abolished protein binding to this element diminished activity of the promoter. Insertion of the poly(dA·dT)-rich element into a plasmid containing the SV40 promoter demonstrated that this element can also enhance the activity of a foreign promoter. Together, the results we have presented here show that the poly(dA·dT)-rich region is important in regulation of NHE1 expression in different cell types.

Generation of Intermediate Porcine iPS Cells Under Culture Condition Favorable for Mesenchymal-to-Epithelial Transition

It has been demonstrated that naïve and primed pluripotency are determined by different extracellular signals. In this study, we investigated whether intermediate pluripotent states could be available by manipulating the culture condition during the process of generating pig induced pluripotent stem cells (piPSCs). By optimizing the culture condition that efficiently promotes mesenchymal-to-epithelial transition (MET), we found that combination of three growth factors (LIF, FGF2 and BMP4) and two inhibitors (2i: CHIR99021 and SB431542) could generate an intermediate pluripotent state of piPSCs, which were named as LFB2i-piPSCs. The LFB2i-piPSCs are stable and fulfill all the criteria of pluripotency, including expression of pluripotent genes, differentiation into three germ layers via embryoid bodies in vitro and teratoma in vivo. More importantly, the mRNA-sequencing data showed that LFB2i-piPSCs had a mixed transcriptome of naïve and primed pluripotency, which featured by expressing high levels of SOX2, L-MYC and ESRRB and relatively low levels of POU5F1, KLF4 and NANOG. Small RNA sequencing also demonstrated that LFB2i-piPSCs had a mixed microRNA profile of naïve and primed pluripotency, which featured by expressing high levels of miR-302b/367 cluster and miR-106a/363 cluster, and low levels of most let-7 family members and miR-17/92 cluster. Altogether, the LFB2i-piPSCs represent a stable intermediate pluripotent state with unique transcriptome and microRNA signatures. The LFB2i-piPSCs will provide a new tool to explore the mechanisms of pluripotency and reprogramming on pig species.

Derivation and characterization of human embryonic germ cells: serum-free culture and differentiation potential

This study examined the effects of a chemically defined culture medium supplement, knock-out serum replacement (KSR), on the growth and differentiation of human embryonic germ cells (hEgc) and found that the efficiency of the initial establishment of hEGC lines in KSR medium was significantly higher than in fetal calf serum (FCS) medium. The percentage of undifferentiated hEGC colonies growing in KSR medium was significantly higher than in FCS-based medium (P < 0.05). The hEGC colonies showed typical mouse embryonic germ cell-like morphology. They showed normal and stable diploid karyotype and expressed alkaline phosphatase (AP), stage-specific embryonic antigens (SSEA) and other specific markers of pluripotent cells. In addition, hEGC could form simple and cystic embryoid bodies (EB) that consisted of various cell types including neural, epithelial and rhythmically beating cardiac cells, even sperm-like and oocyte-like cells. Tumour-like outgrowths were formed in nude mice and found to contain a variety of cell types, including uterine epithelium, adipocytes, squamous tissue and skin structures. In conclusion, an appropriate serum-free culture system has been developed for the establishment of hEGC lines. This may provide an in-vitro model to study differentiation and can be used as a potential source of therapy for infertility and regenerative medicine.