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.

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).

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.

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).

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.

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.

Does cell therapy and tissue engineering represent a promising treatment of diabetic foot ulcers?

Diabetes mellitus is one of the most severe and costly chronic disease of our time. Approximately 2-3% of diabetics have an active foot ulcer, and 15% of all patients with diabetes will develop an ulcer during their lifetime. Treatment of foot complications is one of the main items in the absorption of enormous economic and health resources addressed to the diabetic patients. Advances in basic science, tissue culture techniques and cell therapy promise to improve the treatment of diabetes as well as its complications, i.e. also the ischemic ulcers of the foot. At present, the isolation of any specific type of cells, their in vitro expansion and biological characterization of acquired cell population are possible. For the healing process in ischemic diabetic ulcers, stem cells, endothelial progenitor cells and fibroblasts, both in suspension or placed on an extracellular scaffold are used. This process is focused on stimulating the new blood vessels formation. This is stimulated by the paracrine secretion of multiple growth factors and their receptors. Verified are the vascular endothelial growth factor and its receptor, fibroblast growth factor, interleukin-8 and proangiogenic cytokines (Ref. 62).

Diabetes--adult stem cells as an future alternative therapy?

In both types of the diabetes mellitus, the lack of functional beta-cells is crucial, leading to complications associated with development of hyperglycaemia. One way to achieve a constant normoglycemic state without hypoglycemic episodes is either whole pancreas transplantation, or transplantation of isolated islets of Langerhans. Another approach to correct the beta-cell deficit is the stimulation of beta-cells in pancreas to regeneration. The development of new diabetes therapy is the main goal for many scientists around the world. This article is focused on the stem cells and their potential for clinical applications (Ref. 47).

Autologous biograft and mesenchymal stem cells in treatment of the diabetic foot.

OBJECTIVES: This study was performed to test a new technique for treatment of chronic non-healing wound (diabetic ulcer) using autologous biograft composed of autologous skin fibroblasts on biodegradable collagen membrane (Coladerm) in combination with autologous mesenchymal stem cells (MSC) derived from the patient's bone marrow. DESIGN: The bone marrow aspirate of the patient with diabetic foot was applied directly to the wound and injected into the edges of the wound, finally covered with prepared autologous biograft. The patient received two additional treatments with cultured MSC on day 7 and 17. RESULTS: The wound showed a steady overall decrease in wound size and an increase in the vascularity of the dermis and in the dermal thickness of the wound bed after 29 days of combined treatment. CONCLUSIONS: Closing and healing of the non-healing diabetic ulcer was achieved by using the given combined therapy.

In vitro cytotoxicity testing of coladerm membrane.

At present, biodegradable and biocompatible membranes based on collagen and glycosaminoglycans play an important role in substitutive medicine. Modern biomaterials use a chemically modified collagen-based matrix for implants with programmable biodegradability as a substitute of buccal mucosa, skin, cartilage, etc. Besides the requirements for biocompatibility and biodegradability, the membranes must be also non-toxic. Therefore, cytotoxicity testing of these materials in vitro is an integral part of introducing newly developed types of membranes into clinical practice. As a biological model for the tested COLADERM membrane, cell cultures from human embryonic fibroblasts (B-HEF-2) were used for both cytotoxicity testing as well as in tests to assess the ability of cells to proliferate on this membrane. Along with the ability of cells to grow on the surface and inside the membrane, immunohistochemical examination and scanning electron microscopy (SEM) were performed as well. The obtained results have shown that the COLADERM membrane is non-toxic with suitable structural and biological properties for clinical application as a substitute of buccal mucosa following surgical ablation of malignant tissues from the oral cavity.