A comparative study on the effects of adipose tissue derived and bone marrow mesenchymal stem cells on neurons/glial cells viability, proliferation and differentiation

[Excerpt] It is known that both Mesenchymal Stem Cells (MSCs)and Adipose derived Stem Cells (ASCs) are able to ameliorate the CNS condition upon injury. However it is still not clear whether they have the similar or opposite effects on the different CNS derived cell populations. In this sense the objective of the present work was to understand if ASCs and MSCs preferentially act on different CNS derived cell populations. Hippocampal neurons and glial cells were exposed to MSCs and ASCs conditioned media (CM) (obtained 24, 48, 72 and 96 after 3 days of culture of HUCPVCs) for 1 week. Cell viability experiments (MTS test) revealed that CM obtained for both cell populations at all time points did not cause any deleterious effects on neurons and glial cells. [...]info:eu-repo/semantics/publishedVersio

The secretome of stem cells isolated from the adipose tissue and wharton jelly acts differently on central nervous system derived cell populations

Introduction: It is hypothesized that administration of stromal/stem cells isolated from the adipose tissue (ASCs) and umbilical cord (HUCPVCs) can ameliorate the inured CNS. However it is still not clear whether they have similar or opposite effects on primary cultures of neuronal populations. The objective of the present work was to determine if ASCs and HUCPVCs preferentially act, or not, on specific cell populations within the CNS. Methods: Primary cultures of hippocampal neurons were exposed to ASCs and HUCPVCs conditioned media (CM) (obtained 24, 48, 72 and 96 hours after 3 days of culture) for 1 week. Results: Cell viability experiments (MTS test) revealed that CM obtained from both cell populations at all time points did not cause any deleterious effects on neuronal cells. In fact, it was determined that whenever the ASCs CM were supplemented with bFGF and B27, there was a significant increase on the metabolic viability and neuronal cell density of the cultures. On the other hand in the absence of CM supplementation, it was the HUCPVCs secretome that had the highest impact on the metabolic viability and cell density. In an attempt to unveil which factors could be involved in the observed effects, a screening for the presence of basic fibroblast growth factor (bFGF), nerve growth factor (NGF), stem cell factor (SCF), hepatocyte growth factors (HGF) and vascular endothelial growth factor (VEGF) in the CM was performed. Results revealed the presence of all these factors in ASCs CM, except bFGF; in contrast, in HUCPVCs CM it was only possible to detect robust NGF expression. Conclusions: Overall the results herein confirm important differences on the secretome of ASCs and HUCPVCs, which leads to distinct effects on the metabolic viability and neuronal cell densities in primary cultures of hippocampal neurons; however, the factor(s) that promote the stronger effect of the HUCPVCs CM in neuronal survival is (are) still to be identified.Pennington Biomedical Research FoundationFoundation Calouste de Gulbenkian - The Gulbenkian Programme to Support Research in the Life Sciences and Ciência 2007 ProgramFundação para a Ciência e a Tecnologia (FCT

Adipose tissue derived stem cells secretome: soluble factors and their roles in regenerative medicine

Stem cells have been long looked at as possible therapeutic vehicles for different health related problems. Among the different existing stem cell populations, Adipose derived Stem Cells (ASCs) have been gathering attention in the last 10 years. When compared to other stem cells populations and sources, ASCs can be easily isolated while providing higher yields upon the processing of adipose tissue. Similar to other stem cell populations, it was initially thought that the main potential of ASCs for regenerative medicine approaches was intimately related to their differentiation capability. Although this is true, there has been an increasing body of literature describing the trophic effects of ASCs on the protection, survival and differentiation of a variety of endogenous cells/tissues. Moreover, they have also shown to possess an immunomodulatory character. This effect is closely related to the ASCs’ secretome and the soluble factors found within it. Molecules such as hepatocyte growth factor (HGF), granulocyte and macrophage colony stimulating factors, interleukins (ILs) 6, 7, 8 and 11, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), adipokines and others have been identified within the ASCs’ secretome. Due to its importance regarding future applications for the field of regenerative medicine, we aim, in the present review, to make a comprehensive analysis of the literature relating to the ASCs’ secretome and its relevance to the immune and central nervous system, vascularization and cardiac regeneration. The concluding section will highlight some of the major challenges that remain before ASCs can be used for future clinical applications

Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT)

Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population.Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature.In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction.The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.Background aims: Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Methods: Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. Results: In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. Conclusions: The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters. \ua9 2013, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved

Tools for the identification of bioactives impacting the metabolic syndrome: Screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis. © 2012 Elsevier Inc

Comprehensive homing endonuclease target site specificity profiling reveals evolutionary constraints and enables genome engineering applications

Homing endonucleases (HEs) promote the evolutionary persistence of selfish DNA elements by catalyzing element lateral transfer into new host organisms. The high site specificity of this lateral transfer reaction, termed homing, reflects both the length (14–40 bp) and the limited tolerance of target or homing sites for base pair changes. In order to better understand molecular determinants of homing, we systematically determined the binding and cleavage properties of all single base pair variant target sites of the canonical LAGLIDADG homing endonucleases I-CreI and I-MsoI. These Chlorophyta algal HEs have very similar three-dimensional folds and recognize nearly identical 22 bp target sites, but use substantially different sets of DNA-protein contacts to mediate site-specific recognition and cleavage. The site specificity differences between I-CreI and I-MsoI suggest different evolutionary strategies for HE persistence. These differences also provide practical guidance in target site finding, and in the generation of HE variants with high site specificity and cleavage activity, to enable genome engineering applications

Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

Background: Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Methods/Principal Findings: Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm 2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm 2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Conclusions/Significance: We conclude that CTGF is important in the regulation of cytoskeletal tension mediated AS

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells

Additional file 3: Figure S3. No observable differences in lnASCs and obASCs during early bone regeneration. Critical size calvarial defects were created in the parietal bone of nude mice and assessed after 2 weeks. (A) Representative images of microCT scanning. (B) Quantification of microCT. Scale bar represents 1 mm. Bars, Âą SEM

Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins