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The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2+ oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

Spinal cord injuries (SCIs) are followed by a complex series of events that contribute to the failure of regeneration. To date, there is no robust treatment that can restore the injury-induced loss of function. Since damaged spinal axons do not spontaneously regenerate in their native inhibitory microenvironment, a combined application of biomaterials and neurotrophic factors that induce nerve regeneration emerges as an attractive treatment for SCIs. In this study, we report the novel use of a three-dimensional (3D) hybrid scaffold to provide contact guidance for regrowth of axons in vivo. The scaffold comprises 3D aligned sparsely distributed poly(ε-caprolactone-co-ethyl ethylene phosphate) nanofibers that are supported and dispersed within a collagen hydrogel. Neurotrophin-3 was incorporated into the scaffold as an additional biochemical signal. To evaluate the efficacy of the scaffold in supporting nerve regeneration after SCIs, the construct was implanted into an incision injury, which was created at level C5 in the rat spinal cord. After 3 months of implantation, scaffolds with NT-3 incorporation showed the highest average neurite length (391.9 ± 12.9 μm, p ≤ 0.001) as compared to all the other experimental groups. In addition, these regenerated axons formed along the direction of the aligned nanofibers, regardless of their orientation. Moreover, the presence of the hybrid scaffolds did not affect tissue scarring and inflammatory reaction. Taken together, these findings demonstrate that our scaffold design can serve as a potential platform to support axonal regeneration following SCIs. NMRC (Natl Medical Research Council, S’pore) Accepted version

Targeted drug delivery to specific neural cells within the central nervous system (CNS) plays important roles in treating neurological disorders, such as neurodegenerative (e.g. targeting neurons) and demyelinating diseases (e.g. targeting oligodendrocytes (OLs)). However, the presence of many other cell types within the CNS, such as microglial and astrocytes, may lead to non-specific uptake and subsequent side effects. As such, exploring an effective and targeted drug delivery system is of great necessity. Synthetic micro-/nano-particles that have been coated with biologically derived cellular membranes have emerged as a new class of drug delivery vehicles. However, the use of neural cell-derived membrane coatings remains unexplored. Here, we utilized this technique, and demonstrated the efficacy of targeted delivery by using four types of cell membranes that were derived from the CNS, namely microglial, astrocytes, oligodendrocyte progenitor cells (OPCs) and cortical neurons. Successful cell membrane coating over poly(-caprolactone) nanoparticles (NPs) was confirmed by dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). Subsequently, an extensive screening of these cell membrane coated NPs was carried out on various CNS cells. Results suggested that microglial and OLs were the most sensitive cell types towards cell membrane coated NPs. Specifically, cell membrane coated NPs significantly enhanced the uptake efficiency of OLs (p < 0.001). Additionally, a temporal uptake study indicated that the OLs took up microglial membrane coated NPs (DPP-PCL-M Mem) most efficiently. Besides that, coating the NPs with four types of CNS cell membrane did not result in obvious specific uptake in microglial but reduced the activation of microglial, especially for DPP-PCL-M Mem (p < 0.01). Taken together, DPP-PCL-M Mem were uptaken most efficiently in OLs and did not induce significant microglial activation and may be most suitable for CNS drug delivery applications. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version This work is supported by the National Research Foundation, Singapore, under its Intra-CREATE Thematic Grant Programme (NRF2019-THE002-0001) and the MOE Tier 1 grants (RG38/19 and RG37/20).

Current treatment approaches toward spinal cord injuries (SCI) have mainly focused on overcoming the inhibitory microenvironment that surrounds lesion sites. Unfortunately, the mere modulation of the cell/tissue microenvironment is often insufficient to achieve desired functional recovery. Therefore, stimulating the intrinsic growth ability of injured neurons becomes crucial. MicroRNAs (miRs) play significant roles during axon regeneration by regulating local protein synthesis at growth cones. However, one challenge of using miRs to treat SCI is the lack of efficient delivery approaches. Here, a 3D fiber‐hydrogel scaffold is introduced which can be directly implanted into a spinal cord transected rat. This 3D scaffold consists of aligned electrospun fibers which provide topographical cues to direct axon regeneration, and collagen matrix which enables a sustained delivery of miRs. Correspondingly, treatment with Axon miRs (i.e., a cocktail of miR‐132/miR‐222/miR‐431) significantly enhances axon regeneration. Moreover, administration of Axon miRs along with anti‐inflammatory drug, methylprednisolone, synergistically enhances functional recovery. Additionally, this combined treatment also decreases the expression of pro‐inflammatory genes and enhance gene expressions related to extracellular matrix deposition. Finally, increased Axon miRs dosage with methylprednisolone, significantly promotes functional recovery and remyelination. Altogether, scaffold‐mediated Axon miR treatment with methylprednisolone is a promising therapeutic approach for SCI., A novel approach of targeting axon local protein synthesis to promote the intrinsic growth ability of neurons by scaffold‐mediated microRNA delivery is introduced. The biofunctional 3D fiber‐hydrogel scaffold provides topographical cues that direct axon regrowth and a novel microRNA cocktail (miR‐132/miR‐222/miR‐431), which in the presence of methylprednisolone, enhances both nerve regeneration and functional recovery after spinal cord injury.

Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.

Codelivery of CRISPR-Cas9 and chlorin e6 achieved spatially controlled tumor-specific gene editing with synergistic drug effects., Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)– and reducing agent–responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid–decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator Nrf2, enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and Nrf2 gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP− is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAP-coated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Accepted version

Assessment of intracellular mRNA expression is invaluable for understanding cellular signaling activities, identifying disease stages, and monitoring the gene expression pattern of therapeutic cells during their culture, expansion and/or differentiation process. Previous methods suffer from the need to disrupt the biological samples to perform polymerase chain reaction analysis which can be laborious, fragmented and destructive. Herein, we develop a mRNA nanosensor based on the sustained release of mRNA-specific molecular beacons (probes that fluoresce upon hybridization) from the biodegradable poly(d,l-lactide-co-glycolide) nanoparticles. Post cellular internalization, the particles gradually degrade and release the encapsulated probes which are initially weakly fluorescent. When the released probes meet and hybridize with target mRNA, they restore pre-quenched fluorescence. By virtue of quantifying the fluorescence intensity, we can estimate the cellular mRNA expression. As a case study, β-actin mRNA expression in mesenchymal stem cells cultured on a 3D matrix was monitored and compared with those cultured on a 2D plate for one week. Critically, the observed expression profile shows a great correlation with the established quantitative polymerase chain reaction analysis.

Drivers of RNA interference are potent for manipulating gene and protein levels, which enable the restoration of dysregulated mRNA expression that is commonly associated with injuries and diseases. This review summarizes the potential of targeting neuroglial cells, using RNA interference, to treat nerve injuries sustained in the central nervous system. In addition, the various methods of delivering these RNA interference effectors will be discussed. Ministry of Education (MOE) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: SY Chew and J Lin would like to acknowledge partial funding support from the Singapore National Research Foundation under its National Medical Research Council Cooperative Basic Research Grant (NMRC-CBRG, 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). This research was supported in part by the National Research Foundation (NRF), Republic of Korea (2015K1A1A2032163, 2018K1A4A3A01064257, 2018R1A2B3003446).

Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host‐implant integration. Here, electrospun poly(caprolactone‐co ‐ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2‐inducing miRs, Let‐7c and miR‐124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1‐inducing miR, Anti‐Let‐7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR‐124 treatment group, followed by Anti‐Let‐7c and Let‐7c treatment groups. Based on immunohistochemical quantifications, these miR‐encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host‐implant integration at longer time points. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version

MicroRNAs effectively modulate protein expression and cellular response. Unfortunately, the lack of robust nonviral delivery platforms has limited the therapeutic application of microRNAs. Additionally, there is a shortage of drug‐screening platforms that are directly translatable from in vitro to in vivo. Here, a fiber substrate that provides nonviral delivery of microRNAs for in vitro and in vivo microRNA screening is introduced. As a proof of concept, difficult‐to‐transfect primary neurons are targeted and the efficacy of this system is evaluated in a rat spinal cord injury model. With this platform, enhanced gene‐silencing is achieved in neurons as compared to conventional bolus delivery (p < 0.05). Thereafter, four well‐recognized microRNAs (miR‐21, miR‐222, miR‐132, and miR‐431) and their cocktails are screened systematically. Regardless of age and origin of the neurons, similar trends are observed. Next, this fiber substrate is translated into a 3D system for direct in vivo microRNA screening. Robust nerve ingrowth is observed as early as two weeks after scaffold implantation. Nerve regeneration in response to the microRNA cocktails is similar to in vitro experiments. Altogether, the potential of the fiber platform is demonstrated in providing effective microRNA screening and direct translation into in vivo applications. MOH (Min. of Health, S’pore) NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Reductionist approaches based on 3D fibers reveal that single-cell migration along fibers is driven by lateral actin-based waves for various cell types., In vivo, cells migrate on complex three-dimensional (3D) fibrous matrices, which has made investigation of the key molecular and physical mechanisms that drive cell migration difficult. Using reductionist approaches based on 3D electrospun fibers, we report for various cell types that single-cell migration along fibronectin-coated nanofibers is associated with lateral actin-based waves. These cyclical waves have a fin-like shape and propagate up to several hundred micrometers from the cell body, extending the leading edge and promoting highly persistent directional movement. Cells generate these waves through balanced activation of the Rac1/N-WASP/Arp2/3 and Rho/formins pathways. The waves originate from one major adhesion site at leading end of the cell body, which is linked through actomyosin contractility to another site at the back of the cell, allowing force generation, matrix deformation and cell translocation. By combining experimental and modeling data, we demonstrate that cell migration in a fibrous environment requires the formation and propagation of dynamic, actin based fin-like protrusions.

Our ability to rescue functional deficits after demyelinating diseases or spinal cord injuries is limited by our lack of understanding of the complex remyelination process, which is crucial to functional recovery. In this study, we developed an electrospun suspended poly(e-caprolactone) microfiber platform to enable the screening of therapeutics for remyelination. As a proof of concept, this platform employed scaffold-mediated non-viral delivery of a microRNA (miR) cocktail to promote oligodendrocyte precursor cells (OPCs) differentiation and myelination. We observed enhanced OPCs differentiation when the cells were transfected with miR-219 and miR-338 on the microfiber substrates. Moreover, miRs promoted the formation of MBP+ tubular extensions around the suspended fibers, which was indicative of myelination, instead of flat myelin membranes on 2D substrates. In addition, OPCs that were transfected with the cocktail of miRs formed significantly longer and larger amounts of MBP+ extensions. Taken together, these results demonstrate the efficacy of this functional screening platform for understanding myelination. Statement of Significance The lack of understanding of the complex myelination process has hindered the discovery of effective therapeutic treatments for demyelinating diseases. Hence, in vitro models that enable systematic understanding, visualization and quantification of myelination are valuable. Unfortunately, achieving reproducible in vitro myelination by oligodendrocytes (OLs) remains highly challenging. Here, we engineered a suspended microfiber platform that enables sustained non-viral drug/gene delivery to study OL differentiation and myelination. Sustained drug delivery permits the investigation of OL development, which spans several weeks. We show that promyelinogenic microRNAs promoted OL differentiation and myelination on this platform. Our engineered microfiber substrate could serve as a drug/gene screening platform and facilitate future translation into direct implantable devices for in vivo remyelination purposes.

The use of human induced pluripotent stem cell-derived neural progenitor cells (hiPSC-NPCs) is an attractive therapeutic option for damaged nerve tissues. To direct neuronal differentiation of stem cells, we have previously developed an electrospun polycaprolactone nanofiber scaffold that was functionalized with siRNA targeting Re-1 silencing transcription factor (REST), by mussel-inspired bioadhesive coating. However, the efficacy of nanofiber-mediated RNA interference on hiPSC-NPCs differentiation remains unknown. Furthermore, interaction between such cell-seeded scaffolds with injured tissues has not been tested. In this study, scaffolds were optimized for REST knockdown in hiPSC-NPCs to enhance neuronal differentiation. Specifically, the effects of two different mussel-inspired bioadhesives and transfection reagents were analyzed. Scaffolds functionalized with RNAiMAX Lipofectamine-siREST complexes enhanced the differentiation of hiPSC-NPCs into TUJ1+ cells (60% as compared to 22% in controls with scrambled siNEG after 9 days) without inducing high cytotoxicity. When cell-seeded scaffolds were transplanted to transected spinal cord organotypic slices, similar efficiency in neuronal differentiation was observed. The scaffolds also supported the migration of cells and neurite outgrowth from the spinal cord slices. Taken together, the results suggest that this scaffold can be effective in enhancing hiPSC-NPC neuronal commitment by gene-silencing for the treatment of injured spinal cords. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

Axons damaged by traumatic injuries are often unable to spontaneously regenerate in the adult central nervous system (CNS). Although the peripheral nervous system (PNS) has some regenerative capacity, its ability to regrow remains limited across large lesion gaps due to scar tissue formation. Nucleic acid therapy holds the potential of improving regeneration by enhancing the intrinsic growth ability of neurons and overcoming the inhibitory environment that prevents neurite outgrowth. Nucleic acids modulate gene expression by over-expression of neuronal growth factor or silencing growth-inhibitory molecules. Although in vitro outcomes appear promising, the lack of efficient non-viral nucleic acid delivery methods to the nervous system has limited the application of nucleic acid therapeutics to patients. Here, we review the recent development of efficient non-viral nucleic acid delivery platforms, as applied to the nervous system, including the transfection vectors and carriers used, as well as matrices and scaffolds that are currently used. Additionally, we will discuss possible improvements for localised nucleic acid delivery. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

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.

Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Correction for ‘Scaffold mediated gene knockdown for neuronal differentiation of human neural progenitor cells’ by Wai Hon Chooi et al., Biomater. Sci., 2018, 6, 3019–3029.

At present, the recovery prospect for patients with chronic neurodegenerative diseases or acute trauma in the central nervous system is sub-optimal. The controlled differentiation of neural stem/progenitor cells (NPCs) to functional neurons is a possible treatment strategy. In contrast to the classical approach of biochemicals supplementation for guided stem cell commitment, this study explores the feasibility of directing neuronal differentiation through synergistic integration of three-dimensional nanofibrous topographical cues and scaffold-mediated knockdown of RE-1 silencing transcription factor (REST) in mouse NPCs. Taking advantage of the strong adhesive property and latent reactivity of mussel-inspired polydopamine (PD) coating, electrospun polycaprolactone (PCL) nanofibers were successfully functionalized with REST siRNAs (denoted as siREST PD-fiber). Sustained REST knockdown in NPCs was achieved for up to five days in vitro and the silencing efficiency was significantly higher than that mediated through siRNA adsorption onto non-PD coated sample controls. The silencing of REST, together with nanofiber topographical effect, significantly enhanced NPC neuronal commitment (57.5% Map2(+) cells in siREST PD-fiber vs. 43.5% in siREST PD-film vs. 50% in PD-fiber controls, p 0.05) while reducing astrocytic and oligodendrocytic differentiation (10.7% O4(+) cells vs. ∼30% in siREST PD-film, p 0.01). Taken together, the synergistic effects of scaffold-mediated REST knockdown and topographical cues from PD-modified nanofibers may be a useful strategy for generating functional neurons for therapeutic purposes.

Directing stem cell fate remains a major area of interest and also a hurdle to many, particularly in the field of regenerative medicine. Unfortunately, conventional methods of over-expressing inductive factors through the use of biochemical induction cocktails have led to sub-optimal outcomes. A potential alternative may be to adopt the opposite by selectively silencing genes or pathways that are pivotal to stem cell differentiation. Indeed, over recent years, there have been an increasing number of studies on directing stem cell fate through gene knockdown via RNA interference (RNAi). While the effectiveness of RNAi in controlling stem cell differentiation is evident from the myriad of studies, a chaotically vast collection of gene silencing targets have also been identified. Meanwhile, variations in methods of transfecting stem cells have also affected silencing efficiencies and the subsequent extent of stem cell differentiation. This review serves to unite the pioneers who have ventured into the emerging field of RNAi-enhanced stem cell differentiation by summarizing and evaluating the current approaches adopted in utilizing gene silencing to direct stem cell fate and their corresponding outcomes.

Nerve regeneration after spinal cord injuries (SCI) remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of its biomimicking architecture. In this study, we investigated the feasibility of incorporating neurotrophin-3 (NT-3) and chondroitinase ABC (ChABC) onto electrospun collagen nanofibers for SCI treatment. By using microbial transglutaminase (mTG) mediated crosslinking, proteins were loaded onto electrospun collagen nanofibers at an efficiency of ∼45–48%. By combining NT-3 with heparin during the protein incorporation process, a sustained release of NT-3 was obtained (∼96% by day 28). As indicated by dorsal root ganglion outgrowth assay, NT-3 incorporated collagen scaffolds supported neuronal culture and neurite outgrowth for a longer time period than bolus delivery of NT-3. The presence of heparin also protected ChABC from degradation. Specifically, as evaluated by dimethylmethylene blue assay, bioactive ChABC was detected from collagen scaffolds for at least 32 days in vitro in the presence of heparin (∼32% of bioactivity retained). In contrast, ChABC bioactivity was only ∼1.9% by day 22 in the absence of heparin. Taken together, these results clearly demonstrated the feasibility of incorporating NT-3 and ChABC via mTG immobilization to produce protein-incorporated collagen nanofibers. Such biofunctional nanofiber constructs may find useful applications in SCI treatment by providing topographical signals and multiple biochemical cues that can promote nerve regeneration while antagonizing axonal growth inhibition for CNS regeneration.

SiRNA delivery has found useful applications particularly as therapeutic agents against genetic diseases. Currently, the delivery of siRNA typically takes the form of nanoparticles. In order to expand the applications of these potent but labile molecules for long-term use required by tissue engineering and regenerative medicine, alternative delivery vehicles are required. This work presents a scaffold-mediated approach to siRNA delivery. By encapsulating siRNA within polycaprolactone (PCL) nanofibers (300-400nm in diameter) controlled release of intact siRNA could be achieved for at least 28days under physiological conditions. The successful transfection of HEK 293 cells with GAPDH siRNA released from fibrous scaffolds at day 5, 15 and 30 demonstrated that the encapsulated molecules remained bioactive throughout the period of sustained release, providing silencing efficiency of 61-81% that was comparable to conventional siRNA transfection. Direct seeding of cells on these biofunctional scaffolds, with and without transfection reagent, demonstrated enhanced cellular uptake and efficient GAPDH gene-silencing. This work demonstrates the potential of nanofibrous scaffold-mediated siRNA delivery for long-term gene-silencing applications. The combination of topographical features provided by nanofibrous scaffolds may provide synergistic contact guidance and biochemical signals to mediate and support cellular development in regenerative medicine.

Peripheral nerve regeneration can be enhanced by the stimulation of formation of bands of Büngner prior to implantation. Aligned electrospun poly(epsilon-caprolactone) (PCL) fibers were fabricated to test their potential to provide contact guidance to human Schwann cells. After 7 days of culture, cell cytoskeleton and nuclei were observed to align and elongate along the fiber axes, emulating the structure of bands of Büngner. Microarray analysis revealed a general down-regulation in expression of neurotrophin and neurotrophic receptors in aligned cells as compared to cells seeded on two-dimensional PCL film. Real-time-PCR analyses confirmed the up-regulation of early myelination marker, myelin-associated glycoprotein (MAG), and the down-regulation of NCAM-1, a marker of immature Schwann cells. Similar gene expression changes were also observed on cells cultured on randomly oriented PCL electrospun fibers. However, up-regulation of the myelin-specific gene, P0, was observed only on aligned electrospun fibers, suggesting the propensity of aligned fibers in promoting Schwann cell maturation.

Effective remyelination in the central nervous system (CNS) facilitates the reversal of disability in patients with demyelinating diseases such as multiple sclerosis. Unfortunately until now, effective strategies of controlling oligodendrocyte (OL) differentiation and maturation remain limited. It is well known that topographical and biochemical signals play crucial roles in modulating cell fate commitment. Therefore, in this study, we explored the combined effects of scaffold topography and sustained gene silencing on oligodendroglial precursor cell (OPC) development. Specifically, microRNAs (miRs) were incorporated onto electrospun polycaprolactone (PCL) fiber scaffolds with different fiber diameters and orientations. Regardless of fiber diameter and orientation, efficient knockdown of differentiation inhibitory factors were achieved by either topography alone (up to 70%) or fibers integrated with miR-219 and miR-338 (up to 80%, p < 0.05). Small fiber promoted OPC differentiation by inducing more RIP+ cells (p < 0.05) while large fiber promoted OL maturation by inducing more MBP+ cells (p < 0.05). Random fiber enhanced more RIP+ cells than aligned fibers (p < 0.05), regardless of fiber diameter. Upon miR-219/miR-338 incorporation, 2 μm aligned fibers supported the most MBP+ cells (∼17%). These findings indicated that the coupling of substrate topographic cues with efficient gene silencing by sustained microRNA delivery is a promising way for directing OPC maturation in neural tissue engineering and controlling remyelination in the CNS. NMRC (Natl Medical Research Council, S’pore) Accepted version

While substrate topography influences cell behavior, RNA interference (RNAi) has also emerged as a potent method for understanding and directing cell fate. However, the effects of substrate topography on RNAi remain poorly understood. Here, we report the influence of nanofiber architecture on siRNA-mediated gene-silencing in human somatic and stem cells. The respective model cells, human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs), were cultured onto aligned or randomly oriented electrospun poly(e-caprolactone) fibers of different average diameters (300 nm, 700 nm and 1.3 μm). In HDFs, decreasing fiber diameter from 1.3 μm to 300 nm improved Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Collagen-I silencing efficiencies by ∼ 3.8 and ∼4.4 folds respectively (p < 0.05) while the effective siRNA uptake pathway was altered from clathrin-dependent endocytosis to macropinocytosis. In MSCs, aligned fibers generated significantly higher level of gene silencing of RE-1 silencing transcription factor (REST) and green fluorescent protein (GFP) (∼1.6 and ∼1.5 folds respectively, p < 0.05), than randomly-oriented fibers. Aligned fiber topography facilitated functional siRNA uptake through clathrin-mediated endocytosis and membrane fusion. Taken together, our results demonstrated a promising role of three-dimensional fibrous scaffolds in modulating siRNA-mediated gene-silencing and established the critical synergistic role of these substrates in modulating cellular behavior by RNAi.

The treatment of an injured central nervous system using stem-cell-based regenerative medicine still faces considerable hurdles that need to be overcome. Chief among which is the lack of efficient strategies to generate functional neurons from stem cells. The sustained delivery of biochemical cues and synergistic topographical signaling from electrospun nanofibrous scaffolds may be a potential strategy to enhance neuronal differentiation of stem cells for therapeutic purposes. In this study, retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) were encapsulated into a copolymer of ε-caprolactone and ethyl ethylene phosphate to form a multifunctional, electrospun nanofibrous scaffold. Sustained release of RA and BDNF was achieved for at least 7 and 14 days, respectively. Despite lower cumulative release of drugs as compared to bolus delivery to plain nanofibers (at least 2× and 50× lower for RA and BDNF, respectively), nanofiber-mediated delivery of RA and/or BDNF resulted in similar capacity for neuronal differentiation of mouse neural progenitor cells (NPCs). In addition, nanofiber topography significantly increased neuronal differentiation (with BDNF, 47.4 % Map2+ cells on 2D vs. 53.4 to 56.5 % on nanofibers, p

The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA-PCLEEP (poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of Collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or cell penetrating peptides (CPPs), CADY or MPG, within the nanofibers (550–650 nm in diameter), a sustained release of siRNA was obtained for at least 28 days (loading efficiency ~ 60–67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2–3 times as compared to conventional bolus delivery of siRNA (14 days vs 5–7 days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p < 0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.

Directing differentiation of neural stem/progenitor cells (NPCs) to produce functional neurons is a promising remedy for neural pathological conditions. The major challenge, however, lies in the effective and efficient generation of a sizable population of neurons. A potential strategy is to incorporate RNA interference (RNAi) during directed stem cell differentiation to recapitulate the complex cell-signaling cascades that often occurs during the process. In this study, in vitro silencing of RE1-silencing transcription factor (REST) was carried out using small-interfering RNAs (siRNAs) to evaluate the efficacy of combining REST knockdown with conventional differentiation approaches to enhance neurogenesis. While earlier studies have demonstrated enhanced neuronal lineage commitment from embryonic stem cells and mesenchymal stem cells upon REST knockdown, the effects of REST silencing during other stages of neural development have not been extensively evaluated. We hypothesize that REST knockdown would enhance NPC development to mature neurons and that induced REST silencing can serve as a potential biochemical approach to direct cell fate. Under nonspecific induction conditions, REST knockdown induced eightfold higher Tuj1 mRNA expression at day 14 compared with untransfected cells and cells subjected to scrambled-siRNA treatment (controls). Immunostaining also revealed greater percentage of Tuj1 positive cells with REST knockdown. Combined with neuronal induction, REST silencing enhanced the kinetics of neuronal differentiation and the rate of maturation of committed neuronal cells. Specifically, upregulation of MAP2 occurred as early as 3 days after induction with REST silencing and the expression was comparable to the controls at day 14. Likewise, downregulation of REST generated more than twice the percentage of Tuj1 and MAP2 positive cells compared with controls at day 5 (p

For the development of vascular tissue engineering, the impact of endothelial cells (ECs) on smooth muscle cell (SMC) spreading, proliferation, and differentiation is explored in the current study using a coculture model. In this coculture model, SMCs were encapsulated in a biomimetic hydrogel based on methacrylated dextran-graft-lysine (Dex-MA-LA) and methacrylamide-modified gelatin (Gel-MA), and exposed to a monolayer of ECs. With EC coculture, SMC proliferation in 3D hydrogel was promoted at initial period, and the formation of denser cellular networks was enhanced. ECs dynamically modulated SMC phenotype by promoting a more contractile SMC phenotype initially (on day 2), indicated by the upregulated expression of contractile genes α-actin, calponin, smooth muscle-myosin heavy chain (SM-MHC), and smoothelin; however, the onset of maximum expressions was delayed by ECs. Full differentiation of SMCs was not obtained even with EC coculture. Higher level of platelet-derived growth factor (PDGF)-BB and latent transforming growth factor (TGF)-β1 were detected in medium of coculture. These biochemical cues together with the physical cue of tensional force within cellular networks may be responsible for the dynamic modulation of SMC phenotype in coculture. Synthesis of elastin was promoted by ECs at transcriptional level. The formation of denser cellular networks and synthesis of elastin suggest that coculture with ECs is a potential method to construct functional vessel media layer in vitro.

Stem cells hold great promise in enhancing nerve regeneration. In particular, human mesenchymal stem cells (MSC) represent a clinically viable cell source due in part to their abundance and accessibility. Unfortunately, current methods to direct the fate of stem cells remains largely limited to biochemical-based approaches on two-dimensional substrates with restricted efficacies. Here we have evaluated a scaffold-based approach to directing stem cell differentiation. We demonstrate the combined effects of nanofiber topography and controlled drug release on enhancing MSC neural commitment. By encapsulating up to 0.3 wt.% retinoic acid (RA) within aligned poly(e-caprolactone) (PCL) nanofibers (average diameter ∼270 nm, AF750), sustained released of RA was obtained for at least 14 days (∼60% released). Compared with tissue culture polystyrene (TCPS), the nanofiber topography arising from plain PCL nanofibers significantly up-regulated the expressions of neural markers, Tuj-1, MAP2, GalC and RIP at the mRNA and protein levels. Combined with sustained drug availability, more significant changes in cell morphology and enhancement of neural marker expression were observed. In particular, scaffold-based controlled delivery of RA enhanced MAP2 and RIP expression compared with bolus delivery despite lower amounts of drug (>8 times lower). The generally higher expression of the mature neuronal marker MAP2 compared with glial markers at the mRNA and protein levels suggested an enhanced potential of MSC neuronal differentiation. In addition, positive staining for synaptophysin was detected only in cells cultured on aligned scaffolds in the presence of RA. Taken together, the results highlight the advantage of the scaffold-based approach in enhancing the potential of MSC neuronal differentiation and demonstrated the importance of the drug delivery approach in directing cell fate. Such biomimicking drug-encapsulating scaffolds may permit subsequent direct cell transplantation and provide guidance cues to control the fate of endogenously recruited stem cells.