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11IMPROVING STEM CELL DIFFERENTIATION USING NEAR-INFRARED AND GREEN IRRADIATION
ANINE CROUS, DANIELLA DA SILVA, MADELEEN JANSEN VAN RENSBURG and HEIDI ABRAHAMSE
1Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, South Africa
acrous@uj.ac.za
ABSTRACT
Degenerative disorders, which include osteoporosis and Alzheimer's disease, are characterized by a progressive decline in normal cell or tissue function. Transdifferentiation of adipose-derived mesenchymal stem cells (ADMSCs) into multiple cell lineages, including neurons and osteoblasts, is possible. It has been demonstrated that photobiomodulation can optimize this process (PBM). The use of genetically modified immortalized ADMSCs (iADMSCs) cells provides accessibility by overcoming ethical problems and facilitates continuous cell culture proliferation. In this comparative study, the effects of PBM, using 5 J/cm2 at 825 nm and/or 525 nm consecutively were determined on the differentiation potential of iADMSCs to determine the most effective wavelength of PBM to induce differentiation into neurons and osteoblasts. iADMSCs were characterized using their surface protein markers CD44/90/166 and induced to differentiate into neuronal cells or osteoblasts. iADMSCs stem cell markers were characterized using flow cytometry. Morphological changes were examined, and biochemical assays performed including LDH cytotoxicity assay, reactive oxygen species (ROS) production, trypan blue viability and ATP proliferation. Transdifferentiation of iADMSCs using various transdifferentiation inducers and PBM therapy showed promise. Neuronal transdifferentiation was observed through the decrease in stem cell markers after characterization. Observations of morphology showed changes in PBM treated groups from that of the standard, where the cells showed rounding and synaptic protrusion development. There was an overall increase in cytotoxicity indicative of membrane permeabilization when using all irradiation parameters, signifying transdifferentiation. An increase in ROS production was noted when using 525 nm irradiation, cell viability was maintained throughout the experimental groups, with a significant increase when using consecutive irradiation. Cell proliferation was increased when subjected to 825 nm irradiation, however the use of 525 nm and the consecutive use of 825 nm and 525 nm did lead to a decrease in proliferation, signifying differentiation. Osteoblast transdifferentiation was also seen by a decrease in stem cell marker expression. A noticeable change in cell morphology started to occur for all experimental groups after 24 hours, over time it was seen that 525 nm and consecutive PBM treatments had the most change in morphology seen by a rounded-up cell shape like that of osteoblasts. Biochemical analysis revealed no increase in cytotoxicity or ROS for all experimental groups after 48 hours, after 7 days significant increases where noted, that can be ascribed to contact inhibition. Cell viability was maintained over time and a decrease in cell proliferation was noted in experimental samples where 525 nm and consecutive irradiation had the greatest decrease in proliferation. We suggest that consecutive use of green and NIR PBM along with growth inducers offer a better solution of transdifferentiating iADMSCs to neurons and osteoblasts. Findings from this research will serve as contribution toward validating stem cell technology for application in in vivo, pre-clinical and clinical research settings.
KEYWORDS: Photobiomodulation; Transdifferentiation; Adipose Derived Mesenchymal Stem Cells; Osteoblasts; Neurons
ACKNOWLEDGEMENTS
This work was supported by the National Research Foundation (NRF) S&F -Scarce Skills Postdoctoral Fellowship (Grant no: 120752) received by Anine Crous; the Global Excellence and Stature, Fourth Industrial Revolution (GES 4.0) Postgraduate Scholarship received by Madeleen Jansen van Rensburg and the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (SARChI/NRF-DST) (Grant no: 98337), received by Heidi Abrahamse and Daniella Da Silva.
