(496f) 3D Graphene Foam Based Scaffolds to Control Transdifferentiation of MSCs into Schwann Cell-like Phenotypes Via Electrical Stimuli for Peripheral Nerve Regeneration

Multipotent mesenchymal stem cells (MSCs), isolated and derived from various connective tissue sources and transdifferentiated into Schwann cells (SCs), hold considerable potential for cell-based peripheral nerve regeneration therapies using autologous transplantation via nerve guidance conduits (NGCs).¹ However, current problems regarding; 1) the efficiency of conventionally applied reversible and unscalable, chemical stimuli-based transdifferentiation protocols; 2) controlling final fate of the implanted cell population; and 3) design of 3D microstructure of scaffolds completely mimicking the extracellular matrix, limit the clinical use of transdifferentiated MSCs.¹ Therefore, there is a critical need for alternative approaches to gain mechanistic understanding of transdifferentiation in 3D scaffold microenvironment that can enable precise control on the final fate of the implanted cell population, enhanced paracrine activity, myelination, axonal regeneration and functional clinical outcomes.¹

In previous studies, we have successfully demonstrated transdifferentiation of MSCs to SCs fates displaying a high degree of SC-markers immunolabeling and neurotrophic factor paracrine activity.^2-5 We showed MSCs to SCs transdifferentiation via a chemical induction paradigm on 2D Poly-L-Lactic Acid (PLLA) films.^{3, 6} In addition, we also demonstrated that PLLA-based 2D platforms with surface micropatterns and nerve growth factor (NGF) gradients can be used as depots for the controlled release of NGF, mimicking NGF secretion at physiological levels and promoting directed neurite extension from PC12 cells.⁶ Considering that 3D platforms, mimicking the natural ECM microenvironment, would help to obtain more realistic information, we fabricated gelatin conduits possessing different 3D microstructures and mechanical properties to control the direct transdifferentiation of MSCs into SCs within the 3D conduit matrix via chemical induction.⁴ Recently, using comparative proteomics, we identified significantly regulated proteins and related cellular pathways upon transdifferentiation of MSCs into SCs via chemical stimuli on 2D substrates.⁵ To eliminate the need for expensive chemicals/growth factors and provide a more precise platform to control MSCs transdifferentiation and fate commitment, we demonstrated the first proof-of-concept for the MSCs to SCs transdifferentiation via sole electrical stimulation (100 mV, 50 Hz, 10 min per day during 15 days), free from any chemical stimuli, using conductive graphene-based 2D substrates.

Based on this background, in this study, an alternative strategy based on PLLA films with 3D porous microstructure, surface micropatterns and integrated graphene micro-circuits is used in combination with 3D conductive graphene foams possessing desirable 3D microstructural/mechanical properties to provide an available cellular microenvironment and electrical transdifferentiation. Briefly, PLLA films with micropatterns were prepared as mentioned previously⁶ and graphene-based integrated circuit designs are transferred to the film surface. Then, 3D graphene foam, prepared using chemical vapor deposition (CVD) is integrated into graphene circuits on the film surface via in situ adhesion. The films can easily be rolled into conduits in such a way that the surface with micro-circuits and graphene foam becomes the inner lumen and may be used for in vivo applications. The graphene foams and PLLA films in combination provides a desirable 3D microstructure and a permissive microenvironment for cells and enables precise control in electrical stimuli through the integrated micro-circuits and conductive foams for cellular transdifferentiation. Our results indicated that MSCs seeded on graphene foams showed good attachment, proliferation and growth within the 3D foam microstructure for 7 days with high cell viability (~97% cell survival – based on Trypan blue staining). The seeded MSCs were transdifferentiated into SCs via chemical⁴ or electrical stimuli² as described previously. The immunocytochemical (ICC) studies indicated that ~80% of the cells showed successful transdifferentiation (labeling for SC markers s100, s100β and p75) for both chemical and electrical stimuli compared to the non-transdifferentiated control MSCs. The transdifferentiated MSCs secreted significantly higher amount of growth factors as determined by ELISAs (particularly NGF: ~ 2.5 pg/mL per cell) for both chemical and electrical stimuli based transdifferentiation approaches, which was significantly higher than the non-transdifferentiated control groups. These results were considerably better than the electrically transdifferentiated cells on graphene-based 2D substrates (~75% of the MSCs differentiated into SCs like phenotypes and ~0.55 pg/mL per cell of NGF secretion) as reported in our previous work² and comparable to the chemically transdifferentiated MSCs within 3D gelatin scaffolds (~85% of the MSCs differentiated into SCs like phenotypes and ~2.5 pg/mL per cell of NGF secretion) as indicated in our previous work.⁴ The ICC and ELISA findings are further supported by RT-PCR and Western Blot (WB) experiments performed using selected primers and antibodies to detect the regulated genes and proteins. The co-culture of MSCs seeded in the proposed conduit design with PC12 cells is also evaluated to assess the functional biological activity of secreted growth factors. Overall, these results suggest that the electrical stimuli applied within the 3D graphene matrix enables comparable differentiation and paracrine activity with transdifferentiation procedures involving expensive chemical stimuli applied in 3D scaffolds⁴ or 2D substrates², ultimately leading to development of novel and promising nerve regeneration strategies.

References:

1. Uz, M.; Das Suprem, R.; Ding, S.; Sakaguchi Donald, S.; Claussen Jonathan, C.; Mallapragada Surya, K., Advances in Controlling Differentiation of Adult Stem Cells for Peripheral Nerve Regeneration. Advanced Healthcare Materials 0 (0), 1701046.
2. 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, n/a-n/a.
3. Sharma, A. D.; Zbarska, S.; Petersen, E. M.; Marti, M. E.; Mallapragada, S. K.; Sakaguchi, D. S., Oriented growth and transdifferentiation of mesenchymal stem cells towards a Schwann cell fate on micropatterned substrates. Journal of bioscience and bioengineering 2016, 121 (3), 325-35.
4. 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.
5. Sharma, A. D.; Wiederin, J.; Uz, M.; Ciborowski, P.; Mallapragada, S. K.; Gendelman, H. E.; Sakaguchi, D. S., Proteomic analysis of mesenchymal to Schwann cell transdifferentiation. Journal of Proteomics 2017, 165, 93-101.
6. Uz, M.; Sharma, A. D.; Adhikari, P.; Sakaguchi, D. S.; Mallapragada, S. K., Development of multifunctional films for peripheral nerve regeneration. Acta Biomater 2016.