Lubos Danisovic

The tissue engineering of articular cartilage: cells, scaffolds and stimulating factors:

Damage or loss of articular cartilage as a consequence of congenital anomaly, degenerative joint disease or injury leads to progressive debilitation, which has a negative impact on the quality of life of affected individuals in all age groups. Classical surgical techniques for hyaline cartilage reparation are frequently insufficient and in many cases it is not possible to obtain the expected results. For this reason, researchers and surgeons are forced to find a method to induce complete cartilage repair. Recently, the advent of tissue engineering has provided alternative possibilities for the treatment of these patients by application of cell-based therapy (e.g. chondrocytes and adult stem cells) combined with synthetic substitutes of the extracellular matrix and bioactive factors to prepare functional replacement of hyaline cartilage. This communication is aimed at a brief review of the current status of cartilage tissue engineering and recent advances in the field.

Mesenchymal stem cells for chronic wounds therapy

Abstract Wound healing is a complex process that involves interaction of soluble mediators, extracellular matrix and infiltrating blood cells. Chronic and non-healing skin defects contribute significantly to morbidity and mortality of many patients. Recently, despite the current medical progress, the chronic and non-healing wounds still represent a serious medical problem. In many cases, conventional therapeutic approaches, such as dermal substitutes and growth factor therapy failed and do not produce the expected results, patients are exposed to a high risk of infection, sepsis or amputation. For that reason clinicians and researchers are forced to searching for alternative methods to induce healing process which may result into complete wound closure. Mesenchymal stem cells (MSCs) represent a unique tool of tissue engineering and regenerative medicine and a promising therapeutic strategy. Due to their unique biological properties, MSCs seem to be the perspective modality method for these patients. Many preclinical and clinical studies suggest the possibility of using these cells in tissue regeneration, healing acute and chronic wounds and scar remodelling. The objective of the present review is to summarize the current information and preclinical data about MSCs, their biological characteristics and mode of action during regenerative and healing processes, as well as their clinical application in chronic wounds treatment.

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.

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.

Ultrastructural analysis of different human mesenchymal stem cells after in vitro expansion: a technical review

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.

Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes

Carnosines (CARN) anti-inflammatory potential in autoimmune diseases has been but scarcely investigated as yet. The aim of this study was to evaluate the therapeutic potential of CARN in rat adjuvant arthritis, in the model of carrageenan induced hind paw edema (CARA), and also in primary culture of chondrocytes under H2O2 injury. The experiments were done on healthy animals, arthritic animals, and arthritic animals with oral administration of CARN in a daily dose of 150 mg/kg b.w. during 28 days as well as animals with CARA treated by a single administration of CARN in the same dose. CARN beneficially affected hind paw volume and changes in body weight on day 14 and reduced hind paw swelling in CARA. Markers of oxidative stress in plasma and brain (malondialdehyde, 4-hydroxynonenal, protein carbonyls, and lag time of lipid peroxidation) and also activity of gamma-glutamyltransferase were significantly corrected by CARN. CARN also reduced IL-1alpha in plasma. Suppression of intracellular oxidant levels was also observed in chondrocytes pretreated with CARN. Our results obtained on two animal models showed that CARN has systemic anti-inflammatory activity and protected rat brain and chondrocytes from oxidative stress. This finding suggests that CARN might be beneficial for treatment of arthritic diseases.

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.

The normal human newborns thymus.

The thymic microenvironment constitutes a unique cell environment composed of thymic epithelial cells, myoid cells, and bone marrow-derived accessory cells for the differentiation, maturation and selection of T lymphocytes. The histological feature of thymus is markedly dependent on the age of individual and on various negative stimuli. Our study group consisted of fourteen newborns whose thymuses were removed during surgery performed for various congenital heart defects. We used a palette of seven monoclonal antibodies for exact localization of different cells creating the thymic microenvironment (cytokeratin AE1/AE3, desmin, actin, S100 protein, CD68, CD20, and CD45RO) as well as three monoclonal antibodies against proteins regulating the process of apoptosis (bcl2 oncoprotein, p53 protein, and survivin). We described and microphotographically illustrated the localization of thymic cytokeratin AE1/AE3-positive epithelial cells (subcapsular part of the cortex and medulla, especially Hassalls corpuscles), dendritic cells (medulla, often inside the Hassalls corpuscles), thymic myoid cells (medulla, often in close contact with Hassalls corpuscles), macrophages (mostly cortex, but also medulla and inside the Hassalls corpuscles), B lymphocytes (thymic medulla) and CD45RO-positive T lymphocytes (mostly thymic cortex). We found p53-positive thymic epithelial cell nuclei in subcapsular part of cortex and in outer epithelial cell layer of Hassalls corpuscles (very similar to the basal layer of epidermis). Bcl2 positive lymphocytes were mostly localized in thymic medulla, especially nearby Hassalls corpuscles. The thymuses were mostly survivin-negative with exception of round cells in border between cortex and connective tissue septa (probably migrating progenitor cells) (Tab. 1, Fig. 14, Ref. 66).

Cardiac telocytes as principal interstitial cells for myocardial reparation and regeneration after infarction – our hope

According to our knowledge, this is the first research experiment that focuses on the study of the distribution of c-kit positive cells at the sites of myocardial infarction in human hearts (Fig. 3, Ref. 16).