Jana Karbanová

Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

Haematopoietic stem cell differentiation promotes the release of prominin‐1/CD133‐containing membrane vesicles—a role of the endocytic–exocytic pathway

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co‐culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome‐mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin‐1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol‐binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein–lipid assemblies might be the essential structural determinant in the release process of prominin‐1. Collectively, these data support the concept that prominin‐1‐containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium.

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

Human dental pulp stem cells--isolation and long term cultivation.

Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflicks limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60-90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflicks limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

Prominin-1 (CD133) is not restricted to stem cells located in the basal compartment of murine and human prostate

Rodent and human prominin‐1 are expressed in numerous adult epithelia and somatic stem cells. A report has shown that human PROMININ‐1 carrying the AC133 epitope can be used to identify rare prostate basal stem cells (Richardson et al., J Cell Sci 2004; 117:3539–3545). Here we re‐investigated its general expression in male reproductive tract including mouse and human prostate and in prostate cancer samples using various anti‐prominin‐1 antibodies.

Mixed phenotype hepatocellular carcinoma after transarterial chemoembolization and liver transplantation.

We investigated the phenotype of hepatocellular carcinoma (HCC) in livers removed during transplantation after local ablation therapy by transarterial chemoembolization (TACE). This study involved 80 HCC nodules (40 treated with TACE and 40 not treated with local ablation before transplantation) observed in 64 explanted livers and included clinicopathological evaluations as well as single and double immunohistochemistry and reverse‐transcription polymerase chain reaction (RT‐PCR) for cytokeratin 19 (CK19), epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM), and CD133. HCCs with complete necrosis post‐TACE without viable tumors were excluded from the analysis. Cholangiolar, glandular, or spindle cell areas suggestive of a mixed hepatocholangiocellular phenotype were seen in 14 post‐TACE HCCs and in none of the non‐TACE HCCs (P < 0.001). According to single‐epitope immunohistochemistry of post‐TACE HCCs, CD133, CK19, EpCAM, and NCAM were expressed in 14 (35%), 8 (20%), 12 (30%), and 8 (20%), respectively. Only EpCAM was detected in 4 non‐TACE HCC cases (10%). RT‐PCR experiments using tissues obtained by laser microdissection showed that 4 of 5 investigated post‐TACE HCCs expressed at least 1 of the markers, which were coexpressed in 3 of 5 tumors, whereas CD133 and EpCAM were individually expressed in 2 non‐TACE HCCs. Double immunostaining showed that CD133+ cells frequently coexpressed CK19, EpCAM, or NCAM. Interestingly, the recurrence rate for patients with CD133+ post‐TACE HCC was significantly higher than the rate for patients with CD133− post‐TACE HCC (P = 0.025). In conclusion, HCC with the combined hepatocholangiocellular phenotype appears to be more frequent in post‐TACE HCC versus untreated HCC. Further studies are needed to investigate the potential relationships between TACE and HCC subpopulations with a chemoembolization‐resistant phenotype and their clinical significance. Liver Transpl 17:943–954, 2011.

Expression of intermediate filament nestin in blood vessels of neural and non-neural tissues.

Our previous findings performed in rat tissues demonstrated that intermediate filament nestin is expressed in endothelial cells of newly formed blood vessels of developing organs and neural transplants. The aim of the present study was to identify other cellular markers expressed in nestin-positive (nestin+) blood vessels. To reach this goal we performed double immunofluorescent study to co-localize nestin with endothelium-specific markers (CD31, CD34 II, vimentin) or markers of perivascular cells (GFAP, SMA) in paraffin-embedded sections of normal human brain tissue, low- and high-grade gliomas, postinfarcted heart and samples of non-neural tumours. Our findings documented that all the samples examined contained blood vessels with different ratio of nestin+ endothelial cells. Double immunostaining provided unambiguous evidence that endothelial cells expressed nestin and allowed them to distinguish from other nestin+ elements (perivascular astrocytic endfeet, undifferentiated tumour cells, smooth muscle cells and pericytes). Nestin+ endothelium was not confined only to newly formed capillaries but was also observed in blood vessels of larger calibres, frequently in arterioles and venules. We conclude that nestin represents a reliable vascular marker that is expressed in endothelial cells. Elevation of nestin expression likely corresponds to reorganization of intermediate filament network in the cytoskeleton of endothelial cells in the course of their maturation or adaptation to changes in growing tissues.

Histological and histochemical analysis of embryoid bodies.

We examined the histological structure of embryoid bodies arising from aggregation of mouse embryonic stem (ES D3) cells after 7, 12, 18 and 26 days of in vitro culture. Morphology of originally solid embryoid bodies was affected by the process of cavitation that resulted in formation of cystic embryoid bodies and by spontaneous differentiation of the ES D3 cells. We applied in situ immunophenotyping to characterise cell populations that spontaneously differentiated inside the embryoid bodies in the various stages. The most distinct cell populations that were found inside embryoid bodies were alpha-fetoprotein-positive endodermal cells and myogenic cells that expressed desmin, myogenin or smooth muscle actin. ES D3-derived endothelial cells generated during vasculogenesis inside the embryoid bodies differed from mature endothelial cells because they did not stain for von Willebrand factor. These cells also differed from endothelial cell that were generated during angiogenesis since they did not stain for the intermediate filament nestin. Our results demonstrate the usefulness of this in vitro model for studying early embryogenesis.

Telomere Attrition Occurs during Ex Vivo Expansion of Human Dental Pulp Stem Cells

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

CD133 and membrane microdomains: old facets for future hypotheses.

Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important, not only from a clinical point of view, but also in terms of our basic knowledge of cellular transformation. By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1), novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade. Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.