Differentiation Potential Of Adipose-derived Stem Cells Research Articles

184 Effect of selenium on the differentiation of porcine adipose-derived stem cells into osteoblasts

Bone damage is a common occurrence and although traditional bone healing methods work well, they are not always able to fix all issues pertaining to the injury. However, recent research on stem cell therapies using adipose-derived stem cells (ASC) has shown that ASC that differentiate into osteoblasts can be used as a potentially better solution for bone injuries. Selenium is a nutritional trace element that has been found to be essential in the production of selenoproteins and bone growth. Selenium plays an integral role in osteoblast cell differentiation and proliferation. The aim of this experiment was to test whether adding selenium to osteogenic influences the differentiation potential of ASC into osteoblasts. Porcine ASC were isolated as described (Monaco et al. 2009, Open Tissue Eng. Regen. Med. J. 2, 20-33). Seven different treatments were given to the cells: a negative control of DMEM, a positive control of osteogenic medium, and 5 concentrations of selenium in the osteogenic medium (10, 5, 1, 0.5, and 0.1 µM). Medium was changed twice a week for 4 weeks, at the end of which the cells were stained with Alizarin Red S stain. The number of osteoblastic nodules in each well were counted and divided into the categories of “forming” and “formed.” A generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Chicago, IL, USA) was used to analyse the data. The least statistical difference (l.s.d.) post hoc test was used to perform statistical multiple comparison with an α-level of 0.05. The results showed (Table 1) that medium containing selenium concentrations of 0.1, 0.5, and 1.0 µM were able to develop more nodules than the osteogenic medium. There was no statistical difference in the total amount of forming and formed nodules in those 4 groups; however, there was a statistical tendency for a difference (P=0.06) between the medium with 1 µM selenium and the osteogenic medium. Medium containing 10 or 5 µM selenium had lower nodule counts (both forming and formed) than osteogenic medium. There was no statistical difference between 10 µM and DMEM. These results suggest that low concentrations of selenium have a positive effect on nodule formation, whereas higher concentrations are detrimental. Table 1.The average number (standard deviations in parentheses) of formed, forming, and total osteoblastic nodules in each of the different treatments

Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells.

Background and ObjectivesSchwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated.Methods and ResultsASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×103, 4×103 and 8×103 cells/cm2 an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75NTR mRNA, another SC marker, were significantly higher at the density of 8×103 cells/cm2 when compared with the other cell densities groups (p<0.001).ConclusionsThe results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×103 cells/cm2, possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

Optimizing Hydrocortisone Concentration for Skeletal Muscles Differentiation of ADSCs on PLLA Nano-Scaffolds

Skeletal muscle is a highly differentiated tissue with very specific functions which has low potential of regeneration. Skeletal muscle injuries especially in athletes almost have lead to muscular dysfunctions and healing may be prolonged for several years. Therefore, working on skeletal muscle differentiation remained an importance in biomedical researches. Adipose derived stem cells (ADSCs) are novel source of mesenchymal stem cells which are an excellent alternative for satellite cells in in-vitro skeletal muscle differentiation. Differentiation potential of ADSCs on both tissue culture plate (TCP) and also on Poly l-lactide acid (PLLA) electrospun fibrous nano-scaffold which now is widely used at tissue engineering investigations has studied in this research. Scanning electron microscopy (SEM) and Tensile test were performed for evaluating scaffold properties. Hydrocortisone has considered a critical factors for skeletal muscle differentiation while, the recommended concentrations of it for inducing myogenesis in stem cells is yet discussing. Statistical analysis of our results from colorimetric MTT assay for various concentrations of hydrocortisone showed that the concentration of 10 -7 mol/L is the optimum dose for myogenic differentiation of murine ADSCs which was used on both TCP and PLLA scaffolds and skeletal myosin fiber formations was confirmed with immunocytochemistry. DAPI staining proved myocytes nuclei and syncytium formations. Our results also showed that ADSCs and PLLA nano-scaffolds are the suitable biomaterials for engineering skeletal muscle tissue.

Systems biology approach to identify alterations in the stem cell reservoir of subcutaneous adipose tissue in a rat model of diabetes: effects on differentiation potential and function

Autologous progenitor cells represent a promising option for regenerative cell-based therapies. Nevertheless, it has been shown that ageing and cardiovascular risk factors such as diabetes affect circulating endothelial and bone marrow-derived progenitor cells, limiting their therapeutic potential. However, their impact on other stem cell populations remains unclear. We therefore investigated the effects of diabetes on adipose-derived stem cells (ASCs) and whether these effects might limit the therapeutic potential of autologous ASCs. A systems biology approach was used to analyse the expression of genes related to stem cell identification in subcutaneous adipose tissue (SAT), the stromal vascular fraction and isolated ASCs from Zucker diabetic fatty rats and their non-diabetic controls. An additional model of type 2 diabetes without obesity was also investigated. Bioinformatic approaches were used to investigate the biological significance of these changes. In addition, functional studies on cell viability and differentiation potential were performed. Widespread downregulation of mesenchymal stem cell markers was observed in SAT of diabetic rats. Gene expression and in silico analysis revealed a significant effect on molecules involved in the maintenance of pluripotency and self-renewal, and on the alteration of main signalling pathways important for stem cell maintenance. The viability and differentiation potential of ASCs from diabetic rats was impaired in in vitro models and in in vivo angiogenesis. The impact of type 2 diabetes on ASCs might compromise the efficiency of spontaneous self-repair and direct autologous stem cell therapy.

Differentiation of Human Adipose-derived Stem Cells Co-cultured With Urothelium Cell Line Toward a Urothelium-like Phenotype in a Nude Murine Model

To investigated the urothelium differentiation potential of adipose-derived stem cells (ASCs) that were coimplanted with the immortalized human bladder urothelium cell line (SV-HUC-1) into the subcutaneous tissue of athymic mice. The ASCs were isolated from the human adipose tissue of patients undergoing liposuction procedures and were expanded in vitro. After labeling with CM-DiI, the ASCs were mixed with SV-HUC-1 and implanted into the subcutaneous tissue of athymic mice for 2 and 4 weeks. The urothelium-specific markers uroplakin-Ia and uroplakin-II were detected by immunofluorescence. The transformation rate of ASCs into the urothelium phenotype was evaluated at each measurement point. We found that 25.87% ± 1.38% of ASCs expressed the urothelium-specific marker uroplakin-Ia and 23.60% ± 2.57% of ASCs expressed uroplakin-II 2 weeks after coimplantation with SV-HUC-1 in vivo. After 4 weeks, 70.07% ± 3.84% of ASCs expressed uroplakin-Ia and 65.56% ± 2.94% expressed uroplakin-II. However, no obvious organizational multilayered urothelium structure, such as that of the native bladder mucosa, was found in the subcutaneous tissues of the athymic mice. The results of our study have demonstrated that ASCs could be differentiated toward the urothelium-like phenotype when they were coimplanted in direct contact with cells of a mature urothelium cell line, and the proportion of differentiated cells increased from 2 to 4 weeks. The differentiation potential of ASCs toward the urothelial cell type suggests that ASCs might have potential to be used in urinary tract repair with a tissue engineering approach in the future.

The effect of age on osteogenic, adipogenic and proliferative potential of female adipose-derived stem cells

Human adipose tissue is an ideal source of autologous cells that is both plentiful and easily obtainable in large quantities through the simple surgical procedure of liposuction. The stromal vascular fraction of adipose tissue contains a stem cell population, adipose-derived stem cells (ASCs), capable of adipogenic, osteogenic, myogenic and chondrogenic differentiation. These cells have already been recognized to possess great therapeutic potential in tissue engineering and regeneration. In this study, we sought to determine the effect of donor age on the growth kinetics and differentiation potential of ASCs. For this, ASCs were isolated from liposuctioned adipose tissue obtained from female patients in the age range 20-58 years. Population doubling time was calculated over 2 weeks and differentiation potential was determined by assaying for adipogenesis and osteogenesis. ASCs obtained from older donors appeared to have a slower rate of proliferation, but this relationship was not significant. While adipogenic potential was unrelated to donor age, a distinct relationship between donor age and osteogenic potential was observed. The aetiology of this age-dependent change in osteogenic potential was not due to any changes in the number of precursors with osteogenic capacity in the adipose sample. These findings have important implications for emerging cell-based therapeutic strategies, such as tissue engineering, in addition to treatment of various metabolic bone disorders including osteoporosis.

Regulation of α-Smooth Muscle Actin Protein Expression in Adipose-Derived Stem Cells

The objective of this work was to study the response of adipose-derived stem cells (ASCs) to exogenous biochemical stimulation, and the potential of ASCs to differentiate toward the smooth muscle cell (SMC) lineage. Immunofluorescence staining and Western blot analysis detected protein expression of the early SMC marker α-smooth muscle actin (α-SMA) in both control and experiment groups. Expression of α-SMA in ASCs significantly increased when treated with transforming growth factor-β₁, while α-SMA expression only slightly increased in the presence of retinoic acid (RA), β-mercaptoethanol and ascorbic acid. Treatment with platelet-derived growth factor-BB, RA and dibutyryl-cyclic adenosine monophosphate decreased the expression of α-SMA significantly. While β-mercaptoethanol and ascorbic acid, as well as RA have resulted in increased α-SMA expression in marrow-derived mesenchymal stem cells and other progenitor cells, our results demonstrate that these treatments do not significantly increase α-SMA expression, indicating that the differentiation potential of ASCs and mesenchymal stem cells may be fundamentally different.

202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 �M dexamethasone, 0.5 mM isobutylmethylxantine, 10 �M insulin and 200�M indomethacin) and osteogenic (0.1 �M dexamethasone, 10 mM �-glycerophosphate, and 50�M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro. This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).