Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling tim

A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks

Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent “xylene” and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent “xylene”. Second minimize the time required to perform the extraction. Here we describe a method where: • The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol • The entire procedure takes about 15 min • Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot. With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years

An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis

Th17 immune response to adipose tissue‐derived mesenchymal stromal cells

Adipose tissue‐derived mesenchymal stromal cells (ASCs) hold the promise of achieving successful immunotherapeutic results due to their ability to regulate different T‐cell fate. ASCs also show significant adaptability to environmental stresses by modulating their immunologic profile. Cell‐based therapy for inflammatory disease requires a detailed understanding of the molecular relation between ASCs and Th17 lymphocytes taking into account the influence of inflammation and cell ratio on such interaction. Accordingly, a dose‐dependent increase in Th17 generation was only observed in high MSC:T‐cell ratio with no significant impact of inflammatory priming. IL‐23 receptor (IL‐23R) expression by T cells was not modulated by ASCs when compared to levels in activated T cells, while ROR‐γt expression was significantly increased reaching a maximum in high (1:5) unprimed ASC:T‐cell ratio. Finally, multiplex immunoassay showed substantial changes in the secretory profile of 15 cytokines involved in the Th17 immune response (IL‐1β, IL‐4, IL‐6, IL‐10, IL‐17A, IL‐17F, IL‐22, IL‐21, IL‐23, IL‐25, IL‐31, IL‐33, IFN‐γ, sCD40, and TNF‐α), which was modulated by both cell ratio and inflammatory priming. These findings suggest that Th17 lymphocyte pathway is significantly modulated by ASCs that may lead to immunological changes. Therefore, future ASC‐based immunotherapy should take into account the complex and detailed molecular interactions that depend on several factors including inflammatory priming and cell ratio

Transforming growth factor-β1 inhibits interleukin-1β-induced expression of inflammatory genes and Cathepsin S activity in human vascular smooth muscle cells

Objective and design This study investigated the opposite mechanisms by which IL-1β and TGF-β1 modulated the inflammatory and migratory phenotypes in cultured human intimal vascular smooth muscle cells vSMCs. Materials and treatment Primary human vSMCs, obtained from twelve hypertensive patients who underwent carotid endarterectomy, were incubated for 24 hours with either 40 pM TGF-β1, or 1 nmol/L IL-1β, or their combination in presence or absence of anti-TGF-β neutralizing antibody. Methods The expression levels of matrix metalloproteases and their inhibitors, and the elastolytic enzyme cathepsin S (CTSS) and its inhibitor cystatin C were evaluated with RT-PCR. CTSS activity was measured by fluorometry. Results TGF-β1 reversed IL-1β-induced expression of iNOS, CXCL6, IL1R1, MMP12, and CTSS, while upregulated TIMP2 expression. Furthermore, anti-TGF-β neutralizing antibody abrogated TGF-β effects. Combination with IL-1β and TGF-β1 induced the expression of IL1α, IL1β, IL1R1, and CTSS, but suppressed CST3 expression. CTSS expression in the combination treatment was higher than that of cells treated with anti-TGF-β antibodies alone. Moreover, IL-1β-induced CTSS enzymatic activity was reduced when human vSMCs were co-treated with TGF-β, whereas this reduction was abrogated by anti-TGF-β neutralizing antibody. Conclusion TGF-β1 abrogated IL-1β-induced expression of inflammatory genes and elastolytic activity in cultured human vSMCs. Thus, TGF-β1 can play a crucial role in impairing IL-1β-induced vascular inflammation and damage involved in the etiology of cardiovascular diseases.The authors would like to thank the staff of the vascular surgery department in Hôpital Nord-Ouest for providing us with the samples.Scopu

In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed

Immuno-biological comparison of hepatic stellate cells in a reverted and activated state.

Human hepatic stellate cells (HSCs) demonstrated great immunological plasticity with important consequences for liver cell therapy. Activated HSCs (aHSCs) are in vitro reverted (rHSCs) to a quiescent-like phenotype with potential benefit to reduce liver fibrosis. The goal of this study is to establish and compare the immunological profile of activated and in vitro reverted HSCs and to investigate the impact of inflammatory priming on the immunobiology of both HSCs populations. The distribution of inflammatory primed activated and reverted HSCs across the different phases of the cell cycle is assessed by flow cytometry. In addition, Flow analysis was done to assess the expression level of neuronal, endothelial and stromal markers, cell adhesion molecules, human leucocyte antigens, co-stimulatory molecules, immunoregulatory molecules and natural killer ligands. Our results showed that the cell cycle distribution of both HSCs populations is significantly modulated by inflammation. Accordingly, activated HSC that were in G1 phase switch to S- and G2 phases when exposed to inflammation, while reverted HSCs mostly redistribute into sub-G0 phase. In a HSC state dependent manner, inflammatory priming modulated the expression of the stromal marker CD90, biological receptors (CD95 and CD200R), cell adhesion molecules (CD29, CD54, CD58, CD106 and CD166), human leucocyte antigen HLA-G, co-stimulatory molecules (CD40 and CD252), as well as the immunoregulatory molecules (CD200 and CD274). In conclusion, the immunologic profile of HSCs is significantly modulated by their activation state and inflammation and is important for the development of novel HSC liver cell-based therapy.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A Maladaptive Role for EP4 Receptors in Podocytes

Inhibition of p38 mitogen-activated protein kinase and cyclooxygenase-2 reduces albuminuria in models of chronic kidney disease marked by podocyte injury. Previously, we identified a feedback loop in podocytes whereby an in vitro surrogate for glomerular capillary pressure (i.e., mechanical stretch) along with prostaglandin E2 stimulation of its EP4 receptor induced cyclooxygenase-2 in a p38-dependent manner. Here we asked whether stimulation of EP4 receptors would exacerbate glomerulopathies associated with enhanced glomerular capillary pressure. We generated mice with either podocyte-specific overexpression or depletion of the EP4 receptor (EP4pod+ and EP4pod−/−, respectively). Glomerular prostaglandin E2-stimulated cAMP levels were eightfold greater for EP4pod+ mice compared with nontransgenic (non-TG) mice. In contrast, EP4 mRNA levels were >50% lower, and prostaglandin E2-induced cAMP synthesis was absent in podocytes isolated from EP4pod−/− mice. Non-TG and EP4pod+ mice underwent 5/6 nephrectomy and exhibited similar increases in systolic BP (+25 mmHg) by 4 weeks compared with sham-operated controls. Two weeks after nephrectomy, the albumin-creatinine ratio of EP4pod+ mice (3438 μg/mg) was significantly higher than that of non-TG mice (773 μg/mg; P < 0.0001). Consistent with more severe renal injury, the survival rate for nephrectomized EP4pod+ mice was significantly lower than that for non-TG mice (14 versus 67%). In contrast, 6 weeks after nephrectomy, the albumin-creatinine ratio of EP4pod−/− mice (753 μg/mg) was significantly lower than that of non-TG mice (2516 μg/mg; P < 0.05). These findings suggest that prostaglandin E2, acting via EP4 receptors contributes to podocyte injury and compromises the glomerular filtration barrier

Influence of Polyacrylamide Hydrogel Stiffness on Podocyte morphology, phenotype and mechanical properties.

International audienceChronic kidney disease (CKD) is characterized by a gradual decline in renal function that progresses toward end-stage renal disease (ESRD). Podocytes are highly specialized glomerular epithelial cells, which form with the glomerular basement membrane (GBM) and capillary endothelium, the glomerular filtration barrier (GFB). GBM is an extracellular matrix (ECM) that acts as a mechanical support and provides biophysical signals that control normal podocytes behavior in the process of glomerular filtration. Thus the modulus of elasticity E or stiffness of “ECM” represents an essential characteristic that controls podocyte functions. The biophysical properties of hydrolyzed Polyacrylamide (PAAm) gels resemble to in vivo ECM and thus provide an opportunity to be applied as ECM-like membranes to study cellular behaviors. Therefore, hydrolyzed PAAm hydrogels were investigated for their potential use as new ECM-like constructs to engineer a basement membrane that form with cultured human podocytes a functional glomerular-like filtration barrier. Such ECM-like polyacrylamide hydrogel construct will provide the unique opportunity of understanding in an in vivo-like setting podocyte cells biological responses by controlling the physical properties of the PAAm membranes. In this work, several PAAm hydrogel layers were prepared by changing the crosslinker concentration. The macromolecular microstructure and stiffness were evaluated by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) techniques respectively. Accordingly, the mechanical properties and the polymeric network porosity can be effectively controlled by modulating the crosslinker concentration as well as the swelling degree. Moreover, modulating gel stiffness significantly influenced podocyte behavior including morphology, actin cytoskeleton reorganization. In conclusion, podocytes response to the variation of the mechanical properties of the membranes correlated with the hydrogels stiffness. This work addresses the complexity of podocytes behavior which will further enhance our knowledge to develop a kidney-on-chip model much needed to study kidney function in both health and disease states

Human hepatic stellate cells and inflammation: A regulated cytokine network balance

AIM: Uncertainty about the safety of cell therapy continues to be a major challenge to the medical community. Inflammation and the associated immune response represent a major safety concern hampering the development of long-term clinical therapy. In vivo interactions between the cell graft and the host immune system are mediated by functional environmental sensors and stressors that play significant roles in the immunobiology of the graft. Within this context, human liver stellate cells (HSC) demonstrated marked immunological plasticity that has main importance for future liver cell therapy application. METHODS: By using qPCR technique, we established the cytokine gene expression profile of HSCs and investigated the effect of an inflammatory environment on the immunobiology of HSCs. RESULTS AND DISCUSSION: HSCs present a specific immunological profile as demonstrated by the expression and modulation of major immunological cytokines. Under constitutive conditions, the cytokine pattern expressed by HSCs was characterized by the high expression of IL-6. Inflammation critically modulated the expression of major immunological cytokines. As evidenced by the induction of the expression of several inflammatory genes, HSCs acquire a pro-inflammatory profile that ultimately might have critical implications for their immunological shape. CONCLUSION: These new observations have to be taken into account in any future liver cell therapy application based on the use of HSCs