Methods in Molecular Biology (2015) 1307: 281–287 DOI 10.1007/7651_2014_81 © Springer Science+Business Media New York 2014 Published online: 24 May 2014
Microgrooved Surface Modulates Neuron Differentiation in Human Embryonic Stem Cells David Lu, Chi-Shuo Chen, Chao-Sung Lai, Sushant Soni, Taranze Lam, Clarence Le, Eric Y.-T. Chen, Thien Nguyen, and Wei-Chun Chin Abstract Stem cell-based therapies have drawn intensive attention in the neuronal regenerative fields. Several studies have revealed that stem cells can serve as an inexhaustible source for neurons for transplantation therapies. However, generation of neurons and directionality has not yet been fully investigated. Herein, we investigate the mechanical ramifications of surface topography on human embryonic cell differentiation. Microgrooved surfaces with various pitches were applied to modulate the neuron differentiation. Our protocol showed that neuron differentiation increased as grove pitch decreased. The results indicated that 2 μm microgrooves can improve neuron growth by ~1.7-fold. Our results indicate the importance of mechanotransduction on neuronal differentiation and highlight the feasibility of manipulating the neuronal differentiation with surface topography, providing new perspectives for accommodating clinical transplantation. Keywords: Stem cell, hESC, Neuron, Differentiation, Microgrooves, PDMS
1
Introduction Neuron-related diseases and injury are an increasingly pressing issue in our society (1, 2). Currently, there are more than 2.5 million people suffering from spinal cord injuries (SCI) worldwide (1). Furthermore, the increasing patient population of neurological disorders, such as Parkinson’s disease, has caught the publics’ attention (3, 4). Due to the low regenerative capacity of neuronal cells, various molecular and cellular therapies have been conducted in an attempt to help bridge the injury sites and promote the neuronal conductions (1, 2). For instance, molecular therapies, such as brain-derived neurotrophic factors (BDNF) and nerve growth factor (NGF), have been utilized to induce axon growth (1, 5). Recently, with the developments of stem cell-based approaches, stem cell therapy provides the advantages of both cellular and molecular therapies; additional investigations have addressed these potential applications of stem cell therapies for neuro-regenerative medicine (2, 6).
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Fig. 1 Effect of surface topography on orientation of neurons differentiated from hESCs. SEM images of (a) microgroove; Scale bar: 10 μm. Fluorescence images of neuron alignment (b) regulated by surfaces (a). Neuron axons were indentified with β-tubulin; Scale bar: 200 μm
Fig. 2 Human embryonic stem cells (hESCs) derived neuronal progenitor with varying microgroove pitches. Immunofluorescence of β-III tubulin of neurons differentiated on 2 μm, 5 μm, 10 μm microgroove pitches and a flat surface
The unique self-renewal and pluripotent properties of human embryonic stem cells (hESCs) have been specifically targeted for potential treatments in neuron regenerative medicine (6–8). To enhance the efficacy of stem cell therapies, various protocols have been developed for guiding hESCs to differentiate into various neuronal lineages (2). The impacts of physical microenvironments, such as surface topography and substrate elasticity, on stem cell fates have been investigated (9, 10). For example, synthetic nanofiber matrixes have been designed to promote rapid neuron differentiation (11), and different types of differentiated neuronal cells were found on nanofibers of different diameters (12, 13). Herein, we demonstrate that the physical features can be utilized to guide specific directional cell growth with microgrooves (Fig. 1). Additionally, the surface microgrooves can also be applied to physically modulate the specific differentiation of neurons from hESCs. Our results have shown that the neurons grown on the microgrooves are uniform in direction and increased in neuronal differentiation as the microgroove pitches decreased (Fig. 2). 2 μm microgrooves have shown the best results in neuron differentiation, ~1.7-fold than neuron differentiation on regular flat surfaces, and
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directionality. Our method of microgrooved differentiated hESC into neurons could contribute to neuronal cell therapies and the understanding of mechanotransduction impacts on stem cell fate determination.
2
Materials
2.1 Polydimethylsiloxane (PDMS)
SYLGARD 184 SILICONE ELASTOMER KIT (Dow Corning). 1. SYLGARD 184 Silicone Elastomer Base. 2. SYLGARD 184 Silicone Elastomer Curing Agent. 3. Soft lithography.
2.2 Feeder Cell Components
1. Mouse embryonic fibroblasts (MEFs, Millipore). 2. MEFs medium: Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 20 % Fetal Bovine Serum (Invitrogen), 1 mM L-glutamine (Invitrogen), 1 % nonessential amino acids (Invitrogen). Filter solution with 0.22 μm filter into a bottle and store at 4 C. Should make fresh every 2 weeks. 3. Dissolve 2 mg Mitomycin C (Sigma-Aldrich) in 200 ml of MEFs medium. Mitomycin C is light sensitive so please avoid direct light. 4. Tissue culture plates.
2.3 Human Embryonic Stem Cell Components
1. Human embryonic stem cells (hESCs): (H9, WiCell, Madison, WI). 2. hESCs medium: 20 % KO serum replacement (Invitrogen), 1 % nonessential amino acids (Invitrogen), 1 mM L-glutamine (Invitrogen), Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 4 ng/ml FGF-2 (Sigma-Aldrich). Filter solution with 0.22 μm filter into a bottle and store at 4 C. Should make fresh every 2 weeks. 3. Dissolve 1 mg/ml Dispase (Invitrogen, CA) into Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen). 4. Embryonic Bodies (EBs) medium: hESCs Medium without FGF-2. 5. Neuron induction medium: Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 1 % nonessential amino acids (Invitrogen), 1 % Sodium Pyruvate (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (SigmaAldrich), and 4 ng/ml FGF-2 (Sigma-Aldrich), 1 % N2 supplement (Invitrogen), and FGF2 (20 ng/ml, Invitrogen). 6. Ultra-low attachment dish: (Costar, Fisher). 7. Laminin (Sigma-Aldrich).
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2.4 Immunohistochemical Staining
1. Phosphate buffer saline (PBS; Fisher). 2. 4 % Paraformaldehyde: 2 g paraformaldehyde in 100 ml PBS. Heat mixture to 55 C on a hot plate with a stir bar to incorporate the mixture. Add 4–5 drops of 10 N NaOH and allow the mixture to clear up. Neutralizes the pH to 7.3 with HCl and filter solution with 0.22 μm filter to remove particulates. Should make fresh every 2 weeks. CAUTION: NaOH and HCl are highly corrosive so use safety precaution while using these chemical. Paraformaldehyde is a fixative, so use safety precaution while handling these chemical. 3. 0.1 % Triton X-100 (Sigma-Aldrich) in PBS. 4. 2 % Bovine serum albumin (BSA) in PBS. 5. PBST: PBS with 0.05 % Tween 20. 6. Primary antibodies: Anti-β-tubulin III (1:500, Millipore), Anti-Olig2 fluorescent phalloidin (A12379, Invitrogen), Focal adhesion kinase (FAK, Millipore), vinculin (Millipore). 7. Secondary antibodies: Alexa Fluor 488 (1:500, Invitrogen) and Alexa Fluor 555 (1:500, Invitrogen).
3 3.1
Methods PDMS
To prepare desired amount of PDMS mixture. 1. Microstructure molds (microgroove with 3 μm height and 2, 5, or 10 μm pitches, respectively) are fabricated on silicon wafers with SU-2050 by standard photolithographic techniques. 2. Combine SYLGARD 184 Silicone Elastomer Base and SYLGARD 184 Silicone Elastomer Curing Agent in a 10:1 ratio then mix thoroughly (see Notes 1 and 2). 3. Cast mixture into desired mold and adjust thickness to specific application. 4. Place mold onto platform rocker and rock at intermediate-high speed for 45 min. 5. Place molds into 37 C incubator overnight to allow consolidation (see Note 3).
3.2 Human Embryonic Stem Cell Culture
Cell culture and treatments are incubated at 37 C. Highly recommend that all cell work should be done in a biosafety hood to prevent contamination and use proper sterilization techniques. 1. Amplify and culture MEFs cells with MEFs medium. Then Mitomycin C treat the MEFs for 2 h (see Note 4). 2. Replate and seed MEFs at 12,000 cells/cm2. Allow cells to attach in the incubator overnight.
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3. Wash MEFs plate with PBS to remove residual MEFs medium and aspirate the solution. 4. Plate H9 hESCs line with hESCs medium on MEFs plate. Change medium everyday to ensure a healthy culture and minimal differentiation. 5. Subculture hESCs every 7 days on to new MEFs plates to maintain culture by manually cutting and selecting healthy and undifferentiated colonies to be subcultured (see Note 5). 6. Generation of Embryonic Bodies (EBs): Incubate Dispase with the hESCs for 10–15 min to allow colonies to detach from the MEFs feeder layer (see Note 6). 7. Plate hESC colonies into the ultra-low attachment plate and culture for 5–7 days with EBs medium. Colonies should form into a sphere-like shape called an embryoid body (see Note 7). 3.3 Microgroove Stem Cell Differentiation
1. Once desired microgroove is formed and sterilized with 70 % ethanol, then coat PDMS in 100 μg/ml laminin solution overnight. 2. Wash PDMS with PBS and seed EBs on to PDMS substrate with Neuron induction medium. Incubate for 7 days while changing medium every day (see Note 8).
3.4 Immunohistochemical Staining
All incubation time was done at room temperature. 1. Once the hESCs are done incubating on the PDMS substrate for 7 days. Wash with PBS and fix cells with 4 % paraformaldehyde for 35 min at room temperature and wash with PBS. 2. Treat cells to 0.1 % Triton X-100 for 10 min and wash with PBST. 3. Cells were then blocked with 2 % BSA for 1 h and then washed with PBST. 4. Cells were then incubated with primary antibodies for 1 h then washed with PBST 3 times (see Note 9). 5. Incubate cells with secondary antibodies for 1 h and wash cells with PBST 3 times. 6. Store samples in PBS and cells are ready for imaging.
4
Notes 1. If mixture is too viscous, reduce amount of SYLARD 184 Silicone Elastomer Curing Agent. 2. Place combined mixture in vacuum hood until all air bubbles within mixture removed—approximately 1–2 h depending on concentration of air bubbles within mixture.
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3. Once in incubator, mold can last for 2 weeks but will not exhibit changes beyond that point. Storing prepared mold longer than 2 weeks, though, is not recommended. 4. Make a large batch of MEFs so that you can freeze the cells after Mitomycin C treatment to have cells ready when needed. Mitomycin C is light sensitive so when making the solution, avoid light and wrap bottle in foil to store. 5. Shake plate well to disperse new colonies to allow ample growth and minimize differentiation. 6. Check plate at 10 min to see if the hESC colonies are lifting off by the edge of the colonies. Colonies should be easy to remove by pipetting solution over the colonies, careful not to shred the colonies while pipetting. Beware of incubating with dispase for too long, because MEFs layer will detach from the plate and cause difficulty when trying to remove only the hESCs colonies. 7. Separate the colonies when they are forming EBs, EBs will fuse together if the EBs are in close proximity. 8. Seed EBS apart from each other to allow maximum growth of axons in the microgrooves. 9. When washing cells with PBST, for best result place cells on a shaker to slowly agitate the solution. This will help remove nonspecific staining.
Acknowledgments We would like to acknowledge Muscular Dystrophy Association (MDA), UC Merced GRC Summer Fellowships, and Chang Gung University CMRP D1C0031 for support and funding. We would also like to thank Philip Lee and Catherine Le for their assistance. References 1. Thuret S, Moon LDF, Gage FH (2006) Therapeutic interventions after spinal cord injury. Nat Rev Neurosci 7:628–643 2. Lindvall O, Kokaia Z (2006) Stem cells for the treatment of neurological disorders. Nature 441:1094–1096 3. Dorsey ER, Constantinescu R, Thompson JP, Biglan KM, Holloway RG, Kieburtz K et al (2007) Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030. Neurology 68:384–386 4. Hirtz D, Thurman DJ, Gwinn-Hardy K, Mohamed M, Chaudhuri AR, Zalutsky R
(2007) How common are the “common” neurologic disorders? Neurology 68:326–337 5. Sasaki M, Radtke C, Tan AM, Zhao P, Hamada H, Houkin K et al (2009) BDNFhypersecreting human mesenchymal stem cells promote functional recovery, axonal sprouting, and protection of corticospinal neurons after spinal cord injury. J Neurosci 29: 14932–14941 6. Gage FH (2000) Mammalian neural stem cells. Science 287:1433–1438 7. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines
Neural Differentiation with Microgrooved Surface from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404 8. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 9. Martinez E, Engel E, Planell JA, Samitier J (2009) Effects of artificial micro- and nanostructured surfaces on cell behaviour. Ann Anat 191:126–135 10. Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS (2009) Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5:17–26
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11. Silva GA, Czeisler C, Niece KL, Beniash E, Harrington DA, Kessler JA et al (2004) Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 303:1352–1355 12. Lee MR, Kwon KW, Jung H, Kim HN, Suh KY, Kim K et al (2010) Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays. Biomaterials 31:4360–4366 13. Christopherson GT, Song H, Mao HQ (2009) The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 30:556–564
Microgrooved Surface Modulates Neuron Differentiation in Human Embryonic Stem Cells David Lu, Chi-Shuo Chen, Chao-Sung Lai, Sushant Soni, Taranze Lam, Clarence Le, Eric Y.-T. Chen, Thien Nguyen, and Wei-Chun Chin Abstract Stem cell-based therapies have drawn intensive attention in the neuronal regenerative fields. Several studies have revealed that stem cells can serve as an inexhaustible source for neurons for transplantation therapies. However, generation of neurons and directionality has not yet been fully investigated. Herein, we investigate the mechanical ramifications of surface topography on human embryonic cell differentiation. Microgrooved surfaces with various pitches were applied to modulate the neuron differentiation. Our protocol showed that neuron differentiation increased as grove pitch decreased. The results indicated that 2 μm microgrooves can improve neuron growth by ~1.7-fold. Our results indicate the importance of mechanotransduction on neuronal differentiation and highlight the feasibility of manipulating the neuronal differentiation with surface topography, providing new perspectives for accommodating clinical transplantation. Keywords: Stem cell, hESC, Neuron, Differentiation, Microgrooves, PDMS
1
Introduction Neuron-related diseases and injury are an increasingly pressing issue in our society (1, 2). Currently, there are more than 2.5 million people suffering from spinal cord injuries (SCI) worldwide (1). Furthermore, the increasing patient population of neurological disorders, such as Parkinson’s disease, has caught the publics’ attention (3, 4). Due to the low regenerative capacity of neuronal cells, various molecular and cellular therapies have been conducted in an attempt to help bridge the injury sites and promote the neuronal conductions (1, 2). For instance, molecular therapies, such as brain-derived neurotrophic factors (BDNF) and nerve growth factor (NGF), have been utilized to induce axon growth (1, 5). Recently, with the developments of stem cell-based approaches, stem cell therapy provides the advantages of both cellular and molecular therapies; additional investigations have addressed these potential applications of stem cell therapies for neuro-regenerative medicine (2, 6).
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Fig. 1 Effect of surface topography on orientation of neurons differentiated from hESCs. SEM images of (a) microgroove; Scale bar: 10 μm. Fluorescence images of neuron alignment (b) regulated by surfaces (a). Neuron axons were indentified with β-tubulin; Scale bar: 200 μm
Fig. 2 Human embryonic stem cells (hESCs) derived neuronal progenitor with varying microgroove pitches. Immunofluorescence of β-III tubulin of neurons differentiated on 2 μm, 5 μm, 10 μm microgroove pitches and a flat surface
The unique self-renewal and pluripotent properties of human embryonic stem cells (hESCs) have been specifically targeted for potential treatments in neuron regenerative medicine (6–8). To enhance the efficacy of stem cell therapies, various protocols have been developed for guiding hESCs to differentiate into various neuronal lineages (2). The impacts of physical microenvironments, such as surface topography and substrate elasticity, on stem cell fates have been investigated (9, 10). For example, synthetic nanofiber matrixes have been designed to promote rapid neuron differentiation (11), and different types of differentiated neuronal cells were found on nanofibers of different diameters (12, 13). Herein, we demonstrate that the physical features can be utilized to guide specific directional cell growth with microgrooves (Fig. 1). Additionally, the surface microgrooves can also be applied to physically modulate the specific differentiation of neurons from hESCs. Our results have shown that the neurons grown on the microgrooves are uniform in direction and increased in neuronal differentiation as the microgroove pitches decreased (Fig. 2). 2 μm microgrooves have shown the best results in neuron differentiation, ~1.7-fold than neuron differentiation on regular flat surfaces, and
Neural Differentiation with Microgrooved Surface
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directionality. Our method of microgrooved differentiated hESC into neurons could contribute to neuronal cell therapies and the understanding of mechanotransduction impacts on stem cell fate determination.
2
Materials
2.1 Polydimethylsiloxane (PDMS)
SYLGARD 184 SILICONE ELASTOMER KIT (Dow Corning). 1. SYLGARD 184 Silicone Elastomer Base. 2. SYLGARD 184 Silicone Elastomer Curing Agent. 3. Soft lithography.
2.2 Feeder Cell Components
1. Mouse embryonic fibroblasts (MEFs, Millipore). 2. MEFs medium: Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 20 % Fetal Bovine Serum (Invitrogen), 1 mM L-glutamine (Invitrogen), 1 % nonessential amino acids (Invitrogen). Filter solution with 0.22 μm filter into a bottle and store at 4 C. Should make fresh every 2 weeks. 3. Dissolve 2 mg Mitomycin C (Sigma-Aldrich) in 200 ml of MEFs medium. Mitomycin C is light sensitive so please avoid direct light. 4. Tissue culture plates.
2.3 Human Embryonic Stem Cell Components
1. Human embryonic stem cells (hESCs): (H9, WiCell, Madison, WI). 2. hESCs medium: 20 % KO serum replacement (Invitrogen), 1 % nonessential amino acids (Invitrogen), 1 mM L-glutamine (Invitrogen), Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 4 ng/ml FGF-2 (Sigma-Aldrich). Filter solution with 0.22 μm filter into a bottle and store at 4 C. Should make fresh every 2 weeks. 3. Dissolve 1 mg/ml Dispase (Invitrogen, CA) into Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen). 4. Embryonic Bodies (EBs) medium: hESCs Medium without FGF-2. 5. Neuron induction medium: Dulbecco’s modified Eagle’s medium (DMEM/F12; Invitrogen), 1 % nonessential amino acids (Invitrogen), 1 % Sodium Pyruvate (Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (SigmaAldrich), and 4 ng/ml FGF-2 (Sigma-Aldrich), 1 % N2 supplement (Invitrogen), and FGF2 (20 ng/ml, Invitrogen). 6. Ultra-low attachment dish: (Costar, Fisher). 7. Laminin (Sigma-Aldrich).
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2.4 Immunohistochemical Staining
1. Phosphate buffer saline (PBS; Fisher). 2. 4 % Paraformaldehyde: 2 g paraformaldehyde in 100 ml PBS. Heat mixture to 55 C on a hot plate with a stir bar to incorporate the mixture. Add 4–5 drops of 10 N NaOH and allow the mixture to clear up. Neutralizes the pH to 7.3 with HCl and filter solution with 0.22 μm filter to remove particulates. Should make fresh every 2 weeks. CAUTION: NaOH and HCl are highly corrosive so use safety precaution while using these chemical. Paraformaldehyde is a fixative, so use safety precaution while handling these chemical. 3. 0.1 % Triton X-100 (Sigma-Aldrich) in PBS. 4. 2 % Bovine serum albumin (BSA) in PBS. 5. PBST: PBS with 0.05 % Tween 20. 6. Primary antibodies: Anti-β-tubulin III (1:500, Millipore), Anti-Olig2 fluorescent phalloidin (A12379, Invitrogen), Focal adhesion kinase (FAK, Millipore), vinculin (Millipore). 7. Secondary antibodies: Alexa Fluor 488 (1:500, Invitrogen) and Alexa Fluor 555 (1:500, Invitrogen).
3 3.1
Methods PDMS
To prepare desired amount of PDMS mixture. 1. Microstructure molds (microgroove with 3 μm height and 2, 5, or 10 μm pitches, respectively) are fabricated on silicon wafers with SU-2050 by standard photolithographic techniques. 2. Combine SYLGARD 184 Silicone Elastomer Base and SYLGARD 184 Silicone Elastomer Curing Agent in a 10:1 ratio then mix thoroughly (see Notes 1 and 2). 3. Cast mixture into desired mold and adjust thickness to specific application. 4. Place mold onto platform rocker and rock at intermediate-high speed for 45 min. 5. Place molds into 37 C incubator overnight to allow consolidation (see Note 3).
3.2 Human Embryonic Stem Cell Culture
Cell culture and treatments are incubated at 37 C. Highly recommend that all cell work should be done in a biosafety hood to prevent contamination and use proper sterilization techniques. 1. Amplify and culture MEFs cells with MEFs medium. Then Mitomycin C treat the MEFs for 2 h (see Note 4). 2. Replate and seed MEFs at 12,000 cells/cm2. Allow cells to attach in the incubator overnight.
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3. Wash MEFs plate with PBS to remove residual MEFs medium and aspirate the solution. 4. Plate H9 hESCs line with hESCs medium on MEFs plate. Change medium everyday to ensure a healthy culture and minimal differentiation. 5. Subculture hESCs every 7 days on to new MEFs plates to maintain culture by manually cutting and selecting healthy and undifferentiated colonies to be subcultured (see Note 5). 6. Generation of Embryonic Bodies (EBs): Incubate Dispase with the hESCs for 10–15 min to allow colonies to detach from the MEFs feeder layer (see Note 6). 7. Plate hESC colonies into the ultra-low attachment plate and culture for 5–7 days with EBs medium. Colonies should form into a sphere-like shape called an embryoid body (see Note 7). 3.3 Microgroove Stem Cell Differentiation
1. Once desired microgroove is formed and sterilized with 70 % ethanol, then coat PDMS in 100 μg/ml laminin solution overnight. 2. Wash PDMS with PBS and seed EBs on to PDMS substrate with Neuron induction medium. Incubate for 7 days while changing medium every day (see Note 8).
3.4 Immunohistochemical Staining
All incubation time was done at room temperature. 1. Once the hESCs are done incubating on the PDMS substrate for 7 days. Wash with PBS and fix cells with 4 % paraformaldehyde for 35 min at room temperature and wash with PBS. 2. Treat cells to 0.1 % Triton X-100 for 10 min and wash with PBST. 3. Cells were then blocked with 2 % BSA for 1 h and then washed with PBST. 4. Cells were then incubated with primary antibodies for 1 h then washed with PBST 3 times (see Note 9). 5. Incubate cells with secondary antibodies for 1 h and wash cells with PBST 3 times. 6. Store samples in PBS and cells are ready for imaging.
4
Notes 1. If mixture is too viscous, reduce amount of SYLARD 184 Silicone Elastomer Curing Agent. 2. Place combined mixture in vacuum hood until all air bubbles within mixture removed—approximately 1–2 h depending on concentration of air bubbles within mixture.
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3. Once in incubator, mold can last for 2 weeks but will not exhibit changes beyond that point. Storing prepared mold longer than 2 weeks, though, is not recommended. 4. Make a large batch of MEFs so that you can freeze the cells after Mitomycin C treatment to have cells ready when needed. Mitomycin C is light sensitive so when making the solution, avoid light and wrap bottle in foil to store. 5. Shake plate well to disperse new colonies to allow ample growth and minimize differentiation. 6. Check plate at 10 min to see if the hESC colonies are lifting off by the edge of the colonies. Colonies should be easy to remove by pipetting solution over the colonies, careful not to shred the colonies while pipetting. Beware of incubating with dispase for too long, because MEFs layer will detach from the plate and cause difficulty when trying to remove only the hESCs colonies. 7. Separate the colonies when they are forming EBs, EBs will fuse together if the EBs are in close proximity. 8. Seed EBS apart from each other to allow maximum growth of axons in the microgrooves. 9. When washing cells with PBST, for best result place cells on a shaker to slowly agitate the solution. This will help remove nonspecific staining.
Acknowledgments We would like to acknowledge Muscular Dystrophy Association (MDA), UC Merced GRC Summer Fellowships, and Chang Gung University CMRP D1C0031 for support and funding. We would also like to thank Philip Lee and Catherine Le for their assistance. References 1. Thuret S, Moon LDF, Gage FH (2006) Therapeutic interventions after spinal cord injury. Nat Rev Neurosci 7:628–643 2. Lindvall O, Kokaia Z (2006) Stem cells for the treatment of neurological disorders. Nature 441:1094–1096 3. Dorsey ER, Constantinescu R, Thompson JP, Biglan KM, Holloway RG, Kieburtz K et al (2007) Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030. Neurology 68:384–386 4. Hirtz D, Thurman DJ, Gwinn-Hardy K, Mohamed M, Chaudhuri AR, Zalutsky R
(2007) How common are the “common” neurologic disorders? Neurology 68:326–337 5. Sasaki M, Radtke C, Tan AM, Zhao P, Hamada H, Houkin K et al (2009) BDNFhypersecreting human mesenchymal stem cells promote functional recovery, axonal sprouting, and protection of corticospinal neurons after spinal cord injury. J Neurosci 29: 14932–14941 6. Gage FH (2000) Mammalian neural stem cells. Science 287:1433–1438 7. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines
Neural Differentiation with Microgrooved Surface from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404 8. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 9. Martinez E, Engel E, Planell JA, Samitier J (2009) Effects of artificial micro- and nanostructured surfaces on cell behaviour. Ann Anat 191:126–135 10. Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS (2009) Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell 5:17–26
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11. Silva GA, Czeisler C, Niece KL, Beniash E, Harrington DA, Kessler JA et al (2004) Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 303:1352–1355 12. Lee MR, Kwon KW, Jung H, Kim HN, Suh KY, Kim K et al (2010) Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays. Biomaterials 31:4360–4366 13. Christopherson GT, Song H, Mao HQ (2009) The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 30:556–564
