Introduction: Neurodegenerative diseases are accompanied by loss of neuronal function and integrity. Stem cell therapy is utilized to regenerate neurons to repair the damaged area. Regeneration potential of stem cells can be enhanced by using chemicals with known bioactive properties. In the current study, two bioactive compounds, α-pinene (AP) and thymoquinone (TQ) were explored for their neuronal differentiation potential of rat bone marrow mesenchymal stem cells (MSCs). Methods: MSCs were isolated, cultured and characterized immunocytochemically for the presence of specific surface markers. Optimized concentrations of both compounds (20 µM AP and 12 µM TQ) as determined by MTT assay, were used to treat MSCs in separate and combined groups. All groups were assessed for the presence of neuronal, astroglial, and germ layer markers through qPCR. Neuronal and glial protein expression were analyzed by immunocytochemistry. Results: Both compounds alone and in combination induced differentiation in MSCs with significant gene expression of neuronal markers i.e. neuron specific enolase (NSE), nestin, microtubule-associated protein 2 (MAP2), neurofilament light chain (Nefl) and Tau, and astroglial marker i.e. glial fibrillary acidic protein (GFAP). AP treated group also showed significant upregulation of endodermal and mesodermal markers indicating transition of ectoderm towards the other two germ layers. Conclusion: This study concludes that AP and TQ potentially differentiate MSCs into neuronal and astroglial lineages. However, AP treated group followed germ layer transition. Expression of neuronal as well as glial markers indicate that the differentiated neurons are at the neuroprogenitor stage and can be potential candidates for cellular therapeutics against neurodegenerative disorders.
Central nervous system anomalies give rise to neuropathological consequences with immense damage to the neuronal tissues. Cell based therapeutics have the potential to manage several neuropathologies whereby the differentiated cells are explored for neuronal regeneration. The current study analyzes the effect of a bioactive compound, alpha terpineol (AT) on the differentiation of rat bone marrow derived mesenchymal stem cells (BM-MSCs) toward neuronal lineage, and explores regulation of differentiation process through the study of Wnt pathway mediators. BM-MSCs were cultured and characterized based on their surface markers and trilineage differentiation. Safe dose of AT as optimized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide assay, was used for the treatment of MSCs. Treated cells were analyzed for the neuronal, astroglial and germ layer transition markers at the gene and protein levels, by quantitative polymerase chain reaction and immunocytochemistry, respectively. Temporal expression of Wnt pathway genes was assessed during the course of neuronal differentiation. AT treated group showed significant upregulation of neuron specific (NSE, MAP2, Tau, Nestin, and NefL) and astroglial (GFAP) genes with positive expression of late neuronal markers. Germ layer transition analysis showed the overexpression of ectodermal markers (NCAM, Nestin, and Pax6), whereas endodermal (AFP, MixL1, and Sox17),and mesodermal (Mesp1 and T Brachyury) markers were also found to be upregulated. Wnt signaling pathway was activated during the initial phase (30 min) of differentiation, which later was downregulated at 1, 3, and 5 h. AT efficiently induces neuronal differentiation of BM-MSCs by regulating Wnt signaling. Overexpression of both early and late neuronal markers indicate their neuro-progenitor state and thus can be utilized as a promising approach in cellular therapeutics to treat various neurodegenerative ailments. In addition, exploration of the molecular pathways may be helpful to understand the mechanism of cell-based neuronal regeneration.
BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options. Cell-based therapies using mesenchymal stem cells (MSCs) emerged as an alternative approach to support hepatic regeneration. In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage. Chemical compounds of the triterpene class; glycyrrhizic acid (GA) and 18β-glycyrrhetinic acid (GT) possess diverse therapeutic properties including hepato-protection and anti-fibrosis characteristics. They are capable of modulating several signaling pathways that are crucial in hepatic regeneration. Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes. AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs (hUC-MSCs). METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules. Isolated cells were treated with GA, GT, and their combination for 24 h and then analyzed at three time points; day 7, 14, and 21. qRT-PCR was performed for the expression of hepatic genes. Expression of hepatic proteins was analyzed by immunocytochemistry at day 21. Periodic acid Schiff staining was performed to determine the functional ability of treated cells. RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control, eventually progressed towards the polygonal morphology of hepatocytes with the passage of time. The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation. Preconditioned cells showed positive expression of hepatocyte-specific proteins. The results were further corroborated by positive periodic acid Schiff staining, indicating the presence of glycogen in their cytoplasm. Moreover, bi-nucleated cells, which is the typical feature of hepatocytes, were also seen in the preconditioned cells. CONCLUSION Preconditioning with glycyrrhizic acid, 18β-glycyrrhetinic acid and their combination, successfully differentiates hUC-MSCs into hepatic-like cells. These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.
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