The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

• MeSH
• Acetylcysteine administration & dosage   MeSH
• Actinin genetics   MeSH
• Embryonic Stem Cells cytology   MeSH
• Homeobox Protein Nkx-2.5 genetics   MeSH
• Myocytes, Cardiac cytology   MeSH
• Culture Media, Serum-Free * MeSH
• Cells, Cultured MeSH
• Myosins genetics   MeSH
• Mice MeSH
• In Vitro Techniques MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetylcysteine MeSH
• Actinin MeSH
• Homeobox Protein Nkx-2.5 MeSH
• Culture Media, Serum-Free * MeSH
• Myosins MeSH
• Nkx2-5 protein, mouse MeSH Browser

Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: Neurotransplantation has great potential for future treatments of various neurodegenerative disorders. Preclinically, the Lurcher mutant mouse represents an appropriate model of genetically-determined olivocerebellar degeneration. The aim of the present study was to assess survival of naïve and neurally differentiated P19 carcinoma stem cells following transplantation into the cerebellum of Lurcher mice and wild type littermates. MATERIAL/METHODS: Adult normal wild type (n=51) and Lurcher mutant mice (n=87) of the B6CBA strain were used. The mean age of the animals at the time of transplantation was 261.5 days. Suspension of naive and neurally differentiated P19 carcinoma stem cells was injected into the cerebellum of the mice. In the Lurcher mutants, 2 depths of graft injection were used. Three weeks after implantation the brains of experimental animals were examined histologically. RESULTS: Survival of neuroprogenitor grafts at a depth of 1.6 mm was significantly higher in wild type vs. Lurcher mutant mice. In wild type mice, the typical graft localization was in the middle of the cerebellum, whereas in Lurcher mice the graft was never found inside the degenerated cerebellum and was primarily localized in the mesencephalon. CONCLUSIONS: We conclude that the appearance and low survival rate of cerebellar P19 carcinoma stem cell grafts in the Lurcher mutant mice weigh against the therapeutic value of this cell line in preclinical studies of neurodegeneration.

• MeSH
• Cerebellum cytology   MeSH
• Mice, Mutant Strains MeSH
• Mice MeSH
• Neoplastic Stem Cells cytology   MeSH
• Neural Stem Cells cytology   MeSH
• Graft Survival MeSH
• Stem Cell Transplantation * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND METHODS: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells. RESULTS AND CONCLUSIONS: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.

• MeSH
• Models, Biological MeSH
• Cell Differentiation * MeSH
• Cell Lineage * MeSH
• Cyclin A metabolism   MeSH
• Cyclin D MeSH
• Cyclin E metabolism   MeSH
• Cyclin-Dependent Kinase 4 metabolism   MeSH
• Cyclin-Dependent Kinase 6 metabolism   MeSH
• Cyclins metabolism   MeSH
• Embryo, Mammalian cytology   metabolism   MeSH
• Embryonic Stem Cells metabolism   MeSH
• G1 Phase MeSH
• Cyclin-Dependent Kinase Inhibitor p27 metabolism   MeSH
• Intracellular Space metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Cell Proliferation MeSH
• S Phase MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK4 protein, human MeSH Browser
• Cyclin A MeSH
• Cyclin D MeSH
• Cyclin E MeSH
• Cyclin-Dependent Kinase 4 MeSH
• Cyclin-Dependent Kinase 6 MeSH
• Cyclins MeSH
• Cyclin-Dependent Kinase Inhibitor p27 MeSH

Mammalian heterochromatin protein 1 (HP1alpha, HP1beta, HP1gamma subtypes) and transcriptional intermediary factor TIF1beta play an important role in the regulation of chromatin structure and function. Here, we investigated the nuclear arrangement of these proteins during differentiation of embryonal carcinoma P19 cells into primitive endoderm and into the neural pathway. Additionally, the differentiation potential of trichostatin A (TSA) and 5-deoxyazacytidine (5-dAzaC) was studied. In 70% of the cells from the neural pathway and in 20% of TSA-stimulated cells, HP1alpha and HP1beta co-localized and associated with chromocentres (clusters of centromeres), which correlated with clustering of TIF1beta at these heterochromatic regions. The cell types that we studied were also characterized by a pronounced focal distribution of HP1gamma. The above-mentioned nuclear patterns of HP1 and TIF1beta proteins were completely different from the nuclear patterns observed in the remaining cell types investigated, in which HP1alpha was associated with chromocentres while HP1beta and HP1gamma were largely localized in distinct nuclear regions. Moreover, a dispersed nuclear distribution of TIF1beta was observed. Our findings showed that the nuclear arrangement of HP1 subtypes and TIF1beta is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes.

• MeSH
• Azacitidine analogs & derivatives   pharmacology   MeSH
• Cell Differentiation drug effects   physiology   MeSH
• Cell Nucleus metabolism   MeSH
• Centromere metabolism   MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Decitabine MeSH
• Chromobox Protein Homolog 5 MeSH
• Immunohistochemistry MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Histone Deacetylase Inhibitors MeSH
• Nuclear Proteins metabolism   MeSH
• Microscopy, Confocal MeSH
• Hydroxamic Acids pharmacology   MeSH
• DNA Methylation drug effects   MeSH
• Cell Line, Tumor MeSH
• Protein Subunits genetics   metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Transcription Factors metabolism   MeSH
• Blotting, Western MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Azacitidine MeSH
• Chromosomal Proteins, Non-Histone MeSH
• Decitabine MeSH
• Chromobox Protein Homolog 5 MeSH
• Enzyme Inhibitors MeSH
• Histone Deacetylase Inhibitors MeSH
• Nuclear Proteins MeSH
• Hydroxamic Acids MeSH
• Protein Subunits MeSH
• Recombinant Fusion Proteins MeSH
• transcriptional intermediary factor 1 MeSH Browser
• Transcription Factors MeSH
• trichostatin A MeSH Browser
• Green Fluorescent Proteins MeSH