Three-Dimensional Microscaffolds for Enrichment and Transplantation of iPSC-Derived Neurons

Introduction:  Human induced pluripotent stem cells (iPSCs) can be directly converted into neurons by ectopic expression of neuronal transcription factors, providing a cell source for in vitro disease modeling, drug screening, and regenerative therapies. Cell transplantation therapies present a strategy to treat the functional deficits of neurodegenerative diseases, such as Parkinson’s disease, however the poor survival rates of transplanted cells in vivo limits the efficacy of this treatment. In contrast to the traditional delivery of dissociated cells, we hypothesize that using fibrous microscaffolds to transplant adherent, functional networks of neurons will enhance cell survival and engraftment in vivo. In this study, we evaluated the ability of fibrous 3-D microscaffolds to support the growth and maturation of iPSC-induced neurons (iNs) in vitro, and to enhance survival of iNs transplanted into an ex vivo mouse organotypic brain slice culture. This work serves as a preliminary study for the future in vivo transplantation of microscaffold-seeded dopaminergic neurons into a Parkinson’s disease mouse model.

Materials and Methods:  Fibrous microscaffolds were fabricated by electrospinning sheets of synthetic tyrosine-derived polycarbonate polymers, and sectioning the sheets into squares 100 µm in side length. iPSCs were transfected with the drug-inducible neuronal transcription factor NeuroD1 and were seeded onto microscaffolds shortly after neuronal induction. Maturation and functionality were assessed by immunocytochemistry and calcium imaging. Microscaffolds were used to transplant networks of iNs into ex vivo organotypic mouse brain slices for evaluation of survival and functional integration.

Results and Discussion: Seeding iNs within fibrous microscaffolds yielded an enriched, highly dense population of neurons. The scaffold geometry enabled cell infiltration and cell-cell contact, supporting neuronal maturation. iNs within the microscaffolds expressed βIII-tubulin and MAP2 within 8 days of neural induction, and responded to exogenous electrical stimulation by live cell calcium imaging. Microscaffold-seeded iNs extended significantly longer neurites in an ex vivo organotypic mouse brain slice culture compared to dissociated iNs.

Conclusions: 3-D microscaffolds can be used to enrich the neuronal population of NeuroD1-transfected iPSCs, and these scaffolds support the growth of mature, functional neurons. The use of these scaffolds to enhance neuronal integration into an ex vivo brain slice suggests that this platform will be effective as a transplantation vehicle for in vivo regenerative therapies.

Acknowledgments: Research reported in this publication was supported by NIH T32EB005583, NJCST Core grant, NIH EB000146 RESBIO, NJSCC10-3090-SCR-E-0 and graduate fellowship 11-2957-SCR-E-0, and NSF-IGERT grant 0801620. The authors gratefully acknowledge the RUCDR for the gift of the iPSC line used in this study and the NJCBM for providing the polymers used in this study.