Human Adipose-Derived and Amniotic Fluid-Derived Stem Cells: A Preliminary In Vitro Study Comparing Myogenic Differentiation Capability.

BACKGROUND Around the world, disabilities due to musculoskeletal disorders have increased and are a major health problem worldwide. In recent years, stem cells have been considered to be powerful tools for musculoskeletal tissue engineering. Human adipose-derived stem cells (hADSCs) and amniotic fluid-derived stem cells (hAFSCs) undergo typical differentiation process into cells of mesodermal origin and can be used to treat muscular system diseases. The aim of the present study was to compare the biological characteristic of stem cells isolated from different human tissues (adipose tissue and amniotic fluid) with respect to myogenic capacity and skeletal and smooth muscle differentiation under the same conditions. MATERIAL AND METHODS hAFSCs and hADSCs were isolated during standard medical procedures and widely characterized by specific markers expression and differentiation potential. Both cell types were induced toward smooth and striated muscles differentiation, which was assessed with the use of molecular techniques. RESULTS For phenotypic characterization, both stem cell types were assessed for the expression of OCT-4, SOX2, CD34, CD44, CD45, and CD90. Muscle-specific markers appeared in both stem cell types, but the proportion of positive cells showed differences depending on the experimental conditions used and the source from which the stem cells were isolated. CONCLUSIONS In this study, we demonstrated that hADSCs and hAFSCs have different capability of differentiation toward both muscle types. However, hADSCs seem to be a better source for myogenic protocols and can promote skeletal and smooth muscle regeneration through either direct muscle differentiation or by paracrine mechanism.

Does the Harvesting Technique Affect the Properties of Adipose-Derived Stem Cells?-The Comparative Biological Characterization.

The objective of this study was to evaluate complex biological properties of human stem cells isolated from adipose tissue (ASCs) harvested utilizing different methods: surgical resection (R), power-assisted liposuction (PAL), and laser-assisted liposuction (LAL). ASCs were isolated from healthy donors, due to surgical resection, power-, and laser-assisted liposuction. Isolated cells were characterized by their clonogenicity, proliferation rate, doubling time, multilineage differentiation, and senescence potential. The average number of ASCs from 1g/1 ml of solid adipose tissue/lipoaspirate was 2.9 × 105 ± 2.4 × 105 , 1.1 × 105 ± 0.8 × 105 , and 1.2 × 105 ± 0.7 × 105 , respectively, for ASCsR, ASCsPAL, and ASCsLAL. However, number of colonies formed by ASCsR and ASCsPAL was significantly higher compared to the average number of colonies formed by ASCsLAL. Also, in comparison to other analyzed cell groups, ASCsPAL obtained the highest proliferative activity. All analyzed cells were characterized by stable expression of CD90 and CD44 markers during prolonged culture. Expression of CD34 and CD45 markers was decreasing in subsequent passages. Presented study shows that different ASCs collection method affects some basic characteristics of these cells, such as number of isolated cells, clonogeneity, or doubling time. J. Cell. Biochem. 118: 1097-1107, 2017. © 2016 Wiley Periodicals, Inc.

High Quality Independent From a Donor: Human Amniotic Fluid Derived Stem Cells-A Practical Analysis Based on 165 Clinical Cases.

The aim of the study was to extend the potential use of human stem cells isolated from amniotic fluid in medical applications by confirming their high homogeneity and quality. Amniotic fluid samples were collected during amniocentesis from 165 women during pregnancy. The proliferation rate, clonogenicity, karyotype, aging process, pluripotent cell markers, expression of surface markers, and the potential to differentiate into adipose, bone and cartilage cells of hAFSCs were analyzed. Obtained results revealed that mesenchymal stem cells could be derived successfully from amniotic fluid, which exhibit properties comparable with MSCs of other origins. It is the first study, in which such a large group of patients was involved. Comprehensive statistical and biological analysis were conducted some of which clearly being innovative in relation to human amniotic fluid-derived stem cells. J. Cell. Biochem. 118: 116-126, 2017. © 2016 Wiley Periodicals, Inc.

Adipose-Derived Stem Cells as a Tool in Cell-Based Therapies.

Recent development in stem cell isolation methods and expansion under laboratory conditions create an opportunity to use those aforementioned cells in tissue engineering and regenerative medicine. Particular attention is drawn towards mesenchymal stem cells (MSCs) being multipotent progenitors exhibiting several unique characteristics, including high proliferation potential, self-renewal abilities and multilineage differentiation into cells of mesodermal and non-mesodermal origin. High abundance of MSCs found in adipose tissue makes it a very attractive source of adult stem cells for further use in regenerative medicine applications. Despite immunomodulating properties of adipose-derived stem cells (ASCs) and a secretion of a wide variety of paracrine factors that facilitate tissue regeneration, effectiveness of stem cell therapy was not supported by the results of clinical trials. Lack of a single, universal stem cell marker, patient-to-patient variability, heterogeneity of ASC population combined with multiple widely different protocols of cell isolation and expansion hinder the ability to precisely identify and analyze biological properties of stem cells. The above issues contribute to conflicting data reported in literature. We will review the comprehensive information concerning characteristic features of ASCs. We will also review the regenerative potential and clinical application based on various clinical trials.

Non-alcoholic beverages, unknown influence on cell proliferation - an in vitro study.

INTRODUCTION AND OBJECTIVE: The aim of the presented study was to check differences between 'Diet' and 'non-Diet' soft drinks on cell proliferation. MATERIALS AND METHODS: Coca Cola and Pepsi Cola of different origin and their dietetic versions were examined at concentrations of 2% and 4%. Fructose and glucose as well as medium alone (control) were examined. RESULTS: Cell number was higher in media supplemented with soft drinks, compared to control. Proliferation depended on the soft drink concentration and its origin, but not on sugar and calorific content. CONCLUSIONS: An unknown factor is responsible for the increase in proliferation.

Human amniotic-fluid-derived stem cells: a unique source for regenerative medicine.

INTRODUCTION: The first application of tissue engineering was based on the use of differentiated cells from the adult organism, which was associated with an invasiveness and high risk of diseased cells' transplantation. Over the years, the range of available cell populations for tissue engineering has widened. AREAS COVERED: We review the comprehensive information concerning the characteristic features of amniotic-fluid-derived stem cells (AFSCs). We also review the potential applications of these cells in clinical practice. EXPERT OPINION: AFSCs hold promise for the future treatment of many incurable diseases. However, such cell-based therapies have some limitations, and there are questions relating to the use of stem cells, which should be carefully analyzed before translation of these cells into clinical practice.

Carboplatin and sodium butyrate, separate--yes, but combined--never.

With the object of improving the effectiveness of a malignant melanoma's treatment and a patients' quality of life, there is a serious need to identify new anticancer compounds, for example, among naturally derived compounds such as sodium butyrate. The aim of this study was to assess the combined impact of carboplatin (C) and sodium butyrate on the B16 melanoma viability by in vitro. B16 cell line was exposed to various concentrations of carboplatin (0.001-10 micromol/L) and sodium butyrate (1 to 100 mmol/L) for 24 h. LC10, LC50 and LC90 values were calculated. The influence of carboplatin and sodium butyrate on the cell cycle and apoptosis was assessed. Additionally, magnetic stem cell sorting was performed, positive melanoma CD133 cells were isolated and the effects of carboplatin and sodium butyrate on cell viability with heterogeneous population of melanoma cells (CD133+/CD133-) was compared. For carboplatin LC50 and LC90 were 1.2 micromol/L and 4.58 pmol/L, respectively. For sodium butyrate LC50 and LC90 were 65.73 mmol/L and 275.06 mmol/L. The value for LC10 could not be determined. Sodium butyrate at the highest concentration (100.0 mmol/L) resulted in only 57.36% mortality of cells. A synergistic effect of both compounds was observed in low concentrations of sodium butyrate and carboplatin. That synergism disappeared at concentrations corresponding to LC50. At the concentration corresponding to LC50 C and high concentration of sodium butyrate, a decrease of cell numbers in phase G2/M was observed (r = -0.97). Cells were arrested in phase G1/G0 and S. The presented results exclude the possibility of the combined application of sodium butyrate and carboplatin in cancer therapy.

Ciprofloxacin is a potential topoisomerase II inhibitor for the treatment of NSCLC.

Lung cancer is one of the most common tumors and its treatment is still inefficient. In our previous work we proved that ciprofloxacin has a different influence on five cancer cell lines. Here, we aimed to compare the biological effect of ciprofloxacin on cell lines representing different responses after treatment, thus A549 was chosen as a sensitive model, C6 and B16 as highly resistant. Three different cell lines were analyzed (A549, B16 and C6). The characterization of continuous cell growth was analyzed with the Real-Time Cell Analyzer (RTCA)-DP system. Cytoskeletal changes were demonstrated using immunofluorescence. The cell cycle was analyzed using flow cytometry. Ciprofloxacin was cytostatic only against the A549 cell line. In the case of other tested cell lines a cytostatic effect was not observed. Cytoskeletal analysis confirms the results obtained with RTCA-DP. A549 cells were inhibited in the G2/M phase suggesting a mechanism related to topoisomerase II inhibition. The biological effects of ciprofloxacin support the hypothesis that this drug can serve as an adjuvant treatment for lung cancer, due to its properties enabling topoisomerase II inhibition.

Does aging of mesenchymal stem cells limit their potential application in clinical practice?

Mesenchymal stem cells (MSCs) are in the center of attention of many investigators due to easy isolation from many tissues. MSC capability to differentiate into many cell types makes them a starting point of many new therapies, especially in tissue engineering. However, understanding the process of MSC aging is crucial for selecting donors for cellular therapies, which is necessary for successful treatment. Cell changes can be divided into three major groups. Changes which affect their proliferate rate, differentiation capability and genome stability lead to decrease of their usefulness in new therapies. There are many tools that can be used to describe and measure some features of aging in MSCs but the essence of this process is still unclear. The aim of this review is to take a deep look into the influence of donor age and in vitro aging on MSC properties.

[Identification of circulating tumor cells as a promising method of genitourinary cancer diagnosis]. / Identyfikacja krazacych komórek nowotworowych jako obiecujaca metoda diagnostyki chorób nowotworowych ukladu moczowo-plciowego.

Circulating tumor cells (CTCs) are cells circulating in the blood, which in terms of antigenic or genetic profile correspond to a particular type of cancer. It is suspected that CTCs possess properties of cancer stem cells. Detection, quantification and characterization of CTCs in the peripheral blood can be of great importance for modern oncology. In the case of early-stage disease, CTCs may help in cancer detection, estimation of metastasis risk and treatment prognosis. In advanced cancer patients, CTCs may also have prognostic significance and may facilitate monitoring response to treatment. Identification of CTCs in the circulation and their differentiation from hematopoietic cells and normal epithelial cells could be based on physical and biological properties such as size, density and expression of specific proteins. Immunomagnetic techniques are the most commonly used methods of CTCs isolation. CellSearch System (CSS) is the only test for detecting CTCs in the peripheral blood approved by the Food and Drug Administration (FDA) for clinical use. The paper presents the characteristics of circulating tumor cell isolation methods and the results of studies concerning CTCs isolation in patients with prostate, bladder and kidney cancer. 

The influence of ciprofloxacin on viability of A549, HepG2, A375.S2, B16 and C6 cell lines in vitro.

Ciprofloxacin is a chemotherapeutic agent mainly used in the treatment of the pulmonary and urinary tract infections but is also known for its anticancer properties. The aim of these study was to check the anticancer effect of ciprofloxacin on selected five cell lines. Human non-small cell lung cancer line A549, human hepatocellular carcinoma line HepG2, human and mouse melanoma lines (A375.S2 and B16) and rat glioblastoma line C6 were used for evaluation of cytotoxic properties of ciprofloxacin (in concentration range: 10-1000 microg/mL). Viability was established using trypan blue assay and MTT. Ciprofloxacin induced morphological changes and decreased viability of A549 cells in a concentration and time dependent manner. In case of A375.S2 and B16 cell lines, cytotoxicyty of ciprofloxacin was observed but we were not able to eradicate all cells from A375.S2 and B16 cultures. HepG2 line was sensitive to ciprofloxacin, but this effect was independent from concentration and incubation time. The C6 cells were insensitive to ciprofloxacin. Our results showed that ciprofloxacin can be potentially used for the experimental adjunctive therapy of lung cancer.

Modulating activity of M1 receptor to the reaction of ileal smooth muscle.

BACKGROUND: The subject of the study was determination of the effect of drugs on ileal smooth muscle contraction induced by activation of M(1) type muscarinic receptors. Drugs that have an effect on muscarinic receptors are divided to agonists, with close ties to the receptor and high internal activity and antagonists, with no internal activity. Conducted experiments tested interactions between a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M(1) type, pirenzepine. MATERIAL/METHODS: Testing was conducted on tissues isolated from rat's intestine. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin modification (2006). RESULTS: The purpose of the study was determination of concentration-effect curves for carbachol. This curve was compared with the curve of receptor occupation depending on concentration of this drug. Based on concentration-effect curves, the average value of EC(50) was calculated for carbachol, amounting to 2.44×10(-6) [M/l]. CONCLUSIONS: The results confirmed that atropine is effective in stopping contractions caused by carbachol, meeting the conditions of competitive antagonists. Atropine caused the shift of curves for carbachol to the right. Pirenzepine, selectively blocking muscarinic receptors of M(1) type gave similar results. It was proved that in the preparation of gastric fundus smooth muscle, M(1) type receptors occur not only presynaptically, but also postsynaptically.

Role of M1 receptor in regulation of gastric fundus smooth muscle contraction.

BACKGROUND: The subject of this study is determination of the influence of drugs on gastric fundus smooth muscle contraction induced by activation of muscarinic receptors M1. Experiments tested interactions between a receptor agonist, carbachol and muscarinic receptor antagonists, atropine and pirenzepine. MATERIAL/METHODS: Testing was conducted on tissues isolated from rat's stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification). RESULTS: According to tests, carbachol, in concentrations ranging between 10(-8) M to 10(-4) M, in a dosage-dependent way induces gastric fundus smooth muscle contraction. Presented results indicate that carbachol meets the conditions posed to full agonists. On the other hand, atropine, a non-selective muscarinic receptor antagonist, causes a concentration-dependent shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive antagonists. The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. CONCLUSIONS: From the testing conducted on the preparation of the gastric fundus we can deduce that atropine causes shift of concentration-effect curves for carbachol to the right. A similar effect is released by pirenzepine, selectively blocking muscarinic receptors of M1 type. The results indicate that in the preparation of the gastric fundus smooth muscle, M1 type receptors occur also postsynaptically.

[Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration]. / Mezenchymalne komórki macierzyste narzedziem terapeutycznym w regeneracji tkanek i narzadów.

Tissue engineering is an interdisciplinary field that offers new opportunities for regeneration of diseased and damaged tissue with the use of many different cell types,including adult stem cells. In tissue engineering and regenerative medicine the most popular are mesenchymal stem cells (MSCs) isolated from bone marrow. Bone marrow mesenchymal stem cells are a potential source of progenitor cells for osteoblasts, chondroblasts, adipocytes, skeletal muscles and cardiomyocytes. It has also been shown that these cells can differentiate into ecto- and endodermal cells, e.g. neuronal cells, glial cells, keratinocytes and hepatocytes. The availability of autologous MSCs, their proliferative potential and multilineage differentiation capacity make them an excellent tool for tissue engineering and regenerative medicine. The aim of this publication is to present characteristic and biological properties of mesenchymal stem cells isolated from bone marrow.

The influence of ciprofloxacin on hamster ovarian cancer cell line CHO AA8.

Abstract: Ciprofloxacin is a chinolone antibiotic, which is used mainly in the treatment of urinary tract infections but also in pulmonary tract, prostate gland, bone and bone marrow infection. Ciprofloxacin is also known for its anticancer in vitro properties. In this study hamster ovarian cancer line CHO AA8 was used for evaluation of cytotoxic properties of ciprofloxacin against neoplastic cells. For this purpose we used different concentrations of ciprofloxacin range from 10 to 1000 microg/mL. Cell viability was counted using trypan blue assay. Ciprofloxacin induced morphological changes and decreased viability in a concentration and time dependent manner within CHO AA8 cells. In low concentrations cytotoxic effect of ciprofloxacin is weak only after 24 h incubation. In the highest concentration of ciprofloxacin, after 24, 48 and 72 h incubation only a very small number of living cells (not exceeding 1%) was observed. No living cells were observed after 96 h of incubation times and ciprofloxacin concentrations of 800 and 1000 micrpg/mL. These promising results deserved future studies on chinolones and ovarian cancer.