Current status of the applications of conditioned media derived from mesenchymal stem cells for regenerative medicine.

Recently published studies suggest that the paracrine substances released by mesenchymal stem cells (MSCs) are the primary motive behind the therapeutic action reported in these cells. Pre-clinical and clinical research on MSCs has produced promising outcomes. Furthermore, these cells are generally safe for therapeutic use and may be extracted from a variety of anatomical regions. Recent research has indicated, however, that transplanted cells do not live long and that the advantages of MSC treatment may be attributable to the large diversity of bioactive substances they create, which play a crucial role in the control of essential physiological processes. Secretome derivatives, such as conditioned media or exosomes, may provide significant benefits over cells in terms of manufacture, preservation, handling, longevity of the product, and potential as a ready-to-use biologic product. Despite their immunophenotypic similarities, the secretome of MSCs appears to vary greatly depending on the host's age and the niches in which the cells live. The secretome's effect on multiple biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic, and anti-tumor for tissue maintenance and regeneration has been discovered. Defining the secretome of cultured cultivated MSC populations by conditioned media analysis will allow us to assess its potential as a novel treatment approach. This review will concentrate on accumulating data from pre-clinical and clinical trials pointing to the therapeutic value of the conditioned medium. At last, the necessity of characterizing the conditioned medium for determining its potential for cell-free treatment therapy will be emphasized in this study.

Comparative Analysis of Somatic Stem Cells With Emphasis on Osteochondral Tissue Regeneration.

Congenital anomalies, diseases, and injuries may result in osteochondral damage. Recently, a big hope has been given to somatic stem cells (SSCs) which are characterized as undifferentiated cells with an ability of long-term self-renewing and plasticity. They are adherent with a fibroblast-like morphology in vitro and express various surface markers (e.g. CD29, CD73, CD90, and CD105), but they are negative for CD31, CD34, CD45, and HLA-DR. SSCs secrete various bioactive molecules, which are involved in processes of regeneration. The main goal of the present study was the characterization and comparison of biological properties of SSCs obtained from adipose tissue, dental pulp, and urine concerning osteochondral regeneration. SSCs were maintained in an appropriate growth medium up to the third passage and were analyzed by light and electron microscope. The immunophenotype was analyzed by flow cytometry. The kinetics of proliferation was measured by MTT assay. Human Cytokine/Chemokine Multiplex Assay was used, and SSCs secretory profile was measured by Luminex MAGPIX® Instrument. Pellet cultures and a chondrogenic medium were used to induce chondrogenic differentiation. Osteogenic differentiation was induced by the osteogenic medium. Chondrogenic and osteogenic differentiation was analyzed by real-time PCR. SSCs had similar fibroblast-like morphology. They have similar kinetics of proliferation. SSCs shared the expression CD29, CD44, CD73, CD90, and CD105. They lack expression of CD29 and CD34. SSCs secerned similar levels of IL10 and IL18 while differing in IFN-gamma, IL6, IL8, MCP-1, and RANTES production. SSCs possess a similar capacity for chondrogenic differentiation but slightly differ in osteogenic differentiation. In conclusion, it can be emphasized that SSCs from adipose tissue, dental pulp, and urine share the majority of cellular characteristics typical for SSCs and have great potential to be used in osteochondral tissue regeneration.

Acellular Dermal Matrix in Plastic and Reconstructive Surgery.

Despite significant advances in medical research, plastic surgeons still face a shortage of suitable patient tissues, and soft tissue reconstruction is no exception. In recent years, there has been a rapid boom in the use of acellular dermal matrix (ADM) in reconstructive and aesthetic surgery. ADM is incorporated into the surrounding tissue and gradually replaced by the host's collagen, thus promoting and supporting the healing process and reducing the formation of scar tissue. The main goal of this article is to provide a brief review of the current literature assessing the clinical applications of ADM across a broad spectrum of applications in plastic and reconstructive surgery.

Scanning electron microscopic study of the human uterine tube epithelial lining: surgical biopsy samples and epithelial cell culture.

This article summarizes the importance of the exact morphology of human uterine/fallopian tube epithelium at the scanning electron microscopy (SEM) level for the clinical outcome even nowadays. Visual referential micrographs from SEM reflect two ways to view human epithelial cell lining surfaces: the surface epithelial uterine tube from surgical tissue biopsy and human fallopian tube epithelial cells (HFTEC) culture monolayer surface. One colorized image visualizes ciliated cells, distinguishes them from non-ciliated cells, and provides an educational benefit. A detailed description of the ultrastructure in referential and pathologic human uterine tube epithelium is important in defining the morphological basis of high-grade carcinomas, in the mechanism of pathophysiology, and in discussing options for its prevention. Cell cultures of human fallopian tube epithelial cells offer new approaches in simulating the mechanisms of cancer genesis or may help to elucidate the genetic basis of several diagnoses. New technical approaches in SEM provide higher resolution and detailed surface images. The SEM modality is still one of the current options in diagnostics and may be useful for advancing human reproductive organ cancer research.

Pharmacological Approaches and Regeneration of Bone Defects with Dental Pulp Stem Cells.

Bone defects in the craniomaxillofacial skeleton vary from small periodontal defects to extensive bone loss, which are difficult to restore and can lead to extensive damage of the surrounding structures, deformities, and limited functions. Plenty of surgical regenerative procedures have been developed to reconstruct or prevent alveolar defects, based on guided bone regeneration involving the use of autogenous bone grafts or bone substituents. However, these techniques have limitations in the restoration of morphological and functional reconstruction, thus stopping disease progression but not regenerating lost tissue. Most promising candidates for regenerative therapy of maxillofacial bone defects represent postnatal stem cells, because of their replication potential in the undifferentiated state and their ability to differentiate as well. There is an increased need for using various orofacial sources of stem cells with comparable properties to mesenchymal stem cells because they are more easily available with minimally invasive procedures. In addition to the source of MSCs, another aspect affects the regeneration outcomes. Thermal, mechanical, and chemical stimuli after surgical procedures have the ability to generate pain, usually managed with pharmacological agents, mostly nonsteroidal anti-inflammatory drugs (NSAIDs). Some studies revealed that NSAIDs have no significant cytotoxic effect on bone marrow stem cells from mice, while other studies showed regulation of osteogenic and chondrogenic marker genes in MSC cells by NSAIDs and paracetamol, but no effect was observed in connection with diclofenac use. Therefore, there is a need to focus on such pharmacotherapy, capable of affecting the characteristics and properties of implanted MSCs.

The prognostic value of E-cadherin and Ki-67 compared to standard histopathologic examination in non-muscle invasive bladder cancer.

OBJECTIVES: The objectives of this study were to determine the prognostic value of expression levels of selected biomarkers and their statistical analysis in relation to survival and standard histopathologic examination and other clinicopathologic variables in non-muscle invasive bladder cancer (NMIBC). BACKGROUND: Worldwide, bladder cancer is a frequent malignant disease with rising incidence. Characteristic invasiveness and high recurrence rates call for more diagnostic methods to obtain more accurate information. Prognosis is affected by a significant interpersonal variability of the disease. For this reason, constant search for alternative and better diagnostic methods is essential. METHODS: We analysed cancer tissue from patients with Ta and T1 bladder cancer. E-cadherin and Ki-67 expression levels were analysed using immunohistochemical staining. The expression levels quantified to a percentual amount were statistically analysed in relation to survival and their frequency distribution in the study group. RESULTS: E-cadherin and Ki-67 expression levels show high association with tumor stage and grade         (p<0.001), in contrast, the association with recurrence has proven insignificant. Patients with non-aberrant biomarker expression levels have much higher survival rates than the cases with aberrant expression. CONCLUSION: Low expression levels of Ki-67 and high expression levels of E-cadherin positively affect survival of patients, whereas aberrant expressions pose poorer prognosis (Tab. 2, Fig. 2, Ref. 33).

Tissue engineering of urethra: Systematic review of recent literature.

The purpose of this article was to perform a systematic review of the recent literature on urethral tissue engineering. A total of 31 articles describing the use of tissue engineering for urethra reconstruction were included. The obtained results were discussed in three groups: cells, scaffolds, and clinical results of urethral reconstructions using these components. Stem cells of different origin were used in many experimental studies, but only autologous urothelial cells, fibroblasts, and keratinocytes were applied in clinical trials. Natural and synthetic scaffolds were studied in the context of urethral tissue engineering. The main advantage of synthetic ones is the fact that they can be obtained in unlimited amount and modified by different techniques, but scaffolds of natural origin normally contain chemical groups and bioactive proteins which increase the cell attachment and may promote the cell proliferation and differentiation. The most promising are smart scaffolds delivering different bioactive molecules or those that can be tubularized. In two clinical trials, only onlay-fashioned transplants were used for urethral reconstruction. However, the very promising results were obtained from animal studies where tubularized scaffolds, both non-seeded and cell-seeded, were applied. Impact statement The main goal of this article was to perform a systematic review of the recent literature on urethral tissue engineering. It summarizes the most recent information about cells, seeded or non-seeded scaffolds and clinical application with respect to regeneration of urethra.

Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from different tissues, including bone marrow, adipose tissue, umbilical blood, Wharton's jelly, and dental pulp. Due to their differentiation potential, regenerative and immunosuppressive properties, as well as ability to expand under in vitro conditions, these cells represent a promising therapeutic tool for regenerative medicine. However, the basic prerequisite for the therapeutic utilization of MSCs is obtaining a sufficient amount. While this may be achieved by prolonged cultivation, long-term culture of MSCs is associated with accumulation of morphological and functional changes. In our study, we focused on analyzing morphological and biological changes of cultured adipose tissue-derived stem cells over 30 passages. We performed morphological analysis using light and electron microscopy, as well as analysis of selected biological properties (expression of surface antigens and selected genes involved in cell regulation and apoptosis, cell cycle, and cell senescence) every 5 passages. Our results showed that long-term expansion leads to significant changes in morphology and affects proliferation kinetics and the cell cycle. On the other hand, the MSCs maintained a prototypical immunophenotype, normal cell cycle and apoptosis regulator function, and maintained a low level of telomerase activity during later passages.

Induced pluripotent stem cells in modeling and cell-based therapy of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by neuromuscular degeneration and the progressive loss of upper and lower motor neurons (MNs), causing weakness and paralysis. However, the underlying mechanisms of this disease are still unknown and there is no cure, or even treatment to stop or reverse its pathology. Consequently, most ALS patients die within 3 - 5 years after disease onset. While considerable progress has been made in studying animal models of ALS, they lack clinical suitability due to genetic differences. However, the recent development of induced pluripotent stem cells (iPSCs) has made it possible to study human disease-specific neuronal and glial cells to identify disease mechanisms and develop phenotypic screens for drug discovery. iPSCs provide researchers with a model of naturally occurring pathology under the human genetic background and MNs differentiated from human iPSCs bearing ALS-associated mutations offer a powerful model to study disease pathology. This paper reviews recent methods of differentiating iPSCs into neuronal cells and suggests further applications of these iPSCs-derived cells for ALS disease modeling, drug screening, and possible cell-based therapy.

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).

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.

Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes.

Carnosine's (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.

Recent approaches and challenges in iPSCs: modeling and cell-based therapy of Alzheimer's disease.

The lack of effective therapies for different neurodegenerative disorders has placed huge burdens on society. To overcome the restricted capacity of the central nervous system for regeneration, the promising alternative would be to use stem cells for more effective treatment of chronic degenerative and inflammatory neurological conditions and also of acute neuronal damage and from injuries or cerebrovascular diseases. The generation of induced pluripotent stem cells from somatic cells by the ectopic expression of specific transcription factors has provided the regenerative medicine field with a new tool for investigating and treating neurodegenerative diseases, including Alzheimer's disease (AD). This technology provides an alternative to traditional approaches, such as nuclear transfer and somatic cell fusion using embryonic stem cells. However, due to a problem in standardization of certain reprogramming techniques and systems research, the induced pluripotent stem cell-based technology is still in its infancy. The present paper is aimed at a brief review of the current status in modeling and cell-based therapies for AD.

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 tissuederived 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.

Biomarkers for determination prostate cancer: implication for diagnosis and prognosis.

Prostate cancer (PCa) belongs to most common cancers and it is the second leading cause of cancer death in men. A genetic predisposition or acquired genetic and epigenetic changes with effect of other factors, such as advanced age, race and environmental factors contribute to PCa development. PCa is a very heterogeneous disease that is characterized by different clinical behavior, from indolent, slow-growing tumors to aggressive, fast-growing tumors with lethal progression. Early diagnostics and identification of PCa type are crucial prerequisites for efficient treatment of patients. Recently, the diagnostics of early stages of PCa is based mostly on evaluation of prostate-specific antigen (PSA) in serum of patients. Men with high levels of PSA undergo biopsy in order to determine histopatological grading of PCa - Gleason scoring which classifies tumors from most to least differentiated as well as staging - determination of the status of their primary tumors, with or without lymph node involvement. The results from this screening diagnosis lead into conventional treatment, including radical prostatectomy and brachytherapy. In case of advanced PCa, conventional treatment continues with androgen deprivation therapy. However, in many cases the cancer recurs. Therefore, the clinicians and researchers are forced to find more precise and sensitive biomarker suitable for PCa diagnostics as well as prognostics and therapy. This paper provides review of current most promising molecular and immunohistochemical biomarkers in PCa diagnosis, prognosis and clinical behavior.

The effect of salivary gland extract of Lucilia sericata maggots on human dermal fibroblast proliferation within collagen/hyaluronan membrane in vitro: transmission electron microscopy study.

BACKGROUND: Lucilia sericata maggots are applied to chronic wounds to aid healing when conventional treatments have failed. After their application into a necrotic wound, they potentially influence wound healing with a combination of specific proteinases that are involved in the remodeling of extracellular matrix. These proteases cause changes in fibroblast adhesion and spread upon extracellular matrix protein surfaces, affecting integrity of the protein surfaces-especially fibronectin-while maintaining cell viability. OBJECTIVE: This study focused on in vitro monitoring of the effect of homogenate substances prepared from maggot salivary gland of L sericata on the ultrastructure of human neonatal fibroblasts. METHODS: Collagen/hyaluronan membrane was used as the synthetic substitute of extracellular matrix. The cultured human neonatal fibroblasts B-HNF-1 were seeded on the surface of the collagen/hyaluronan membrane and cultured with maggot salivary gland extract (SGE) at a concentration of 2.4 glands/1 mL. RESULTS: The authors observed increased cell metabolism and protein production (euchromatic nucleus, voluminous nuclear membrane, large reticular nuclei, distended and filled cisterns of rough endoplasmic reticulum, Golgi apparatus with saccules, and vesicles packed with fine fibrillar material) after incubating the cells in culture medium with SGE. CONCLUSION: The authors believe that increased cell metabolism and protein production corresponded with formation of microfibrillar net used for migration of fibroblasts in culture, but mainly for proper production of extracellular matrix. The authors suggest that their results may help explain the effect of SGE on wound healing and support implementation of maggot therapy into human medicine.

Perspectives of induced pluripotent stem cells for cardiovascular system regeneration.

Induced pluripotent stem cells (iPSCs) hold great promise for basic research and regenerative medicine. They offer the same advantages as embryonic stem cells (ESCs) and moreover new perspectives for personalized medicine. iPSCs can be generated from adult somatic tissues by over-expression of a few defined transcription factors, including Oct4, Sox2, Klf4, and c-myc. For regenerative medicine in particular, the technology provides great hope for patients with incurable diseases or potentially fatal disorders such as heart failure. The endogenous regenerative potentials of adult hearts are extremely limited and insufficient to compensate for myocardial loss occurring after myocardial infarction. Recent discoveries have demonstrated that iPSCs have the potential to significantly advance future cardiovascular regenerative therapies. Moreover, iPSCs can be generated from somatic cells of patients with genetic basis for their disease. This human iPSC derivates offer tremendous potential for new disease models. This paper reviews current applications of iPSCs in cardiovascular regenerative medicine and discusses progress in modeling cardiovascular diseases using iPSCs-derived cardiac cells.

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 Hassall's corpuscles), dendritic cells (medulla, often inside the Hassall's corpuscles), thymic myoid cells (medulla, often in close contact with Hassall's corpuscles), macrophages (mostly cortex, but also medulla and inside the Hassall's 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 Hassall's corpuscles (very similar to the basal layer of epidermis). Bcl2 positive lymphocytes were mostly localized in thymic medulla, especially nearby Hassall's 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).

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.

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.