Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation

Abstract

Background: Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Methods: Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. Results: The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. Conclusion: The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

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

“…Furthermore, the dendrite extensions are present in most cells. VPA does not have an influence on the death rate, the cell population has had minimal variation over the treatment time points as opposed to studies with other small chemicals, such as BME and DMSO [2,3,23]. The cell counts and subsequent ALP and Reazurin assays support the stability of the cell culture population with nominal changes over time.…”

“…Bioplex analysis was performed as per Santos et al [3] with 500 µL aliquots collected at timepoints and controls as follows: DMEM control, starve, B27 control, 0, 3, 6, and 24 h. The assay was performed with Bioplex human 27-plex (M50-0KCAF0Y Bio-Rad Laboratories, Hercules, CA, USA). The data analysis was completed in DanteR software (DanteR version 1.0.0.10.…”

“…Several studies have explored the effect of small molecule chemical inducers on the potential to drive neurogenic differentiation in stem cells. At optimized concentrations and short treatment times, chemicals such as beta-mercaptoethanol (BME) and dimethylsulfoxide (DMSO), have shown the potential to induce a structural and molecular phenotype in stem cells that resemble differentiating neural cells [2,3]. Not surprisingly, these molecules had negative effects inducing a range of stress and apoptotic markers with an increasing treatment time frame.…”

A Comparison of Proteins Expressed between Human and Mouse Adipose-Derived Mesenchymal Stem Cells by a Proteome Analysis through Liquid Chromatography with Tandem Mass Spectrometry

“…We found that 92% (401 type proteins) of the proteins expressed by ADSCs in humans and mice were consistent. Recently, in a comprehensive protein expression analysis using the iTRAQ measurement method [ 15 ], in which peptide fragments were tagged before performing an analysis similar to LC-MS/MS, it was reported that more than 2000 types of proteins were identified from hADSCs [ 16 ]. In this study, 1653 proteins with one or more peptide fragments was identified in hADSCs, and 1623 were identified in mADSCs.…”

Selectively-Packaged Proteins in Breast Cancer Extracellular Vesicles Involved in Metastasis

“…All samples were centrifuged at 20,000× g for 10 min to remove any cellular debris. Assays were performed according to the manufacturer's instructions, as previously described [62]. Images of results were generated in MS Excel, DanteR (Version 1.0.0.10, Richland, WA, USA) and GraphPad Prism (Version 8.2.1, San Diego, CA, USA).…”