RESUMO
Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.
Assuntos
Embrião de Mamíferos/citologia , Genoma Humano/genética , Mutação , Células-Tronco/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , DNA/genética , DNA/metabolismo , Metilação de DNA , DNA Mitocondrial/química , Humanos , Regiões Promotoras GenéticasRESUMO
Human embryonic stem cells (hESCs) can differentiate into a variety of specialized cell types. Thus, they provide a model system for embryonic development to investigate the molecular processes of cell differentiation and lineage commitment. The development of the cardiac lineage is easily detected in mixed cultures by the appearance of spontaneously contracting areas of cells. We performed gene expression profiling of undifferentiated and differentiating hESCs and monitored 468 genes expressed during cardiac development and/or in cardiac tissue. Their transcription during early differentiation of hESCs through embryoid bodies (EBs) was investigated and compared with spontaneously differentiating hESCs maintained on feeders in culture without passaging (high-density (HD) protocol). We observed a larger variation in the gene expression between cells from a single cell line that were differentiated using two different protocols than in cells from different cell lines that were cultured according to the same protocol. Notably, the EB protocol resulted in more reproducible transcription profiles than the HD protocol. The results presented here provide new information about gene regulation during early differentiation of hESCs with emphasis on the cardiomyogenic program. In addition, we also identified regulatory elements that could prove critical for the development of the cardiomyocyte lineage.
Assuntos
Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , Miócitos Cardíacos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Análise por Conglomerados , Células-Tronco Embrionárias/citologia , Humanos , Miócitos Cardíacos/citologiaRESUMO
Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using one standardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.
Assuntos
Técnicas de Cultura de Células/normas , Meios de Cultura/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Inativação Metabólica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cariotipagem , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/enzimologia , Cultura Primária de Células , Reprodutibilidade dos TestesRESUMO
This chapter describes the principles for derivation and maintenance of human embryonic stem cells. Detailed protocols are outlined and researchers who are generally skilled in mammalian cell culture should be able to repeat the processes successfully. Further, the protocols are intended for scientists who do not have access to advanced IVF equipment and therefore cannot perform, e.g. assisted hatching. In addition to derivation, we also discuss characterisation and banking of hES cells.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Blastocisto/citologia , HumanosRESUMO
This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Bancos de Tecidos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais/citologia , Colágeno/farmacologia , Dissecação , Combinação de Medicamentos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Laminina/farmacologia , Camundongos , Proteoglicanas/farmacologia , Controle de Qualidade , Telomerase/metabolismoRESUMO
Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células-Tronco Embrionárias/citologia , Tripsina/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Cromossomos Humanos/genética , Células Clonais , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Células-Tronco Pluripotentes/citologia , Reprodutibilidade dos TestesRESUMO
Housekeeping genes (HKGs) are involved in basic functions needed for the sustenance of the cell and are assumed to be constitutively expressed at a constant level. Based on these features, HKGs are frequently used for normalization of gene expression data. In the present study, we used the CodeLink Gene Expression Bioarray system to interrogate changes in gene expression occurring during differentiation of human ESCs (hESCs). Notably, in the three hESC lines used for the study, we observed that the RNA levels of 56 frequently used HKGs varied to a degree that rendered them inappropriate as reference genes. Therefore, we defined a novel set of HKGs specifically for hESCs. Here we present a comprehensive list of 292 genes that are stably expressed (coefficient of variation <20%) in differentiating hESCs. These genes were further grouped into high-, medium-, and low-expressed genes. The expression patterns of these novel HKGs show very little overlap with results obtained from somatic cells and tissues. We further explored the stability of this novel set of HKGs in independent, publicly available gene expression data from hESCs and observed substantial similarities with our results. Gene expression was confirmed by real-time quantitative polymerase chain reaction analysis. Taken together, these results suggest that differentiating hESCs have a unique HKG signature and underscore the necessity to validate the expression profiles of putative HKGs. In addition, this novel set of HKGs can preferentially be used as controls in gene expression analyses of differentiating hESCs.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Genes Essenciais , Animais , Biomarcadores/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.
Assuntos
Fibrose Cística/metabolismo , Intestino Delgado/metabolismo , Mucina-1/química , Mucina-1/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Mucina-1/análise , Mucinas/metabolismo , Solubilidade , Regulação para CimaRESUMO
There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs). Using light microscopy and immunohistochemistry, we observed that morphological changes of differentiating hESCs precede any major alterations in the expression of several commonly used hESC markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog). In an attempt to quantify the changes during stochastic differentiation of hESCs, we developed a robust and sensitive multi-marker quantitative real-time polymerase chain reaction (QPCR) method. To maximize the sensitivity of the method, we measured the expression of up- and downregulated genes before and after differentiation of the hESCs. Out of the 12 genes assayed, we found it clearly sufficient to determine the relative differentiation state of the cells by calculating a collective expression index based on the mRNA levels of Oct-4, Nanog, Cripto, and alpha-fetoprotein. We evaluated the method using different hESC lines maintained in either feeder-dependent or feeder-free culture conditions. The QPCR method is very flexible, and by appropriately selecting reporter genes, the method can be designed for various applications. The combination of QPCR with hESC-based technologies opens novel avenues for high-throughput analysis of hESCs in, for example, pharmacological and cytotoxicity screening.