Agnieszka Śmieszek

The Effect of Age on Osteogenic and Adipogenic Differentiation Potential of Human Adipose Derived Stromal Stem Cells (hASCs) and the Impact of Stress Factors in the Course of the Differentiation Process

Human adipose tissue is a great source of autologous mesenchymal stem cells (hASCs), which are recognized for their vast therapeutic applications. Their ability to self-renew and differentiate into several lineages makes them a promising tool for cell-based therapies in different types of degenerative diseases. Thus it is crucial to evaluate age-related changes in hASCs, as the elderly are a group that will benefit most from their considerable potential. In this study we investigated the effect of donor age on growth kinetics, cellular senescence marker levels, and osteogenic and adipogenic potential of hASCs. It also has been known that, during life, organisms accumulate oxidative damage that negatively affects cell metabolism. Taking this into consideration, we evaluated the levels of nitric oxide, reactive oxygen species, and superoxide dismutase activity. We observed that ROS and NO increase with aging, while SOD activity is significantly reduced. Moreover cells obtained from older patients displayed senescence associated features, for example, β-galactosidase activity, enlarged morphology, and p53 protein upregulation. All of those characteristics seem to contribute to decreased proliferation potential of those cells. Our results suggest that due to aging some cellular modification may be required before applying aged cells efficiently in therapies such as tissue engineering and regenerative medicine.

The production and distribution of IL-6 and TNF-α in subcutaneous adipose tissue and their correlation with serum concentrations in Welsh ponies with equine metabolic syndrome

A main symptom of equine metabolic syndrome (EMS) in ponies is pathological obesity characterized by abnormal accumulation of fat deposits and inflammation. In this study, we analyzed the expression of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in subcutaneous adipose tissue and the correlation with serum concentrations in peripheral blood of Welsh ponies. Based on clinical examination findings, the animals were divided into two groups: ponies affected with EMS (n = 8) and obese ponies (n = 8). The adipose tissue was examined using immunohistochemical analysis while concentrations IL-6 and TNF-α were measured using enzyme-linked immunosorbent assays (ELISAs). Additionally, histological characterization of the adipose tissue was performed. The results obtained showed that IL-6 expression in adipose tissue biopsies derived from animals with EMS was enhanced while TNF-α levels of both groups were comparable. Compared to the obese ponies, EMS animals also had significantly elevated levels of serum IL-6 and TNF-α. Histological analysis revealed macrophage infiltration and fibrosis in adipose tissue preparations from the EMS group. These data suggest that IL-6 may play a key role in the course of EMS in Welsh ponies. Our findings also demonstrated that analysis of pro-inflammatory cytokines levels in serum may serve as an additional tool for diagnosing EMS.

Biological effects of sol–gel derived ZrO2 and SiO2/ZrO2 coatings on stainless steel surface—In vitro model using mesenchymal stem cells

The objective of this study was to determine biocompatibility of zirconia-based coatings obtained by the sol–gel method. Two matrices, ZrO2 and SiO2/ZrO2, were created and applied on stainless steel type 316L with dip-coating technique. The morphology and topography of biomaterials’ surface were characterized using energy-dispersive X-ray spectroscopy and atomic force microscopy, while chemical composition was analyzed by Raman spectroscopy. Additionally, wettability and surface free energy were characterized. Biocompatibility of obtained biomaterials was evaluated using an in vitro model employing mesenchymal stem cells (MSCs) of adipose and bone marrow origin. Biological analysis included determination of proliferation activity and morphology of MSCs in cultures on synthesized biomaterials. Osteoinductive properties of biomaterials were determined both in non-osteogenic, as well as osteogenic conditions. The results showed that investigated biomaterials exerted different impact on MSCs. Biomaterial with ZrO2 layer was more biocompatible for adipose-derived MSCs, while SiO2/ZrO2 layer promoted proliferation of bone marrow derived MSCs. Moreover, hybrid coating exhibited greater osteoinductive properties than ZrO2 coating, both on cultures with adipose-derived stromal (stem) cells and bone marrow stromal cells. Observed biological effects may result not only from different chemical composition, but also from diverse wettability. The ZrO2 coating was characterized as hydrophobic layer, while SiO2/ZrO2 exhibited hydrophilic properties. The results obtained suggest that behavior of MSCs in response to the biomaterial may vary depending on their origin, therefore we postulate, that screening analysis of implants’ biocompatibility, should incorporate model applying both adipose- and bone marrow derived MSCs.

Endurance Exercise Mobilizes Developmentally Early Stem Cells into Peripheral Blood and Increases Their Number in Bone Marrow: Implications for Tissue Regeneration.

Endurance exercise has been reported to increase the number of circulating hematopoietic stem/progenitor cells (HSPCs) in peripheral blood (PB) as well as in bone marrow (BM). We therefore became interested in whether endurance exercise has the same effect on very small embryonic-like stem cells (VSELs), which have been described as a population of developmentally early stem cells residing in BM. Mice were run daily for 1 hour on a treadmill for periods of 5 days or 5 weeks. Human volunteers had trained in long-distance running for one year, six times per week. FACS-based analyses and RT-PCR of murine and human VSELs and HSPCs from collected bone marrow and peripheral blood were performed. We observed that endurance exercise increased the number of VSELs circulating in PB and residing in BM. In parallel, we observed an increase in the number of HSPCs. These observations were subsequently confirmed in young athletes, who showed an increase in circulating VSELs and HSPCs after intensive running exercise. We provide for the first time evidence that endurance exercise may have beneficial effects on the expansion of developmentally early stem cells. We hypothesize that these circulating stem cells are involved in repairing minor exercise-related tissue and organ injuries.

Macroautophagy and Selective Mitophagy Ameliorate Chondrogenic Differentiation Potential in Adipose Stem Cells of Equine Metabolic Syndrome: New Findings in the Field of Progenitor Cells Differentiation

Equine metabolic syndrome (EMS) is mainly characterized by insulin resistance, obesity, and local or systemic inflammation. That unfriendly environment of adipose tissue has huge impact on stem cells population (ASC) residing within. In the present study, using molecular biology techniques and multiple imaging techniques (SEM, FIB-SEM, and confocal microscopy), we evaluated the impact of EMS on ASC viability and chondrogenic differentiation. Moreover, we visualized the mitochondrial network and dynamics in ASCCTRL and ASCEMS during control and chondrogenic conditions. In control conditions, ASCEMS were characterized by increased mitochondrial fission in comparison to ASCCTRL. We found that extensive remodeling of mitochondrial network including fusion and fission occurs during early step of differentiation. Moreover, we observed mitochondria morphology deterioration in ASCEMS. These conditions seem to cause autophagic shift in ASCEMS, as we observed increased accumulation of LAMP2 and formation of multiple autophagosomes in those cells, some of which contained dysfunctional mitochondria. “Autophagic” switch may be a rescue mechanism allowing ASCEMS to clear impaired by ROS proteins and mitochondria. Moreover it provides a precursors-to-macromolecules synthesis, especially during chondrogenesis. Our data indicates that autophagy in ASCEMS would be crucial for the quality control mechanisms and maintenance of cellular homeostasis ASCEMS allowing them to be in “stemness” status.

The morphology, proliferation rate, and population doubling time factor of adipose-derived mesenchymal stem cells cultured on to non-aqueous SiO2, TiO2, and hybrid sol-gel-derived oxide coatings.

In recent years, much attention has been paid to the development of tissue engineering and regenerative medicine, especially when stem cells of various sources are concerned. In addition to the interest in mesenchymal stem cells isolated from bone marrow, recently more consideration has been given to stem cells isolated from adipose tissue (AdMSCs), due to their less invasive method of collection as well as their ease of isolation and culture. However, the development of regenerative medicine requires both the application of biocompatible material and the stem cells to accelerate the regeneration. In this study, we investigated the morphology, proliferation rate index (PRi), and population doubling time factor of adipose-derived mesenchymal stem cells cultured on non-aqueous sol-gel-derived SiO2, TiO2, and SiO2/TiO2 oxide coatings. The results indicated an increase in PRi of AdMSCs when cultured on to titanium dioxide, suggesting its high attractiveness for AdMSCs. In addition, the proper morphology and the shortest doubling time of AdMSCs were observed when cultured on titanium dioxide coating.

Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line.

Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure.

Physical Activity Increases the Total Number of Bone-Marrow-Derived Mesenchymal Stem Cells, Enhances Their Osteogenic Potential, and Inhibits Their Adipogenic Properties.

Aging and sedentary lifestyle are common nowadays and are associated with the increasing number of chronic diseases. Thus, physical activity is recommended as one of three healthy behavior factors that play a crucial role in health prophylaxis. In the present study, we were interested whether physical activity influences the number and potential of bone-marrow-derived mesenchymal stem cells BMMSCs. In this study, four-week-old male C57Bl/6 mice were trained on a treadmill at progressive speeds over a 5-week period. Comparisons made between exercised (EX) and sedentary animal groups revealed (i) significantly higher number of MSCs in EX animals, (ii) elevated alkaline phosphatase (ALP) activity, (iii) increased level of osteopontin (OPN) and osteocalcin (OCL), and (iv) reduced marrow cavity fat. The results obtained support the thesis that EX may play a substantial role in the regeneration of mesenchymal tissues. Therefore, EX may represent a novel, nonpharmacological strategy of slowing down age-related decline of the musculoskeletal functions.

The effect of the bioactive sphingolipids S1P and C1P on multipotent stromal cells – new opportunities in regenerative medicine

Abstract Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.

The Influence of Spirulina platensis Filtrates on Caco-2 Proliferative Activity and Expression of Apoptosis-Related microRNAs and mRNA

Spirulina platensis (SP) is a blue-green microalga that has recently raised attention not only as a nutritional component, but also as a source of bioactivities that have therapeutic effects and may find application in medicine, including cancer treatment. In the present study we determined the cytotoxic effect of S. platensis filtrates (SPF) on human colon cancer cell line Caco-2. Three concentrations of SPF were tested—1.25%, 2.5%, and 5% (v/v). We have found that the highest concentration of SPF exerts the strongest anti-proliferative and pro-apoptotic effect on Caco-2 cultures. The SPF negatively affected the morphology of Caco-2 causing colony shrinking and significant inhibition of metabolic and proliferative activity of cells. The wound-healing assay showed that the SPF impaired migratory capabilities of Caco-2. This observation was consistent with lowered mRNA levels for metalloproteinases. Furthermore, SPF decreased the transcript level of pro-survival genes (cyclin D1, surviving, and c-Myc) and reduced the autocrine secretion of Wnt-10b. The cytotoxic effect of SPF involved the modulation of the Bax and Bcl-2 ratio and a decrease of mitochondrial activity, and was related with increased levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Moreover, the SPF also caused an increased number of cells in the apoptotic sub-G0 phase and up-regulated expression of mir-145, simultaneously decreasing expression of mir-17 and 146. Obtained results indicate that SPF can be considered as an agent with anti-cancer properties that may be used for colon cancer prevention and treatment.