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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. [ABSTRACT FROM AUTHOR]

In this study we characterized regulation of the Na+/H+ exchanger promoter in several tissue types. A conserved poly (dA:dT) region was important in regulation of the promoter. Nuclear extracts from rat myocardium and from mouse proximal tubule cells protected the poly (dA:dT) region of the NHE1 promoter. A protein from nuclear extracts also bound to the poly (dA:dT) element in gel mobility shift binding assays. The binding was specific and was removed by mutations in the poly (dA:dT) region. Characterization of the binding to the poly (dA:dT) region in gel mobility shift assays showed that it was reduced by high concentrations of the divalent cations Mg++ and Mn++. The inhibition by divalent cations was reduced by decreasing the pH of the binding assay. N-terminal sequencing of the poly (dA:dT) binding protein showed that it was a member of the HMG (high mobility group) family of nuclear proteins which are important in cell growth and proliferation. The results are the first direct detection of a protein that regulates the NHE1 promoter. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem- and induced pluripotent stem-like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage-specific cells. The 'Yamanaka factors' was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage-specific media for 1-2 weeks, the cardiomyocyte-like cells and neurocyte-like cells were generated and confirmed by the quantitative RT-PCR and immunocytochemical staining. The metastable-state cells could also be converted into oocyte-like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2-3 weeks. The generated OLCs were surrounded by cumulus granulosa cell-like cells and formed a structure resembling goat cumulus-oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell-specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage-specific cells can facilitate stem cell research in domestic animals. [ABSTRACT FROM AUTHOR]

At present, the experimental technique to produce human red blood cells in vitro is complicated, and in order to optimize the induction steps, human pluripotent stem cells were differentiated into red blood cells through two induction steps. First, human pluripotent stem cells (including Rh negative A type umbilical cord mesenchymal stem cells (hUCMSCRh-A) and human iPS cells (hiPS)) were differentiated into CD31+ and CD34+ cells in BVF medium. PCR and flow cytometry were used to exam the expression of CD31 and CD34. We found that hUCMSCRh-A derived CD31+ and CD34+ cells were 5.3% and 22.7%, respectively; hiPS derived CD31+ and CD34+ cells were 31.2% and 8.2%, respectively. For the second induction step, the obtained CD31+ and CD34+ cells were differentiated into mature erythrocytes for 36 days under the addition of various growth factors. Through Giemsa staining, we found that the obtained mature erythrocytes were similar in morphology and size to normal human erythrocytes, and some obtained erythrocytes were enucleated. Globin expression was detected by real time RT-PCR, and the expression of β-globin was more than 20%. The obtained erythrocytes are collected into the centrifuge tube, and then erythrocytes were naturally settled and showed the red color. Our findings provide a novel and effective method for the quantity generation of human red blood cells in vitro.

In post-transcriptional mRNA modification, m⁶A has been observed in a wide range of eukaryotes. METTL3, as a component of methyltransferase complex for m⁶A modification, regulates mouse naïve pluripotency and influences mRNA stability, especially affecting the expression level of the key pluripotent transcription factors. To reveal the expression pattern of the porcine METTL3 gene, we analyzed METTL3 expression level in different porcine tissues, somatic cells, and induced pluripotent stem cells (piPSCs) by RT-PCR. To identify the function of METTL3 for regulation of the expression of porcine pluripotent genes, we cloned a 1 859-bp coding sequence of METTL3 and synthesized a shRNA against METTL3. When knocking down METTL3 expression in piPSCs, the cell type of piPSCs became naïve-like morphology, alkaline phosphatase activity was increased, and expression level of pluripotent genes NANOG, OCT4 and LIN28A was significantly elevated. In addition, piPSCs cultured in medium containing 10 mmol/L cycloleucine for 48 h exhibited the similar result as that knocked down METTL3. These findings set the stage for optimization of piPS culture condition and further study on the roles of m6A in piPSCs.

Laminin (LN) proteins are important components of extracellular matrix. These proteins regulate cell proliferation, differentiation, migration, and tissue repair. The LN family has 12 genes that encode 5 α, 4 β, and 3 γ proteins. LamininA5 (LAMA5) as an important gene can support pluripotent cell growth and have been widely studied. However, porcine LAMA5 is absent in all tested porcine genomic databases so far. In this study, we confirmed for the first time the existence of porcine LAMA5 through bioinformatics analysis, and verified this result by cDNA cloning and sequencing. To reveal the expression pattern of Laminin gene family, we detected the expression of Laminin genes in porcine tissues, somatic cells, and porcine induced pluripotent stem cells (piPSCs). The results showed that an alternative splicing variant of Laminin B1 (LAMB1-a) was found exclusively in all tested piPSCs. The expression of this alternative splicing variant is positively correlated with the pluripotent state of piPSCs. The above findings provide evidences and foundations for the father use of LN as extracellular matrix to facilitate the derivation and culture of porcine pluripotent stem cells.

The estrogen related receptor family member Esrrb (Estrogen related receptor β) is a gene that expresses in the early stage of embryo and plays an important role in the core pluripotent network. Its function has been analyzed in human and mouse, although no report so far related to pig. Therefore, to explore its mechanism of transcriptional regulation and expression pattern, we cloned a 3.3 kb pig ESRRB promoter by PCR and constructed the green fluorescence protein (GFP) reporter vector pE3.3. We used these vectors to study the ESRRB expression pattern in 293T, Hela and C2C12. Sequence was analyzed for regulatory elements that share homology to known transcription factor binding sites by TFSEARCH and JASPER program. Some pluripotency related genes such as SMAD, STAT3, MYC, KLF4 and ESRRB have been found within the 3.3 kb sequence by co-transfected pig ESRRB promoter and these potential regulators. We found that ESRRB only expressed in 293T and SMAD could activate ESRRB expression obviously. To determine the core promoter region, a series of ESRRB promoter fragments with gradually truncated 5'-end were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure these promoter activities. The result suggested that the core promoter of pig ESRRB located within -25 bp to -269 bp region. These results suggest that these transcription factor binding sites and the core promoter region may be essential for transcriptional regulation of pig ESRRB gene.

Oocyte-like cells (OLC) can be generated by stem cells after the induction and differentiation in vitro, and maturated when transplanted in vivo to improve the development potential. Human amniotic fluid stem cells (hAFSC) were cultured for 10 days in porcine follicle fluid (pFF) that was extracted from the medium follicle with high levels of hormones and Bmp 15 protein. After the induction, the cell aggregates showed the germ cell-like cells and produced the germ cell marker oct4, and triggered epigenetic changes with high expression of methylation transferase gene dnmt3b. The cell aggregates were packaged into porcine theca folliculi to form grafts, which were then transplanted into mouse renal capsule. After one month of transplantation, the morphology of OLC from a graft was not only similar to oocytes, but also expressed the germ cells markers (oct4, nanog, stella, ifitm3, dazl, nanos3, bmp15, and gd9). The results demonstrate that the in vivo differentiation model was useful for OLC development.

The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

The octamer-binding transcription factor 4 gene (Oct4) plays a critical role in maintaining pluripotency during early mammalian embryonic development and self-renewal of embryonic stem (ES) cells. In this study, we cloned the Oct4 cDNA and 2.8-kb regulatory region upstream of the start codon in Guanzhong dairy goat ( Capra hircus ). The comparative sequence analysis of Oct4 cDNA showed that it was highly conserved among six mammalian species. The goat Oct4 5' regulatory regions were homologous to the corresponding regions of Oct4 in other species and were functional in directing the expression of luciferase in mouse P19 embryonic carcinoma cells and mouse J1 ES cells. Furthermore, the methylation levels in the goat Oct4 minimal promoter and proximal enhancer in the fetal genital ridge were lower than those in the heart. Additionally, two processed pseudogenes that shared high homology with goat Oct4 cDNA were identified and characterized.

The aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells (pAFS). For cardiac differentiation, the cells were formed embryoid bodies (EBs) firstly, then cultured in induced-medium including 5-azacytidine (5-aza) and vitamin C (Vc). We detected the specific markers of cardiomyocyte by immunocytochemistry, RT-PCR and transmission electron microscope. The results showed that some embryoid bodies beat rhythmically after 10 days of induction. Furthermore, analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest (33%) when induction using 0.1 mmol/L Vc and 5 micromol/L 5-aza. Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressed alpha-actin, Tnni3. RT-PCR analysis also illustrated that TbX5, Gata4, alpha-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters. Especially, we observed basic structures of myocardium, such as myofilament, glycogen granule and so on by transmission electron microscope. In conclusion, 5-azacytidine and vitamin C could promote differentiation of pAFS into myocardium.

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.

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo, we engineered mice with a 160-kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species-specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. In addition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1-induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.

Placental choriocarcinoma is a highly malignant tumor of the female reproductive organs. Even with chemotherapy, placental choriocarcinoma has a 20% mortality rate due to its indistinct pathogenesis and the absence of efficient therapeutic methods for this cancer. Wnt proteins and Wnt signaling are involved in a variety of developmental and cellular processes, and aberrant activation of Wnt signaling is linked to carcinogenesis in many types of tissue. In the present study, using immunostaining and reverse transcription PCR, we found that Wnt5a was prominently expressed in human primary cytotrophoblast cells, but absent in the human choriocarcinoma cell line JAR. This implies that there is a previously unknown correlation between Wnt5a and placental choriocarcinoma. Furthermore, we found that the addition of exogenous recombinant Wnt5a protein to the JAR cells repressed their proliferation and induced apoptosis, indicating that Wnt5a has a tumor suppressive effect in placental choriocarcinoma. In conclusion, the results of the present study suggest that there is a correlation between Wnt5a and human placental choriocarcinoma; therefore, Wnt5a has a potential use in the treatment of this malignancy.

Currently, animal somatic cell reprogramming into the induced pluripotent stem cell (iPS) is one of the hottest research target in the field of cell biology. We focused on the analysis of telomerase reverse transcriptase (TERT) gene expression during goat somatic fibroblasts reprogramming, and investigated the relationship between the expression of TERT and the pluripotency of reprogrammed cells. RNA samples of fetal tissues isolated from Guanzhong milk goat fetus, and the induced goat reprogramming cell clones were used to determine the relative expression levels of TERT by the real-time RT-PCR method. Goat embryonic fibroblasts (GEF) collected from the Guanzhong milk goat with normal karyotype were induced by 4 transcription factors to become reprogramming cells. The expression of TERT in reprogramming cells was detected by Real-time RT-PCR. The results showed that the expression of TERT in testis tissue was higher than that in epithelial tissues (P < 0.01). The expression level of TERT was higher in AP staining positive cells than that in AP staining negative cells (P < 0.01). This result indicated that TERT activity played an important role in cell reprogramming.

Homologous recombination is an important technique that is used to modify mammalian genome. Here, we constructed an efficient common gene targeting vector based on the plasmid pBS246. The vector consisted two positive selection markers, neomycin resistance gene (neo) and enhanced green fluorescent protein gene (EGFP) flanked by locus of X-over P1 (LoxP) sites. Two synthesized multiple cloning sequences MCS-1 and MCS-2 that contain several "8 bp cutter" enzyme sites were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk (herpes simplex virus thymidine kinase) gene was located adjacent to MCS-1 site. The constructed vector was named pGT-V1, and its functions were characterized in C2C12 cells. The vector had the following unique features: 1) EGFP was used to monitor instantly the transfection rate that was essential for increasing the efficiency of gene knockout (KO); 2) The EGFP marker located between two LoxP sites was able to be removed from KO positive cells to avoid the potential damage of selection markers to the recipient cells. The process could be monitored visually and the positive cells without selecting markers (the loss of green fluorescent cells) could be sorted out by either flow cytometry or immunomagnetic beads; 3) "8 bp cutter" restriction sites were embedded in MCS sequences, which then enhanced the versatility of this vector. In summary, the constructed plasmid optimized the vector of gene targeting and provided a new technique means for the transgenic animal research.

Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO., (Copyright 2010 Elsevier Inc. All rights reserved.)

The somatic cells can be induced into ES-like stem cells when retrovirally infected the defined transcription factors including Oct4, Sox2, Klf4 and c-Myc. These ES-like cells are named induced pluripotent stem (iPS) cells and this method is called iPS technology. Until the end of 2009, iPS cell lines have been generated in various animal species, such as mouse, human, rhesus monkey, rat and pig. Mouse iPS cells are also used to generate chimera mice and viable mice through the tetraploid complementation. Although iPS cells are extremely similar to ES cells in both morphology and growth features, to generate iPS cells do need the defined culture procedures. Based on the update global iPS technology development and the iPS studies in our laboratory, this paper focused on the establishment of iPS cell lines and improvement of iPS cell culture condition.

We constructed shRNA vectors with different stem length, and tested the silencing effectiveness in mouse cells and embryos. We designed interfering RNAs with stems of 21 bp, 27 bp, and 29 bp. The enhanced green fluorescent protein gene was used as target gene. The synthesized single strands were annealed and cloned into psiSTRIKE and the recombinant plasmids (EGFP-21 siRNA, EGFP-27 siRNA, and EGFP-29 siRNA) were transfected into the mouse embryonic fibroblast with lipofection. The mRNA expression level of the enhanced green fluorescent protein gene was checked by real-time quantitative PCR. The silencing effectiveness of the 29 bp shRNA vector was stronger than which of the 21 bp and 27 bp. The findings in this study are of interest for selecting the hairpins for mouse individuals.

Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.

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.

The integrity and transparency of the cornea plays a key role in preserving vision. This paper reports a procedure to create an artificial sheet of corneal epithelium from cryopreserved limbal stem cells (LSCs) and to use this for corneal transplantation. Corneal LSCs were isolated from biopsy specimens of rabbit limbal lamellar and cryopreserved in liquid nitrogen at 2-4 passages. The cells were grown in culture medium for 12-14 days on top of a cell-free human amniotic membrane framed on a nitrocellulose sheet. The corneal epithelium generated was transplanted into the right eyes of 14 LSC deficient (LSCD) rabbits (seven experimental animals, seven controls) with corneal damage. The seven LSCD rabbits in the experimental group were transplanted with a corneal epithelial sheet generated from the cryopreserved corneal LSCs. Four LSCD rabbits were used as the vehicle control and were transplanted with a cell-free amniotic membrane, and the remaining three LSCD rabbits were negative controls without transplantation. Over a 2-month recovery period, 2/7 animals in the experimental group recovered completely, four recovered partially and one did not respond. In the control groups, three negative controls and three vehicle controls lost their vision completely, and one of the vehicle controls partially recovered transparency of the cornea Following treatment, corneal transparency of the experimental rabbits was significantly improved compared to controls (P<0.05). The results indicated that cryopreserved corneal LSCs can repair damaged rabbit cornea, suggesting a possible new clinical approach to reconstruction of corneal epithelium.

Embryonic stem cells provide one potential source of cardiomyocytes for cardiac transplantation; however, after differentiation of stem cells in vitro, cardiomyocytes usually account for only a minority of cells present. To gain insights into improving cardiomyocyte development from stem cells, we examined the role of the Na(+)/H(+) exchanger isoform 1 (NHE1) in cardiomyocyte differentiation. NHE1 protein and message levels were induced by treatment of CGR8 cells to form embryoid bodies and cardiomyocytes. The NHE1 protein was present on the cell surface and NHE1 inhibitor-sensitive activity was detected. Inhibition of NHE1 activity during differentiation of CGR8 cells prevented cardiomyocyte differentiation as indicated by decreased message for transcription factors Nkx2-5 and Tbx5 and decreased levels of alpha-myosin heavy chain protein. Increased expression of NHE1 from an adenoviral vector facilitated cardiomyocyte differentiation. Similar results were found with cardiomyocyte differentiation of P19 embryonal carcinoma cells. CGR8 cells were treated to induce differentiation, but when differentiation was inhibited by dispersing the EBs, myocardial development was inhibited. The results demonstrate that NHE1 activity is important in facilitating stem cell differentiation to cardiomyocyte lineage. Elevated NHE1 expression appears to be triggered as part of the process that facilitates cardiomyocyte development.

Sodium/proton exchanger 1 (NHE1) plays an important role in the cardiomyocyte development. To study the effect of NHE1 activity in stem cells differentiation into cardiomyocytes, we treated P19 stem cells with dimethyl sulfoxide (DMSO) to initiate cardiomyocyte differentiation. In separate experiments, P19 cells were incubated with NHE1 specific inhibitor EMD87580 during the DMSO induction. The formed embryoid bodies (EBs) were detected with cell morphology detection, immunohistochemisty staining and RT-PCR analysis of expression of cardio-specific gene markers. Results showed that P19 cells were able to differentiate into cardiomyocytes and form the beating cell clusters. However, when cells treated with NHE1 inhibitor EMD87580, they could still form the EBs and proliferate when cell clusters adhered on the culture plate, but cells were unable to differentiate. This observation indicates that inhibition of NHE1 activity affected P19 stem cells differentiating into cardiomyocytes.

To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.