Stefan Polak

Growth factors and chondrogenic differentiation of mesenchymal stem cells

The main purpose of the article is to review recent knowledge about growth factors and their effect on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions. Damaged or lost articular cartilage leads to progressive debilitation, which have major impact on the life quality of the affected individuals of both sexes in all age groups. Mature hyaline cartilage has a very low self-repair potential due to intrinsic properties - lack of innervation and vascular supply. Another limiting factor is low mitotic potential of chondrocytes. Small defects are healed by migration of chondrocytes, while large ones are healed by formation of inferior fibrocartilage. However, in many cases osteoarthritis develops. Recently, cellular therapy combining mesenchymal stem cells and proper differentiation factors seems to be promising tool for hyaline cartilage defects healing.

Ki67, PCNA, and MCM proteins: Markers of proliferation in the diagnosis of breast cancer

The proliferative activity of tumour cells represents an important prognostic marker in the diagnosis of cancer. One of the methods for assessing the proliferative activity of cells is the immunohistochemical detection of cell cycle-specific antigens. For example, Ki67, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance (MCM) proteins are standard markers of proliferation that are commonly used to assess the growth fraction of a cell population. The function of Ki67, the widely used marker of proliferation, still remains unclear. In contrast, PCNA and MCM proteins have been identified as important participants of DNA replication. All three proteins only manifest their expression during the cell division of normal and neoplastic cells. Since the expression of these proliferative markers was confirmed in several malignant tumours, their prognostic and predictive values have been evaluated to determine their significance in the diagnosis of cancer. This review offers insight into the discovery of the abovementioned proteins, as well as their current molecular and biological importance. In addition, the functions and properties of all three proteins and their use as markers of proliferation in the diagnosis of breast cancer are described. This work also reveals new findings about the role of Ki67 during the mitotic phase of the cell cycle. Finally, information is provided about the advantages and disadvantages of using all three antigens in the diagnosis of cancer.

Fluorescence of hematoxylin and eosin-stained histological sections of the human spleen

A major problem in the morphometric evaluation of human spleen is the simple but reliable determination of the border between T-cell and B-cell dependent areas, and other structures of the spleen. It was investigated whether cryostat sections of frozen surgical specimens of the human spleen and sections of paraffin-embedded specimens could be used for this purpose after being stained with haematoxylin and eosin and mounted in autofluorescence-free medium for fluorescence microscopical evaluation. Comparison was made with sections that were immunohistochemically-stained for fibronectin and collagens type II and type IV. Both in cryostat sections and paraffin sections, fluorescence was found in circumferential reticulum of periarterial lymphatic sheets, arterial terminals, arterial walls and walls of red pulp sinuses in the spleen. Evaluation was hindered by fluorescence of erythrocytes in paraffin sections but not in cryostat sections. Results were similar as those obtained with immunohistochemical fibronectin staining and are sufficient for morphometric evaluation or orientation in the tissue in case of neoplasia.

iPS cell technologies and their prospect for bone regeneration and disease modeling: A mini review

Graphical abstract

Induced pluripotent stem cells and their implication for regenerative medicine

Abstract In 2006 Yamanaka’s group showed that stem cells with properties similar to embryonic stem cells could be generated from mouse fibroblasts by introducing four genes. These cells were termed induced pluripotent stem cells (iPSCs). Because iPSCs avoid many of ethical concerns associated with the use of embryonic material, they have great potential in cell-based regenerative medicine. They are suitable also for other various purposes, including disease modelling, personalized cell therapy, drug or toxicity screening and basic research. Moreover, in the future, there might become possible to generate organs for human transplantation. Despite these progresses, several studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to immunogenicity of some cells differentiated from iPSCs. Recent methodological improvements are increasing the ease and efficacy of reprogramming, and reducing the genomic modification. However, to minimize or eliminate genetic alternations in the derived iPSC line creation, factor-free human iPSCs are necessary. In this review we discuss recent possibilities of using iPSCs for clinical applications and new advances in field of their reprogramming methods. The main goal of present article was to review the current knowledge about iPSCs and to discuss their potential for regenerative medicine.

The fluid of female urethral expulsions analysed by histochemical electron-microscopic and other methods

The fluid of female urethral expulsions (female ejaculate), obtained by applying the digital stimulation technique (Grafenberg, 1950; Zavia~i~ et al., 1983b) was analysed in 10 women patients of the 2nd Hospital of Gynaecology and Obstetrics in Bratislava, Czechoslovakia. In the majority of the patients, collections of the fluid were repeated on different days of their menstrual cycle. With the aim of determining standard values of exfoliated squamous cells, which are a constant component of the fluid of female urethral expulsions (Zavia~i~ et al., i983a,b), we were the first to study this kind of material by using not only cytological methods (Koss, 1979) but also histochemical methods for the determination of enzymes, mucosubstances and lipids (Pearse, 1972; Lojda et al., 1976). Transmission (TEM) and scanning electron microscopic (SEM) studies were carried out to detect differences between the ultrastructure and the spatial configuration of squamous cells during the proliferative and secretory phases of the cycle. On examining the smears by cytological methods, characteristic differences were observed in the configuration and eosinophilia of the cells on different days of the menstrual cycle (Zavia~i~ et al., 1983a,b). Large flat squamous cells of the superficial type exhibited only minimal or no activity of hydrolytic enzymes acid and alkaline phosphatases, non-specific esterase and succinate and ~-glycerophosphate dehydrogenases. Distinct, minimal, activities were seen in intermediary cells, especially during the secretory phase of the cycle. In the cytoplasm of squamous cells, non-polar lipid drops staining with Fat Red 7B (Serva, Heidelberg) were found sporadically. During the proliferative phase of the cycle and during ovulation, SEM and TEM examinations revealed squamous cells occurring mostly isolated or in small clusters arranged in a slate-like pattern with overlapping edges. An elevated knob-like nucleus was seen in their centre. Microridges were observed on the flat surface of the cells. More prominent ridges marked the cell junctions. On the epithelial cells, and also beyond them, clumps of mucosubstances were visualized which covered the cells surfaces to a varying extent (Fig. 1). In samples collected before and during ovulation, marked

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells.

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterisation showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.

Morphological characterization of in vitro expanded human dental pulp-derived stem cells

Stem cells play pivotal role in the development of tissues and organs, as well as they maintain homeostasis and integrity of multicellular organisms. Human dental pulp-derived stem cells are capable of differentiation into osteoblasts, odontoblasts, adipocytes and neuronal-like cells and can have potential use in tissue regeneration. The aim of this study was a detailed description of human dental pulp-derived stem cells (HDPSCs) cultivated in vitro ultrastructural morphology. HDPSCs were isolated from the dental pulp of impacted third molars from healthy donors. Transmission electron microscopic analysis showed typical ultrastructural morphology similar to mesenchymal stem cells from other organs. The morphology of HDPSCs cells reflects their proteosynthetic and metabolic activity. Each cell contained spherical or irregularly-shaped large pale nucleus with a large amount of euchromatine. Nuclei had noticeable nucleolus (or two nucleoli) located nearby the nuclear envelope. Abundant cisterns of rough endoplasmic reticulum and numerous coated matrix vesicles as well as granules of glycogen were present in their cytoplasm. Nearby the nucleus small, elongated mitochondria were placed. Most of HDPSCs created and secreted vesicles; in plasmalemma bounded amorphous electron-lucide granules and also few glycogen granules. These secretory vesicles had around 0.5 μm in diameter. We assume that it can be a special type of communication between cells, probably paracrine type of cell signalling, but its real function is still unknown.

Rhythmic changes of human female uroepithelial squamous cells during menstrual cycle. Transmission and scanning electron microscopic study.

The cellular component of the fluid of female urethral expulsions was analysed in two healthy women (37-year-old multipara and 27-year-old nullipara) on days 1, 10, 12, 14, 26 and 27 of their menstrual cycle. Transmission and scanning electron microscopy were used to determine standard values of uroepithelial squamous cells of the fluid of female urethral expulsions on different days of the menstrual cycle. In agreement with the known changes of these cells found in cytological smears during the cycle, electron microscopy confirmed characteristic changes in the appearance of the fine cell structure and the space configuration of the squamous cells. Changes in the desmosomal junctions among squamous cells during the cycle were found. Striking adherence of bacteria and mucosubstances to the squamous cell surfaces were observed in the first half of the menstrual cycle and at the time of ovulation.

Toxicity testing and drug screening using iPSC-derived hepatocytes, cardiomyocytes and neural cells

Unexpected toxicity in areas such as cardiotoxicity, hepatotoxicity, and neurotoxicity is a serious complication of clinical therapy and one of the key causes for failure of promising drug candidates in development. Animal studies have been widely used for toxicology research to provide preclinical security evaluation of various therapeutic agents under development. Species differences in drug penetration of the blood-brain barrier, drug metabolism, and related toxicity contribute to failure of drug trials from animal models to human. The existing system for drug discovery has relied on immortalized cell lines, animal models of human disease, and clinical trials in humans. Moreover, drug candidates that are passed as being safe in the preclinical stage often show toxic effects during the clinical stage. Only around 16% drugs are approved for human use. Research on induced pluripotent stem cells (iPSCs) promises to enhance drug discovery and development by providing simple, reproducible, and economically effective tools for drug toxicity screening under development and, on the other hand, for studying the disease mechanism and pathways. In this review, we provide an overview of basic information about iPSCs, and discuss efforts aimed at the use of iPSC-derived hepatocytes, cardiomyocytes, and neural cells in drug discovery and toxicity testing.