Proteomic analysis of cyclic ketamine compounds ability to induce neural differentiation in human adult mesenchymal stem cells

Neural regeneration is of great interest due to its potential to treat traumatic brain injuries and diseases that impact quality of life. Growth factor mediated differentiation can take up to several weeks to months to produce the cell of interest whereas chemical stimulation may be as minimal as a few hours. The smaller time scale is of great clinical relevance. Adipose derived stem cells (ADSCs) were treated for up to 24 h with a novel differentiation media containing the cyclic ketamine compounds to direct neurogenic induction. The extent of differentiation was investigated by proteome changes occurring during the process. The treatments indicated the ADSCs responded favorably to the neurogenic induction media by presenting a number of morphological cues of neuronal phenotype previously seen and a higher cell population post induction compared to previous studies. Furthermore, approximately 3500 proteins were analyzed and identified by mass spectrometric iTRAQ analyses. The bioinformatics analyses revealed hundreds of proteins whose expression level changes were statistically significant and biologically relevant to neurogenesis and annotated as being involved in neurogenic development. Complementing this, the Bioplex cytokine assay profiles present evidence of decreased panel of stress response cytokines and a relative increase in those involved in neurogenesi

In vitro bioactivity and stem cells attachment of three-dimensionally ordered macroporous bioactive glass incorporating iron oxides

Three-dimensionally ordered macroporous bioactive SiO2-CaO-Na2O-P2O5 glass (3DOM-BG) is synthesized by using the sol-gel method. After an in vitro test in simulated body fluid (SBF), the hydroxyapatite (HAp) crystalline phase is clearly formed on its surface as confirmed by X-ray diffractometry (XRD) and Raman spectroscopy. Magnetic 3DOM-BG/Fe samples are synthesized by partial substitution of SiO2 with iron oxide. Whilst the HAp layer is not confirmed, energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and XRD analysis reveal calcium phosphate layer on the surface of 3DOM-BG/Fe samples after the SBF soaking. The growth of HAp-like layer is slower with increasing iron oxides. The initial mechanism that thought to induce bone formation is reduced due to the replacement of Ca2+ with Fe ions in the glass network. The formation of HAp-like layer is modified by the sedimentation of Ca and P while the nonmagnetic 3DOM-BG forms the calcium phosphate by the ionic exchange following the Hench mechanism. The adult human adipose tissue-derived stem cells (hADSCs) can be closely attached and well spread on the flat-plate of all 3DOM-BG/Fe and 3DOM-BG. Without detectable cytotoxicity possibly induced by iron oxides, the osteoblast can be grown and proliferated. In addition to these bioactivity and biocompatibility, porous structures can allow their possible use in targeted drug delivery and magnetic properties of 3DOM-BG/Fe can essentially be implemented in hyperthermia therapy

Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes

The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation

Molecular Dynamics of Cytokine Interactions and Signalling of Mesenchymal Stem Cells Undergoing Directed Neural-like Differentiation

Mesenchymal stem cells are a continually expanding area in research and clinical applications. Their usefulness and capacity to differentiate into various cells, particularly neural types, has driven the research area for several years. Neural differentiation has considerable usefulness. There are several successful differentiation techniques of mesenchymal stem cells that employ the use of small molecules, growth factors and commercially available kits and supplements. Phenotyping, molecular biology, genomics and proteomics investigation revealed a wealth of data about these cells during neurogenic differentiation. However, there remain large gaps in the knowledge base, particularly related to cytokines and how their role, drive mechanisms and the downstream signalling processes change with their varied expression throughout the differentiation process. In this study, adult mesenchymal stem cells were induced with neurogenic differentiation media, the cellular changes monitored by live-cell microscopy and the changes in cytokine expression in the intracellular region, secretion into the media and in the extracellular vesicle cargo were examined and analysed bioinformatically. Through this analysis, the up-regulation of key cytokines was revealed, and several neuroprotective and neurotrophic roles were displayed. Statistically significant molecules IFN-G, IL1B, IL6, TNF-A, have roles in astrocyte development. Furthermore, the cytokine bioinformatics suggests the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is upregulated, supporting differentiation toward an astroglial lineage

A proteomic investigation of multi-lineage differentiated adult adipose-derived stem cells

Empirical thesis.Bibliography: leaves 223-249.Chapter 1. A review of in-vitro differentiated adipose-derived stem cells and proteomic characterisation -- Chapter 2. Preliminary proteomic comparison of multi-lineage differentiated adipose-derived stem cells -- Chapter 3. proteomic analysis of human adipose derived stem cells during in vitro neuronal differentiation with β-mercaptoethanol -- Chapter 4. Proteomic analysis of human adipose derived stem cells during novel in vitro neuronal differentiation with cyclic ketamines.Regenerative medicine and stem cell therapies has rapidly come into vogue and remained in the public and research spotlight for the last decade due to the bountiful applications it promises. A large number of clinical applications now exist for a wide variety of injuries or disease states, ranging from skin damage in burn victims to degenerative joints in aged patients and, in a limited capacity, the repair of neuronal tissue.Notwithstanding these advances, there is an insufficiency in the knowledge base regarding a stem cell’s fate and characterisation of the extent of differentiation. This thesis presents the first investigation of its type, a broad proteomic investigation of differentiated stem cells derived from both rat and human adipose tissue.The breadth of this body of work investigated the cellular and secreted proteome changes of homogenous ADSC cultures directed toward various phenotypic lineages by means of induction media. These differentiated lineages included osteocytes, chondrocytes, myocytes, adipocytes and neuronal phenotypes. For a marked comparison, primary derived cells from the relevant mature tissue were used as a benchmark measure for the extent of differentiation in the majority of the differentiation experiments. These comparisons aimed to expand our current knowledge about the depth of change occurring during each ADSC differentiation with respect to the mature primary derived cells. Furthermore this study queried the validity of currently employed markers and the possible alternative proteins identified which could be considered for future work.The core chapters focused on the extent of neurogenic differentiation with various chemical inducers. The initial chemical investigated, the previously published β-mercaptoethanol which acted as an inducer, was found to have an overall toxic effect on cells, indicated by the up-regulation of stress proteins, if in contact for up to 24 hours. However the chemical initiated a morphological change that mimicked neuronal cells which furthermore expressed key neuronal protective and restructuring proteins. Nonetheless the cell’s proteome indicated that β-mercaptoethanol induced an overall cellular distress.Novel to this study was the investigation of a naturally occurring chemical alternative; cyclic ketamine class chemicals that was theorised to have a less toxic effect. The cyclic ketamines investigated proved to be a superior induction chemical producing a higher population of cells that exhibited neuronal morphological properties as well as a wide variety of neuronally related proteins. These differentiations proved to be far gentler with a marked decrease in expression of stress related proteins compared to the β-mercaptoethanol treated cells. Due to the novel application and findings of the cyclic ketamine based neuronal induction a patent was filed around the method for the future development into a clinical and pharmaceutical application.Mode of access: World wide web1 online resource (xxi, 249 leaves) illustrations (some colour

Valproic Acid Promotes Early Neural Differentiation in Adult Mesenchymal Stem Cells Through Protein Signalling Pathways

Regenerative medicine is a rapidly expanding area in research and clinical applications. Therapies involving the use of small molecule chemicals aim to simplify the creation of specific drugs for clinical applications. Adult mesenchymal stem cells have recently shown the capacity to differentiate into several cell types applicable for regenerative medicine (specifically neural cells, using chemicals). Valproic acid was an ideal candidate due to its clinical stability. It has been implicated in the induction of neural differentiation; however, the mechanism and the downstream events were not known. In this study, we showed that using valproic acid on adult mesenchymal stem cells induced neural differentiation within 24 h by upregulating the expression of suppressor of cytokine signaling 5 (SOCS5) and Fibroblast growth factor 21 (FGF21), without increasing the potential death rate of the cells. Through this, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is downregulated, and the mitogen-activated protein kinase (MAPK) cascade is activated. The bioinformatics analyses revealed the expression of several neuro-specific proteins as well as a range of functional and structural proteins involved in the formation and development of the neural cells

Molecular Mechanisms Involved in Neural Substructure Development during Phosphodiesterase Inhibitor Treatment of Mesenchymal Stem Cells

Stem cells are highly important in biology due to their unique innate ability to self-renew and differentiate into other specialised cells. In a neurological context, treating major injuries such as traumatic brain injury, spinal cord injury and stroke is a strong basis for research in this area. Mesenchymal stem cells (MSC) are a strong candidate because of their accessibility, compatibility if autologous, high yield and multipotency with a potential to generate neural cells. With the use of small-molecule chemicals, the neural induction of stem cells may occur within minutes or hours. Isobutylmethyl xanthine (IBMX) has been widely used in cocktails to induce neural differentiation. However, the key molecular mechanisms it instigates in the process are largely unknown. In this study we showed that IBMX-treated mesenchymal stem cells induced differentiation within 24 h with the unique expression of several key proteins such as Adapter protein crk, hypoxanthine-guanine phosphoribosyltransferase, DNA topoisomerase 2-beta and Cell division protein kinase 5 (CDK5), vital in linking signalling pathways. Furthermore, the increased expression of basic fibroblast growth factor in treated cells promotes phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) cascades and GTPase–Hras interactions. Bioinformatic and pathway analyses revealed upregulation in expression and an increase in the number of proteins with biological ontologies related to neural development and substructure formation. These findings enhance the understanding of the utility of IBMX in MSC neural differentiation and its involvement in neurite substructure development

Molecular Dynamics of Cytokine Interactions and Signalling of Mesenchymal Stem Cells Undergoing Directed Neural-like Differentiation

Mesenchymal stem cells are a continually expanding area in research and clinical applications. Their usefulness and capacity to differentiate into various cells, particularly neural types, has driven the research area for several years. Neural differentiation has considerable usefulness. There are several successful differentiation techniques of mesenchymal stem cells that employ the use of small molecules, growth factors and commercially available kits and supplements. Phenotyping, molecular biology, genomics and proteomics investigation revealed a wealth of data about these cells during neurogenic differentiation. However, there remain large gaps in the knowledge base, particularly related to cytokines and how their role, drive mechanisms and the downstream signalling processes change with their varied expression throughout the differentiation process. In this study, adult mesenchymal stem cells were induced with neurogenic differentiation media, the cellular changes monitored by live-cell microscopy and the changes in cytokine expression in the intracellular region, secretion into the media and in the extracellular vesicle cargo were examined and analysed bioinformatically. Through this analysis, the up-regulation of key cytokines was revealed, and several neuroprotective and neurotrophic roles were displayed. Statistically significant molecules IFN-G, IL1B, IL6, TNF-A, have roles in astrocyte development. Furthermore, the cytokine bioinformatics suggests the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is upregulated, supporting differentiation toward an astroglial lineage

EMPREGABILIDADE ACIMA DOS 40 ANOS NA REGIÃO DO CAPÃO REDONDO, ZONA SUL DA CAPITAL DE SÃO PAULO

O principal objetivo deste artigo é avaliar a empregabilidade do cidadão acima dos 40 anos de idade, na região do Capão Redondo, Zona Sul da Capital de São Paulo. Entender como vê sua empregabilidade, a quem atribui ser a responsabilidade para manter-se empregado. Foi realizada uma pesquisa de campo, exploratória e descritiva, qualitativa e quantitativa. Utilizando uma amostragem de 30 profissionais de diversas áreas, que responderam a 11 questões simples e diretas. Os principais resultados obtidos foram a constatação de que os colaboradores preparam-se para enfrentar esse novo desafio profissional, mantendo-se atualizados através de cursos, buscando sempre novos conhecimentos. Pode-se notar que ao atingir os 40 anos, não é o fim e sim o começo de uma nova etapa. Ao longo do tempo esse indivíduo adquiriu maturidade, conhecimento, experiência, determinação, segurança, responsabilidade e comprometimento. Fatores importantes para a realização de atividades a serem desenvolvidas nas organizações.&nbsp