Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

• Keywords
• Embryonic stem (ES) cells, Mitosis length, Multipolar division, Single-cell tracking, Spindle assembly checkpoint (SAC), Time-lapse microscopy,
• Publication type
• Journal Article MeSH

Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.

• Keywords
• acentrosomal, cell cycle, centriole, centrosome, differentiation, pluripotency, self-renewal, stem cell,
• MeSH
• Cell Differentiation * MeSH
• Cell Self Renewal * MeSH
• Centrosome metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Intracellular Signaling Peptides and Proteins antagonists & inhibitors   metabolism   MeSH
• Humans MeSH
• Mitosis MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Cell Proliferation MeSH
• Protein Serine-Threonine Kinases antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Intracellular Signaling Peptides and Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• PLK4 protein, human MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• STIL protein, human MeSH Browser

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

• MeSH
• Principal Component Analysis MeSH
• Cell Line MeSH
• Mass Spectrometry methods   MeSH
• Calibration MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Human Embryonic Stem Cells physiology   MeSH
• Multivariate Analysis MeSH
• Mice MeSH
• Neural Networks, Computer * MeSH
• Specimen Handling MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). METHODS: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. RESULTS: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. CONCLUSION: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

• MeSH
• Cell Differentiation * genetics   MeSH
• Chemokine CXCL1 pharmacology   MeSH
• Adult MeSH
• Epidermal Cells * MeSH
• Epidermis pathology   MeSH
• Fibroblast Growth Factor 2 pharmacology   MeSH
• Interleukin-8 pharmacology   MeSH
• Keratin-10 metabolism   MeSH
• Keratin-14 metabolism   MeSH
• Keratinocytes cytology   drug effects   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Melanocytes metabolism   MeSH
• Melanoma metabolism   pathology   MeSH
• Neoplasm Metastasis MeSH
• Cell Communication * MeSH
• Cell Line, Tumor MeSH
• S100 Proteins metabolism   MeSH
• Aged MeSH
• Gene Expression Profiling MeSH
• Vascular Endothelial Growth Factor A pharmacology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chemokine CXCL1 MeSH
• Fibroblast Growth Factor 2 MeSH
• Interleukin-8 MeSH
• Keratin-10 MeSH
• Keratin-14 MeSH
• S100 Proteins MeSH
• Vascular Endothelial Growth Factor A MeSH

Malignant melanoma is a highly aggressive tumor with increasing incidence and high mortality. The importance of immunohistochemistry in diagnosis of the primary tumor and in early identification of metastases in lymphatic nodes is enormous; however melanoma phenotype is frequently variable and thus several markers must be employed simultaneously. The purposes of this study are to describe changes of phenotype of malignant melanoma in vitro and in vivo and to investigate whether changes of environmental factors mimicking natural conditions affect the phenotype of melanoma cells and can revert the typical in vitro loss of diagnostic markers. The influence of microenvironment was studied by means of immunocytochemistry on co-cultures of melanoma cells with melanoma-associated fibroblast and/or in conditioned media. The markers typical for melanoma (HMB45, Melan-A, Tyrosinase) were lost in malignant cells isolated from malignant effusion; however, tumor metastases shared identical phenotype with primary tumor (all markers positive). The melanoma cell lines also exerted reduced phenotype in vitro. The only constantly present diagnostic marker observed in our experiment was S100 protein and, in lesser extent, also Nestin. The phenotype loss was reverted under the influence of melanoma-associated fibroblast and/or both types of conditioned media. Loss of some markers of melanoma cell phenotype is not only of diagnostic significance, but it can presumably also contribute to biological behavior of melanoma. The presented study shows how the conditions of cultivation of melanoma cells can influence their phenotype. This observation can have some impact on considerations about the role of microenvironment in tumor biology.

• MeSH
• Models, Biological MeSH
• Cell Culture Techniques MeSH
• Fibroblasts cytology   drug effects   metabolism   MeSH
• Immunophenotyping MeSH
• Immunohistochemistry MeSH
• Coculture Techniques MeSH
• Culture Media, Conditioned pharmacology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• MART-1 Antigen metabolism   MeSH
• Melanoma metabolism   pathology   MeSH
• Melanoma-Specific Antigens metabolism   MeSH
• gp100 Melanoma Antigen MeSH
• Biomarkers, Tumor metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Cells, Cultured MeSH
• Tumor Microenvironment drug effects   MeSH
• Skin Neoplasms metabolism   pathology   MeSH
• Nestin metabolism   MeSH
• S100 Proteins metabolism   MeSH
• Monophenol Monooxygenase metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Culture Media, Conditioned MeSH
• MART-1 Antigen MeSH
• Melanoma-Specific Antigens MeSH
• gp100 Melanoma Antigen MeSH
• Biomarkers, Tumor MeSH
• Nestin MeSH
• PMEL protein, human MeSH Browser
• S100 Proteins MeSH
• Monophenol Monooxygenase MeSH