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The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) National Medical Research Council (NMRC) Accepted version Partial funding support from the Singapore National Research Foundation under its National Medical Research Council-Cooperative Basic Research Grant (NMRC-CBRG) grant (NMRC/CBRG/0096/2015) and administered by the Singapore Ministry of Health’s National Medical Research Council; Ministry of Education Tier 1 grant (RG38/19); and A*Star BMRC Singapore-China 12th Joint Research Programme Grant (Project No: 1610500024) are acknowledged. Jiah Shin Chin would like to thank the NTU Interdisciplinary Graduate Research Officer’s scheme for supporting her through this work. We would also like to thank New England Biolabs for providing the Cas9 proteins.

International audience; Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a poly-saccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force micros-copy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modu-lus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline ace-tyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during elec-trospinning and suggests the feasibility of using such poly-saccharide nanofibers in supporting fMSC neuronal commitment.

In this study, we promote neuronal differentiation of human mesenchymal stem cells (MSCs) through scaffold-mediated sustained release of siRNA targeting RE-1 silencing transcription factor (REST). Poly (ϵ-caprolactone) nanofibers were surface modified with mussel inspired DOPA-melanin (DM) coating for adsorption of REST siRNA. DM modification increased siRNA-loading efficiency and reduced the initial burst release. Fiber alignment and DM modification enhanced REST knockdown efficiencies. Under non-specific differentiation condition, REST silencing and fiber topography enhanced MSC neuronal markers expressions and reduced glial cell commitment. Such scaffolds may find useful applications in enhancing MSCs neuronal differentiation under non-specific conditions such as an in vivo environment. NMRC (Natl Medical Research Council, S’pore) Accepted version

The potential of electrospun pullulan/dextran (P/D) nanofibers (average diameter = 323 nm) for vascular tissue engineering applications is explored. The mechanical properties of the nanofibers are of the same order of magnitude as that of human arteries (Young's modulus ≈0.88 MPa; tensile strength ≈0.35 MPa). It is demonstrated that the nanofiber topography enables cell adhesion and that the endothelial phenotype is maintained on the nanofibers. Moreover, P/D nanofibers support a stable confluent monolayer of endothelial cells over 14 d. SMCs seeded on nanofibers display similar levels of alpha smooth muscle actin and a lower proliferation rate than cells on 2D cultures. The observations suggest that nanofibers promote a shift to a quiescent contractile phenotype in SMCs.

Fibre structures represent a potential class of materials for the formation of synthetic nerve conduits due to their biomimicking architecture. Although the advantages of fibres in enhancing nerve regeneration have been demonstrated, in vivo evaluation of fibre size effect on nerve regeneration remains limited. In this study, we analyzed the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration across a 15-mm critical defect gap in a rat sciatic nerve injury model. By using an electrospinning technique, fibrous conduits comprised of aligned electrospun poly (ε-caprolactone) (PCL) microfibers (981 ± 83 nm, Microfiber) or nanofibers (251 ± 32 nm, Nanofiber) were obtained. At three months post implantation, axons regenerated across the defect gap in all animals that received fibrous conduits. In contrast, complete nerve regeneration was not observed in the control group that received empty, non-porous PCL film conduits (Film). Nanofiber conduits resulted in significantly higher total number of myelinated axons and thicker myelin sheaths compared to Microfiber and Film conduits. Retrograde labeling revealed a significant increase in number of regenerated dorsal root ganglion sensory neurons in the presence of Nanofiber conduits (1.93 ± 0.71 x 103 vs. 0.98 ± 0.30 x 103 in Microfiber, p

Astrocyte-nanofiber interactions are studied by culturing primary rat cortical astrocytes on poly[caprolactone-co-(ethyl ethylene phosphate)] electrospun nanofibers and solvent-cast films (two-dimensional control). The results indicate that nanofiber topography significantly suppresses astrocyte proliferation and enhances apoptosis, without altering cellular activation as compared to films. Moreover, nanofiber topography enhances gene-silencing efficiency in astrocytes. The results suggest that nanofibers may serve as potential substrates for nerve regeneration by suppressing astrocyte growth and may further facilitate the use of gene-silencing to enhance CNS regeneration.

This project embarked with the following specific aims: Synthesize and characterize electrospun scaffolds that are functionalized through the encapsulation of siRNA. a. Establish a robust system to incorporate and release siRNA in a sustained manner from poly(ε-caprolactone) (PCL) scaffolds made up of aligned and/ or randomly-oriented micro- and nano-fibers using electrospinning. b. Characterize the distribution; in vitro release kinetics; and bioactivity of the incorporated siRNA. c. Characterize the physical properties of electrospun scaffolds, such as scaffold morphology, and fiber diameter with respect to the inclusion of siRNA. d. Characterize the transfection efficiency of siRNA-encapsulated electrospun scaffolds with respect to the architecture of the scaffolds (scaffolds with aligned fibers vs. randomly-oriented fibers). RG 121/06