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Remyelination in the central nervous system (CNS) is critical in the treatment of many neural pathological conditions. Unfortunately, the ability to direct and enhance oligodendrocyte (OL) differentiation and maturation remains limited. It is known that microenvironmental signals, such as substrate topography and biochemical signaling, regulate cell fate commitment. Therefore, in this study, we developed a nanofiber-mediated microRNA (miR) delivery method to control oligodendroglial precursor cell (OPC) differentiation through a combination of fiber topography and gene silencing. Using poly(ɛ-caprolactone) nanofibers, efficient knockdown of OL differentiation inhibitory regulators were achieved by either nanofiber alone (20–40%, p < 0.05) or the synergistic integration with miR-219 and miR-338 (up to 60%, p < 0.05). As compared to two-dimensional culture, nanofiber topography enhanced OPC differentiation by inducing 2-fold increase in RIP+ cells (p < 0.01) while the presence of miRs further enhanced the result to 3-fold (p < 0.001). In addition, nanofiber-mediated delivery of miR-219 and miR-338 promoted OL maturation by increasing the number of MBP+ cells significantly (p < 0.01). Taken together, the results demonstrate the efficacy of nanofibers in providing topographical cues and microRNA reverse transfection to direct OPC differentiation. Such scaffolds may find useful applications in directing oligodendrocyte differentiation and myelination for treatment of CNS pathological conditions that require remyelination.

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.

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.

Although stem cell therapy holds tremendous promise in tissue regeneration and disease treatment, its full potential may only be realized through the thorough understanding and capability in specifically directing stem cell fate commitment. A scaffold-based approach of imparting physical and biochemical cues appears to be a logical method in reconstructing the complex three-dimensional configuration of stem cell niches. In fact, interest in this area has gained significant momentum over the recent years. This review summarizes and evaluates the recent outcomes of studies associated with a scaffold-based approach to directing and understanding stem cell neural and cardiovascular differentiation.

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 < 0.05) and reduced glial cell differentiation. BDNF was a more potent inducer of neuronal differentiation than RA. RA supplementation alone resulted in minimal effect on NPC differentiation, and dual supplementation of RA and BDNF did not further enhance the neuronal differentiation of NPCs. Collectively, the results suggest that synergistic effects of nanofiber topography and sustained delivery of RA and/or BDNF may contribute towards the design of a multifunctional artificial stem cell niche for NPC neuronal differentiation.

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 < 0.05). Morphologically, REST-silencing enhanced the number and length of neurite extensions from Tuj1 positive cells ( p < 0.05), which was not evaluated in previous differentiation studies with REST knockdown. Taken together, these results demonstrate the efficacy of combining REST silencing during directed NPC differentiation to enhance the rate of differentiation and subsequent maturation of NPCs. This study also highlights the potential of RNAi as a biomedical strategy for guided stem cell differentiation.

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