RAMAN SPECTROSCOPY: COMPARING THE “FINGERPRINTS” OF С6 GLIOMA AND MESENCHYMAL STEM CELLS

Endothelial Differentiation of Adipose-Derived Stem Cells: Effects of Endothelial Cell Growth Supplement and Shear Force

Growing evidence indicates that adipose tissue (AT) represents a potential source of pluripotent mesenchymal stem cells. However, the mechanisms underlying the lineage-specific commitment of human adipose-derived stem cells (ASC) remain still not fully elucidated. Oxysterols are cholesterol oxide products resulting from non-enzymatic (ie, 7-Ketocolesterol) or enzymatic (ie, 5,6-Secosterol) oxidation of cholesterol, which are now emerging as reliable markers of adipose “oxidative stress” in vivo. Recent data suggest that, by regulating the adipogenic differentiation of ASC, lipid peroxidation products may play an important role in linking the adipose dysfunction with impairment of glucose homeostasis. In this study we combined a lipidomic approach with the subcutaneous (sc) microdialysis technique to characterize the adipose-derived profile of fatty acids (FA) and oxysterols in vivo. ASC were isolated from abdominal sc, mesemteric (MES) and omental (OM) fat specimens obtained from obese nondiabetic (OB) and type 2 diabetic (OBT2D) patients. Flow cytometry (FC) was used for the evaluation of cell viability, mitochondrial status and cell immunophenotyping. In AT interstitial fluid, abundant concentrations of oxysterols (7κC and 5,6-S) and fatty acids (lipokines) were found. Experimental challenging with 7κC and 5,6-S showed a different time-dose-response effect. Indeed, the MTT assay, we found that in ASC isolated from the sc depot 5,6-S (50 and 10μM) reduced cell viability after 24, 48 and 72 h, respectively. In contrast, in the same cell-type population, the effect of 7κC at 10μM was observed only after 72 h. FC analysis indicated a similar effect of both oxysterols even after short-time exposure either in ASC from the MES or the OM fat depot. Notably, cell challenge with 7κC and 5,6-S at 10, 5 and 1 μM, respectively, was accompanied by an impairment of mitochondrial status only in OM, but not in MES. Furthermore, both the oxysterols (10μM) downregulated the expression of stemness surface markers suggesting a different “susceptibility” of the ASC to lipid peroxidation cell damage. Accordingly, 7κC at 10 and 1μM impaired the adipogenic differentiation of sc and OM ASC isolated from either OB or T2DOB, and demodulated the mitochondrial activity of the differentiated adipocytes. Altogether, our results suggest human AT as a critical compartment for storage and secretion of lipokines and oxysterols, which, when in excess, appear to detrimentally modulate the mitochondrial activity and the adipogenic differentiation of adipose precursor cells. This work was partly funded from Fondazione Cassa di Risparmio di Perugia, project 2010.020.0098, “Ricerca Scientifica e Tecnologica”

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu2+) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco’s Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.

Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus. Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR). Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by approximately 8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbalpha gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs. These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose–derived Stem Cells With Forced Expression of MyoD

Adipose‑derived stem cells(ASCs) can easily be obtained and expanded invitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion(DRG) neurons were co‑cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co‑culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co‑culture and compared with single‑culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co‑culture with ASCs, resulting in increased neurite length after 24h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell‑to‑cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue‑engineered constructs may be promising for peripheral nerve repair.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

The current epidemic of obesity and overweight has caused a surge of interest in the study of adipose tissue formation. Much progress has been made in defining the transcriptional networks controlling the terminal differentiation of preadipocytes into mature adipocytes. However, the earliest steps that direct mesenchymal stem cells located in adipose tissue (adipose-derived stem cells (ASCs)) down the adipocyte lineage remain largely unknown. In this chapter, we first focus on the identification of intrinsic and extrinsic factors regulating thebalance between human ASC proliferation and differentiation. We point out the role of factors secreted by undifferentiated ASCs, such a FGF2, activin A, Hedgehog molecules, or secreted by adipose tissue macrophages. We also outline the role of miRNAs in these processes. In a second part, the developmental origin of adipocytes during embryogenesis is discussed. We summarize the surprising findings that have recently emerged from in vivo lineage tracing studies, unravelling unsuspected developmental origins for white adipocytes. We examine the possibility that the differential developmental origin of adipocytes could also reflect functional site-specific regulations of adipose tissue. Finally, we present human-induced pluripotent stem cells as a novel model for investigating the earliest steps of human adipocyte development.

Adipose tissue is widely recognized as an extremely active endocrine organ producing adipokines as leptin that bridge metabolism and the immune system. Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) is a ubiquitous homeodomain transcription factor involved in the adipogenic differentiation and insulin-sensitivity processes. Leptin, as pleiotropic adipokine, and TGF-β, known to be expressed by primary pre-adipocytes [adipose-derived stem cells (ASCs)] and mature differentiated adipocytes, modulate inflammatory responses. We aimed to assess for the first time if leptin and TGF-β interfere with PREP1 expression in both ASCs and mature differentiated adipocytes. Human ASCs were isolated from subcutaneous adipose liposuction and, after expansion, fully differentiated to mature adipocytes. In both ASCs and adipocytes, leptin and TGF-β1 significantly decreased the expression of PREP1, alone and following concurrent Toll-like receptor 4 (TLR4) activation. Moreover, in adipocytes, but not in ASCs, leptin increased TLR4 and IL-33 expression, whereas TGF-β1 enhanced TLR4 and IL-6 expression. Taken together, we provide evidence for a direct regulation of PREP1 by leptin and TGF-β1 in ASCs and mature adipocytes. The effects of leptin and TGF-β1 on immune receptors and cytokines, however, are limited to mature adipocytes, suggesting that modulating immune responses depends on the differentiation of ASCs. Further studies are needed to fully understand the regulation of PREP1 expression and its potential for the development of new therapeutic approaches in obesity-related diseases.

Hydrogel from Acellular Porcine Adipose Tissue Accelerates Wound Healing by Inducing Intradermal Adipocyte Regeneration

The histone deacetylases SIRT1 and SIRT2 have been shown to be involved in the differentiation of rodent adipocyte precursors. In light of the differences in gene expression and metabolic function of visceral (V) and subcutaneous (S) adipose tissue (AT) and their resident cells, the aim of this study was to investigate the role of SIRT1 and SIRT2 in the differentiation of adipose stem cells (ASCs) isolated from SAT and VAT biopsies of nondiabetic obese and nonobese individuals. Human ASCs were isolated from paired SAT and VAT biopsies obtained from 83 nonobese and 92 obese subjects and were differentiated in vitro. Adipogenesis was evaluated by analyzing the lipid deposition using an image processing software, and gene expression by RT-qPCR. SIRT1 and SIRT2 protein expression was modified by using recombinant adenoviral vectors. Visceral but not subcutaneous ASCs from obese subjects showed an intrinsic increase in both adipogenesis and lipid accumulation when compared with ASCs from nonobese subjects, and this was associated with reduced SIRT1 and SIRT2 mRNA and protein levels. Moreover, adipose tissue mRNA levels of SIRT1 and SIRT2 showed an inverse correlation with BMI in the visceral but not subcutaneous depot. Overexpression of SIRT1 or SIRT2 in visceral ASCs from obese subjects resulted in inhibition of adipocyte differentiation, whereas knockdown of SIRT1 or SIRT2 in visceral ASCs from nonobese subjects enhanced this process. Changes in SIRT1 or SIRT2 expression and adipocyte differentiation were paralleled by corresponding changes in PPARG, CEBPA, and other genes marking terminal adipocyte differentiation. SIRT1 and SIRT2 modulate the differentiation of human ASC. Reduced expression of SIRT1 and SIRT2 may enhance the differentiation capacity of visceral ASC in human obesity, fostering visceral adipose tissue expansion.

Background: Adipose derived stem cells (ADSCs) are a new type of mesenchymal stem cells (MSC) resemble bone marrow stem cells in their biologic activities. DScs is multipotent stem cells can differentiate into multiple cell types including osteoblasts, chondrocytes, adipocytes, myocytes, vascular endothelial cells, and neurons. Stromal vascular fraction (SVF) corresponds to ADSC and describes the cells obtained immediately from adipose tissues after the digestion of collagenase. Adipose tissue is a promising source of adult mesenchymal stem cells for therapeutic applications because it is available in large amounts (100 ml up to 1 liter) through liposuction and with minimal morbidity. Aim: To isolate and characterize the adipose-derived stem cells from human liposuction aspirate. Material and Methods: liposuction aspirate was used for the extraction of the stromal vascular fragment (SVF). SVF was used for culturing and isolation of hADSCs.ADSCs were cultured in tissue-culture flasks using collagenase digestion method. Flow cytometric analysis was used to identify ADSCs markers using fluorescence-activated cell sorting (FACS)against CD44, CD90, and CD45. Results: Human ADSCs were isolated successfully using collagenase digestion method. After 4 days the cells appeared to be spindle in shape and formed symmetric colonies. After 7 days, the cell sheet formed 60% confluence and completed to 90% confluence after 10 days incubation. FACS analysis of ADSCs surface markers revealed expression of CD44, and CD90 markers in most of the adherent cells. In contrast, most adherent cells were negative for CD45. Conclusion: Human ADSCs were isolated successfully from human lipoaspirate. Keywords: ADSCs; MSC; SVF

DNA Methylation Plasticity of Human Adipose-Derived Stem Cells in Lineage Commitment

Human adipose-derived stem cells possess a lot of stem cell characteristics, so they may be considered a source of stem cell population. On the basis of that, we have investigated the hepatic potential of adipose-derived stem cells, obtained from liposuction, following two differentiation protocols. In the first procedure, medium was supplemented with epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF) and nicotinamide; the second involved the addition of factors such as dexametasone, EGF, insulin-transferrin-sodium selenite, HGF, dimethyl sulfoxide and oncostatin. In parallel, we carried out our study in the Hep G2 cell line, as human hepatic differentiated in vitro model. Immunocytochemical analysis and RT-PCR were performed using hepatic markers to evaluate cell differentiation. DNA content, MTT test and carboxyl fluorescein succinimidyl ester staining were carried out to evaluate cell proliferation. We reported the evidence of basal hepatic marker in undifferentiated adipose-derived stem cells, which confirmed their multipotency. A strong expression of albumin and α-fetoprotein was observed in hepatic-induced adipose-derived stem cells following both differentiation procedures. Morphological aspects of the two types of hepatic adipose-derived stem cells were alike. Proliferation index suggested that the first differentiation procedure promoted better growth than the second. These preliminary findings suggest adipose-derived stem cells may be induced into hepatic lineage, and the most significant difference between the two standard differentiation procedures concerns proliferation rate. This aspect is to be considered when adipose-derived stem cells are employed in research and clinical studies.

Adipose-derived stem cells (ASCs) are considered a great alternative source of mesenchymal stem cells (MSCs). Unlike bone marrow stem cells (BMSCs), ASCs can be retrieved in high numbers from lipoaspirate, a by-product of liposuction procedures. Given that ASCs represent an easily accessible and abundant source of multipotent cells, ASCs have garnered attention and curiosity from both scientific and clinical communities for their potential in clinical applications. Furthermore, their unique immunobiology and secretome are attractive therapeutic properties. A decade since the discovery of a stem cell reservoir residing within adipose tissue, ASC-based clinical trials have grown over the years around the world along with assessments made on their safety and efficacy. With the progress of ASCs into clinical applications, the aim towards producing clinical-grade ASCs becomes increasingly important. Several countries have recognised the growing industry of cell therapies and have developed regulatory frameworks to assure their safety. With more research efforts made to understand their effects in both scientific and clinical settings, ASCs hold great promise as a future therapeutic strategy in treating a wide variety of diseases. Therefore, this review seeks to highlight the clinical applicability of ASCs as well as their progress in clinical trials across various medical disciplines.