Jaroslav Mokry

Signaling involved in hair follicle morphogenesis and development.

Hair follicle morphogenesis depends on Wnt, Shh, Notch, BMP and other signaling pathways interplay between epithelial and mesenchymal cells. The Wnt pathway plays an essential role during hair follicle induction, Shh is involved in morphogenesis and late stage differentiation, Notch signaling determines stem cell fate while BMP is involved in cellular differentiation. The Wnt pathway is considered to be the master regulator during hair follicle morphogenesis. Wnt signaling proceeds through EDA/EDAR/NF-κB signaling. NF-κB regulates the Wnt pathway and acts as a signal mediator by upregulating the expression of Shh ligand. Signal crosstalk between epithelial and mesenchymal cells takes place mainly through primary cilia. Primary cilia formation is initiated with epithelial laminin-511 interaction with dermal β-1 integrin, which also upregulates expression of downstream effectors of Shh pathway in dermal lineage. PDGF signal transduction essential for crosstalk is mediated through epithelial PDGF-A and PDGFRα expressed on the primary cilia. Dermal Shh and PDGF signaling up-regulates dermal noggin expression; noggin is a potent inhibitor of BMP signaling which helps in counteracting BMP mediated β-catenin inhibition. This interplay of signaling between the epithelial and dermal lineage helps in epithelial Shh signal amplification. The dermal Wnt pathway helps in upregulation of epithelial Notch expression. Dysregulation of these pathways leads to certain abnormalities and in some cases even tumor outgrowth.

Gamma radiation induces senescence in human adult mesenchymal stem cells from bone marrow and periodontal ligaments.

Purpose: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. Methods: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co60 and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. Results: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21Cip1/Waf1) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16INK4a) and increased activity of senescence-associated β-galactosidase. Conclusion: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.

Irradiation of Adult Human Dental Pulp Stem Cells Provokes Activation of p53, Cell Cycle Arrest, and Senescence but Not Apoptosis

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-β-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.

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.

Morphological and functional changes in P-glycoprotein during dexamethasone-induced hepatomegaly

1 The effect of dexamethasone on hepatic and renal P‐glycoprotein (P‐gp) expression, localization and activity was investigated in rats after 4 days oral administration of two dose regimens (1 or 25 mg/kg per day). Simultaneous increases in liver weight were evaluated by quantitative histological examination. 2 In the liver, dexamethasone pretreatment produced hepatomegaly as a consequence of extensive periportal fat accumulation, which was quantified by densitometry of oil red O‐stained liver sections. Quantitative immunohistochemical analysis revealed preferential periportal zonation of P‐gp in control animals. Dexamethasone pretreatment resulted in spatially disproportional induction of P‐gp protein expression within the liver acinus characterized by preferential increase in pericentral areas, with consequent uniform panlobular distribution. Western blot analysis confirmed these results, showing increases in P‐gp protein. Quantitative reverse transcription–polymerase chain reaction analysis revealed no statistically significant change in liver mdr1b mRNA expression after either dexamethasone treatment regimen. The expression of mdr1a mRNA was significantly decreased by 85–87%. 3 In the kidney, dexamethasone reduced mdr1a mRNA expression by 69–89%, whereas mdr1b mRNA expression was increased in a dose‐dependent manner. However, despite tendencies, no significant increases in P‐gp expression were observed at the protein level. 4 The in vivo function of P‐gp was evaluated by measuring renal and biliary secretion of rhodamine‐123 (Rho123) under a steady state plasma concentration. The biliary, renal and tubular secretory clearance of Rho123 was significantly increased only after high‐dose dexamethasone. 5 In conclusion, the present study suggests that drug interactions observed during corticosteroid therapy may be mediated, at least in part, through increased biliary, and also renal, excretion of P‐gp substrates. Expression of P‐gp in the liver showed primary periportal zonation with differential changes during induction. Accompanying hepatomegaly may be explained by severe microvesicular steatosis selectively localized to the periportal areas.

Breast cancer and cancer stem cells: a mini-review.

Breast cancer is the most common type of malignant disease in women worldwide. In developing countries the past few years have sustained an increasing incidence of this type of cancer. Currently, breast cancer is the second leading cause of death due to cancer in women. In 2008 alone it was diagnosed in more than 1 million patients and each year the number of breast cancer-related deaths is estimated to be ~450,000. The mortality rate in breast cancer patients has been decreasing over the years thanks to the development of early diagnostic methods and more effective treatments. But despite the new advances in cancer diagnosis and treatment, the risk of recurrence and metastasis is ever present. It has been theorized that cancer stem cells are involved in the process of tumor growth and metastases. Due to their self-renewing and differentiation capabilities, they are now considered the underlying factor in tumor recurrence and the main reason for therapy resistance. Therefore, the characterization of cancer stem cells may contribute to the development of more effective treatment strategies that should make it possible to eliminate cancer stem cells in order to prevent tumor relapse and metastasis in diagnosed patients.

A novel myoepithelial/progenitor cell marker in the breast?

Dear Editor, Neuroepithelial stem cell protein (Nestin) is an intermediate filament (IF) protein considered to be a marker of neural stem cells. Nestin expression has been confirmed in stem/ progenitor cells of the dermis, hair follicles, intestine, pancreas, bone marrow as well as in neural, muscle and other tissues (reviewed in [7]). Its expression is down regulated in the course of differentiation and subsequently replaced by another type of IF [3, 10]. Nestin expression has also been detected in the endothelium [11, 12], and we have noted the activation of nestin expression in newly formed human blood vessels [7]. The human nestin molecule has a homology with rodent nestin confirming its highly conserved nature (Fig. 2c) [5]. The myoepithelial cells of the breast form an outer layer of the terminal duct lobular unit. They play an active part in mammary gland branching morphogenesis [6], and their correct recognition and detection is crucial in the diagnosis of a number of pathological breast lesions [4, 8]. Several markers have been reported for the immunohistochemical detection of breast myoepithelial cells such as α-smooth muscle actin, smooth muscle myosin heavy chain, calponin, h-caldesmon, S100 protein, p63, CD10, maspin and specific cytokeratins 5, 7, 15 and 17 (reviewed in [2, 8]). However, the specificity and sensitivity of these markers vary widely. Of these, maspin [9] and p63 [1, 2, 13] are generally considered the most promising. In histopathological practice it is very often necessary to confirm a diagnosis using a wide battery of immunohistological stainings. Undoubtedly, the identification of new markers is desirable. We analysed the expression of nestin by an indirect immunohistochemical method (primary monoclonal antinestin antibody, clone 10C2, cat. no. MAB5326, Chemicon International, diluted 1:200; secondary staining system EnVision, Dako; antigen retrieval by microwave generator in citrate buffer pH 7.0; diaminobenzidine as chromogen) in 300 archival formalin-fixed and paraffin-embedded tissue samples of breast cancer and surrounding mammary tissue as well as in tissue microarrays consisting of 109 cases of breast cancer, developed for another project. Nestin expression of various intensities was seen in the cytoplasm of myoepithelial cells of almost all normal ducts and lobular acini found in the vicinity of tumours (Fig. 1a and b). The intensity of expression varied from strong/ marked (32 cases, ∼10%), to moderate/clearly visible (91 cases, ∼29%) to low/visible (113 cases, ∼38%) and to very low/visible in less than 50% cells only (64 cases, ∼21%). In our opinion, the reasons for the lower positive intensity in some cases can be explained by the fixation conditions. The specimens obtained by excision were generally more positive than specimens from the centre of the mastectomy. Pathological lesions like adenosis with atypical hyperplasia and in situ carcinoma exhibited very strong positivity (Fig. 1c and d), and the detection of nestin could also be used for the diagnosis of pseudo-invasion in some controversial cases (Fig. 1e). In invasive carcinomas, we found nestin positivity in a portion of epithelial cells in a minority (∼1/3) of cases. The positivity in the epithelial component was confirmed by simultaneous AE1/AE3 Virchows Arch (2007) 450:607–609 DOI 10.1007/s00428-007-0403-x

Induced Pluripotent Stem Cells and Their Use in Cardiac and Neural Regenerative Medicine

Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson’s, Alzheimer’s and Huntington’s disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges.

Re-expression of nestin in the myocardium of postinfarcted patients.

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.

Zonation of multidrug resistance‐associated protein 2 in rat liver after induction with dexamethasone

Background and Aim:  The present study was aimed to evaluate the hepatic zonation of multidrug resistance‐associated protein 2 (mrp2), an important drug transporter, and its potential changes during the induction of its expression by known inducer, dexamethasone (DEX).