WO2016148656A1 - Neurogenesis of dopaminergic neurons - Google Patents

Abstract

The invention relates to a method of enhancing the differentiation of cells into neurons, wherein the cells are selected from the group multipotent stem cells, pluripotent stem cells and somatic cells, comprising seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium; said substrates; a composite comprising at least one of said substrate, wells or chambers and either a plurality of said multipotent or pluripotent cells or a plurality of multipotent or pluripotent cell progeny, including cells differentiated therefrom; and a drug screening method involving the use of said composite.

Description

NEUROGENESIS OF DOPAMINERGIC NEURONS

FIELD OF THE INVENTION

The present invention relates to novel substrates that facilitate the differentiation of muitipotent or pluripotent cells, such as stem cells or progenitor cells and somatic cells; wells or chambers containing said substrates, particularly, but not exclusively, for culturing said cells; a method for the differentiation of muitipotent, pluripotent or somatic cells involving use of said substrates, wells or chambers; a composite comprising at least one of said substrates, wells or chambers and either a plurality of said muitipotent, pluripotent or somatic cells or a plurality of muitipotent, pluripotent or somatic cell progeny, including cells differentiated therefrom; and a drug screening method involving the use of said composite.

BACKGROUND OF THE INVENTION

Stem cell research could potentially impact the development of disease-modifying therapies, such as those designed to treat Parkinson's disease (PD), which has been hampered by the lack of predictive and progressive cellular models. Parkinson's disease, which affects more than 2% of the population over 65 years of age, and is associated with motor and cognitive deficits, has been attributed to the loss of midbrain dopaminergic (DA) neurons within the pars compacta of the substantia nigra [Damier P, et al., Brain 122: 1437-1448 (1999)]. The development of new in vitro models of the disease, such as using patient-derived pluripotent stem cells, would enable drugs against disease pathology to be screened more efficiently. These methods can then be used to evaluate environmental and genetic factors implicated in PD, as well as elucidate the underlying biological mechanisms associated with this disease.

Although considerable progress has been made in deriving DA neuronal cells from stem cells, more work must be done to derive DA cells that are robust and reproducible in quality and quantity, and that can be enriched for selective subtypes of DA neurons. The desired cell population must be clearly defined and stringently purified before stem cell-derived DA neurons can be used for future clinical applications. Deriving midbrain DA neurons in vitro requires several biochemical cues including a variety of growth factors and patterning neurotrophic factors to increase derivation efficiency [Chambers SM, et al., Nat Biotechnol 27: 275-280, (2009); Kriks S, et al., Nature 480: 547-551 (2011 )]. However, this can be impractical and expensive for large-scale production of neurons. Although methods have been devised using fewer growth factors [Erceg S, et al., PLoS ONE 3: e2122 (2008)], such methods yield lower subtype specific neurons, requiring purification of the cell population, which further reduces the cell yield.

Interactions between cells and their mechanical microenvironment play important roles in determining cell fate and cell behaviour. Cell survival, proliferation and their propensity to differentiate into specific cell types are affected by extracellular biochemical and biophysical signals [Dingal P, Discher D. Nat Mater 13: 532-537 (2014)]. Accumulating evidence demonstrates that the topography of substrates that cells adhere to influence their response [Ankam S, et al., Acta Biomater 9: 4535-4545 (2013); Hoffman-Kim D, et al., Annu Rev Biomed Eng 12: 203-231 (2010); Koo S, et al., Acta Biomater 10: 1975-1984 (2014); Yim E, et al., Biomaterials 31 : 1299-1306 (2010); Lim J, et al., Biomacromolecules 6: 3319-3327 (2005); Hamilton D, et al., Cell Motil Cytoskeleton 66: 260-271 (2009); Mahoney M, et al., Biomaterials 26: 771-778 (2005); Rajnicek A, McCaig C. J Cell Sci 1 0 (23): 2915-2924 (1997); Gerecht S, et al., Biomaterials 28: 4068-4077 (2007)]. In our previous work, we have shown that patterned nanostructures are able to induce neuronal marker expression in human mesenchymal stem cells [Yim E, et al., Exp Cell Res 313: 1820-1829 (2007)]. Neural stem cells are also influenced by nanoscale patterned substrates, which for instance, cause them to elongate along poly (L- lactic acid) fibres with significant outgrowth of neurites in the direction of the fibres [Yang F, et al., Biomaterials 26: 2603-2610 (2005)]. Similarly, other groups have shown that aligned topographical structures are able to promote differentiation and neurite alignment as well as influence cell maturation [Chew S, et al., Biomaterials 29: 653-661 (2008); Christopherson G, et al., Biomaterials 30: 556-564 (2009); Johansson F, et al., Biomaterials 27: 1251-1258 (2006)].

By taking into account biophysical influences in neuronal differentiation, other studies have shown that certain topographies can enhance differentiation of neural progenitors toward neural lineages [Moe AAK, et al., Small 8: 3050-3061 (2012); Migliorini E, et al., Biotechnol Bioeng 108: 2736-2746 (2011 )]. Additional studies have also shown that topography can enhance neuronal differentiation of other stem cell types [Migliorini E, et al., Biotechnol Bioeng 108: 2736-2746 (2011 ); Pan F, et al., Biomaterials 34: 8131-8139 (2013); Yang K, et al. ACS Applied Materials & Interfaces 5: 10529-10540 (2013); Lee MR, et al., Biomaterials 31 : 4360-4366 (2010); Chan LY, et al., Biomaterials 34: 382-392 (2013)]. However, differentiation of these stem cells into particular neuronal subtypes remains unexplored for specific applications, such as the treatment of PD. Here, we demonstrate that certain topographies improve the efficiency of DA neuron derivation. We used an 18-pattern Multi-Architecture (MARC) chip with a combination of micro- or nano- patterned substrates to optimize and identify patterns or structures that can best improve the efficiency of neural progenitor cell differentiation into the midbrain DA neuronal subtype. We showed that, for neural progenitor cells, gratings with specific dimensions improve derivation as well as maturation of the DA neuron subtype, with an associated alignment of these neurons along the grating patterns. We also showed that, for pluripotent stem cells, a 2 stage method involving the use of a separate grating in each stage resulted in the production of morphologically and electrophysiologically mature dopaminergic neurons. We also show that the transdifferentiation of somatic cells into neuronal lineage can be significantly improved on patterned substrates. The results of this study thus reveal how topographical cues can be used to generate neurons, particularly DA neurons for cellular models of PD.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a method of enhancing the differentiation of cells into neurons, wherein the cells are selected from the group multipotent stem cells, pluripotent stem cells and somatic cells, comprising seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro- grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium.

In a preferred embodiment the method of enhancing the differentiation of cells into neurons comprises two stages; i) seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium, and ii) removing the cells from the substrate of stage i) after a period of contact with medium and re-seeding the removed cells onto a substrate patterned with nano- and/or micro gratings or pillars, and contacting the cells with medium. According to a further aspect of the invention there is provided a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, wherein the dimensions of the gratings are 250 nm to 2 pm wide with 250 nm to 2 pm spacing and 250 nm to 2 pm height. According to a further aspect of the invention there is provided the use of dopaminergic neurons produced according to any aspect of the invention for the treatment of Parkinson's disease.

According to a further aspect of the invention there is provided a method of treatment of Parkinson's disease, comprising administering to a subject in need thereof an efficacious amount of dopaminergic neurons produced according to any aspect of the invention. According to a further aspect of the invention there is provided the use of dopaminergic neurons produced according to any aspect of the invention to screen for compounds with activity in ameliorating or treating Parkinson's disease, in vitro testing or drug screening related to Parkinson's disease or diseases related to dopaminergic neurons.

According to a further aspect of the invention there is provided a method of testing compounds for activity in ameliorating or treating Parkinson's disease, comprising the steps:

(a) contact dopaminergic neurons obtained according to any aspect of the invention with the test compound, and

(b) quantitate the level of any one or more from the group comprising dopamine production, TH expression, Lmxla expression, Foxa2 expression, PITX3 expression, MAP2 expression, NURR1 expression, ALDH1a1 expression, SCN1a expression, VMAT2 expression, Pax6 expression and neuron complexity; and/or

(c) measure the electrophysiological profile of said neurons; and

(d) compare the level quantitated in (b) and/or measured in (c) with the level in untreated dopaminergic neurons, wherein a difference in level of dopamine production, TH expression, Lmxla expression, Foxa2 expression PITX3 expression, MAP2 expression, NURR1 expression, ALDH1a1 expression, SCN1a expression, VMAT2 expression, Pax6 expression, neuron complexity and/or electrophysiological profile indicates the compound has Parkinson's disease- ameliorating activity. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Shows the dimensions, construction (A) and arrangement (B) of the MARC chip used to determine the effect of 3-dimensional topographies on cell differentiation.

Figure 2: Neural progenitor cells (passages 12-19) isolated from the hippocampus of 5-day old mice were seeded onto laminin-coated plates and characterized by their immune-reactivity against nestin and SOX2 (A) and GFAP (B, C), and their ability to differentiate into neurons and astrocytes.

Figure 3: Controlled differentiation of murine neural progenitor cells (mNPCs) to dopaminergic neurons on poly-dimethylsiloxane (PDMS). (A) Timeline of differentiation of mNPCs to dopaminergic neurons and the composition of the media for induction and patterning. (B) Scanning electron microscope images of the gratings of size 250nm with 250 nm pitch and 250 nm height, 2pm with 2pm width and 2pm height, and hierarchical grating of size 2*2*2 pm gratings with hierarchical perpendicular 250*250x250 nm grating on the ridge (2*2*2 pm gratings -¹- 250*250*250 nm lines) replicated with high fidelity onto PDMS substrates. No topographical features were observed on the unpatterned control.

Figure 4: Controlled differentiation of human induced pluripotent stem cells (iPSCs) to midbrain dopaminergic neurons on patterned poly-dimethylsiloxane (PDMS). (A) Timeline of differentiation of human iPSCs to dopaminergic neurons and the composition of the media at different stages. Modifications and optimization to the protocol based on dual SMAD inhibition method (Kirkeby A, et al. Cell reports 1 : 703-714 (2012)) were made incorporating the use of topographical patterns for dopaminergic neuronal differentiation. The size of embryoid bodies was standardized on fabricated PDMS chambers consisting of microwells, whereupon they were harvested for a two-stage differentiation process on fabricated patterned chambers involving the expansion of neural progenitor cells (stage 1) and the terminal differentiation of neurons (stage 2). Concentrations of PLO/FN/Lam were optimized on the substrates for cell attachment. (B) Brightfield images of embryoid bodies in microwells and on patterned substrates at stage 1. Cells were observed to be aligned on the grating axis (arrow) at stage 1. Scale bar: 100 pm (C) Immunofluorescence images of cells expressing tyrosine hydroxylase (TH), a dopaminergic neuron marker at stage 2 on patterned substrates. Scale bar: 50 pm. (EB: embryoid body; NB: neurobasal medium; PLO: poly-L-ornithine; FN: fibronectin; Lam: laminin; SHH: sonic hedgehog; BDNF: brain-derived neurotrophic factor; GDNF: glial cell derived neurotrophic factor; TH: tyrosine hydroxylase; BF: brightfield). Figure 5: Scanning electron microscopy images (left to right) of an unpatterned surface, gratings of size 2 pm with 2 pm width and 2 pm height, 250 pm with 250 nm pitch and 250 nm height and hierarchical gratings of size 2 pm with 250 nm perpendicular gratings.

Figure 6: Results comparing gene expression upon differentiation on patterned PDMS surfaces. (A) TH is a marker of dopaminergic neurons and (B) PITX3 is a marker of nigral dopaminergic cells.

Figure 7: Screening with 18 pattern Multi-Architecture (MARC) chip. (A) Quantification and data analysis of GFAP+ (glial fibrillary acidic protein) (astrocytes) and TUJ1 + (β-lll-tubulin) (neurons) cells on 18 pattern MARC chip. 2pmx2pmx2pm gratings (1), 250 nm*250 nmx250 nm gratings (5), and 2x2*2 pm gratings -¹- 250x250x250 nm lines (6) produced significantly higher percentage of TUJ1 -positive cells compared to the unpatterned substrate (n=3). *p<0.05, **p<0.01 are compared to unpatterned control. p<0.05, ^##p<0.01 are compared between different patterns. (B) Quantification and data analysis of TH+ (tyrosine hydroxylase) (dopaminergic) cells on 18 pattern MARC chip. 2pmx2pmx2pm gratings (1 ), 250 nmx250 nmx250 nm gratings (5), and 2^χ2 2 pm gratings -¹- 250x250x250 nm lines (6) produced significantly higher percentage of TH-positive cells compared to the unpatterned substrate (n=3). **p<0.01 , ***p<0.001 are compared to unpatterned control. Comparison between all the patterns is tabulated in Supplementary Table 2. (C) Quantification and data analysis of TH-positive cells over TUJ1-positive cells on 18 pattern MARC chip (n=3). All data are represented as data ± standard error mean (SEM) of three independent experiments. *p<0.05 compared to unpatterned control. p<0.05, ^# p<0.01 compared between different patterns.

Figure 8: Quantitative real-time PCR for Gene Expression Analysis. (A) Relative gene expression of tyrosine hydroxylase (TH) after 11 days of differentiation. (B) Relative gene expression of pituitary homeobox 3 (PITX3) after 19 days of differentiation. Figure 9: Immunofluorescence staining of dopaminergic neuronal markers on the differentiated murine neural progenitor cells on PDMS 2x2*2 pm gratings, 250x250x250 nm gratings, hierarchical gratings, and unpatterned PDMS substrates. Neuronal markers tyrosine hydroxylase (TH), microtubule associated protein 2 (MAP2), nuclear receptor related protein 1 (NURR1 ), voltage gated sodium channels (SCN1a), and vesicular monoamine transporter 2 (VMAT2), aldehyde dehydrogenase 1 family member A1 (ALDH1a1), pituitary homeobox 3 (PITX3) in green. Nuclei were counter-stained with DAPI (blue). Arrows indicate the direction of the major grating axis. Figure 10: Population counts of stained murine neural progenitor cells on single patterned or unpatterned PDMS substrates after differentiation. (A) Quantification and data analysis of ΤΉ- positive (dopaminergic) cells (n=3) after 19 day differentiation on 2μιη^χ2μιη^χ2μΐ7Ί gratings and unpatterned PDMS substrates. (B) Quantification and data analysis of MAP2+ (microtubule associated protein 2) cells (n=4) after 11 day differentiation on 2μΓη^χ2μιτι^χ2μηΓΐ gratings and unpatterned PDMS substrates. (C) Quantification and data analysis of ALDH1a1+ (aldehyde dehydrogenase 1 family member A1) (dopaminergic precursor) cells on 2μΐ7ΐ^χ2μιη^χ2μΓη gratings, 250 nmx250 nmx250 nm gratings, and unpatterned PDMS substrates (n=3). (D) Quantification and data analysis of LMX1a+ (LIM homeobox transcription factor 1 alpha) (dopaminergic precursor) and PITX3+ (pituitary homeobox 3) (nigral neurons) cells on 2μπι 2μηι^χ2μιη gratings, 250 nm ^χ250 nmx250 nm gratings, and unpatterned PDMS substrates (n=3). All data are represented as data ± SEM of n independent experiments. *p<0.05 ^**p<0.01 , ^***p<0.001 compared to unpatterned control. Scale bar, 50 μηι.

Figure 11: Elongation and alignment of neurons on single patterned PDMS substrates. (A) Immunofluorescence staining of TUJ1 and GFAP in cells on 2^χ2^χ2 μητι gratings, 250x250x250 nm gratings, hierarchical gratings (2^χ2^χ2 μιη gratings -¹- 250x250x250 nm lines), and unpatterned PDMS substrates. Cells were more elongated and aligned on the 2 2 2 μηι and hierarchical gratings axis. Arrows indicate the direction of the major grating axis. Scale bar: upper panel 100 μιη; bottom panel, 50 μηη. (B) Neurons were more elongated when differentiated on 2^χ2^χ2 μιη gratings and hierarchical gratings than unpatterned control. (C) Significantly more neurons were aligned on the grating axis on the 2 2 2 μιη gratings and hierarchical gratings than on the 250x250x250 nm gratings. All data are represented as mean ± SEM and experiments were carried out in duplicate or more with over 200 cells measured on each sample. Figure 12: Stiffness measurement by atomic force microscopy (AFM) showed the elastic modulus of PDMS (10:1 base/curing ratio) and TCPS to be 3.27 ± 0.133 MPa and 2149 ± 285 MPa respectively, indicating a considerable difference in stiffness between these two substrates.

Figure 13: Population counts of stained cells on single patterned or unpatterned TCPS substrates after 11 days of differentiation. (A) Immunofluorescence staining of TUJ1 , GFAP and MAP2 in cells on 2 2 2 μιτι gratings, 250x250x250 nm gratings, and unpatterned TCPS substrates. Arrows indicate the direction of the grating axis. Scale bar, 100 pm. (B) 2 2 2 pm gratings generated higher neuron-to-astrocyte ratios than unpatterned control based on staining of TUJ1 (neurons) and GFAP (astrocytes). Data are represented as data ± SEM of at least two independent experiments. (C) Quantification and data analysis of MAP2-positive (mature neuron) cells on 2μηη^χ2μΓη^χ2μηη gratings, 250 nmx250 nmx250 nm gratings, and unpatterned TCPS substrates. Both gratings produced more MAP2+ cells than unpatterned control. Data are represented as data ± SEM of at least three independent experiments. ^*p<0.05, ^**p<0.01 compared to unpatterned control.

Figure 14: Immunostaining of dopaminergic neuronal markers on human iPSC-derived cells on unpatterned, gratings, and pillars PDMS substrates. Beta-Ill tubulin (TUJ1), microtubule associated protein 2 (MAP2), pituitary homeobox 3 (PITX3), LIM homeobox transcription factor 1 alpha (LMX1a), forkhead box protein A2 (FOXA2) were all robustly expressed in patterned substrates.

Figure 15: Quantification and data analysis of stained cells on patterned and unpatterned substrates after 21 day differentiation. (A) Quantification and data analysis of TUJ1-positive (neuronal) cells after 21 day differentiation on unpatterned, gratings and pillars PDMS substrates. Both gratings and pillars have higher percentages of TUJ1 -positive cells than unpatterned (n=5). (B) Quantification and data analysis of TH-positive (dopaminergic) cells after 21 day differentiation on unpatterned, gratings and pillars PDMS substrates (n=5). (C) Percentage of TH-positive cells normalized on unpatterned substrates for each experiment. Gratings have about two-fold increase in TH-positive cells than unpatterned (n=5). (D) Quantification and data analysis of TH/TUJ1 cells on unpatterned, gratings, and pillars PDMS substrates (n=5). (E) Quantification and data analysis of FOXA2-positive (midbrain DA) cells on unpatterned, gratings, and pillars PDMS substrates. Gratings and pillars have more cells expressing FOXA2 than unpatterned (n=3). All data are represented as data ± SEM of n independent experiments. At least five fields of view were taken for analysis and over 200-1000 cells were counted for each sample. ^*p<0.05 ^**p<0.01 compared to unpatterned control. Scale bar, 100 pm. (TUJ1 : beta-Ill tubulin; TH: tyrosine hydroxylase; FOXA2: forkhead box A2).

Figure 16: Morphology of TH neurons on patterned and unpatterned substrates after 21 day differentiation. (A) Representative images of human iPSC-derived TH neurons on unpatterned, gratings and pillared PDMS substrates. Images were digitally stitched from overlapping fields of view to capture the entire length of neurites. Cells on gratings were aligned on the gratings axis (arrows). Scale bar: 100 pm (B) Average neurite length per neuron. TH neurons were more elongated when differentiated on gratings than pillars and unpatterned control. (C) Number of branch points per neuron (D) Sholl analysis. TH neurons have significantly more branching and increased neuronal complexity when differentiated on pillars than gratings and unpatterned control. (E) Number of terminals per neuron is significantly greater when differentiated on pillars. (F) Graph shows percentage of spontaneous postsynaptic current is higher in TH +ve neurons differentiated on pillars. (G) Representative trace shows spontaneous synaptic activity (arrowhead) can be detected as early as 4 weeks post differentiation. Scale bar: 10 pA (vertical) and 2 s (horizontal). Inset (red box) scale bar: 10 pA (vertical) and 0.2 s (horizontal). All data are represented as data ± SEM of three independent experiments with over 30 TH +ve neurons analyzed on each pattern. ^*p<0.05 ^**p<0.01 ***p<0.001 ****p<0.0001. Figure 17: Electrophysiology properties of human iPSC-derived neurons on unpatterned, gratings, and pillars PDMS substrates. (A) Shows representative tracings of firing patterns on substrates at 4 and 5 weeks post differentiation. Neurons were capable of firing repetitive action potential as a response to current injection as early as 4 weeks post differentiation. (B) Percentage of differentiated neurons capable of repetitive firing at 5 weeks post differentiation. More neurons on pillars were capable of repetitive firing than when on unpatterned and gratings. Five to nine neurons were recorded from three experiments per condition.

Figure 18: Immunostaining of dopaminergic neuronal markers on human iPSC-derived cells from Parkinson's disease patient with LRRK2 mutation on unpatterned, gratings, and pillars PDMS substrates. TUJ1 , TH and LMX1a were robustly expressed indicating patient-derived iPS cells can be successfully differentiated to midbrain dopaminergic neurons on patterned substrates.

Figure 19: Electrophysiology properties of human induced pluripotent stem cell (iPSC)-derived cells from PD patient with LRRK2 mutation on unpatterned, gratings, and pillars PDMS substrates. (A) Representative tracings of firing patterns on substrates at 4 and 5 weeks post differentiation. Neurons were capable of firing a repetitive action potential as a response to current injection as early as 4 weeks post differentiation. (B) Shows the percentage of differentiated cells capable of repetitive firing at 5 weeks post differentiation. More neurons on gratings and pillars were capable of repetitive firing than on unpatterned PDMS. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Certain terms employed in the specification, examples and appended claims are collected here for convenience. Reference herein to a biologically inert polymer is reference to a polymer that is chemically inactive, at least during the period of use for the differentiation of multipotent or pluripotent cells, thus the invention also includes polymers that, after use, are biodegradable.

Further, reference herein to multipotent or pluripotent cells, is reference to stem cells, progenitor cells and induced pluripotent stem cells. As used herein, reference to a "grating" is reference to a micro or nanoscale structure of a series of parallel beams with crests and troughs resulting in three dimensional grooves of specific space(s) between the walls of each groove, height(s) of the wall of the groove and width(s) of the walls of the groove.

As used herein, reference to a "hierarchical grating" is reference to a grating with an overlay of set(s) of micro and/or nanoscale three dimensional structures with dimensions smaller than that of the first resulting in a grating with indentations or undulations on its walls and/or the width of its walls and/or within the space between the walls.

As used herein, reference to "pillars" is reference to a series of microscale or nanoscale upright shafts or structures relatively slender in proportion to their height, and of any shape in section In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the term "comprising" as used in the context of the invention refers to where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term "comprising" encompasses the more restrictive terms "consisting essentially of and "consisting of." word "comprises", or variations such as "comprised" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.

According to a preferred aspect, the present invention provides a method of enhancing the differentiation of cells into neurons, wherein the cells are selected from the group multipotent stem cells, pluripotent stem cells and somatic cells, comprising seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro- grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium.

Preferably, the dimensions of the gratings are 250 nm to 2 pm wide with 250 nm to 2 pm spacing and 250 nm to 2 pm height, and the hierarchical secondary structures are gratings with dimensions of 100 nm to 250 nm wide with 100 nm to 250 nm spacing and 100 nm to 250 nm height; and the pillars are 250 nm to 40 pm diameter with a 1 pm to 15 pm pitch. It is preferred that the pillars are 0.5 pm to 10 pm diameter, more preferably 0.5 pm to 5 pm, with a 5 pm to 12 pm pitch; even more preferably the pillars are 1 pm to 2 pm in diameter with an 8 pm to 12 pm pitch. It would be understood that intermediate sizes not particularly specified herein may be suitable according to the invention. In particular, it is preferred that the gratings according to any aspect of the invention are parallel. It is also preferred that the hierarchical secondary gratings according to any aspect of the invention are perpendicular to the longitudinal axis of the gratings.

In another preferred embodiment, the gratings have an aspect ratio of 1 (grating width: spacing: height of 1 :1 :1 ). In a preferred embodiment, the method of enhancing the differentiation of cells into neurons further comprises the steps; a) maintaining the seeded cells in medium; b) inducing neuronal lineage phenotype by withdrawal of growth factors and/or inhibitors; c) replacing the medium of b) with neural patterning medium.

In a preferred embodiment the method of enhancing the differentiation of cells into neurons comprises two stages; i) seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium, and ii) removing the cells from the substrate of stage i) after a period of contact with medium and re-seeding the removed cells onto a substrate patterned with nano- and/or micro gratings or pillars, and contacting the cells with medium.

In this two stage method, pluripotent stem cells such as, for example, induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) may be seeded onto a substrate patterned with gratings in step i) in order to increase lineage commitment towards midbrain dopaminergic neurons. After a period of culture on the substrate, the cells may be removed from the gratings and re-seeded onto a substrate patterned with pillars to promote neurite branching and a more differentiated morphology with an increased capability of firing repetitive action potentials. These cells also showed increased spontaneous postsynaptic activity and better electrophysiology profile. See, for example, Figures 17 and 19 herein.

In a preferred embodiment of the invention, there is provided a two stage method of enhancing the differentiation of embryoid body (EB) cells, derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), into dopaminergic neurons comprising; i) seeding dissociated embryoid body (EB) cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with parallel grating structures, and contacting the cells with medium comprising SMAD inhibitor, Noggin, sonic hedgehog (SHH) and CT99021 ; and ii) removing the cells from the substrate of stage i) after a period of contact with said medium and re-seeding the removed cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with gratings or pillars, and contacting the cells with medium comprising BDNF, GDNF and Ascorbic Acid; and adding DAPT to the medium at day 4.

In a more preferred embodiment of the invention, there is provided a two stage method of enhancing the differentiation of embryoid body (EB) cells, derived from human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), into dopaminergic neurons comprising; i) seeding dissociated embryoid body (EB) cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with 2 pm ridge by 2 pm space by 2 pm depth parallel grating structures, and contacting the cells with medium comprising SMAD inhibitor SB431542, Noggin, SHH and CT99021 to promote initial lineage commitment of midbrain DA neurons; and ii) removing the cells from the substrate of stage i) after a period of contact with said medium and re-seeding the removed cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with 2 pm diameter pillars with 2 pm height, and contacting the cells with medium comprising BDNF, GDNF and Ascorbic Acid for 3 days; and adding DAPT to the medium at day 4 for at least another 7 days to promote differentiation into functional midbrain dopaminergic neurons.

In another preferred embodiment, the neural patterning medium in c) comprises Neurobasal and DMEM/F12 in 1 :1 ratio, 1x B27 supplement, 0.25x N2 supplement, 100ng/ml FGF-8, 200ng/ml Sonic Hedgehog and 1pg/mi laminin or, if the cells are pluripotent stem cells, the medium in c) comprises BDNF (20 ng/ml), GDNF (10 ng/ml) and ascorbic acid (200 μΜ) for 3 days, and then A-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1 ,1-dimethylethyl ester (DAPT) (2.5 μΜ) is added to the medium from day 4 of stage 2. According to any aspect of the present invention, the embryoid bodies are preferably formed in microwells having a diameter of about 500 pm to 800 pm. In a preferred embodiment, the embryoid bodies are grown to about 300 pm in diameter before being dissociated and seeded onto substrate in stage (i). According to any aspect of the present invention, the seeded cells are preferably contacted with the neural patterning medium for at least 5 to 30 days, more preferably for at least 7 to 19 days.

Preferably in the two stage method the substrate is PDMS.

DAPT is an inhibitor of γ-secretase, resulting in promotion of neuronal commitment. It would be understood that other γ-secretase inhibitors may be useful in the methods of the invention. In a preferred embodiment of the method, the seeded cells are differentiated into dopaminergic neurons, which typically express tyrosine hydroxylase (TH). More preferably, the cells are differentiated into TH+ neurons which express substantia nigra dopaminergic neuron markers (PITX3+) and/or midbrain dopaminergic neuron markers (FOXA2, NURR1 and LMX1a).

In another aspect of the invention, the cells are somatic cells and they are trans-differentiated by non-viral transfection of one or more transcription factors. Preferably, the somatic cells are fibroblasts. The somatic cells may be derived from a vertebrate. Preferably, the cells are derived from a mammal, such as a mouse. More preferably, the somatic cells are derived from a human. The human may have Parkinson's disease.

In a preferred embodiment of the invention, the hierarchical secondary structures are selected from the group comprising gratings, pillars, columns and lens.

The use of a substrate comprising gratings or hierarchical gratings during transdifferentiation of somatic cells increase the proportion of cells that are differentiated into neurons. For example, as shown herein, the proportion of fibroblasts that can be transdifferentiated into TUJI+ cells is enhanced if they are plated onto gratings, particularly advantageously on hierarchical gratings when compared to those plated onto unpatterned substrate. Preferably, hierarchical gratings, of size 2 pm with 250 nm perpendicular gratings, such as those shown in Figure 5 herein may be used.

Somatic cells that have been transdifferentiated into neurons on a patterned substrate as described supra may be then subjected to a second stage of differentiation on a second patterned substrate as described herein. In a preferred embodiment, the substrate comprises a biologically inert polymer. The said substrate may be made from a silicone compound such as a silicone oil (polymerized siloxane), an example of which would be polydimethylsiloxane (PDMS). PDMS is the most widely used silicon-based organic polymer, it is optically clear, inert, non-toxic and non-flammable. Moreover, it is viscoelastic in nature, which complements the viscoelastic nature of cells. It does not swell with the addition of aqueous media, so it can be used without deformations. PDMS is a polymer that can change stiffness by simply changing the amount of the curing agent used. This means the invention provides a specified topography that can be used with the flexibility of various stiffness ranges. However, those skilled in the art will appreciate that while PDMS is one of the preferred materials for the manufacture of our substrate other polymers that are chemically inert during the period of differentiation or use and which possess and one or more of the following characteristics, including any combination thereof, may be used: clear, inert, non-toxic, nonflammable, does not swell on exposure to aqueous media, it is an easy material to work, and it can change stiffness by simply changing the way it is made.

In an alternative embodiment of the invention said substrate is made from polystyrene, ideally tissue culture polystyrene (TCPS). The incorporation of nano-topography on tissue culture polystyrene, ideally, but not exclusively, with the help of nano-imprint lithography, allows for the use of this vastly accepted biomaterial in a novel way for the differentiation of multipotent or pluripotent cells and so allows for the generation of, for example, dopaminergic neurons.

According to a preferred embodiment of the invention, the substrate is selected from the group comprising polymethylsiloxane (PDMS) and tissue culture polystyrene (TCPS). According to a preferred embodiment of the invention, the PDMS grating structures are produced by soft lithography and the TCPS grating structures are produced by heat embossing. In another preferred embodiment, the substrate is coated with extracellular matrix proteins prior to seeding. Preferably, the extracellular matrix proteins are selected from the group comprising poly-L-ornithine, fibronectin and laminin. One or more of these matrix proteins may be used for coating the substrate, and the particular repertoire used may depend on the type of cells to be differentiated, or the type of differentiated cells required. For example, as shown herein, mNPCs may be seeded onto substrate coated with laminin for neural induction and differentiation, whereas human iPSCs may be seeded onto substrate coated with laminin, fibronectin and poly- L-ornithine in the two stage method described. As herein disclosed, the amount of laminin used to coat substrates for differentiation of mNPCs may be 5 pg/ml - 50 pg/ml. As herein disclosed, the amount of laminin, fibronectin and poly-L-ornithine used to coat substrates for differentiation of iPSCs may be 5 pg/ml - 50 pg/ml, 5 pg/ml - 50 pg/ml and 33 pg/ml - 100 pg/ml, respectively. Preferably, the amount of laminin, fibronectin and poly-L-ornithine is 20 pg/ml, 20 pg/ml and 33 pg/ml, respectively. It would be understood by the person skilled in the art that the amounts of extracellular matrix proteins to coat the substrate can vary from the stated amounts and still be effective, and determined without undue experimentation. Moreover, it would be understood that additional extracellular matrix proteins may be used with those specifically described herein.

According to a further aspect of the invention there is provided a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, wherein the dimensions of the gratings are 250 nm to 2 pm wide with 250 nm to 2 pm spacing and 250 nm to 2 pm height. Preferably the nano- and/or micro-grating structures are hierarchical gratings. In a preferred embodiment of the substrate, the hierarchical secondary structures are selected from the group comprising gratings, pillars, columns and lens. More preferably the secondary structures are gratings or pillars. More particularly, the secondary structures are gratings.

More preferably, the hierarchical secondary structures are gratings with dimensions of 100 nm to 250 nm wide with 100 nm to 250 nm spacing and 100 nm to 250 nm height. Preferably the hierarchical secondary structure gratings are parallel.

In another preferred embodiment of the substrate, the hierarchical secondary structure gratings are perpendicular to the longitudinal axis of the gratings. Hierarchical gratings of this form have been shown herein to significantly increase the yields of subtype-specific and region-specific dopaminergic neurons compared to unpatterned substrates and gratings without secondary structure. In addition, we have found that the hierarchical gratings promote neuron differentiation and maturation as well as transdifferentiation.

More preferably still, the gratings have an aspect ratio of 1 (grating width: spacing: height of 1 :1 :1).

According to a further aspect of the invention there is provided the use of dopaminergic neurons produced according to any aspect of the invention for the treatment of Parkinson's disease. According to a further aspect of the invention there is provided a method of treatment of Parkinson's disease, comprising administering to a subject in need thereof an efficacious amount of dopaminergic neurons produced according to any aspect of the invention. The subject may be a vertebrate. Preferably the subject is a mammal, such as a mouse. More particularly the subject is a human.

According to a further aspect of the invention there is provided the use of dopaminergic neurons produced according to any aspect of the invention to screen for compounds with activity in ameliorating or treating Parkinson's disease, in vitro testing or drug screening related to Parkinson's disease or diseases related to dopaminergic neurons. According to a further aspect of the invention there is provided a method of testing compounds for activity in ameliorating or treating Parkinson's disease, comprising the steps:

(a) contact dopaminergic neurons obtained according to any any aspect of the invention with the test compound, and

(b) quantitate the level of any one or more from the group comprising dopamine production, TH expression, Lmxla expression, Foxa2 expression PITX3 expression, MAP2 expression,

NURR1 expression, ALDH1a1 expression, SCN1a expression, VMAT2 expression, Pax6 expression and neuron complexity; and/or

(c) measure the electrophysiological profile of said neurons; and

(d) compare the level quantitated in (b) and/or measured in (c) with the level in untreated dopaminergic neurons, wherein a difference in level of dopamine production, TH expression, Lmxla expression, Foxa2 expression PITX3 expression, MAP2 expression, NURR1 expression, ALDH1a1 expression, SCN1a expression, VMAT2 expression, Pax6 expression, neuron complexity and/or electrophysiological profile indicates the compound has Parkinson's disease-ameliorating activity. It would be understood by the skilled person that other markers or characteristics of dopaminergic neuron function may be suitable to quantitate in the aforesaid method of testing.

In a preferred embodiment of any aspect of the invention, the gratings are 2 pm wide with 2 pm spacing and 2 pm height, the hierarchical secondary gratings are 250 nm wide with 250 nm spacing and 250 nm height and are perpendicular to the longitudinal axis of said gratings and the pillars are 1 pm. EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001 ). Materials and Methods

Fabrication of MARC chip

The Multi-Architecture (MARC) chip is a versatile customizable topography microarray with a size of 2.2 cm ^χ 2.2 cm which can incorporate different topographies [Moe AAK, et al., Small 8: 3050-3061 (2012)]. Each of the different topographies has a field area of approximately 4 mm². The MARC chip used in this study consists of 8 distinct surface topographies of 6 ^χ 6 array in duplicates and an unpatterned polycarbonate film, serving as the unpatterned control field. The dimensions, construction and arrangement of the MARC chip are presented in Table 1 and Figure 1. In the texts henceforth, we would use the codes labelled in Table 1 when referring to the topographies. Fabrication and replication of PDMS and TCPS substrates

Polydimethylsiloxane (PDMS) substrates were fabricated using the Sylgard 184 Silicone Elastomer kit (Dow Corning, Michigan, USA) via soft lithography. The silicon molds used consist of the 18-pattern MARC chip with the patterns as listed in Table 1. The Sylgard 184 polymer (PDMS) was mixed with the accompanying curing agent in a 10:1 ratio and desiccated under vacuum for 30 min. The mixture was then poured into the silicon molds or unpatterned culture dishes, desiccated for another 30 min and placed into a 70°C oven to cure for 2 hours before demolding. On the other hand, the tissue culture polystyrene (TCPS) replicas were prepared by heat embossing.

TABLE 1 : List of multi-architectural patterns on the MARC chip replicas used in the murine neural progenitor cell (mNCP) culture.

Preparation of substrates for murine neural progenitor cell (mNPC) culture The PDMS or TCPS substrates were cut to fit into the culture plates and air-plasma treated (Harrick Plasma, Ithaca, NY, USA) for 120 seconds at 29.6 W. After washing in 100% ethanol, the substrates were placed under ultraviolet light for 40 min and coated with 33 pg/ml poly-L- ornithine solution (Sigma-Aldrich, Missouri, USA) overnight. The substrates were then washed twice with sterile water and coated with 20 pg/nnl laminin (Life Technologies, California, USA) overnight for controlled differentiation of mNPC. After each coating, vacuum degassing was applied for a short period to force the extracellular matrix proteins into the patterns. Culture of murine neural progenitor cells (mNPCs)

The mNPCs (passages 12-19) were isolated from the hippocampus of 5-day old mice [20] and maintained on 8 pg/ml of laminin (Life Technologies) in a neural progenitor expansion medium. This medium contained Dulbecco's modified Eagle medium (DMEM)/nutrient mixture F12 (Biological Industries, Israel) in a 1 :1 ratio, 1 * N2 supplements (Life Technologies) and 1 * penicillin-streptomycin (Caisson Biotech, Texas, USA). 20 ng/ml basic fibroblast growth factor (bFGF) (Life Technologies) and 20 ng/ml epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN, USA) were supplemented daily, with a complete medium change every two days. Upon achieving 80-90% confluence, cell passaging was performed by detaching with Accutase (Innovative Cell Technologies Inc, California, USA) and incubating at 37°C for 3 min. The cell suspension was centrifuged at 1 100 rpm for 5 min and seeded onto laminin-coated plates at an approximately 1 :3 ratio. Primary mNPCs were characterized by their immune- reactivity against nestin and SOX2, and their ability to differentiate into neurons and astrocytes (Figure 2). All procedures and care of animals were in accordance with the local Institutional Animal Care and Use Committee (IACUC) guidelines.

Controlled differentiation of mNPCs into midbrain dopaminergic neurons

The mNPCs were seeded at 15,000 cells per cm² on the PDMS or TCPS substrates in the neural progenitor expansion medium. After allowing attachment of cells for 24 hours, the growth factors were withdrawn to induce differentiation into neuronal lineage. The medium was then changed to neural induction medium containing DMEM/F12, 1 * N2 supplement, 1 * penicillin- streptomycin solution, 5 ng/ml bFGF (Life Technologies) and 1 pg/ml laminin. Half of the medium was replaced every two days. On the 7^th day of culture, the medium was changed to neural patterning medium containing Neurobasal (Life Technologies) and DMEM/F12 in 1 :1 ratio, 1 * B27 supplement (Life Technologies), 0.25* N2 supplement and 1 * penicillin- streptomycin. The medium was supplemented with 100 ng/ml fibroblast growth factor 8 (FGF-8) (Sigma-Aldrich), 200 ng/ml recombinant Sonic Hedgehog (SHH) (R&D Systems) as well as 1 g/ml laminin, and was replaced every two days. Differentiation was halted on the 1 1^th or 19^th day of culture. A graphical representation of the protocol is shown in Figure 3.

Controlled differentiation of human neural progenitor cells on substrate topography

The preparation of patterned substrates was as described above. Human iPSCs or human ESCs derived from a normal subject or a PD patient were cultured using a feeder-free protocol. The pluripotent stem cells were expanded on Matrigel (Life Technologies) coated polystyrene culture plates with mTeSR 1 medium (Stemcell Technologies). Embryoid bodies (EBs) were formed using microwells (AggreWell, Stemcell Technologies), or other possible EB formation methodologies. Typically 1.5 * 10⁶ cells are needed for a single microwell chamber. EB medium (DME /F-12 (24.25ml), Neurobasal medium (24.25ml), 1x N2 supplement, 1x B27 supplement and L-glutamine in 50 ml total) was supplemented with SB431542 (10 μΜ) (Cellagen Technology), Noggin (200 ng/ml) (R&D Systems), SHH-C24II (200 ng/ml) (R&D Systems), CT99021 (0.8 μΜ) (Cellagen Technology) and Y-27632 (10 μΜ) (Tocris Bioscience) to each well of the microwell chambers that will be used. The EBs were incubated at 37°C with 5% C0₂ and 95% humidity for 4 days with a daily medium change. Aggregates formed were dislodged by carefully drawing up the medium and firmly pipette it back into the middle and around the wells. Dissociated EBs cells were seeded on 2 pm ridge by 2 μητι space by 2 pm depth grating pattern or unpatterned controls with poly-L- Ornithine (33 pg/ml) (Sigma-Aldrich), fibronectin (20 pg/ml) (Biological Industries) and laminin (20 μg/ml) (Life Technologies) coating, for 7 days in supplemented media with dual SMAD inhibitor (SB431542 (10 μΜ) (Cellagen Technology), Noggin (200 ng/ml) (R&D Systems), SHH- C24II (200 ng/ml) (R&D Systems), CT99021 (0.8 μΜ) (Cellagen Technology) (Chamber S et al. Nat Biotechnol. 2009 Mar; 27(3): 275-280). Daily supplemented media changes were performed for the next 4 days followed by media change without supplements for another 2 days.

Terminal differentiation to dopaminergic neurons on substrate topography

The neural progenitor cells were then detached using Accutase (Stem Cell Technologies) and re-plated on either 2 pm ridge by 2 pm space by 2 pm depth grating pattern, 1 pm pillars or unpatterned controls with poly-L-Ornithine (33 pg/ml) (Sigma-Aldrich), fibronectin (20 pg/ml) (Biological Industries) and laminin (20 pg/ml) (Life Technologies) coating. Daily media change was performed in the presence of BDNF (20 ng/ml) (Life Technologies) / GDNF (10 ng/ml) (Life Technologies) / ascorbic acid (200 pM) (Sigma-Aldrich) for 3 days followed by the addition of DAPT (2.5 μΜ) (Tocris Bioscience) for at least another 7 days (Figure 4). Differentiation can be terminated starting from day 21 for downstream applications. Non-viral direct transdifferentiation of somatic cells to neuronal cells

The preparation of patterned substrates was described above. Molecular cloning and plasmid purification.

As published in Adler A and Grigsby C et al., (Molecular Therapy Nucleic Acids 1 : e32; doi:10.1038/mtna.2012.25 (2012), pmax-Brn2 (4,154 bp), pmax-AscH (3,497 bp), and pmax- Mytl l (6,359 bp), which express the mouse transcription factors Brn2, AscH , and Mytl l, respectively, under control of the CMV promoter, were generated by first excising the GFP coding sequence from pmax-GFP (3486 bp; Amaxa, Cologne, Germany) with Sacl digestion, blunting by DNA polymerase I Klenow fragment, Nhel-HF digestion (NEB, Ipswich, MA), and gel extraction (QIAquick Gel Extraction Kit; QIAGEN, Hilden, Germany).

Then, Brn2, AscH , and Mytl l inserts were prepared by digestion of the lentiviral vectors Tet-O- FUW-Brn2 (Addgene 27151 ), Tet-0-FUW-Ascl1 (Addgene 27150), and Tet-0-FUW-Myt11 (Addgene 27152) with EcoRV and Nhel-HF (NEB) and gel extraction, and were subsequently ligated into the empty pmax vector with T4 DNA ligase (NEB). When used together, these three plasmids are abbreviated as pmax-BAM. pUN01-mAscl1 (3,892 bp; InvivoGen, San Diego, CA) and pUN01-mMyt1 lb (6,744 bp; InvivoGen), expressing mouse AscH and Mytl l under control of the EF1a/HTLV promoter, were used in conjunction with pmax-Brn2, and when used together are abbreviated as pUNO-AM/pmax-B. Plasmids were propagated in Escherichia coli DH5a (Invitrogen, Carlsbad, CA) and purified with EndoFree Plasmid Mega and Maxi kits (QIAGEN). Plasmid DNA concentrations were quantified by measurement of absorbance at 260 nm with a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA). Non-viral transfection

While the Brn2, AscH and Mytl l plasmid could be transfected to the fibroblast using commercially available liposomal vehicle such as lipofectamine 2000 (Life Technologies), in this study, a polymeric vehicle was used.

Poly(CBA-ABOL) synthesis and polyplex formation. Poly(CBA-ABOL) was synthesized by Michael polyaddition of 3.67 g N,N- cystaminebisacrylamide (CBA) (Polysciences, Warrington, PA) and 1.26 g 4-amino-1-butanol (ABOL) (Sigma-Aldrich, St Louis, MO) as described by Lin et al., (Bioconjug Chem 18: 138-145 (2007)) and Adler A and Grigsby C et al., (Molecular Therapy Nucleic Acids 1 : e32; doi:10.1038/mtna.2012.25 (2012)). The reaction product was purified by dialysis (3.5 kDa cutoff) in acidic deionized water (pH 4) and then lyophilized. The polymer was collected in its HCI-salt form (1.63 g, 33% yield). p(CBA-ABOL)/DNA nanocomplexes (polyplexes) were synthesized at a polymer:DNA mass ratio of 45:1 , based on previous studies. Polyplexes were prepared by adding a HEPES buffer solution (20 mmol/l HEPES, 5 wt % glucose, pH 7.4) of p(CBA-ABOL) (900 pg/ml) to a HEPES buffer solution (20 mmol/l HEPES, 5 wt % glucose, pH 7.4) of plasmid DNA (75 pg/ml), followed immediately by vortexing for 20 seconds. Cell culture and transfection.

A total of 40,000 cells (80,000 cells) passage 6-8 PMEF-HLs (Millipore) were seeded per well in 24-well TCPS plates (BD, Franklin Lakes, NJ) at 37 °C and 5% C0₂ in complete PMEF medium: Dulbecco's Modified Eagle's Medium with 4.5 g/l glucose (GIBCO 11960-044) (Invitrogen), 10% FBS (Atlanta Biologicals), 25 pg ml^"1 gentamicin (Invitrogen), and 1 * GlutaMAX, nonessential amino acids, sodium pyruvate, and β-mercaptoethanol (Invitrogen). In most experiments, poly- D-lysine/laminin-coated PDMS with 2 Mm gratings, hierarchical gratings, or unpatterned PDMS were tested; as control samples glass coverslips, or bottom of TCPS wells were tested. Twenty- four hours after seeding, PMEFs were transfected with pmax-BAM or pUNO-AM/pmax-B plasmid cocktails for induced neuronal transdifferentiation. BAM factor plasmids were delivered at an equimolar ratio in all cases. A 2:1 ratio of Lipofectamine 2000 (Invitrogen) volume (μΙ) to DNA mass (pg) was used for flow cytometry experiments. All transfections were carried out in serum- and antibiotic-free OptiMEM (Invitrogen). OptiMEM was replaced with complete PMEF medium 4 hours after the onset of transfection. Serial transfections for neuronal transdifferentiation were performed in a similar protocol at day 3 and day 5. Forty-eight hours after the final transfection (which was at day 5) was completed, PMEF medium was replaced with N3 neural induction medium containing: DMEM/F-12 (Invitrogen), 25 pg ml^"1 bovine insulin (Gemini Bio-Products, West Sacramento, CA), 50 pg ml^-1 human apo-transferrin, 30 nmol/l sodium selenite, 20 nmol/l progesterone, 100 pmol/l putrescine (Sigma-Aldrich), 10 ng ml^"1 human bFGF2 (Stemgent, Cambridge, MA), and 25 pg mf¹ gentamicin (Invitrogen). Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde for 15 min and subsequently blocked with 10% goat serum (Life Technologies) or donkey serum (Merck Millipore, Germany) and permeabilized with 0.1% Triton-X for 60 min. Primary antibodies were diluted in 1% goat or donkey serum and incubated overnight at 4°C followed by overnight incubation with the appropriate secondary antibodies at 4^°C. The secondary antibodies used were goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa Fluor 488, goat anti-mouse Alexa Fluor 546, goat anti-rabbit Alexa Fluor 546 (Life Technologies), donkey anti-goat FITC or goat anti-rat FITC (Abeam, England, UK) at the concentration of 1 :500. The cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min before mounting with Prolong Gold anti-fade mounting media (Life Technologies). The images of the cells were taken with inverted fluorescence DMIRM microscope (Leica, Germany) or confocal microscope (Leica SP5, Germany). Table 2. List of antibodies for staining

Analysis of gene expression

Total RNA was extracted from the cells using the RNeasy Mini Kit according to manufacturer's instructions (Qiagen, Germany). The RNA quantity and concentration were determined using Nanodrop ND-1000. cDNA synthesis was performed with 100 ng of total RNA using Superscript VILO cDNA Synthesis Kit (Life Technologies) and the reverse transcription was carried out at 42°C for 90 min. Real-time quantitative PCR was carried out using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems). TaqMan probes (Applied Biosystems) were used to analyze the following genes of interest: TH (Assay ID: Mm00447557_m), Pitx3 (assay ID: Mm01194166_g1 ) and GAPDH (assay ID: Mm99999915_g1 ). Relative quantification was calculated using the AACt method which was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene.

Measurement of neuron elongation and alignment The elongation and alignment of the neurons were calculated from the TUJ1 + population. The elongation parameter E is calculated as the ratio of the long axis over the short axis on an approximated ellipse [Wong S, et al., Biomech Model Mechanobiol 13: 27-39 (2013)] by using ImageJ (NIH). The percentage of aligned cells was obtained by looking at the number of cells that had E > 3 and angle of the long axis and the grating < 15. For each sample, over 200 cells were measured.

Scanning electron microscopy (SEM) of the substrate replicas

Scanning electron microscopy (SEM) of the replicas was carried out at various magnifications in high vacuum after sputter-coating the samples with platinum at 30 mA for 30 sec. The protein- coated substrates were firstly fixed with 2% gluteraldehyde in 0.1 M sodium cacoylate and 3 mM CaCI₂, followed by serial dehydration in ethanol and exposure to increasing concentrations of hexamethyldisilazane (HMDS). Lastly, the substrates were dried overnight before SEM imaging.

Statistical analysis

Data was presented as the mean and standard error of mean where appropriate. All experiments were carried out in duplicates or more. Data analysis was performed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's test for multiple comparisons of at least three groups or unpaired Student's t-test for two groups using Prism 6.0 (Graphpad Software Inc.). p values of < 0.05 were considered statistically significant.

RESULTS

Characterization of PDMS and TCPS replicas Fidelity of replication with and without extracellular matrix coating was checked using a scanning electron microscope (SEM). The 2*2x2 pm, 250x250*250 nm, and hierarchical grating patterns of PDMS or TCPS were replicated with good fidelity (Figure 5 and Figure 6). Unpatterned controls replicated from PDMS and TCPS were also examined using SEM to confirm the absence of topographical features. Effect of MARC topographical cues on differentiation of neural progenitor cells

In order to identify the topographies that would lead to increased midbrain dopaminergic neuron yield, mNPCs were allowed to differentiate into neurons on PDMS replicas of 18-pattern MARC for 19 days. After this 19 day period screening was conducted to identify the percentage of TUJ1+ (β-lll-tubulin) (immature neurons) and GFAP+ (glial fibrillary acidic protein) (astrocytes) cells (Figure 7A). The 18-pattern MARC chip yielded three topographies with significantly higher numbers of TUJ1+ cells compared to the unpatterned control. These were the 2 μιη gratings with 2 pm spacing and 2 pm height (2x2*2 pm gratings or Grating 1 ), the 250 nm gratings with 250 nm spacing and 250 nm height (250*250x250 nm gratings or Grating 5, and the 2*2 2 pm gratings with hierarchical perpendicular 250x250*250 nm grating in the groove (2*2*2 pm gratings 250*250*250 nm lines or Hierarchical 6). These gratings yielded 47.6 ± 7.0 % (p < 0.05), 53.0 ± 2.0 % (p<0.01 ) and 45.1 ± 12.5 % (p < 0.05) of total cells, respectively.

Two topographies generated high neuron-to-astrocyte (N:A) ratios as compared to the unpatterned control. These were 250*250*250 nm gratings (Grating 5) and Hierarchical 6 which produced N:A ratios of 1.9 ± 0.3 (p < 0.05) and 2.5 ± 0.9 (p < 0.01), respectively, compared to an N:A of 0.4 ± 0.1 on the unpatterned control. On the other hand, the 2*2*2 pm gratings with hierarchical parallel 250*250*250 nm grating in the groove (2*2*2 pm gratings || 250*250*250 nm lines or Hierarchical 7) produced significantly higher GFAP+ cells than TUJ1+ cells (p < 0.01 ). Next, we performed another screen to analyze the percentage of tyrosine hydroxylase (TH+) (dopaminergic-committed neuron) cells after 19 days of differentiation (Figure 7B and Table 3). Five topographical patterns showed significantly higher numbers of TH expressing cells than unpatterned control. These were 2*2*2 pm gratings (Grating 1 ) (26.3 ± 3.6 %, p < 0.001 ), 250*250*250 nm gratings (Grating 5) (25.9 ± 3.8 %, p < 0.001), Hierarchical 6 (34.1 ± 5.2 %, p < 0.001), Hierarchical 7 (24.1 ± 0.5 %, p < 0.01), and Microlens 16, which is a convex microlens of 1.8 pm diameter with 2 pm pitch (18.9 ± 3.0 %, p < 0.05). To examine TH+ expression in the TUJ1+ immature neuronal sub-population, TH+ TUJ1+ cells were quantified (Figure 7C). Among the topographies, only Microlens 15, which has a 1 pm pitch concave microlens, produced a lower proportion of TH+ expressing cells than the unpatterned control (p < 0.05). No significant differences in the proportion of TH+ expressing cells among the TUJ1+ population was observed on other topographies compared to the unpatterned control. Table 3: Comparison between topographical patterns which have significant differences in percentage of TH+ cells.

Compared Patterns Significance P-value

Unpatterned vs 1 ** <0.01

Un patterned vs 5 ** <0.01

Unpatterned vs 6 **** <0.0001

Unpatterned vs 7 **

<0.01

1 vs 9 * <0.05

1 vs 10 * <0.05

1 vs 13 *** <0.001

1 vs 15 *** <0.001

2 vs 6 ** <0.01

3 vs 6 ** <0.01

4 vs 6 <0.05

5 vs 9 *

<0.05

5 vs 10 * <0.05

5 vs 13 ** <0.01

5 vs 15 *** <0.001

6 s 8 ** <0.01

6 vs 9 **** <0.0001

vs 10 **** <0.0001

vs 11 *** <0.001

vs 12 ** <0.01 Compared Patterns Significance P-value

6 s 13 **** <0.0001

6 vs 14 *** <0.001

6 vs 15 ****

<0.0001

6 s 16 *

<0.05

6 vs 17 *

<0.05

6 vs 18 * <0.05

7 vs 13 **

<0.01

7 vs 15 ** O.01

Analysis of differentiation of neural progenitor cells on single pattern PDMS substrates

From the results of the 18-pattern MARC chip, we selected and focused on two single-imprinted patterns, 2*2*2 pm gratings and 250x250*250 nm gratings, as well as the double-imprinted pattern, Hierarchical 6, for closer examination of mNPCs differentiation.

We firstly performed quantitative PCR to analyze the gene expression of TH and Pitx3 on the patterned substrates after 11 days and 19 days of differentiation, respectively (Figure 8). The mNPCs differentiated on both grating patterns with the hierarchical grating being associated with a higher expression of TH and Pitx3 than unpatterned substrates. Among the three patterns, the cells that differentiated on Hierarchical 6 had the highest expression of TH, while those that had differentiated on 2*2*2 pm gratings had the highest expression of Pitx3.

The presence of proteins involved in the maturation and function of dopaminergic neurons were further evaluated by immunofluorescence staining (Figure 9). Neurons were stained with nuclear receptor related protein 1 (NURR1 ), sodium channel SCN1a, vesicular monoamine transporter 2 (VMAT2), aldehyde dehydrogenase 1 family member A1 (ALDH1a1 ), and pituitary homeobox 3 (PITX3). The presence of NURR1 indicated that the neurons generated were likely to be midbrain dopaminergic neurons (Figure 9). These neurons also possessed the sodium channels SCN1a and VMAT2, which are membrane proteins that transport dopamine into synaptic vesicles (Figure 9). These markers were robustly expressed in both the single-imprinted (2*2*2 μιτι gratings and 250x250x250 nm gratings) and the double-imprinted (Hierarchical 6) substrates.

Image analyses performed on the single-imprinted patterned substrates showed that the percentage of TH+ (dopaminergic-committed) cells were significantly higher on 2χ2χ2 pm gratings (25.0 ± 0.3 % of total cells; p < 0.05) compared to the unpatterned control (Figure 10A). This result is consistent with the data from the MARC chip. After 11 days of differentiation, the percentage of microtubule-associated protein 2 (MAP2+, marker for mature neurons) cells was highest on 2χ2χ2 pm gratings (23.3 ± 2.0 %; p < 0.001 ) amongst the three substrates (Figure 10B). 250x250x250 nm gratings also produced significantly more MAP2+ cells (20.4 ± 1.3 %) than the unpatterned control (10.1 ± 1.1 %; p < 0.01 ).

Differentiation on both grating substrates produced more cells that expressed ALDH1a1 (a marker for dopaminergic-committed population) than the unpatterned control, with 40.6 ± 1.1 % (p < 0.01 ) on 2x2x2 pm gratings and 30.6 ± 4.0 % (p < 0.05) on 250x250x250 nm gratings (Figure 10C). Compared to the unpatterned control, we also observed more cells expressing the LIM homeobox transcription factor 1 alpha (LMX1 a, a marker for dopaminergic-committed population) on 2χ2 2 pm gratings (37.3 ± 2.7 %; p < 0.001 ) and 250x250x250 nm gratings (26.3 ± 2.2 %; p < 0.001 ) (Figure 10D). 2 2^χ2 pm gratings also produced significantly more pituitary homeobox 3 (PITX3+, a marker for substantia nigra dopaminergic neurons) cells (18.3 ± 1.4 %; p < 0.01) than the unpatterned control (Figure 10D). All TH+ cells were positive for ALDH1a1 and LMX1 a, and about half of TH+ cells expressed PITX3.

Analysis of elongation and alignment of neurons on patterned PDMS substrates

Longer neurites are correlated with the maturation of the differentiated cells, and thus neuron elongation was quantified using the E value. Compared to the unpatterned control, the neurons were significantly more elongated when differentiated on 2 2χ2 pm gratings and Hierarchical 6 with an E value of 10.4 ± 0.8 (p < 0.0001 ) and 8.6 ± 0.7 (p < 0.0001 ), respectively (Figure 11A and Figure 11 B). In addition, neurite alignment also influences axon guidance towards successful synaptic circuitry. Therefore, the alignment of neurons was compared between the grating patterns. We found that the alignment on 2^χ2 2 pm gratings and Hierarchical 6 were more extensive than 250x250x250 nm gratings, with 65.9 % and 93.1 % of the cells aligned along the grating axis, respectively (Figure 1 1A and Figure 1 1C). In comparison, only 18.8 % of the cells were aligned along the grating axis on 250x250x250 nm gratings. Analysis of differentiation of neural progenitor cells on TCPS substrates

We also differentiated mNPCs on TCPS substrates with 2x2*2 μιτι gratings and 250x250x250 nm gratings to determine if the effects of these gratings on the mNPC differentiation apply to different substrate rigidities. Stiffness measurement by atomic force microscopy (AFM) showed the elastic modulus of PDMS (10:1 base/curing ratio) and TCPS to be 3.27 ± 0.133 MPa and 2149 ± 285 MPa respectively, indicating a considerable difference in stiffness between these two substrates (Figure 12). The percentage of immature neurons and astrocytes (expressing TUJ1 and GFAP, respectively), and the percentage of mature neurons (expressing MAP2) were analyzed after 1 1 days of differentiation on the TCPS substrates (Figure 13A). 2 2χ2 μηη gratings generated a significantly higher N:A ratio (0.73 ± 0.05, p < 0.05) than the unpatterned control (0.45 ± 0.04) (Figure 13B). A significantly higher percentage of MAP2+ cells was observed on both 2χ2χ2 pm gratings (45.1 ± 0.9 %; p < 0.01 ) and 250*250x250 nm gratings (42.2 ± 2.0 %; p < 0.05) compared to the unpatterned control (Figure 13C).

Human iPSC-derived neurons display increased neuronal complexity and branching when grown on a combination of PDMS substrates comprising gratings and pillars

The two stage differentiation method as shown in Figure 4A, using embryoid bodies produced from human iPSCs, produced expanded neural progenitor cells when cultured on substrates with gratings in stage 1 (Figure 4B) and terminally differentiated midbrain dopaminergic neurons on substrates with pillars in stage 2 (Figure 4C). Figure 4B shows bright field images of embryoid bodies in microwells and on patterned substrates at stage 1. Cells were observed to be aligned on the grating axis (arrow) at stage 1 . Figure 4C shows immunofluorescence images of cells expressing tyrosine hydroxylase (TH), a dopaminergic neuron marker at stage 2 on patterned substrates. In particular, when the cells are grown on substrates with pillars in stage 2, they display a highly differentiated morphology with significant neurite branching. Immunostaining for dopaminergic neuronal markers (TUJ1 , MAP2, PITX3, LMX1 a and FOXA2) on human induced pluripotent stem cell (iPSC)-derived cells cultured on unpatterned, gratings, and pillars PDMS substrates in stage 2 showed robust expression when on patterned substrates (Figure 14).

Quantification and data analysis of TUJ1 -positive (neuronal) cells after 21 day differentiation on unpatterned, gratings and pillars PDMS substrates in stage 2 showed that both gratings and pillars produced higher percentages of TUJ1 -positive cells than unpatterned (Figure 15A). Quantification and data analysis of TH-positive (dopaminergic) cells after 21 day differentiation on unpatterned, gratings and pillars PDMS substrates in stage 2 showed differences in percentage TH+ cells (Figure 15B) which was more pronounced when the data was normalised to the unpatterned substrates; significant with respect to gratings (Figure 15C). Both gratings and pillars in stage 2 produced an increased ratio of TH+:TUJI+ cells over unpatterned (Figure 15D). Quantification and data analysis of FOXA2-positive (midbrain DA) cells on unpatterned, gratings, and pillars PDMS substrates in stage 2 showed that substrates with gratings and pillars had significantly more cells expressing FOXA2 than unpatterned substrates (Figure 15E).

Morphology and synaptic activity of TH+ neurons is more mature on stage 2 pillars substrates after 21 day differentiation.

Figure 16A shows representative morphology of cells on the three substrates, whereby cells on gratings were aligned on the gratings axis. The average neurite length per neuron was longer for cells grown on gratings in stage 2 than for those grown on pillars substrates (Figure 16B), whereas the number of branch points (Figure 16C), neuronal complexity (Figure 16D) and number of terminals per neuron (Figure 16E), were significantly greater for neurons grown on pillars substrates compared to gratings (p<0.01). In addition, the percentage of cells with a spontaneous postsynaptic current was higher in TH +ve neurons differentiated on pillars (Figure 16F) and can be detected as early as 4 weeks post differentiation (Figure 16G; arrowhead).

Repetitive action potential capability of TH+ neurons is more pronounced on stage 2 pillars substrates after 5 weeks post differentiation.

Figure 17 shows representative tracings of firing patterns on gratings at 4 weeks and 5 weeks post differentiation. (A) Cells were capable of firing a repetitive action potential as a response to current injection as early as 4 weeks post differentiation. Spontaneous synaptic activity (arrowhead) was recorded as early as 4 weeks post differentiation and is more frequently observed at 5 weeks. (B) The percentage of differentiated neurons capable of repetitive firing at 5 weeks post differentiation was greater for cells on pillars than cells grown in stage 2 on unpatterned substrates or on gratings. Five to nine neurons were recorded from three experiments per condition. Parkinson's disease patient-derived iPSCs can be successfully differentiated to midbrain dopaminergic neurons on patterned substrates.

Figure 18 shows dopaminergic neuronal markers (TUJ1 , TH and LMX1a) are expressed on human iPSC-derived cells from a Parkinson's disease patient with a LRRK2 mutation when grown on gratings, and pillars PDMS substrates. The robust expression indicates that patient- derived iPS cells can be successfully differentiated to midbrain dopaminergic neurons on patterned substrates.

Figure 19 (A) shows representative tracings of firing patterns on gratings at 4 weeks and 5 weeks post differentiation. Neurons were capable of firing a repetitive action potential as a response to current injection as early as 4 weeks post differentiation. (B) The data for the percentage of differentiated cells capable of repetitive firing at 5 weeks post differentiation showed that more neurons differentiated on gratings and pillars were capable of repetitive firing than on unpatterned substrates.

Non-viral direct transdifferentiation of somatic cells to neuronal cells As previously described in Adler A and Grigsby C et al., (Molecular Therapy Nucleic Acids 1 : e32; doi:10.1038/mtna.2012.25 (2012)), somatic cells such as fibroblasts can be transdifferentiated by using plasmids encoding neuronal transcription factors (Brn2, Ascl1 , Mytl l) with a carrier such as bio reducible linear poly(amido amine). With repeated dosing, transdifferentiation can be achieved at low toxicity. As reported in Adler et al., (Molecular Therapy Nucleic Acids, 2012), serial 0.5pg /cm² doses of reprogramming factors delivered at 48-hour intervals (at day 1 , day 3 and day 5) produced up to 7.6% Tuj1+ cells after culturing in N3 medium for 10 days. While an increase of Tuj1+ cells were observed on the 2 pm gratings (to 9%), a significant increase of Tuj1 + cells (to 14%) can be derived on the hierarchical platform shown in Figure 5. DISCUSSION

In our studies, we have demonstrated the importance of topographical cues in promoting neuronal differentiation by adopting three different differentiation methods to derive neurons. Importantly, a 2 stage differentiation involving different topographical cues may lead to greater morphological and functional differentiation of dopaminergic neurons. Based on a systematic screening of the MARC chip under the same differentiation conditions, three topographies were first identified that improved the yield of dopaminergic-committed cells from murine neuronal progenitor cells (mNPCs). These were 2*2x2 pm gratings, 250*250x250 nm gratings, and Hierarchical 6. Between the two single-imprinted grating patterns, the micron- width 2x2x2 pm gratings produced greater yields of subtype-specific and region-specific dopaminergic neurons, as well as longer neurites aligned along the grating axis. Based on this analysis, we found that 2^χ2^χ2 pm gratings is the most effective topography at inducing differentiation of mNPCs to dopaminergic neurons in synergy with the appropriate biochemical cues. The double-imprinted grating pattern, Hierarchical 6, also performed efficiently and was also comparable to 2*2x2 pm gratings.

Substrate topographies were previously demonstrated to affect neuronal specification and differentiation propensity during neural stem/progenitor cell differentiation. [Moe AAK, et al., Small 8: 3050-3061 (2012); Yang K, et al., ACS Applied Materials & Interfaces 5: 10529-10540 (2013)]. Most studies however have focused on comparing single topographical patterns with the same dimension and shape. Considering different topographical patterns play different roles in neural stem cell differentiation, we proposed the importance of identifying specific patterns to improve derivation efficiency of subtype specific neurons. In line with this, the differentiation of mNPC to dopaminergic neuron subtype (TH+, ALDH1a1 +, PITX3+, LMX1a+) was examined on a variety of patterned structures on the MARC chip. In comparison to a related study by Moe et al. [Moe AAK, et al., Small 8: 3050-3061 (2012)], the media composition and the protocol used here for deriving subtype-specific neurons may follow a different neural induction pathway, which is further guided by topographical cues. This led to a different proportion of neuronal cells (TUJ1 + cells) but a consistent yield of astrocytes (GFAP+ cells) on the different patterns, thus reflecting different outcomes in these studies. The aspect ratio of the gratings was shown to be an important determinant of mesenchymal stem cell elongation and alignment [Wong S, et al., Biomech Model Mechanobiol 13: 27-39 (2013); Chua J, et al., Biomaterials 35(27): 7750-61 (2014)]. Therefore, the spacing and height of the gratings play important roles in the topographical construct that determines cell behaviour and cell fate. By comparing grating topographies on the MARC chip, we found that 2x2*2 pm gratings and 250*250*250 nm gratings gave the highest yield of TUJ1 + and TH+ cells, which are attributed to their grating aspect ratio of 1 (grating width : spacing : height of 1 :1 :1 ). By decreasing this aspect ratio (Gratings 2-4) (Figures 7A and 7B) the extent of neural induction, and dopaminergic subtype specification, was reduced, suggesting that the aspect ratio of these patterns is critical in ensuring optimal lineage guidance.

From the MARC chip screening, we identified that Hierarchical 6 yielded significantly more TUJ1+ and TH+ cells, but yielded the least number of astrocytes, compared to the unpatterned control. We focused on single-imprinted patterns (2*2x2 pm gratings and 250x250x250 nm gratings) for single-pattern analysis due to their practical applicability for scaled-up fabrication and manufacturing. However, double-imprinted patterns such as the hierarchical gratings also provided similar enhancement, albeit via potentially distinct mechanisms. Indeed, Hierarchical 6, which is a combination of two grating patterns, showed higher gene expression of TH and Pitx3 and fewer astrocytes than the unpatterned control, suggesting that double-imprinted patterns also provide a significant advantage in the derivation of midbrain dopaminergic neurons. Most methods of DA neuron derivation can require up to 80 days of cell culture and rely heavily on the constant input of expensive biochemical components. The hierarchical gratings can produce a purer population of neurons four times faster and through the reduction of cell culture duration can cut production cost drastically.

Compared to the unpatterned control, there was no significant difference in the proportion of cells showing dopaminergic neuron commitment (TH+ cells) among the population of TUJ1 + cells, and this was regardless of the substrate topography presented. One exception to this was Microlens 15, which seems to have a negative effect on the dopaminergic subtype specification (Figure 7C). Topographical gratings enhance neuronal differentiation and indeed, an increase in the neuronal population was still observed. This indicates a synergistic effect between chemical induction and topographical cues, and this may explain the increase in TH-expressing cells on the topography.

Key midbrain dopamine regulators PITX3 and LMX1a, which play important roles during neurogenesis and neurotransmitter phenotype determination of dopaminergic neurons [Hong S, et al., Stem Cells Dev 23: 477-487 (2014); Cai J, et al., Stem Cells 27: 220-229 (2009); Nunes I, et al., Proc Natl Acad Sci USA 100: 4245-4250 (2003)], were highly expressed in cells differentiated on 2*2x2 pm gratings. Transduction of NURR1 and PITX3 was shown to promote embryonic stem cell differentiation into a dopaminergic phenotype [Martinat C, et al., Proc Natl >4cad Sci USA 103: 2874-2879 (2006)]. Importantly, the expression of ALDH1a1 , which is the transcriptional target of PITX3, was also increased on this topography [Jacobs F, et al., Development 134: 2673-2684 (2007)]. As ALDH1a1 is expressed specifically in dopaminergic neurons in the ventral midbrain and is decreased in Parkinson's disease patients [Gaiter D, et al., Neurobiol Dis 14: 637-647 (2003); Anderson D, et al., Brain Res 1408: 81-87 (2011 )], these results suggest that the dopaminergic phenotype derived here is physiologically relevant as cellular models and can be favourably specified. Late stage markers of dopaminergic neurons including NURR1 , voltage gated sodium channels (SCN1a), and vesicular monoamine transporter 2 (VMAT2) were also expressed in the neurons. This further supports the successful midbrain dopaminergic neuron subtype specification on the topographies. Sodium channel development suggests that the neurons possess the components required for the successful firing of action potentials [Caputi L, et al., Synapse 48: 123-130 (2003)]. Furthermore, 2*2*2 pm gratings produced the highest percentage of MAP2+ cells, as well as dopaminergic precursor markers, which indicate greater differentiation potential into mature neurons with functional components.

Substrate stiffness has been shown to affect differentiation of stem cells towards specific lineages. Engler et al. showed that mesenchymal stem cells were neurogenic when differentiated on substrates of comparable stiffness to the brain tissue (-700 Pa), but myogenic and osteogenic on stiffer substrates [Engler A, et al., Cell 126: 677-689 (2006)]. Similarly, neural stem cell differentiation was also affected by the substrate stiffness, where softer substrates favour neural differentiation and stiffer substrates favour oligodendrocyte differentiation [Leipzig N, Shoichet . Biomaterials 30: 6867-6878 (2009)]. Here, we showed that despite differentiation on stiffer substrates such as TCPS, topography-enhanced differentiation was observed for the mNPCs. The influence of these patterns on neuronal differentiation may thus play a larger role than substrate stiffness effects, which have an inevitable influence to a certain extent and have to be considered along with the chosen pattern.

Other methods to derive midbrain dopaminergic neurons from pluripotent stem cells have obtained higher yields than was observed for our method of mNPC differentiation [Kriks S, et al., Nature 480: 547-551 (2011 ); Perrier A, et al., Proc Natl Acad Sci USA 101 : 12543-12548 (2004)]. However, these studies involved 80 days of culture. Hence, the lower dopaminergic neuron yield in mNPC differentiation, which was observed after 19 days in our study, may due to a premature time point. In addition, the only ventralising factors used in this study to achieve specification were SHH and FGF8. This is opposed to the usual pluripotent stem cell differentiation protocols, which rely on a high dependence of biochemical input, such as the dual SMAD inhibition pathway. Recent studies have shown that non-mesencephalic neural stem cells are more restricted in their developmental capacity to generate midbrain dopaminergic neurons, suggesting differences in the epigenetic state of these cells in the responsiveness to dopaminergic induction signals [Studer L. Prog Brain Res 200: 243-263 (2012); Roftler R, et al., Neuroscience 170: 417-428 (2010); Mohamed A, et al., J Neurosci Res 90: 529-539 (2012)]. Hence the hippocampal origin of mNPCs may limit their potential in developing into mesencephalic neurons. In particular, the epigenetic differences between the subventricular zone and hippocampal neurons account for differences in miRNA regulation of neurogenesis [Mohamed A, et al., J Neurosci Res 90: 529-539 (2012)].

By using human induced pluripotent stem cells (iPSCs), we have also examined the ability of topographical patterns to influence the differentiation of hPSCs into subtype-specific and regionalized dopaminergic (DA) neurons. We have successfully established a step-by-step method to differentiate human pluripotent stem cells into ventral midbrain DA neurons on topographical patterned substrates. We have tested our method on induced pluripotent stem (iPS) cells to show the robustness of the protocol, on normal subjects as well as patient cell lines. Results indicate that these pluripotent stem cells can be differentiated to the midbrain DA neuronal lineage on the optimized two stage differentiation process with the use of topography. Among the different patterned substrates used in the second stage of differentiation (gratings and pillars) that was tested, gratings showed more midbrain DA neurons that were derived from human pluripotent stem cells, suggesting that gratings may be good for initial lineage commitment. For functionality, midbrain DA neurons that were derived on pillars were better, based on the increased neuronal complexity and branching. The electrophysiology studies also indicated that a larger proportion of cells that were differentiated using pillars substrates were more capable of firing repetitive action potentials. Interestingly and notably, adopting this two stage topography protocol for human induced pluripotent stem cells (iPSC)-derived cells from Parkinson's disease with LRRK2 mutation showed robust expression of Beta-Ill tubulin (TUJ1 ), tyrosine hydroxylase (TH), LIM homeobox transcription factor 1 alpha (LMX1a), indicating patient derived iPS cells can be successfully differentiated to midbrain DA neurons on patterned substrates (Figure 18). Examining the electrophysiology properties of human induced pluripotent stem cell (iPSC)-derived cells from PD patient with LRRK2 mutation on unpatterned, gratings, and pillars PDMS substrates demonstrated that a higher percentage of neurons on gratings and pillars were capable of repetitive firing than on unpatterned substrate (Figure 19), showing that patient-derived iPSCs can be matured into DA neurons. Therefore, the use of gratings for the initial lineage commitment of midbrain DA neurons, followed by pillars for the maturation of DA neuron functionality will allow for an accelerated DA differentiation process, the development of robust cell models that will provide novel insights into mechanisms underlying DA neuronal development and ultimately discover new therapeutic approaches for Parkinson's disease.

Besides using induced pluripotent stem cells to generate patient specific dopaminergic neurons [Lu H, et al., Biomaterials 35: 2816-2826 (2014); Hartfield EM, et al., PloS one 9: e87388 (2014); Soldner F, et al., Cell 136: 964-977 (2009)] and also for pluripotent stem cells to engraft into animal models [Kriks S, et al., Nature 480: 547-551 (2011); Kirkeby A, et al., Cell reports 1 : 703- 714 (2012)], new methods are being optimized for the direct reprogramming of fibroblasts into neurons to circumvent the long tedious differentiation process. However, efficiencies of reprogramming have been low, with approximately 1.5% - 2.9% of adult human fibroblasts being converted using miRNA [Ambasudhan R, et al., Cell Stem Cell 9: 113-118 (2011 )]. Other studies have generated dopaminergic neurons directly from mouse or human fibroblasts with efficiency levels of between 1 -5% for TH+ cells [Caiazzo M, et al., Nature 476: 224-227 (2011 )]. Epigenetic regulation was shown to influence cell fate determination and self-renewal, and epigenetic modifications on neural stem cells could also increase neuronal differentiation. However, reprogrammed ceils showed greater efficiency in generating region-specific neuronal subtypes [Deleidi M, et al., PloS one 6: e19926 (2011)]. As topography influences neuronal subtype derivation, it is speculated that topography also play a role in the epigenetic regulation that determines cell fate decisions. The use of topography in a transient or prolonged manner may well be the next step to improve reprogramming efficiencies, and subsequently, differentiation of stem cells into midbrain dopaminergic neurons.

Several mechanisms have been suggested to facilitate topography-induced enhancement of neuronal differentiation. Mechanosensing of topography through the focal adhesion is essential for topography-induced enhancement in neuronal differentiation [Migliorini E, et al., Biotechnol Bioeng 108: 2736-2746 (2011 ); Teo BKK, et al., ACS Nano 7: 4785-4798 (2013); Frey M, et al., Biophys J 90: 3774-3782 (2006)], while the focal adhesion kinase (FAK) is critical in mechanosensing [Teo BKK, et al. ACS Nano 7: 4785-4798 (2013); Wang H, et al., Proc Natl Acad Sci USA 98: 1 1295-11300 (2001 )]. In the case of gratings, the grooves and gaps provide more contact points for the formation of focal adhesions [Yang K, et al., ACS Applied Materials & Interfaces 5: 10529-10540 (2013)]. We observed that cells sit within the grating groove of the 2 2x2 m gratings, which may allow for the enhanced formation of focal adhesion complexes with increased elongation of neurites. Neurons that are more elongated and aligned to the grating patterns may contribute to the development of successful axon guidance towards successful synaptic circuitry.

Cytoskeletal elongation in cells guided by grating patterns has also been proposed to involve the transfer of tensional force to the nucleus, which influences signal transduction and gene expression, and thus stem cell differentiation [Yim E, et al., Exp Cell Res 313: 1820-1829 (2007); Teo BKK, et al., ACS Nano 7: 4785-4798 (2013)]. Other possible reasons why 2*2*2 pm gratings produced the highest yield of dopaminergic neurons are that the collection of extracellular matrix and growth factors secreted by the cells within the grooves provide an ideal microenvironment for the mNPCs. The enhanced neuronal differentiation, which was observed with increasing grating depth, has also been suggested to be due to the depth-sensing ability of the neurites. This ability is facilitated by filopodia adhesion and neurite bending, both of which have been closely associated with neurite alignment and growth [Chua J, et al., Biomaterials S0142-S9612 (2014)].

CONCLUSION We have shown that micro- and nano-topographies influence dopaminergic subtype specification and differentiation, and can be potentially used to improve derivation efficiencies of other subtype specific neurons and adapted for other clinical uses. As the role of topography in aiding cell fate determination is demonstrated, we have begun to understand the mechanisms that drive topography induced downstream signalling and determination of cell fate. We have successfully established a step-by-step method to differentiate human pluripotent stem cells into ventral midbrain DA neurons on topographical patterned substrates. We have tested our method on induced pluripotent stem (iPS) cells to show the robustness of the protocol, on normal subjects as well as patient cell lines. Results indicate that these pluripotent stem cells can be differentiated to the midbrain DA neuronal lineage on the optimized protocol with the use of topography. In different patterned substrates that we have tested, gratings showed more midbrain DA neurons that were derived from human pluripotent stem cells, suggesting that gratings may be good for initial stage 1 lineage commitment. For functionality, midbrain DA neurons that were derived on pillars in stage 2 were better, based on the increased neuronal complexity and branching. Electrophysiology studies also showed that more cells that were differentiated on pillars in stage 2 were capable of firing repetitive action potentials. Therefore, we suggest the use of gratings for the initial lineage commitment of midbrain DA neurons, followed by pillars for the last push of functionality. These midbrain DA neurons can be translated into a wide range of clinical applications, and also provide novel insights into the mechanisms that underlie dopaminergic neuron development in vivo, which are important in the discovery of new therapeutic approaches for Parkinson's Disease.

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Claims

1. A method of enhancing the differentiation of cells into neurons, wherein the cells are selected from the group muitipotent stem cells, pluripotent stem cells and somatic cells, comprising seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium.

2. The method of claim 1 , further comprising the steps; a) maintaining the seeded cells in medium; b) inducing neuronal lineage phenotype by withdrawal of growth factors and/or inhibitors; c) replacing the medium of b) with neural patterning medium.

3. The method of claim 2 comprising two stages; i) seeding the cells onto a substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, and contacting the cells with medium to induce neuronal lineage commitment, and ii) removing the cells from the substrate of stage i) after a period of contact with said medium and re-seeding the removed cells onto a substrate patterned with nano- and/or micro pillars, and contacting the cells with neural patterning medium.

4. The method of claim 2 or 3, wherein the neural patterning medium in c) comprises Neurobasal and DMEM/F12 in 1 :1 ratio, 1x B27 supplement, 0.25x N2 supplement, 100ng/ml FGF-8, 200ng/ml Sonic Hedgehog and 1 pg/ml laminin or, if the cells are pluripotent stem cells, the medium in c) comprises BDNF (20 ng/ml), GDNF (10 ng/ml) and ascorbic acid (200 μΜ) for 3 days, and then DAPT (2.5 μΜ) is added to the medium from day 4 of stage (ii).

5. The method of any one of claims 1 to 4, wherein the seeded cells are differentiated into dopaminergic neurons.

6. The method of any one of claims 3 to 5, wherein the seeded cells are contacted with the neural patterning medium for at least 5 to 30 days, preferably for at least 7 to 19 days.

7. The method of any one of claims 1 to 3, wherein the cells are somatic cells and they are trans-differentiated by non-viral transfection of one or more transcription factors.

8. The method of claim 7, wherein the somatic cells are fibroblasts.

9. The method of any one of claims 1 to 8, wherein the hierarchical secondary structures are selected from the group comprising gratings, pillars, columns and lens.

10. The method of any one of claims 1 to 9, wherein the dimensions of the gratings are 250 nm to 2 pm wide with 250 nm to 2 pm spacing and 250 nm to 2 pm height, and the hierarchical secondary structures are gratings with dimensions of 100 nm to 250 nm wide with 100 nm to 250 nm spacing and 100 nm to 250 nm height; and the pillars are 0.5 pm to 5 pm diameter with 8 pm to 12 pm pitch.

11. The method of any one of claims 1 to 10, wherein the gratings are parallel.

12. The method of any one of claims 1 to 11 , wherein the hierarchical secondary gratings are perpendicular to the longitudinal axis of the gratings.

13. The method of any one of claims 1 to 12, wherein the gratings have an aspect ratio of 1 (grating width: spacing: height of 1 :1 :1).

14. The method of any one of the previous claims, wherein the substrate is coated with extracellular matrix proteins prior to seeding.

15. The method of claim 14, wherein the extracellular matrix proteins are selected from at least one from the group comprising poly-L-ornithine, fibronectin and laminin.

16. The method of any one of claims 3 to 15, comprising a two stage method of enhancing the differentiation of embryoid body (EB) cells, derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), into dopaminergic neurons comprising; i) seeding dissociated embryoid body (EB) cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with parallel grating structures, and contacting the cells with medium comprising SMAD inhibitor, Noggin, sonic hedgehog (SHH) and CT99021 to induce neuronal lineage commitment; and ii) removing the cells from the substrate of stage i) after a period of contact with said medium and re-seeding the removed cells onto a substrate coated with laminin, fibronectin and poly-L-ornithine, wherein the substrate has been patterned with pillars or gratings, and contacting the cells with medium comprising BDNF, GDNF and Ascorbic Acid; and adding DAPT to the medium from day 4.

17. The method of any one of the previous claims, wherein the substrate is selected from the group comprising polymethylsiloxane (PDMS) and tissue culture polystyrene (TCPS).

18. A substrate patterned with nano- and/or micro-grating structures, wherein the nano- and/or micro-grating structures are selected from gratings and hierarchical gratings, wherein said hierarchical gratings comprise said gratings with secondary structures at the base of and within the grooves of said gratings, wherein the dimensions of the gratings are 250nm to 2pm wide with 250nm to 2pm spacing and 250nm to 2pm height.

19. The substrate of claim 18, wherein the hierarchical secondary structures are selected from the group comprising gratings, pillars, columns and lens.

20. The substrate of claim 18, wherein the hierarchical secondary structures are gratings with dimensions of 100nm to 250nm wide with 100nm to 250nm spacing and 100nm to 250nm height.

21. The substrate of claim 18, wherein the gratings are parallel.

22. The substrate of claim 18 or 19, wherein the hierarchical secondary gratings are perpendicular to the longitudinal axis of the gratings.

23. The substrate of any one of claims 18 to 22, wherein the gratings have an aspect ratio of 1 (grating width: spacing: height of 1 :1:1 ).

24. The substrate of any one of claims 18 to 23, wherein the substrate is coated with extracellular matrix proteins.

25. The substrate of claim 24, wherein the extracellular matrix proteins are selected from at least one from the group comprising poly-L-omithine, fibronectin and laminin.

26. The substrate of any one of claims 18 to 25, wherein the substrate is selected from the group comprising polymethylsiloxane (PDMS) and tissue culture polystyrene (TCPS).

27. Use of dopaminergic neurons produced according to any one of claims 1 to 6 and 9 to 17 for the treatment of Parkinson's disease.

28. A method of treatment of Parkinson's disease, comprising administering to a subject in need thereof an efficacious amount of dopaminergic neurons produced according to any one of claims 1 to 6 and 9 to 17.

29. The use of dopaminergic neurons produced according to any one of claims 1 to 6 and 9 to 17 to screen for compounds with activity in ameliorating or treating Parkinson's disease, in vitro testing or drug screening related to Parkinson's disease or diseases related to dopaminergic neurons.

30. A method of testing compounds for activity in ameliorating or treating Parkinson's disease, comprising the steps:

(a) contact dopaminergic neurons obtained according to any one of claims 1 to 6 with the test compound, and

(b) quantitate the level of any one or more from the group comprising dopamine production, TH expression, Lmxla expression, Foxa2 expression PITX3 expression, MAP2 expression, NURR1 expression, ALDH1a1 expression, SCN1a expression,

VMAT2 expression, Pax6 expression and neuron complexity; and/or

(c) measure the electrophysiological profile of said neurons; and

(d) compare the level quantitated in (b) and/or measured in (c) with the level in untreated dopaminergic neurons, wherein a difference in level indicates the compound has Parkinson's disease-ameliorating activity.

31. The method or substrate of any one of the previous claims, wherein the gratings are 2pm wide with 2pm spacing and 2pm height, the hierarchical secondary gratings are 250nm wide with 250nm spacing and 250nm height and are perpendicular to the longitudinal axis of said gratings and the pillars are 2 pm diameter with 12 pm pitch.