Can Vero cell co-culture improve in-vitro maturation of bovine oocytes?

This study was carried out to evaluate the effect of Vero cell co-culture on developmental competence of immature oocytes. Bovine cumulus-oocyte complexes (COC) were matured in presence or absence of Vero cells. Matured oocytes were inseminated and cultured for up to 9 days. Cleavage percentages were recorded on day 2 after insemination and embryos were evaluated on a daily basis. Expanding/expanded and hatching/hatched blastocysts were used for cell number assay. Results indicated a significantly greater cleavage percentage in oocytes matured in presence of Vero cells than control (86% versus 76%, P < or = 0.05). The percentages of advanced embryos appear to be greater on a daily basis in COC matured in presence of Vero cells compared with control. However, these differences were not significant. Blastocysts derived from COC matured in the presence of Vero cells had a significantly higher (P < or = 0.05) number of inner cell mass, trophectoderm and total cell number in expanding/expanded (65.25, 224.5 and 289.7 respectively) and hatching/hatched (67.75, 289.75 and 357.5) embryos in comparison to the control (42, 203.5, 245.5 and 51.3, 265, 316.3 respectively). Results confirm that co-culture of bovine COC during in-vitro maturation, enhances their ability for cleavage and for producing blastocysts with higher quality.

Comparative proteomic analysis of mouse embryonic stem cells and neonatal-derived cardiomyocytes.

Pluripotent embryonic stem cells (ESCs) spontaneously differentiate via embryo-like aggregates into cardiomyocytes. A thorough understanding of the molecular conditions in ESCs is necessary before other potential applications of these cells such as cell therapy can be materialized. We applied two dimensional electrophoresis to analyze and compare the proteome profiling of spontaneous mouse ESC-derived cardiomyocytes (ESC-DCs), undifferentiated mouse ESCs, and neonatal-derived cardiomyocytes (N-DCs). Ninety-five percent of the proteins detected on the ESC-DCs and N-DCs could be precisely paired with one other, whereas only twenty percent of the ESC proteins could be reliably matched with those on the ESC-DCs and N-DCSs, suggesting a striking similarity between them. Having identified sixty proteins in the said three cell types, we sought to provide possible explanations for their differential expression patterns and discuss their relevance to cell biology. This study provides a new insight into the gene expression pattern of differentiated cardiomyocytes and is further evidence for a close relation between ESC-DCs and N-DCSs.

Kinetic properties of extracted lactate dehydrogenase and creatine kinase from mouse embryonic stem cell- and neonatal-derived cardiomyocytes.

Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.

Proteomic signature of human embryonic stem cells.

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.

Generation of insulin-secreting cells from human embryonic stem cells.

A growth factor-mediated selection method was used to obtained insulin-secreting cells from human embryonic stem cells (hESC; Royan H1). Our resultant cells were positive for dithizone, a zinc-chelating agent known to selectively stain pancreatic beta cells and immunoreactive for antibodies against insulin, glucagon, and C-peptide. Semi-quantitative reverse transcription-polymerase chain reaction detected expression of proinsulin, insulin and other pancreatic beta-cell-related genes, such as Nkx6.1, Is11, Glut2, Pax4, and prohormone convertase2 (PC2). Moreover, glucagon, somatostatin, K(ATP)-channel genes KIR6.2 and SUR1, islet amyloid polypeptide (IAPP), PC1/3, and glucokinase (GCK) were expressed in the differentiating hESC in a developmental stage-dependent manner. Also, the addition of glucose to the culture medium triggered insulin release from differentiated cells, but transmission electron microscopy of the differentiated cells did not show typical beta-cell granules, even though secretary granules were detected. The results showed that hESC have the ability to transcribe and process insulin, but further improvements of the current method are required to generate a sufficient source of true beta cells for the treatment of diabetes mellitus.

Generation of new human embryonic stem cell lines with diploid and triploid karyotypes.

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.

The effect of extracellular matrix on embryonic stem cell-derived cardiomyocytes.

Extracellular matrix (ECM) components significantly influence the growth characteristics of cardiomyocytes, development of spontaneous contractile activity and morphologic differentiation. The present study involves characterization of ES cell-derived cardiomyocytes cultured on different ECMs. We hypothesized that cardiogel, which is a naturally occurring ECM containing a complex mixture of laminin, fibronectin may better support ES cell-derived cardiomyocytes maturation in terms of chronotropic characteristics and subcellular structure development. Spontaneously differentiated cardiomyocytes from 7-day embryoid bodies (EBs) derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a fibroblast-derived ECM (cardiogel), and control (without ECM) for 7 + 21 days. The growth characteristics of cardiomyocytes were assessed by immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR), positive and negative chronotropic drugs and transmission electron microscopy (TEM). The spontaneously beating cells expressed markers characteristic of cardiomyocytes including alpha-actinin, desmin, troponin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin-43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF) and myosin light chain isoform-2V (MLC-2V). In addition, spontaneous beating of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel, by carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine and/or Bay-K. Ultrastructure analysis showed myofibrillar bundle organization and exhibited intercalated discs, T-tubules, Z-, A-, I-, H- and M-bands and the showed earlier maturation of the cardiomyocytes on cardiogel. We conclude that substructurally matured cardiomyocytes can be produced from ES cells and the cardiac fibroblast-derived ECM (cardiogel) supported earlier maturation of ES-derived cardiomyocytes, in terms of chronotropic characteristics and subcellular structure development.

Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.