Neuronal Differentiation of Induced Pluripotent Stem Cells on

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Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels Kristan S. Worthington, Brian J. Green, Mary Rethwisch, Luke A. Wiley, Budd A. Tucker, C. Allan Guymon, and Aliasger K. Salem Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00098 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels Kristan S. Worthington,1,2,3 Brian J. Green,1 Mary Rethwisch,1 Luke A. Wiley,2 Budd A. Tucker,2 C. Allan Guymon1 and Aliasger K. Salem3* 1. Department of Chemical and Biochemical Engineering, The University of Iowa, Iowa City, IA 52242 2. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, Iowa 52242 3. Division of Pharmaceutics and Translational Therapeutics, Department of Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa, Iowa City, Iowa 52242

ABSTRACT: The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-crosslinked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that after adequate surfactant removal, these materials are non-toxic, support the attachment

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of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration.

KEYWORDS: Chitosan, Photopolymerization, Surfactant Templating, Induced Pluripotent Stem Cells, Neuronal Differentiation

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INTRODUCTION Retinal degenerative diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are blinding neural degenerative disorders that impair millions of people worldwide.1, 2 As is typical for neurodegenerative disorders, very few clinical treatments, either preventative or restorative, exist.3-5 Evidence suggests that the sensitive layers of the retina can be restored by injection of functional stem-cell derived donor cells, offering hope for the development of successful treatments for these life-altering diseases.6-16 Although promising, the rates of donor cell survival and integration following traditional bolus cell injection is often very poor. Less than 0.1% of injected cells survive longer than a few days in the sub-retinal space, and even fewer integrate with the native tissue, which is necessary to restore retinal function.8, 13 This result can be attributed to high shear forces during and a lack of physical support following donor cell injection. A promising alternative to bolus cell injection is to deliver cells on support scaffolds designed to prevent anoikis (apoptosis resulting specifically from ECM detachment), shield cells against fluid shear stress and function as physical supports to hold cells in the appropriate location post-implantation.17-19 The use of allogenic and xenogenic materials fabricated using collagen and its derivatives have been explored for decades as potential retinal cell delivery vehicles.20-24 These materials have largely failed to produce desirable results without detrimental consequences such as neural rosette formation,25 rapid axonal retraction,26 folding in the subretinal space21 or up-regulation of genes associated with angiogenesis.27 Synthetic scaffolds (degradable and otherwise) which can be sourced from common starting materials using a wide selection of chemistries, have been employed with mild success.28-35 As compared to a bolus cell injection, subretinal implantation of one such cell-laden construct resulted in a 10-fold increase in donor cell survival.33 Unfortunately, synthetic scaffolds are often less than ideal. For example, their compressive moduli are often drastically higher than the retina, which causes mechanical 3


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damage of the remaining host tissues.12,

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Furthermore, for the most commonly studied synthetic

materials, degradation results in acidic byproducts that can accumulate in the sub-retinal space, leading to inflammation and cell death.37,

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The ability to simultaneously control scaffold stiffness,

biocompatibility, and structure using readily available materials remains a major hurdle to effective retinal transplantation. Chitosan, a derivative of one of the most abundant biopolymers on earth (chitin), is an attractive option for biomaterial fabrication. This biopolymer has been repeatedly demonstrated to be biocompatible39-41 and is even widely considered to be antimicrobial.42, 43 Chitosan is often incorporated into medical materials, including drug and gene delivery systems, wound dressings, and tissue engineering scaffolds.44-59 Furthermore, the effectiveness and mechanical properties of chitosan materials can be improved by introducing physical associations or chemical cross-links. For example, reversible physical interactions can readily be formed between chitosan and small anions like sulfates, polyelectrolytes such as poly(acrylic acid), and even some metal ions like Cu2+ to form hydrogels.59-63 However, these physically-associated networks typically have very weak mechanical properties, rendering them difficult to manipulate post-fabrication. In addition, since the associations are reversible, their dissolution is difficult to control in varying environmental conditions.60 Conversely, chemical cross-linking of chitosan chains produces more robust hydrogels with no dissolution. Although this covalent bonding can be accomplished with a variety of crosslinkers,60,

61, 64

cross-linking using

photopolymerization provides the opportunity to control the extent of reaction in both time and space. In this manner, material cross-linking density can be altered simply by adjusting light intensity or exposure time. Material structure can also be controlled by selectively exposing certain areas of the prepolymerization mixture. Although this approach has been used for drug delivery and tissue engineering

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applications, control of the resulting hydrogel structure and mechanical properties as well as application to neuronal differentiation have yet to be fully explored.65-67 Most hydrogels, including those composed primarily of chitosan, have some inherent porosity. Unfortunately this inherent pore structure is often not sufficient to allow adequate transport of nutrients for appropriate cell survivability and growth. Thus, cell scaffolds in particular may benefit by the incorporation of controlled, intentional structure. One promising method of controlling polymer structure uses self-assembling surfactant systems as polymerization templates to direct polymer morphology. This templating process has been utilized to generate structure in a number of polymer systems, resulting in materials with useful property relationships that are often not observed in traditional or isotropic polymer systems.68-74 In some cases, the polymer morphology also facilitates a greater propensity for cell attachment.75 In this approach, organized surfactant molecules are used to control polymer structure by serving as polymerization templates: water- and oil-soluble domains that are inherent in the system act to segregate monomers based on their hydrophobicity. Thus, if a photocrosslinkable polar monomer such as methacrylate-functionalized chitosan (MeCTS) is incorporated into the surfactant phase, the monomer would likely segregate in the water soluble domains of the parent template and may adopt a geometry that resembles that of the self-assembled phase.76 Photopolymerization can then be utilized to crosslink the formulation, potentially allowing for transfer of the order from the parent template to the polymer system. Here we explore the use of surfactant templating in crosslinked MeCTS hydrogel systems, which is novel for biomolecule-based photopolymers. Water uptake of these hydrogels was characterized for two surfactant families using templates of varying molecular weight. For one surfactant from each family, the effect of pH on physical properties was also investigated. Importantly, we examined the efficiency of surfactant removal from the polymer systems and characterized the network architectural changes 5


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brought about by templating. Furthermore, we verified the biocompatibility of the surfactant templated chitosan hydrogels and examined their ability to support the differentiation of induced pluripotent stem cells to neuronal phenotypes. MATERIALS AND METHODS Synthesis of Methacrylated Chitosan. Chitosan was functionalized with methacrylate groups by a Michael-addition reaction; a modified version of a previously reported method.66 First, 1 g of low molecular weight chitosan (96.1% deacetylation, 1% w/v in 1% v/v acetic acid 35 cps, Sigma Aldrich, St. Louis, MO) was dissolved in 50 mL 1% acetic acid with 40 mL ethanol until homogenous. Thereafter, 5 mL of 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AOHPMA, Sigma-Aldrich) was added drop-wise while stirring. This mixture was allowed to react for 48 hours at room temperature. Nuclear Magnetic Resonance (NMR). For reaction confirmation by 1H nuclear magnetic resonance (NMR), the raw reaction products were dialyzed against distilled water for two days. The resulting suspension of chitosan was then frozen and lyophilized. Functionalized chitosan was then dissolved in D2O with 5% DCl. The solutions were analyzed using a Bruker Avance-300 probe with a field strength of 300 MHz. Fabrication of Surfactant-Templated Hydrogels. Based on their previous use in surfactanttemplated polymerization systems, two families of surfactant were used to template chitosan hydrogels: non-ionic polyoxyethylene ethers and cationic quaternary ammonium salts. Namely, polyoxyethylene (4) lauryl ether (Brij 30, Sigma-Aldrich, St. Louis, MO), polyoxyethylene (2) cetyl ether (Brij 52, Sigma-Aldrich) and polyoxyethylene (10) cetyl ether (Brij 56, Sigma-Aldrich) were used for their varying molecular weights and hydrophobic segment lengths. Likewise, dodecyltrimethylammonium bromide (DTAB, Sigma-Aldrich), cetyltrimethylammonium bromide (CTAB, Sigma-Aldrich), tetradecylmethylammonium bromide (TTAB, Sigma-Aldrich) were used to investigate the same factors 6


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as in the non-ionic surfactants, while dodecyltrimethylammonium chloride was used to elucidate the effect of counter ion size. Non-templated MeCTS hydrogels (no surfactant) were also prepared by replacing the surfactant with a comparable amount of dilute acetic acid. In all cases, the water-soluble photoinitiator Irgacure 2959 (BASF, Ludwigshafen, Germany) was used to initiate polymerization. To fabricate hydrogels, MeCTS reaction product (50 wt%) was mixed to homogeneity with 30 wt% surfactant, 19.9 wt% of 1% acetic acid, and 0.1 wt% photoinitiator in a glass vial using vortex mixing and gentle heating, if necessary. About 1 g of each mixture was then transferred by pipette and capillary action to a laminate mold constructed from ®Rain-X pre-treated whole glass slides and glass slide fragment spacers (1 mm thick). Samples were then exposed to broad spectrum UV light (100 W mercury spot lamp, UVP LLC, Upland, CA) with no filter for 10 minutes. After polymerization, the mold was deconstructed and disks were cut from the thin sheet using 5 mm or 8 mm biopsy punches. Samples were transferred immediately to an excess of 50/50 (v/v) 1 M phosphate buffered saline (PBS) and 95% ethanol with continuous mixing by shaking to remove residual surfactant and unreacted species. Surfactant extraction proceeded for one week with the solvent being exchanged on days two, four and six. Thereafter, samples were rinsed three times with an excess of PBS before further analysis. Verification of Surfactant Removal. Immediately following polymerization, three hydrogels (8 mm disks) from each group were transferred to a 2 mL centrifuge tube and allowed to dry at room temperature and pressure for three days. The remaining three underwent the surfactant removal process described above before being dried in the same manner. After drying, each material was crushed to a powder using a laboratory spatula. Each surfactant alone (DTAB and Brij 56) was used as a control. Surfactant removal was determined using a Thermo Electron Nexus 670 Fourier transform infrared spectrometer (FTIR) equipped with an ATR accessory and a nitrogen cooled MCT detector. Prior to sample collection, background was collected on the bare ZnSe crystal. For each run, the sample powder 7


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was spread on the ATR crystal. 128 scans were collected for each sample to create an absorbance spectrum. To facilitate direct comparison between spectra, each dataset was offset by a constant value such that all baselines overlapped. Further, each surfactant-templated spectrum (before and after surfactant removal) was normalized to the non-templated chitosan hydrogel spectrum using the peak height at or near 1730 cm-1, which is present in the chitosan hydrogel spectra and distinctly absent in the surfactant controls. Therafter, each surfactant control spectrum was normalized to its respective ‘before removal’ spectrum using the aliphatic absorption peak height at or near 2930 cm-1. Measurement of Physical Properties. Changes in hydrogel water absorption due to surfactant templating were examined by quantifying sample swelling. 8 mm disk samples were removed from the PBS solution, gently blotted with laboratory tissue paper, and weighed. After drying overnight under vacuum, the samples were weighed again. The water uptake was calculated as follows: − % = 100 × where WW and WD represent the mass of the sample when wet and dry, respectively. The compressive modulus was measured as previously described, using a dynamic mechanical analysis instrument (DMA Q800 V7.0 Build 113, TA Instruments, New Castle, DE) equipped with a submersion compression clamp in static mode.36 Prior to each group of measurements, the drive shaft position, clamp mass, clamp offset, and clamp compliance were calibrated according to suggested protocols. Fully hydrated 8 mm samples were carefully placed in the center of the basin. Once the sample was installed, the top portion of the clamp was gently lowered onto the sample surface, the furnace was closed to maintain constant temperature (20 °C) and prevent disturbances, and a pre-load force of 0.1 mN was applied to the sample. The force was then gradually increased to a final value of 0.2 N at a rate of 0.02 N/min and displacement data were collected every 2 seconds as the sample was compressed. All samples were assumed to be cylindrical with a diameter measured using digital calipers 8


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just prior to mechanical testing. Sample thickness was measured using the initial displacement of the clamp before testing began. One-way analysis of variance and Tukey’s multiple comparison tests were used to determine statistical significance. The properties of functionalized chitosan hydrogels were also studied at a range of pH values. Acetate buffer was prepared at pH 3 and 5 using sodium acetate and acetic acid. Basic buffer was prepared at pH 9 using sodium hydroxide and sodium tetraborate. These solutions were used in place of PBS after surfactant removal. Water uptake and compressive modulus were measured in the same manner as at neutral pH. Two-way analysis of variance and Tukey’s multiple comparison tests were used to determine statistical significance. Characterization of Hydrogel Structure. To preserve hydrogel structure for imaging purposes, materials were dried using CO2 critical point drying (E3100, Quorum Technologies, Laughton, East Sussex, England). After drying, samples were mounted to an aluminum SEM stub using colloidal graphite. A gold-palladium coating was applied using an argon beam K550 sputter coater (Emitech Ltd., Kent, England). Images were captured using a Hitachi S-4800 SEM (Hitachi High-Technologies, Ontario, Canada) at an accelerating voltage of 1 kV. To collect information about pore size, images were processed using ImageJ. Briefly, binary images were created using the software’s automatic threshold algorithm. Thereafter, the cross-sectional area of each dark region (pore) was measured using the particle analysis function, excluding pores at the edge of the image. To avoid biased gating of pore sizes and thereby accurately represent pores of all sizes, the data were plotted as cumulative frequency distributions using GraphPad Prism. Kolmogorov-Smirnov tests were used to determine statistical differences between groups. This analysis was performed at two magnifications to confirm the results’ independence from this variable.

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Cell Culture Materials. Pluripotency media was composed of Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Life Technologies, Gibco, Carlsbad, CA) with 15% fetal bovine serum (FBS, Life Technologies), 1% 100X non-essential amino acids (NEAA, Life Technologies), 0.4 mM L-Glutamine (Life Technologies), 0.1 mg/mL Primocin (InvivoGen, San Diego, CA) and 8.88 ng/mL 2-mercaptoethanol (Aldrich). Just prior to use, 2 units/µL mouse recombinant leukemia inhibitory factor (mLif, ESGRO, EMD Millipore, Billerica, MA) were added and media was warmed to 37 °C. Differentiation media was composed of DMEM media (Life Technologies) with 2% 50X B27 supplement (Life Technologies), 1% 100X N2 supplement (Life Technologies), 0.4 mM L-Glutamine (Life Technologies), 1% 100X NEAA (Life Technologies) and 0.1 mg/mL Primocin (Life Technologies). Just prior to use, 1 ng/mL mouse recombinant Noggin (R&D Systems, Minneapolis, MN), 1 ng/mL mouse recombinant Dkk-1 (R&D Systems), 1 ng/mL mouse recombinant Bfgf (R&D Systems), and 1 ng/mL Igf-1 (R&D Systems) were added and media was warmed to 37 °C. Culture of Mouse Induced Pluripotent Stem Cells. Fibroblasts were isolated from the umbilical cords of dsRed mice and de-differentiated using a retroviral approach to yield murine induced pluripotent stem cells (MiPSCs), as described elsewhere.77,

78

Before beginning differentiation, MiPSCs were gently

thawed from a frozen stock, centrifuged, resuspended in pluripotency media and plated on Matrigel® thin-coated tissue culture treated plates (Corning). Cells were transferred to a new plate at a density of 1:6 three times in one week, with media being replaced daily. After one week, cells were removed from the plate using 0.25% trypsin-EDTA, washed with fresh media and re-plated as needed. The media was replaced every day and all cells were cultured in a humid 37ºC environment with 5% CO2. Evaluation of Cytotoxicity. To test the efficiency of surfactant extraction, the cytotoxicity of leached hydrogel components was evaluated. Briefly, 8 mm samples were either washed for one week to extract surfactant, as described above, or used directly after fabrication. After the allotted wash time (0 or 7 10


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days), samples were briefly rinsed with an excess of 1X PBS three times, cut to a diameter of 5 mm using a biopsy punch, and transferred to a 96-well plate. Pluripotency media (200 µL) was added to each sample and the plate was kept at cell culture conditions for 24 hours. On the same day and in the same plate, one additional well per sample was coated with Matrigel® using a thin-coating method. Briefly, 1 mL Matrigel® was diluted in 50 mL cold DMEM media and added to the wells (200 µL/well). After one hour at room temperature, the excess liquid was removed and cells were seeded at 2 x 104 cells/well. The following day, the conditioned media from the hydrogels was used to feed the cells. After 20 hours of exposure, cell viability was measured using a colorimetric assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, fresh media (100 µL) was placed in each well, followed by 20 µL MTS reagent. The plate was then incubated at 37 °C for 4 hours to allow for color development. Finally, the light absorbance of each well was measured at 492 nm using a spectrophotometer (EZ Read 400 microplate reader, Biochrom, Cambridge, UK). To calculate viability percentage, the mean of each group was normalized to the mean of the control group at each time point. Two-way analysis of variance and Tukey’s multiple comparison tests were used to determine statistical significance. Cell Culture on MeCTS Hydrogels. After surfactant extraction, three PBS washes and cutting to 5 mm, MeCTS hydrogel disks were transferred to a 40 µm nylon mesh insert (five samples per insert, Falcon cell strainer, Corning) and placed in a 6-well plate. In order to facilitate cell attachment, samples were coated with Matrigel by the thin-coating method described above (3 mL per well). After incubating at room temperature for one hour, the inserts were removed from the plate and excess liquid was gently removed by blotting the insert with a laboratory napkin. The inserts were placed in a new 6-well plate and the samples were allowed to air dry for 30 minutes in a sterile flow hood. Finally, 2 x 104 cells in 10 µL pluripotency media were carefully added to each sample, the plate was incubated at 37 °C for thirty 11


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minutes, and 4 mL of fresh media were gently added to each well. Thereafter, pluripotency media was replaced daily for one week, after which it was replaced with differentiation media, which was replaced daily for an additional week. Gene Expression. RNA was isolated from cells at 7 and 14 days post-seeding using an RNeasy Mini Kit (Qiagen, Venlo, Limburg, the Netherlands) and the final concentration determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific). cDNA was produced using a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosytems, Life Technologies), then pluripotency genes (Nanog, Oct4, Sox2, Lin28, Dnmt1 and Klf4) and retinal development genes (Pax6, Otx2 and Rx) were amplified using reverse transcriptase PCR (primer sequences in Table S1). The resulting DNA was characterized by electrophoresis on a 2% agarose gel with a run time of 30 minutes. To confirm each sequence, PCR amplicons were also cleaned using a QIAquick PCR Purification Kit (Qiagen) and then TA cloned into the pCR®2.1 TOPO® Vector using the TOPO® TA Cloning® Kit (Invitrogen) or directly sequenced. Cloning reactions were transformed into One Shot® TOP10 chemically competent cells (Invitrogen)

and

cultured

onto

plates

containing

LB

agar,

ampicillin,

isopropyl

β-D-1-

thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). The pCR®2.1 TOPO® Vector contains beta-galactosidase that enables blue (if vector only) and white (if product is cloned into the vector) colony discrimination. White colonies were selected and cultured overnight in LB broth containing ampicillin at 37 °C with shaking. The following day, bacterial DNA was purified using the QIAprep® Spin Miniprep Kit (Qiagen) and used for bidirectional Sanger sequencing analysis. Immunocytochemistry. Cell protein localization was analyzed 7 and 14 days post-seeding using immunocytochemistry. Samples were fixed in 4% paraformaldehyde, rinsed three times with 1X PBS, and incubated at 4°C overnight in primary antibody solution consisting of mouse anti-Sox2 12


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(#MAB2018; 1:250; R&D Systems) or rabbit anti-Tuj-1 (#T2200-200UL; 1:1000; Sigma-Aldrich) and AlexaFluor® 488-conjugated Phalloidin (#A12379; 1:1000; Life Technologies) in blocking buffer (5% normal goat serum, 0.5% Triton-X and 3% BSA in 1X PBS). The following day, the samples were rinsed three times with PBS and incubated for two hours at room temperature in secondary antibody solution containing fluorescently-conjugated Alexa Fluor® secondary antibodies (1:500, Life Technologies) in blocking buffer. Samples were rinsed three times with PBS again, counterstained with DAPI, and mounted with a cover slip. Immunofluorescent images were collected using a Leica DM 2500 SPE confocal microscope (Leica Microsystems, Wetzlar, Germany). RESULTS AND DISCUSSION In this study, we aimed to explore the effects of hydrogel structure on the physical and biological properties of chitosan hydrogels using a surfactant templating technique (Figure 1). Notably, we synthesized a methacrylated chitosan using Michael-addition and subsequently fabricated hydrogels using photopolymerization and surfactant templating with two families of surfactant. Herein we report the water uptake and compressive modulus of the resulting hydrogels with changes of pH. We also examine surfactant removal and characterize network architecture. Lastly, we elucidate their ability to support the differentiation of murine induced pluripotent stem cells to neuronal phenotypes. Characterization of MeCTS. The functionalization of chitosan was confirmed by the appearance of peaks at δ 5.97 (1H), 5.63 (1H), and 1.76 (3H) ppm corresponding to the hydrogens on the methacrylate group. MeCTS: 1H NMR (D2O/DCl, 300 MHz): δ 5.97 (s, 1H), 5.63 (s, 1H), 5.01 (m, 4H), 4.05 (m,

Figure 1. Schematic of surfactant templating and removal process used to create chitosan hydrogels. 13


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1H), 3.87 (m, 1H), 3.76 (m, 1H), 3.61 (m, 1H), 3.53 (m, 1H), 3.31 (d, 1H), 2.86 (t, 2H), 2.34 (t, 2H), 1.76 (m, 3H).

Figure 2. 1H NMR (300 MHz, D2O/DCl) of functionalized chitosan. Chemical shifts at a) δ 5.97 (1H), b) 5.63 (1H), and c) 1.76 (3H) ppm indicate the presence of methacrylate groups. Physical Properties of Surfactant-Templated MeCTS Hydrogels. Surfactant templating was used to control the physical properties of MeCTS hydrogels, including water uptake. Non-ionic polyoxyethylene cetyl ethers (Brij) have shown promise in imparting structure on polymers and thereby altering their properties. Interestingly, at the concentrations used in this study these surfactants had no significant influence on the water uptake of functionalized chitosan hydrogels, regardless of their molecular weight or hydrophobic segment length (Figure 3A). Previous research suggests that surfactant geometry largely dictates self-assembled feature size and geometry.79-82 In the case of surfactant templating of hydrophilic monomers, agglomerates of a discontinuous phase formed by the surfactant create what later become pores within the network. Thus, the volume occupied by the non-reactive surfactant is expected to correlate with water uptake. In the case of Brij-templated chitosan we describe 14


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here, a complete lack of surfactant selfassembly would be unlikely since the formulation contained a high percentage of surfactant. Rather, the absence of the expected water uptake trends can be attributed to changes in self-assembled geometries

brought

on

by

the

polymerization reaction, as has been observed in many previous systems when the polymer morphology is not influenced by the surfactant templating process.69, 83

72,

Conversely, dramatic and significant

changes in water uptake were observed for chitosan

hydrogels

templated

with

quaternary ammonium surfactants. In fact, Figure 3. Water uptake of MeCTS hydrogels templated

all

of

the

quaternary

with A) non-ionic and B) cationic surfactants with

surfactants studied induced at least a three-

varying molecular weights compared to non-templated

fold increase in water uptake when used as

controls. Error bars represent standard error of the

polymerization

mean, n = 3, *p

Neuronal Differentiation of Induced Pluripotent Stem Cells on

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