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Article
RGD-functionalized nanofibers increase early GFAP expression during neural differentiation of mouse embryonic stem cells Diana L. Philip, Elena A. Silantyeva, Matthew L. Becker, and Rebecca K. Willits Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00018 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019
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Biomacromolecules
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RGD-functionalized nanofibers increase early GFAP
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expression during neural differentiation of mouse
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embryonic stem cells
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Diana L. Philip1†, Elena A. Silantyeva2†, Matthew L. Becker1,2, Rebecca K. Willits1,3
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1 Department
of Biomedical Engineering, 2 Department of Polymer Science, 3Department of
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Mechanical Engineering
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The University of Akron, Akron OH 44325, USA
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Keywords: D3 mouse embryonic stem cells; neural differentiation; RGD; functionalized
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nanofibers; glial fibrillary acidic protein
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Corresponding authors: MLB (
[email protected]) & RKW (
[email protected])
14
†
Both authors contributed equally to this work
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Abstract
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Stem cell differentiation towards a specific lineage is controlled by its microenvironment. Polymer
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scaffolds have long been investigated to provide microenvironment cues; however synthetic
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polymers lack the specific signaling motifs necessary to direct cellular responses on their own. In
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this study, we fabricated random and aligned poly(ɛ-caprolactone) nanofiber substrates, surface-
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functionalized with RGD via strain-promoted azide-alkyne cycloaddition, that were used to
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investigate the role of a covalently tethered bioactive peptide (RGD) and nanofiber orientation on
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neural differentiation of mouse embryonic stem cells. Gene and protein expression showed neural
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differentiation progression over 14 days, with similar expression on RGD random and aligned
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nanofibers for neurons and glia over time. The high levels of glial fibrillary acidic protein
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expression at early time points were indicative of neural progenitors, and occurred earlier than on
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controls or in previous reports. These results highlight the influence of RGD binding versus
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topography in differentiation.
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Biomacromolecules
Introduction
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Embryonic stem cells (ESCs) have unique properties of self-renewal and differentiation, making
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them suitable choices for in vitro models to study various pathologies. With the potential to
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differentiate into a wide variety of lineages, ESCs can be used for tissue culture models of diseases
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or for therapeutic approaches for neurodegenerative diseases1-2. Soluble bioactive agents combined
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with a compatible protein-coated surface are the typical strategy to support and direct
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differentiation. While use of adsorbed protein is convenient, this method suffers from batch-to-
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batch variation and the presentation of bioactive protein motifs is uncontrolled and highly variable.
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However, using rationally designed synthetic or biomimetic materials3, the topographical4-8 and
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bioactive cues5, 8-14 can be controlled tightly to regulate the differentiation of these ESC cultures.
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These physical and biochemical cues, inspired by the native extracellular matrix (ECM), can be
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used to study the differentiation process and design therapeutic devices which take advantage of
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the specific features.
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As the ECM plays a crucial role in regulating cell behavior, synthetic polymer scaffolds have
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been fabricated to mimic ECM topography using nanofibers10, 15. Topographical factors, such as
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nanofiber orientation, have influenced proliferation16-17 and cell function for various tissues, e.g.,
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nerve4, 14 and smooth muscle7. Random fiber orientations mimic the ECM structure more closely4,
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while an aligned fiber topography facilitates contact guidance11, cellular alignment, and directional
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migration, which are desirable responses for neuronal regeneration and neurite outgrowth11.
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Electrospinning of various synthetic polymers, including poly(ɛ-caprolactone) (PCL)9,
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poly(L-lactic acid)14, 19, poly(glycolic acid)19 and poly(DL-lactic-co-glycolic acid)19 has been used
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to develop nanofiber substrates for bone20, vascular tissue7,
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However, the aforementioned synthetic polymer substrates are hydrophobic and lack bioactive
21,
11, 18-19,
and nerve tissue engineering.
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cell-recognition sites, resulting in low cell adhesion and proliferation. Generally, bioactivity is
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added via addition of whole proteins during9, 18, 21,22-24 or after25-26 electrospinning. Although whole
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proteins are useful, as they add multiple binding sites for cells, electrospinning or adsorbing
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proteins eliminates control over the presentation of the bioactive sites, increasing the batch-to-
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batch variation in the preformed substrates. By functionalizing synthetic nanofibers after
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electrospinning with a tethered synthetic peptide, the bioactivity and concentration of the peptide
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can be maintained14, 27-28.
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Covalently tethering bioactive peptides to the surface of the nanofiber is an attractive alternative
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to adsorbed proteins. Strain-promoted azide-alkyne cycloaddition (SPAAC) reactions has been
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used widely to functionalize the surfaces of nanofibers due to the high efficiency, mild reaction
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conditions and orthogonality of the reactants14,
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bioconjugation is scalable and highly reproducible in comparison to other surface-tethering
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techniques such as plasma treatment, wet chemical methods, surface graft polymerization29-30. The
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SPAAC method of surface modification of the nanofibers post electrospinning affords precise
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control over the amount of surface available functionality28. The use of 4-dibenzocyclooctynol
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(DIBO) as an initiator of the ε-caprolactone polymerization results in end-functionalized PCL,
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where the reactive handle survives the electrospinning process27. Strained cyclooctynes, such as
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DIBO, react quickly with azides due to ring strain,31 allowing fast surface functionalization in
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metal-free conditions. In addition, the aromatic rings afford characterization of the peptide surface
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concentration by UV-visible spectroscopy27.
28-29.
This rapid and convenient method of
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We have shown previously11 that aligned nanofibers functionalized with tethered GYIGSR
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peptide mimicked adsorbed laminin during mESC neural differentiation. Therefore, we were
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interested in investigating how other bioactive laminin peptides influenced the neural 4 ACS Paragon Plus Environment
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differentiation process. RGD is an ubiquitous peptide that has been previously shown to mimic
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fibronectin32, but is also found on the α chain of laminin33. The RGD peptide has been used in
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studies of mESC pluripotency24 and neural stem cell differentiation34 and has clear integrin
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interaction sites with cells35. Here, we investigated RGD-tethered nanofibers to compare random
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and aligned nanofiber orientations on mESC neural differentiation.
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Materials and Methods
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Materials used for Nanofiber Synthesis and Cell Study
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Nanofibers
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All materials were used as received unless otherwise stated. Tetrahydrofuran (anhydrous,
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≥99.9%, inhibitor-free), chloroform (anhydrous, contains amylenes as stabilizer, ≥99%), and
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calcium hydride (reagent grade, 95%) were purchased from Sigma-Aldrich (St. Louis, MO).
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Phenylacetaldehyde (98%, stabilized), lithium di-isopropylamide mono(tetrahydrofuran) (1.5 M
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solution in cyclohexane, AcroSeal™), iodotrimethylsilane (95-97%), n-butyllithium (2.5 M
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solution in hexanes, AcroSeal™), hexanes and methylene chloride were purchased from Fisher
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Scientific (Houston, TX). Sodium thiosulfate pentahydrate (Proteomics grade, 99%) was
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purchased from Amresco, LLC (Solon, OH). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was
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purchased from Oakwood Products, Inc. (Estill, SC). Sodium sulfate anhydrous (ACS grade) and
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methanol (ACS grade), hydrochloric acid (36.5-38%, ACS Grade) were purchased from VWR
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International (Radnor, PA).
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Dry toluene (HPLC Grade, 99.7%, Alfa Aesar) for polymerization was purified and dried on an
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Inert Pure Solv system (MD Solvent Purification system, model PS-MD-3) and degassed using
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three cycles of the freeze-vacuum-thaw. ε-Caprolactone (ε-CL, 99%, ACROS Organics™) was 5 ACS Paragon Plus Environment
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dried over calcium hydride under nitrogen overnight and distilled under reduced pressure.
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Magnesium
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dibenzocyclooctynol (DIBO)27 initiator and DIBO-end functionalized poly(ԑ-caprolactone) were
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synthesized using methods described previously11. Resins for peptide synthesis (Novabiochem®)
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were purchased from EMD Millipore (Billerica, MA). Fmoc-amino acids were purchased from
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Aapptec (Louisville, KY).
2,6-di-tert-butyl-4-methylphenoxide
catalyst
[Mg(BHT)2(THF)2]36,
4-
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Square (22 x 22 mm) and round (8 mm) Fisherbrand™ borosilicate cover glasses (#1.5) were
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washed with methanol / toluene / methanol, dried with nitrogen, and cleaned with UV light (355
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nm) for 3 min prior to use. A UVO Cleaner, Model #42A UV light unit was used to clean the glass
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coverslips for nanofiber collection. After nanofibers were collected on the glass coverslips, the
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nanofiber mats were glued to the edges of a glass slide by a silicone sealant and dried under vacuum
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overnight.
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Cell study
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Materials used during this study included the following: D3 mESCs (ATCC); ES-qualified 0.1%
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gelatin (Embryomax, Millipore); D3 growth medium consisted of Dulbecco’s Modification of
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Eagle’s Medium (DMEM) (Corning) prepared with sodium bicarbonate (Sigma), supplemented
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with 10% ES-qualified fetal bovine serum (Millipore), 10-4 M β-mercaptoethanol (Millipore
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Embryomax), 4 mM L-glutamine (Life Technologies), 4.7 mM 4-(2-hydroxyethyl)-1-
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piperazineethanesulfonic acid (Hyclone GE Healthcare), and 1000 units/mL human recombinant
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leukemia inducing factor (GlobalStem); trypsin-EDTA (Sigma Aldrich); flow cytometry
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antibodies: anti-SSEA1 (BioLegend 125606) and isotype control (BioLegend 401611); Neural
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differentiation medium consisted of 70% DMEM/F-12 (Corning), 20% neurobasal medium (Life
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Technologies), 1X N2 supplement, 20% PluriQ serum replacement (GlobalStem), 10-3 M sodium 6 ACS Paragon Plus Environment
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pyruvate (Sigma), 2 mM L-glutamine (Life Technologies), and 2 μM retinoic acid (Sigma). TRIzol
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reagent (Life Technologies); cDNA Synthesis Kit (Quantabio), SYBR Green (Quantabio).
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Paraformaldehyde (Fisher Scientific); Triton-X (Sigma); sodium borohydride (MP Biomedicals);
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Primary antibodies: NES (Abcam 134017; 1:10,000), SSEA1 (DSHB MC480; 1:8), POU5F1
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(Abcam 198857; 1:1000), SOX1 (Cell Signaling Tech 4194; 1:200), GFAP (BioLegend 82401;
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1:1000), and TUBB3 (Abcam 78078; 1:500) for early time points (days 1 and 3); GFAP, TUBB3,
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MAP2 (Abcam 11267; 1:500), GAP43 (Abcam 16053; 1:500), and OLIG1 (Abcam 53041; 1:500).
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Nuclei stain: H33342 (Life Technologies H1399); Secondary antibodies were diluted at 1:400;
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goat anti-mouse IgM AF546 (Life Technologies A21045), donkey anti-rabbit IgG AF647 (Life
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Technologies A31573), goat anti-chicken IgY AF488 (Life Technologies A11039), goat anti-
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mouse IgG2a AF546 (Life Technologies A21133), and goat anti-mouse IgG1 AF546 (Life
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Technologies A21123).
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Fabrication of Synthetic Nanofibers
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Experimental Methods
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Proton 1H nuclear magnetic resonance (NMR) (300 MHz and 500 MHz) spectra were recorded
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on Varian Mercury 300 and 500 spectrometers. The polymers were dissolved in CDCl3 solvent at
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15 mg/mL, the relaxation time was 2 sec with 64 transients. Size exclusion chromatography (SEC)
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was used to determine molecular mass and molecular mass distributions (Đm). Chromatograms
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were collected on a Tosoh EcoSEC HLC-8320GPC using refractive index detector and N,N-
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dimethylformamide (DMF) containing 0.1 M lithium bromide as the eluent at a flow rate of 0.3
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mL/min and 40 °C. The 2 columns were calibrated using narrow molecular mass poly(styrene)
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standards (20 standards from 0.5 kDa to 5,480 kDa). Nanofiber scaffolds were sterilized by
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ethylene oxide using an Anprolene benchtop sterilizer (Anderson Products, Inc., Haw River, NC) 7 ACS Paragon Plus Environment
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according to the manufacturer’s protocol for 12 h at room temperature and 35% humidity
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(concentration of ethylene oxide is about 0.5 g/L), purged for at least 48 h and stored in a vacuum
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desiccator until further use.
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Electrospinning conditions and nanofiber collection
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The electrospinning setup for aligned nanofiber scaffolds is shown in Figure 1 (B, D). For
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aligned fiber scaffolds, the DIBO-terminated PCL was dissolved in HFIP (17% (w/v)) to yield a
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clear, slightly viscous solution. The solution was placed in a 2 mL glass syringe with a 22 gauge
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needle for aligned and 23 gauge needle for random fibers (JG22-0.5X or GJ23-0.5X, Jensen Global
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Dispensing Solutions). A voltage of 15 kV was applied to the solution, and the tip-to-collector
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distance was set to 10 cm. Aluminum foil was used as the grounded collector for random fibers
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and metal plate with gaps (24 x 110 mm) for aligned fibers. Random nanofibers were collected on
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glass cover slides placed on aluminum foil. Aligned nanofibers were collected by placing cover
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glasses in between the gaps of the collector. The collected nanofiber mats were glued to the edges
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of a glass slide with a silicone sealant and dried under vacuum overnight.
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Characterization of diameter and orientation
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Nanofiber dimensions and alignment were imaged by scanning electron microscope (SEM) with
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an applied voltage of 5 kV (JSM-7401F, JEOL, Peabody, MA). Samples were sputter coated for
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30 seconds with silver under nitrogen atmosphere prior to imaging. High voltage power supply
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(ES30P-5W, Gamma High Voltage, Ormond Beach, FL) was used for electrospinning. The
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variation in nanofiber diameters was measured on at least 3 independent samples (5 images of each
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sample with >150 fibers per sample) using ImageJ and reported as an average ± standard deviation.
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The DirectionalityTM plugin of ImageJ was used to quantify the relative degree of alignment of the
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scaffolds by analyzing the angle distribution of fibers. The values are reported as an average ± 8 ACS Paragon Plus Environment
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standard deviation. Fityk 0.9.8 was used to fit a Gaussian function (red curve) and calculate
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average angle as the peak of the distribution fit. Angles for aligned fibers were normalized to 0.
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The highest peak was normalized to 1.
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Solid phase peptide synthesis
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N3-GRGDS and N3-GRGES peptides were synthesized using standard FMOC conditions on a
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CEM Discovery microwave peptide synthesizer. The N-terminus was derivatized with 6-
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azidohexanoic acid11, 37. The peptides were purified by precipitation from trifluoroacetic acid into
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cold diethyl ether and wash 3 times into cold diethyl ether, followed by dialysis against water for
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3 days and lyophilization. The desired peptide product was confirmed by electrospray ionization
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mass spectrometry.
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Nanofiber functionalization
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Nanofiber covered glass slides were dipped into a solution of the respective azide-functionalized
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peptide (1.59 µmol/mL) in 1:2 water/methanol (v/v) solution for 5 min. The cover slips with
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functionalized nanofibers were rinsed with 1:2 water/methanol (v/v) solution, blown with nitrogen
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and dried overnight in a desiccator. Scaffolds were sterilized using an ethylene oxide exposure
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cycle for 12 h, degassed for 2 days and stored in a vacuum desiccator until further use.
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The extent of functionalization with each peptide (reported as an average ± standard deviation)
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was confirmed using UV−visible spectroscopy (SynergyTM MX plate reader from BioTek, with
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spectral resolution 1 nm), using chloroform as a solvent. The peak intensity at 306 nm (which
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corresponds to π-π* transition in alkyne bond in the DIBO-functionalized polymer) decreases after
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reaction with azide-functionalized peptide in comparison with fibers before functionalization. The
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surface concentrations of the GRGDS or GRGES peptide were calculated by dividing number of
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moles of reacted alkyne groups by surface area of the fibers. The surface area of the fibers was 9 ACS Paragon Plus Environment
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calculated by finding the volume of the fibers from measured mass of the fiber mats and measured
2
by SEM fiber diameter. Density of the DIBO-PCL was assumed to be the same as of PCL (1.145
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g/cm3).
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Cell Study
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mESCs Culture
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D3 mESCs were maintained pluripotent on feeder-free gelatin coated culture flasks in an
7
incubator at 37 ºC and 5% CO2. Growth medium for maintaining pluripotent mESCs was changed
8
daily. The cells were passaged once they reached 75-85% confluency; they were washed with 1x
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PBS and incubated in 1x trypsin EDTA for 2 min at 37 ºC. The detached cells were neutralized
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with growth medium and centrifuged (160 g for 5 min at 4 ºC) to collect in a pellet. Cells were
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seeded at 20,000 cells/cm2 for further culture; pluripotency was determined using flow cytometry
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by staining against SSEA1. Anti-SSEA1 was incubated with 250,000 cells for 1 hour at 4 °C and
13
then analyzed using flow cytometry against an isotype control. Cells which expressed an average
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of 98.8% SSEA1+ were used for neural differentiation of mESCs.
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Neural Differentiation
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Pluripotent mESCs were seeded in neural differentiation medium at a seeding density of 125,000
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cells/cm2 on the substrates, and neural differentiation was induced using retinoic acid. The neural
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differentiation profile was analyzed at days 1, 3, 7, and 14 after seeding; pluripotent, neural
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progenitor, neuronal, and glial markers were analyzed using gene and protein expression.
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Gene Expression
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RNA isolation was performed using TRIzol Reagent and reverse transcribed using a cDNA
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synthesis kit, according to the manufacturer’s protocols. Quantitative PCR was performed using
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SYBR Green on pluripotent, neural progenitor, neuronal, and glial genes (Applied Biosciences 10 ACS Paragon Plus Environment
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7500 qPCR system) using MIQE guidelines. Data analysis of ΔCt was calculated by subtracting
2
the Ct of the gene of interest from housekeeping genes (β-actin and Gapdh) at the time point (days
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1, 3, 7, or 14 of differentiation); ΔΔCt was calculated as the difference between the ΔCt(timepoint)
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- ΔCt(pluripotent). Data is represented as log2(fold change). Primer sequences can be found in
5
Table 1. Note that we use standard gene and protein symbols, with italicized symbols indicating
6
genes while proteins are not italicized. Pou5f1 is seen in many reports as Oct4.
7 Table 1: Summary of genes and their sequence utilized for gene analysis Gene Sequence (Reverse) Pluripotent Pou5f1 GAA GCC GAC AAC AAT GAG AAC Nes GGA AAG CCA AGA GAA GCC T Neural Progenitor Sox1 GTA CAG TAT TTA TCG TCC GCA GA Pax6 AAG GGC GGT GAG CAG ATG T Tubb3 GTG GAC TTG GAA CCT GGA AC Neuronal Map2 GAC CCA GAG TGT GTG AGT TTA T Gap43 TCA GGC ATG TTC TTG GTC AG Gfap GCG ATA GTC GTT AGC TTC GTG Glial Olig1 AGC AAC TAC ATC GCT CCT TG β-actin CAC GGT TGG CCT TAG GGT TCA G Housekeeping Gapdh GTG GAG TCA TAC TGG AAC ATG TAG
8 9
Sequence (Forward) GGC ACT TCA GAA ACA TGG TCT CAC CTC AAG ATG TCC CTT AGT C GGC AGT CAT ACA AAA GTT GGC CAT GCT GGA GCT GGT TGG CCT CCG TAT AGT GCC CTT TG CCA CTA ATG CCA GTT TCT CTC T AGG AGG AGA AAG ACG CTG TA CCA CCA GTA ACA TGC AAG AGA TCC AGA CTT CTC TCC CAG AC GCT GTA TTC CCC TCC ATC GTG AAT GGT GAA GGT CGG TGT G
Protein Expression
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At the appropriate timepoint, samples were fixed with freshly prepared 4% buffered
11
paraformaldehyde, washed, and stored in PBS at 4 °C. Cells were permeabilized in 0.5% and 0.1%
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Triton-X for 10 and 5 min respectively, and permeabilized with 1mg/mL of sodium borohydride
13
twice for 4 min. Blocking was performed with BSA and 0.1% Triton-X for 1 hour. The cells were
14
then stained with primary antibodies overnight at 4 °C, followed by staining with the appropriate
15
secondary antibody overnight at 4 °C. Proteins of interest were NES, SSEA1, POU5F1, SOX1,
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GFAP, and TUBB3 for early time points (days 1 and 3) and GFAP, TUBB3, MAP2, GAP43, and
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OLIG1 for later time points (days 7 and 14). 11 ACS Paragon Plus Environment
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Analysis of Images
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Images were captured on an inverted fluorescent microscope at exposure times set by controls
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that had only secondary antibodies and nuclei stain. At least five images were obtained from each
4
sample. Image analysis was done on ImageJ (National Institutes of Health, v1.5h)38.
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Cell Aggregate Size: Using ImageJ, the sizes of mESCs aggregates were measured on at least 5
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images for each sample for days 7 and 14 of neural differentiation. Aggregates were identified by
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nuclei stain and were outlined using either Oval or Polygon Selections tool. The outline was
8
measured to determine the area of the aggregate. Cells that were close but did not touch each other
9
were not considered aggregates.
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Percent Positive Cells: All images were captured at the same exposure as the respective control.
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A threshold for captured control images (no primary antibody) was set at 0.05 was performed to prove that there was no statistical difference
7
between diameters of random and aligned fibers as well as to show statistical difference between
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neurite alignment cultured on different substrates. Two-way ANOVA with Bonferroni post-hoc
9
test was utilized to determine significance between gene expression using ΔΔCt, with p
