Development and Evaluation of a New qPCR Assay for the Detection of <i>Mycoplasma</i> in Cell Cultures

In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor®GeM qEP and Plasmotest® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded

Embryonic Stem Cell-Specific miR302-367 Cluster: Human Gene Structure and Functional Characterization of Its Core Promoter▿ †

MicroRNAs (miRNAs) play a central role in the regulation of multiple biological processes including the maintenance of stem cell self-renewal and pluripotency. Recently, the miRNA cluster miR302-367 was shown to be differentially expressed in embryonic stem cells (ESCs). Unfortunately, very little is known about the genomic structure of miRNA-encoding genes and their transcriptional units. Here, we have characterized the structure of the gene coding for the human miR302-367 cluster. We identify the transcriptional start and functional core promoter region which specifically drives the expression of this miRNA cluster. The promoter activity depends on the ontogeny and hierarchical cellular stage. It is functional during embryonic development, but it is turned off later in development. From a hierarchical standpoint, its activity decays upon differentiation of ESCs, suggesting that its activity is restricted to the ESC compartment and that the ESC-specific expression of the miR302-367 cluster is fully conferred by its core promoter transcriptional activity. Furthermore, algorithmic prediction of transcription factor binding sites and knockdown studies suggest that ESC-associated transcription factors, including Nanog, Oct3/4, Sox2, and Rex1 may be upstream regulators of miR302-367 promoter. This study represents the first identification, characterization, and functional validation of a human miRNA promoter in stem cells. This study opens up new avenues to further investigate the upstream transcriptional regulation of the miR302-367 cluster and to dissect how these miRNAs integrate in the complex molecular network conferring stem cell properties to ESCs

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins

Expression profile of telomere-associated genes in multiple myeloma

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM). © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.Fil: De la Guardia, Rafael Díaz. Universidad de Granada; EspañaFil: Catalina, Purificación. Universidad de Granada; EspañaFil: Panero, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Elosua, Carolina. Universidad de Granada; EspañaFil: Pulgarin, Andrés. Universidad de Granada; EspañaFil: López, María Belén. Universidad de Granada; EspañaFil: Ayllón, Verónica. Universidad de Granada; EspañaFil: Ligero, Gertrudis. Universidad de Granada; EspañaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Leone, Paola E.. Universidad de Granada; Españ

Nodal/Activin Signaling Predicts Human Pluripotent Stem Cell Lines Prone to Differentiate Toward the Hematopoietic Lineage

Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification