Ľuboš Danišovič

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.

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.

In vitro cytotoxicity testing of coladerm membrane

Atpresent, biodegradable and biocompatible membranes based on collagen andglycosaminoglycans play an important role in substitutive medicine. Modernbiomaterials use a chemically modified collagen-based matrix for implants withprogrammable biodegradability as a substitute of buccal mucosa, skin,cartilage,etc. Besides the requirements for biocompatibility and biodegradability, themembranes must be also non-toxic. Therefore, cytotoxicity testing of thesematerials in vitro is an integral part of introducingnewlydeveloped types of membranes into clinical practice. As a biological model forthe tested COLADERM membrane, cell cultures from human embryonic fibroblasts(B-HEF-2) were used for both cytotoxicity testing as well as in tests to assessthe ability of cells to proliferate on this membrane. Along with the ability ofcells to grow on the surface and inside the membrane, immunohistochemicalexamination and scanning electron microscopy (SEM) were performed as well. Theobtained results have shown that the COLADERM membrane is non-toxic withsuitable structural and biological properties for clinical application as asubstitute of buccal mucosa following surgical ablation of malignant tissuesfrom the oral cavity.

Biological and morphological characterization of human neonatal fibroblast cell culture B-HNF-1

In the present study, human neonatal fibroblasts were isolated from a two-month-old human male. The purpose of the present investigation was the analysis of the morphology (light and transmission electron microscopy), karyotype and growth characteristics of the human neonatal fibroblast cell culture B-HNF-1. Moreover, STR typing and mitochondrial DNA amplification and sequencing was also performed. Analysis of chromosomes count showed that B-HNF-1 cell culture is diploid and has normal male karyotype 46, XY, which was stable during cultivation. The transmission electron microscopy demonstrated the ultra-structure of the B-HNF-1 cells; they have typical morphological features of proteosynthesis-active cells. Large number of fibroblasts bearing different shapes and surface characteristics adhered to the substrate with microvilli and filopodia. Our in vitro expanded fibroblasts have a large and irregular nucleus with prevalence of euchromatin. One to three nucleoli are present in each nucleus. The cytoplasm contains a richly developed rough endoplasmic reticulum and prominent Golgi apparatus cisterns. The result of the fragment analysis is a DNA profile defined as multiplex of STR markers and sex determining system. Sequencing analysis of hypervariable segments of control region of mitochondrial DNA showed haplotype that belongs to haplogroup W. In this study, we complexly characterized a new cell culture of human neonatal fibroblasts B-HNF-1 from different points of view. This culture should be used for further biomedical experiments.

Comprehensive characterization of human adipose tissue-derived stem cells expanded in vitro

Adipose tissue seems to be a rich and safe source of mesenchymal stem cells (MSCs). The present study was aimed to investigate the biological and morphological characteristics of human adipose tissue-derived stem cells (ATSCs). Light and transmission electron microscopy were used. Course of proliferation was analyzed by growth curve. Expression of surface antigens was assessed by flow cytometry. Chondrogenic potential was assessed by immunohistochemistry. Obtained results showed morphology typical of fibroblastoid cells. TEM analysis proved ultrastructural morphology similar to MSCs from other sources. ATSCs reflected their proteosynthetic and metabolic activity. Each cell had irregular shape of nucleus with noticeable nucleoli. Abundant cisterns of rough endoplasmic reticulum were present in their cytoplasm. Karyotype mapping showed normal count of human chromosomes (46,XX). The growth curve revealed high capability for proliferation and population doubling time was 27.36 hours. ATSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105 and CD106, but did not express CD14, CD34, CD45 and HLA-DR. It was also proved that ATSCs underwent chondrogenic differentiation in vitro. On the basis of obtained results it should be emphasized that ATSCs are typical MSCs and after further investigations they may be used in tissue engineering and regenerative medicine.

Options for histological study of the structure and ultrastructure of human urinary bladder epithelium

The urothelium lines all urinary passages, with exception of the distal portions of the urethra. For the first time the structure of the human bladder was described by Leonardo Da Vinci in 15th century, however, the exact ultrastructure and function of the bladder’s epithelium have not been fully understood. The aim of our study was to investigate the structure of normal human urinary bladder epithelium with methods of classical histology, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We obtained biopsies from non-tumor areas from the human urinary bladder of tumor-bearing patients during transurethral resections of these tumours in general or spinal anaesthesia. Totally we investigated biopsies from 20 patients, 16 males and 4 females. The mean age of this group of patients was averaged 66.5 years. The urothelium is comprised of three cell types including polyhedral basal cells, piriform intermediate cells, and superficial umbrella cells. In human urinary bladder epithelium we found a direct connection between intermediate cells and the basement membrane. These thin cytoplasmic projections are detectable not only on slides for light microscopy (semi-thin sections), but also in transmission electron-micrographs. In semi-thin sections we found also direct connections between superficial umbrella cells and basement membrane. These connections we were not able to verify via transmission electron-microscopy. Nevertheless our results show that the human urinary bladder urothelium is a special type of pseudostratified epithelium and each cell has a thin cytoplasmic projection with a direct contact with basement membrane.

Adult stem cells derived from skeletal muscle — biology and potential

Skeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.

What happens to an acellular dermal matrix after implantation in the human body? A histological and electron microscopic study

Acellular matrices are used for various purposes and they have been studied extensively for their potential roles in regenerating tissues or organs. The acellular matrix generates physiological cues that mimic the native tissue microenvironment. Acellular dermal matrix (ADM) is a soft connective tissue graft generated by a decellularization process that preserves the intact extracellular skin matrix. Upon implantation, this structure serves as a scaffold for donor-side cells to facilitate subsequent incorporation and revascularization. In breast reconstruction, ADM is used mainly for lower pole coverage and the shaping of a new breast. It helps control the positioning of the implant in the inframammary fold, and prevents the formation of contractile pseudocapsule around the breast implant. In this study, we provide a comprehensive histological description of ADM used for human breast reconstruction over the course of several months following implementation. Using immunohistochemical methods (a panel of 12 antibodies) coupled with optical and transmission electron microscopy, we confirmed that the original acellular dermal matrix became recolonized by fibroblasts and myofibroblasts, and also by various other free cells of the connective tissue (lymphocytes, macrophages and multinucleated giant cells, granulocytes, mast cells) after implantation into the patient’s body. Within the implanted ADM, there was a relatively rapid ingrowth of blood vessels. Lymphatic vessels were only detected in one case 9 months after the implantation of the ADM. These results suggest that lymphangiogenesis is a longer process than angiogenesis.

Histological and immunohistochemical characteristics of capsular synovial metaplasias that form around silicone breast implants

Metaplasia is a reversible phenomenon that usually occurs in response to chronic irritation and/or inflammation. It allows for the substitution of fragile cells with those that are better able to survive under various circumstances. Our study aims to describe the histology of one unusual type of metaplasia results in the formation of a synovial-like membrane typical of joints inside a female patient’s breasts around silicone implants. We analyzed samples from 22 female patients, who underwent delayed-staged breast reconstructions. Attention was paid especially to tissue that was in direct contact with the silicone expander and that was under permanent pressure and friction between the implant and the surrounding tissue. Biopsies of explanted periprosthetic capsules were processed for examination by light microscopy. Immunohistochemical staining (ten different primary antibodies) was performed to examine a variety of cell-specific antigens. At the interface between the tissue capsule and the silicone breast expander, we typically observed a 50–200-μm cellular lining. Thanks to the high cellular density, this cellular layer resembled epithelium. However, there was no basement membrane, and cells were negative for cytokeratin. The cells forming the superficial layer were strongly positive for vimentin and podoplanin and weakly positive for the S100 protein. These cells did not express desmin or smooth muscle actin. Within the most superficial layer (synovial intima), we distinguished two types of cells: phagocytic (CD68-positive) and fibroblast-like synovial cells. We conclude that the cellular lining surrounding silicone breast implants looks like a true synovial membrane resembling a fibrous form of synovium.

Induced pluripotent stem cells for modeling and cell therapy of Parkinson's disease.

Neurodegenerative disorders include a variety of hereditary or sporadic diseases involving the chronic, progressive loss on neural tissue. Parkinsons disease (PD) is the second most common neurodegenerative disease, affecting more than 6 million people worldwide (Wan et al., 2015). Degeneration of nigrostriatal dopamine (DA) neurons is the main pathology in PD, although other dopaminergic and non-dopaminergic systems are also affected. Characteristic symptoms are rigidity, hypokinesia, tremor, and postural instability. The loss of DA neurons is accompanied by lewy bodies and lewy neuritis, which are mainly formed by insoluble aggregates of alpha-synuclein and Tau protein and might restrain the survival and development of newborn neurons. Etiology of PD remains unclear, however interactions between environmental and genetic factors are believed to cause the loss of nigral DA neurons and ensuing locomotor system. Research indicated that increasing level of iron, oxidative stress, mitochondrial and ubiquitin-proteasome system dysfunction, inflammation, and apoptosis may lead to the progression of PD (Pu et al., 2012; Zhu et al., 2016).