Transdifferentiation of Mesenchymal Stem Cells into Schwann Cell-like Phenotypes Using Electrical Stimulation

Maxsam S. Donta¹, Merjem Mededovic¹, Emily Kozik², Metin Uz¹, Donald S. Sakaguchi^{2, 3}, Jonathan Claussen⁴ and Surya K. Mallapragada^{1, 2, 4}

¹ Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011

² Neuroscience Program, Iowa State University, Ames, IA 50011

³ Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011

⁴ Division of Materials Science and Engineering, Ames Laboratory (DOE), Ames, Iowa 50011

Mesenchymal stem cells (MSCs) are stem cells with multipotent properties and as such, can be differentiated into a multitude of different phenotypes including neuroglial cells. The phenotype can be manipulated during the differentiation process by such factors as chemicals, electrical stimulation, or environmental cues; one or a combination of the aforementioned factors may be used to influence the final phenotype.^1,2 In order to examine the effects of differing electrical stimuli on MSC differentiation, electrical stimuli were applied to genetically engineered brain-derived neurotrophic factor (BDNF)-hypersecreting MSCs through different devices – two-dimensional flexible inkjet-printed graphene interdigitated electrode circuits as well as three-dimensional gelatin-based conduits with conductive graphene rods – in order to differentiate them into Schwann cell-like and neuronal cell-like phenotypes. To explore these effects, electrical stimulation of either 25, 50, and 100 mV, at 50 Hz was applied for 10 min/day and for 10 days to MSCs seeded on each respective device. Successful differentiation was evaluated through Immunocytochemistry (ICC) analysis using Schwann cell markers s100, s100β, and p75 as well as the neuronal markers TuJ1, Map2, and GFAP; paracrine activity using enzyme-linked immunosorbent assay (ELISA) was evaluated to detect secreted amounts of BDNF, glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF). ICC results indicate highly efficient differentiation of MSCs into Schwann cell-like phenotypes and a low degree of differentiation into neuronal phenotypes. As voltage was decreased from 100 mV to 25 mV, the percentage of MSCs that differentiated into neuronal phenotypes increased. Voltage was differed for only the 2D circuits; the 3D gelatin conduits stimulation studies were conducted at 100 mV and exhibited s100, s100β, and p75 staining. ELISA results revealed that GDNF release was very low (100-1000 pg/mL) for all voltages, as was expected due to its neuronal-supporting nature. BDNF releases were higher (20,000-25,000 pg/mL) because the cells were genetically engineered to secrete BDNF. NGF had the highest secretion levels (65,000-75,000 pg/mL), which is indicative of Schwann cell-like phenotypes due to NGF’s role in repairing nerve tissue. Furthermore, the NGF levels measured were much higher in the electrically-stimulated cells than the control cases with releases measured at three to four times higher. Moreover the NGF levels secreted from electrically-stimulated cells showed higher or at least similar paracrine activity compared to chemically differentiated cells. The trends were consistent in the 2D devices and the 3D gelatin conduits; however, the 3D conduits with electrical stimuli exhibited lower neurotrophic factor release, which can be attributed to a difference of electrical field between the circuits and conduits. The NGF release in the gelatin conduits was also not as relatively high compared to the chemically differentiated control, but release was still almost twice that as the untreated control cells. Methods performed with the aforementioned devices were contrasted with each other as well as with a control, which utilized established chemical differentiation procedures as well as the same ICC and ELISA tests. BDNF-secreting MSCs that are treated with only electro-stimulus are expected to exhibit equal, if not superior rates of differentiation for both 2D and 3D models than traditional chemical differentiation shown in our previous works.^1,2 Results from these experiments have the potential to revolutionize the way that peripheral nervous system damage is treated.

References

1. Das, S. R.*; Uz, M.*; Ding, S.; Lentner, M. T.; Hondred, J. A.; Cargill, A. A.; Sakaguchi, D. S.; Mallapragada, S.; Claussen, J. C., Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits. Advanced Healthcare Materials 2017, 6 (7). (* indicates equal contribution)
2. Uz, M.; Buyukoz, M.; Sharma, A. D.; Sakaguchi, D. S.; Altinkaya, S. A.; Mallapragada, S. K., Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes. Acta Biomaterialia 2017, 53, 293-306.