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Hybrid Nanofiber Scaffold-Based Direct Conversion of Neural Precursor Cells/Dopamine Neurons

The concept of cellular reprogramming was developed to generate induced neural precursor cells (iNPCs)/dopaminergic (iDA) neurons using diverse approaches. Here, we investigated the effects of various nanoscale scaffolds (fiber, dot, and line) on iNPC/iDA differentiation by direct reprogramming. The generation and maturation of iDA neurons (microtubule-associated protein 2-positive and tyrosine hydroxylase-positive) and iNPCs (NESTIN-positive and SOX2-positive) increased on fiber and dot scaffolds as compared to that of the flat (control) scaffold. This study demonstrates that nanotopographical environments are suitable for direct differentiation methods and may improve the differentiation efficiency.


Introduction
The direct differentiation of somatic cells into tissuespecific cells such as induced neurons (1) and neural precursor cells (2)(3)(4)(5) may serve as an attractive approach in the field of stem cell research. The first study in this direction was reported by Vierbuchen et al. (1) using neuronal lineage-inducing transcription factors such as Brn2, Ascl1, and Myt1L (BAM). These factors converted mouse fibroblasts into neuronal cells without transitioning into a pluripotent state. Since then, several strategies have been employed to perform direct conversion. Direct reprogramming using non-integrating transcription factors (6,7), chemical (small molecule) treatment (8,9), and stromal cell-derived inducing activity (10) has been carried out. The low efficiency of this method is, however, a major limitation. To enhance the conversion efficiency, some studies have suggested that cell topography interactions control cell phenotypes (11,12) and ameliorate tissue-spe- cific cell generation (13,14). Nanotopography may substantially encourage cellular behavior from morphological changes to differentiation. Furthermore, the extracellular environment may provide signaling cues to adherent cells (15). Matrix elasticity may direct human mesenchymal stem cells to differentiate into specific lineages. A soft matrix may induce neurogenic phenotype, while stiffer matrices may induce myogenic and osteogenic phenotypes (16). Engineered nanometer-scale scaffolds hold great promises for stem cell differentiation and transplantation (17). Recent reports have shown that mechanical factors such as stress (18)(19)(20), adhesion area (21,22), substrate elasticity (16), and topography on micro-and nano-scale scaffolds (23)(24)(25) may induce differentiation of stem cells. The development of lithography-based platforms such as silicon-etched, photocured, and replica-molded patterns has enormously contributed to the biological study evaluating cellular behavior on well-ordered surfaces (26). In this study, we hypothesized that topographical cue combined with our previous conversion research method (4) may influence neural precursor cells (iNPCs)/dopaminergic (iDA) neuron generation. iNPC generation was observed earlier and iNPC-derived DA neurons exhibited high capacity to mature on fiber and dot nanoscale structures.

Materials and Methods
Fabrication of electrospun aligned nanofiber scaffold and nanostructured substrates with UV-assisted capillary force lithography (CFL) Polycaprolactone (PCL; MW, 80,000; Sigma-Aldrich, MO, USA) was dissolved in a mixture of chloroform (Samchun Pure Chemical, Korea) and dimethylformamide (Sigma-Aldrich, MO, USA) at a volume ratio of 75：25. Electrospinning PCL solution was used at 20% concentration (weight-to-volume ratio), as standardized by our group to obtain a unidirectionally aligned and homogeneous nanofiber scaffold array. The electrospinning process was conducted as follows: PCL solution was infused with a disposable syringe and a metal needle (23-gauge) at a rate of 1.5 ml/h to form polymer drops. A DC voltage of 15 kV was applied to the tip of the needle to induce electrospinning. The aligned nanofiber was harvested with an inclined-gap method, wherein the ejecting electrospun nanofiber was aligned in an electromagnetic field formed by two aluminum metal strips. The repulsive force between nanofibers allowed the formation of a well-oriented nanofiber scaffold. To control the nanofiber density, we repeatedly transferred the nanofiber array to a punched PDMS slab (2-cm diameter). The electrospinning time for each transfer was ∼1 min and the transfer was repeated for five times to fabricate a single cell culture scaffold. The schematic diagram of the fabrication procedure is illustrated in Fig. 1.
A small amount (0.1∼0.5 ml) of UV curable PUA prepolymer was drop-dispensed on a silicon master with positive patterns (features sticking out), and a supporting polyethylene terephthalate (PET) film was carefully placed on the top surface to allow conformal contact. The PET film used in this study was surface modified with urethane groups to increase adhesion with an acrylate-containing monomer (Minuta Tech., Korea). The silicon masters were prepared by photolithography or electron-beam lithography. The resin was exposed to UV (wavelength: 250∼400 nm) for 17 s at an intensity of 100 mW/cm 2 , and the cured first replica was peeled-off from the silicon master using a sharp tweezer. The first replica was overnight exposed to UV to remove any uncured active groups on the surface. The second replica was prepared using a capillary molding process on glass coverslips with the overcured first PUA as a mold, resulting in a pattern similar to that of the silicon master. After curing, the first replica was removed from the surface using sharp tweezers. The fabricated PUA nanopatterns were sterilized by rinsing with isopropyl alcohol and distilled water, and coated with 0.1% gelatin for 1 h at room temperature prior to cell culture (27). Two types of nanopatterns that varied in size (from 400 nm) were used, including dots (400 nm in diameter) and lines (400 nm in width). For consistency, the pattern height was fixed to 500 nm, as verified by atomic force microscopy measurements (data not shown).

Retroviral production
Retroviral construction and production were performed as previously described (4)

iNPC generation and iDA neuron differentiation
To generate iNPCs, REFs were cultured on gelatin-

Immunocytochemistry and antibodies
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized, and blocked in a blocking solution (10% normal goat serum, Pel-Freez, Rogers, AR, USA), 0.3% Triton X-100 (Sigma-Aldrich in PBS) for 1 h. The samples were stained with primary antibodies for 1 day and then incubated for 1 h with secondary antibodies in the dark. Cells were photographed using an epifluorescence microscope (Leica Micro systems, Wetzlar, Germany). Monoclonal primary antibodies used were as follows: NESTIN (BD Bioscience, Franklin Lakes, NJ), microtubule-associated protein 2 (MAP2; Sigma-Aldrich). Polyclonal primary antibodies used included those against SOX2 (Chemicon, Temecula, CA) and tyrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA).

Cell counting and statistical analysis
Cell counting was performed on a random selection of 5∼10 uniform microscopic fields/wells, with 3∼4 wells per experimental condition. All values were confirmed with at least three independent experiments. Data are expressed as mean±SEM. When more than two groups were compared, a paired t-test was performed using SigmaPlot for Windows, version 10.0 (Systat Software GmbH, Erkrath, Germany) (5).

Nanoscale structures are suitable for cell survival and direct conversion process
We have previously shown that NPCs may be generated from rodent fibroblasts transduced with the ectopic gene expression method (4). The nanoscale pattern protocol for NPC/DA neuron generation from human pluripotent stem cells and somatic cells has been reported (13,14,25). To induce NPCs from fibroblasts, we combined these two protocols, as shown in Fig. 2A. After 3 days of BAMX transduction (DIV 4), cells were transferred on several nanoscale scaffolds, such as fiber, dot, and line. During NPC induction/expansion, cell morphology changed, as evident from the small and slender cells, with no effect on survival and proliferation properties (Fig. 2B).

Discussion
Generation of iNPC/iDA neurons from fibroblasts has been the focus of stem cell regeneration research. To obtain iNPC/iDA neurons in vitro and in vivo, various types of cell sources and materials have been developed. We have successfully demonstrated the generation of expandable iNPCs and DA neurons from somatic cells using exogene expression (4) and stromal cell-derived inducing activity (SDIA) methods (10). Various types of experimental procedures have been developed. A recent technique based on nanoscale pattern structure has gathered considerable attention.
The investigation of cell fate modulated using nanoscale pattern environment is one of the active areas in stem cell engineering because of the possible side-effects caused by biochemical signals and biophysical cues (28). In vivo, all adherent cells encounter topographical features from neighboring cells and the extracellular matrix. The functions of cells, in general, and of neurons, in particular, depend on their shape and polarity (13). Cell polarity is controlled by the confinement of focal adhesions on the gratings. Focal adhesions have been shown to play an important role in topography sensing (19). The surfaces can be altered (activated) at will to tip adhesion/cytoskeletal balance and initiate differentiation, hence better informing biological mechanisms of stem cell growth and direct conversion (29,30). Based on this mechanism, nanotopographical signal exerts synergic effects that may cause biochemical stimulation.
In our work, we combined nanoscale pattern structures (fiber, dot, and line) with the general conversion process. The transduced cells were transferred onto nanoscale pattern substrates, which resulted in the quick transformation of the cells into NPC-like cells without cell death (data not shown). Thus, the nanoscale pattern substrates are nontoxic and may serve as promising tools in conversion material technique. The fiber and dot pattern substrates enhanced the generation of NPCs (NESTIN-and SOX2positive; Fig. 3) and DA neurons (MAP2-and TH-positive; Fig. 4).