Nanofiber-Based in Vitro System for High Myogenic Differentiation of

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Nanofiber-based In Vitro System for High Myogenic Differentiation of Human Embryonic Stem Cells Matthew C. Leung, Ashleigh Cooper, Soumen Jana, Ching-Ting Tsao, Timothy Petrie, and Miqin Zhang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 16 Oct 2013 Downloaded from http://pubs.acs.org on October 23, 2013

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Nanofiber-based In Vitro System for High

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Myogenic Differentiation of Human Embryonic

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Stem Cells

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Matthew Leunga, Ashleigh Cooper1, Soumen Jana1, Ching-Ting Tsao1, Timothy Petrieb, and

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Miqin Zhanga* a

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Department of Materials Science & Engineering, University of Washington, Seattle,

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Washington, USA 98195 b

Department of Pharmacology, University of Washington, Seattle, Washington, USA 98195

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Corresponding Author

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* Department of Materials Science and Engineering, University of Washington, 302L Roberts Hall, Box 352120, Seattle, WA 98195, USA. Telephone: 206-616-9356; Fax: 206-543-3100;

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Email: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT:

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Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide

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unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by

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the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We

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present a novel approach to effectively enhance myogenic differentiation of human embryonic

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stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble

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the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a

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protein. The myogenic differentiation was assessed by cell morphology, gene activities, and

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protein expression. hESCs grown on C-PCL uniaxially-aligned nanofibers in media containing

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Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber

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orientation, with increased expressions of marker genes and proteins associated with myogenic

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differentiation as compared to control substrates. The combination of Wnt3a signaling and

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aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells

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over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of

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cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of

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hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that

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chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system

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for embryonic myogenesis and muscle regeneration.

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Keywords: muscle; stem cell; nanotopography; chitosan; polycaprolactone; scaffold. 2


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INTRODUCTION

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Muscular damage due to injury, surgery or degenerative disease can severely compromise

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patient mobility and reduce quality of life. Treatments using tissue transplants are limited by

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donor tissue availability and donor site morbidity. The regeneration of a large number of

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myoblasts is essential to skeletal muscle repair and may relieve current tissue constraints.1, 2 A

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bioactive cell culture environment combined with relevant cell types, growth factors, and

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topological properties is needed to facilitate the proliferation and differentiation of myoblasts.

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While muscle satellite cells, a myoblast progenitor, have been expanded in vitro, the limited cell

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population is inadequate for successful therapy.3 Mesenchymal stem cells (MSCs) may provide

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sufficient myoblast supply via directed myogenic differentiation; however, only limited progress

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has been made along this path.4 For instance, the application of Wnt3a, a signaling protein

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involved in mesodermal lineage commitment, is reported to induce myogenic differentiation of

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MSCs after 10 days of culture; however, there was only 3.76% of the induced MSCs showed

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myosin-heavy-chain (MHC) expression, suggesting that Wnt3a alone cannot induce the

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differentiation of large myogenic populations.5, 6 This limits the use of MSCs as a cell source.

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Alternatively, pluripotent embryonic stem cells (ESCs) have considerable advantages over

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adult stem cells as a cell source due to their capacity for unlimited proliferation in an 3


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undifferentiated state and their ability to differentiate into various lineages of cells.7, 8 In recent

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studies mouse and human ESCs (hESCs) have been transfected to induce the expression of Pax3,

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a transcript factor responsible for activation of myogenic regulatory factors.9, 10 Nevertheless,

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only 16-21% of the Pax3-expressing hESCs exhibited the terminal myogenic differentiation

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marker MHC. Also, cells modified by gene transfection are not ideal for therapeutic use as gene

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expression is difficult to control.11 Myogenic cells have also been obtained from mESCs induced

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by using retinoic acid12 or by sorting cells with an antibody specific for myogenic cells from

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mouse induced pluripotent stem cells (iPSCs).13 Inspiringly, one study demonstrated the success

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in myogenic differentiation of hESCs using a stroma-free induction system.14 However this

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technique has a very low conversion rate and is time-consuming, and no major advance has been

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made since then.15 Though using feeder layers can increase differentiation efficiency, the process

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is very costly and labor-intensive, and yet bears the risk of transmitting virus and other unwanted

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macromolecules from feeder cells. Therefore, for muscle tissue engineering there is an urgent

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need for a hESC culture system with well-defined cell-culture conditions without using feeder

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layers for enhanced myogenic differentiation.

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The ECM microenvironment plays a key role in directing the myogenic commitment.4, 16-19

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The cell fate is regulated by the ECM molecules, physical characteristics of the

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microenvironment and signaling molecules in culture media.20, 21 An ideal in vitro model of the 4


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myogenic microenvironment should replicate the anisotropic topology of the ECM of native

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muscle tissue. Using nanofibrous scaffolds in inducing myogenic precursors from hESCs is a

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promising approach for muscle regeneration.22-25 We developed a fibrous scaffold with highly-

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aligned chitosan-polycaprolactone (C-PCL) nanofibers supports skeletal muscle cell attachment

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and proliferation, and promotes skeletal muscle morphogenesis and myotube formation.19

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Chitosan, a biodegradable natural polysaccharide derived by the partial deacetylation of chitin,

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shares structural similarities to glycosaminoglycans present in the native ECM. PCL is a

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biocompatible synthetic polymer with excellent stability in vivo.26

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In this study, we investigate the effect of nanotopgraphy of C-PCL nanofibers and

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signaling cues of the culture environment on myogenic differentiation of hESCs to identify an

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appropriate

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commitments of hESCs were investigated in controlled culture conditions with media containing

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different signaling molecules and on substrates of varying compositions and topologies. The

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ability of these tissue engineered micro-environments to regulate hESC myogenic commitment

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was evaluated by cell morphology, gene activities, and protein expression.

micro-environment

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effective

embryonic

myogenesis.

The

myogenic

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EXPERIMENTAL SECTION:

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Cell culture substrate fabrication. 5


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Collagen films were fabricated from freshly-harvested rat tail collagen as previously described.27 Chitosan-PCL (C-PCL) scaffolds with either randomly-oriented or aligned nanofibers and 22

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C-PCL films were fabricated using methods developed previously

. Briefly, 7 wt% chitosan

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(85% deacetylated, medium molecular weight, Aldrich, St Louis, MO) was fully dissolved by

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refluxing the chitosan/trifluoroacetic acid (TFA, Aldrich) mixture for 3 hrs at 70°C. 10 wt% PCL

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solution was prepared by dissolving PCL (80,000 molecular weight, Aldrich) in 2,2,2,-

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trifluoroethanol (TFE, Sigma-Aldrich). The two solutions were then mixed at weight ratio of

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40:60 and vortexed for 2 min to produce a homogeneous mixture solution. The C-PCL mixture

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was electrospun at 22 kV on a rotating grounded drum (200 rpm), or a pair of grounded parallel

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electrodes (separation distance 4 cm), to produce randomly-oriented and aligned nanofibers,

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respectively. The collected nanofibers were attached to 10 mm diameter glass cover slips using

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3.5 wt% poly-L-lactide (Boehringer Ingelheim, Germany) in hexafluoroisopropanol (Sigma-

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Aldrich). To fabricate C-PCL films, dilute solutions of chitosan (1 wt%) and PCL (1.7 wt%)

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were mixed at the same weight ratio of 40:60 as those for nanofiber preparation, cast to form

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two-dimensional films on 10 mm diameter glass cover slips and air-dried. To remove residual

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acids from all C-PCL materials, the substrate-attached cover slips were neutralized with 14%

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ammonium hydroxide for 5 min, followed by rinsing with DI water. 6


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Pure PCL nanofibers were made from 10 wt% PCL (80,000 molecular weight, Aldrich)

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solution prepared by dissolving PCL in 2,2,2,-trifluoroethanol (TFE, Sigma-Aldrich). The PCL

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solution was then electrospun at 22 kV on a rotating grounded drum (200 rpm). The collected

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nanofibers were attached to 10 mm diameter glass cover slips using 3.5 wt% poly-L-lactide

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(Boehringer Ingelheim, Germany) in hexafluoroisopropanol (Sigma-Aldrich). C-PCL nanofibers

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were electrospun into either randomly-oriented multilayer meshes, or uniaxially aligned

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multilayer meshes, as reported previously, with mean nanofiber diameters of 215.79 ± 44.2 nm

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and 175.82 ± 55.95 nm respectively.19 In the aligned C-PCL nanofiber meshes, 90.1% of the

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nanofibers are oriented ± 20° from horizontal, with fibers overlapping each other tightly.22 The

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Young’s modulus of the random and aligned C-PCL nanofibers were measured to be 12.35 ±

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1.38 MPa and 22.41 ±1.11 MPa, respectively.22 By combining natural chitosan with synthetic

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PCL, the material exhibits high-level stability, excellent mechanical properties, and cellular

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compatibility.28 All the samples were sterilized in 70% ethanol and rinsed with PBS before cell

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culture.

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Cell culture.

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Human embryonic stem cells (hESCs), BG01v, were maintained and propagated on human

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foreskin fibroblast (hFF) feeder layers (ATCC, USA) as described previously.29 To study 7


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myogenic differentiation, hESCs were seeded at 100,000 cells/cm2 on each substrate. hESCs

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were first cultured for 24 hrs in standard proliferation culture media consisting of a 1:1 Mixture

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of Dulbecco's Modified Eagles Medium and Ham's F-12 medium (80% volume), knockout

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serum replacement (Invitrogen) (5% volume), and fetal bovine serum (Life Technologies) (15%

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volume). The Ham's F-12 medium contains 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine,

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15 mM HEPES and 0.5 mM sodium pyruvate (Gibco) supplemented with 2.0 mM

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L-Alanyl-L-Glutamine (Gibco), 0.1 mM Non-essential amino acids (Gibco), 0.1 mM

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2-mercaptoethanol (Sigma Aldrich) and 4 ng/ml basic fibroblast growth factor (bFGF) (R&D

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Systems). After cell proliferation study, the proliferation medium was subsequently replaced

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with other media for differentiation study for another 48 hrs. The hESCs were cultured in various

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media including serum-free standard proliferation medium without basic fibroblast growth factor

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(bFGF), and serum-free medium containing 20 nM Wnt3a (R&D Systems), 25 nM retinoic acid

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(Sigma-Aldrich), or 20 nM Pax3 (AbNova) in the absence of bFGF.

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Quantitative RT- PCR.

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Cell-substrate constructs were homogenized by vortexing and passing through QIAshredder

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columns (Qiagen). Total RNAs were isolated using RNeasy, and 30 ng of the total RNA from

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each sample was converted to cDNA using the QuantiTect Reverse Transcription Kit following 8


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the manufacturer’s instructions (Qiagen). SYBR Green PCR Master mix (Qiagen) was used for

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template amplification with a primer for each of the transcripts examined. Thermocycling was

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performed at the following conditions: 95°C for 15 minutes, 45 cycles of denaturation (94°C, 15

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s), annealing (55°C, 30 s), and extension (72°C, 30 s). The reaction was monitored using a

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CFX96 (BioRad) real-time PCR detection system. The relative expression of each gene was

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normalized by GAPDH expression of each sample.

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Primer sequences are listed in Supplementary Table 1.

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Scanning electron microscopy (SEM).

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Samples were removed from cell culture medium, rinsed with PBS, and fixed with

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Karnovsky’s fixative (8% paraformladehyde, 25% glutaraldehyde, 0.2 M cacodylate, 0.2 M

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sucrose) overnight. After fixation, samples were rinsed with DI water 3 times and dehydrated by

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sequential incubation in 25%, 50%, 75%, 95% and 100% ethanol for 10 min each. The samples

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were sputter-coated with Au/Pd before imaging with a scanning electron microscope (JEOL

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7000F).

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To assess hESC alignment, hESCs on nanofibers were imaged using SEM and cell

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alignment was quantified using Photoshop CS3 (Adobe). The angle of cell body relative to the

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horizontal axis was measured. 0º represents that cells are aligned parallel to the orientation of the 9


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aligned nanofibers and 90º represents that cells are aligned perpendicular to the orientation of

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aligned nanofibers.

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Immunocytochemistry.

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For immunocytochemistry studies, the samples were fixed in 4% methanol-free

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formaldehyde in PBS for 15 min, followed by ice-cold PBS washing. The cellular membrane

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was permeabilized with 0.25 v% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. After

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incubated with 10% fetal calf serum (Sigma-Aldrich) in PBS for 30 min to block non-specific

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protein binding, the samples were incubated with primary mouse anti human antibodies for

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MyoD, Myogenin, Myf5, Myf6, and MHC (Abcam) in PBS at a 1:100 dilution with 0.25 vol%

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Triton X-100 for 1 hr at room temperature. The samples were then incubated in a 1:500 dilution

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of FITC-conjugated goat anti mouse secondary antibody (Abcam) in PBS for 1 hr at room

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temperature. The samples were rinsed 3 times with PBS for each step. For confocal imaging, the

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samples were mounted onto a cover slip with Prolong Gold Antifade reagent with DAPI

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(Invitrogen), and then imaged with a Zeiss Meta 510 confocal microscope (Zeiss).

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Morphological Analysis.

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For cell morphology analysis, fluorescent images of cells undergoing myogenic

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differentiation and expressing MHC were analyzed in Photoshop CS3 Extended (Adobe Systems)

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following the reported methods.30,

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perpendicular to the length of the cell. Myotube length was measured from the furthest points

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along the length of the cell. Nuclei were selected with the magic wand tool, and circularity was

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measured with the formula 4π * (nuclear area/ nuclear perimeter2), with 1 corresponding to a

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perfect circle, and values approaching 0 indicating increasing elongation. Myotube frequency

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was determined as the percentage of MHC expressing cells over the total cells in the image.

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Three random fields of the view were assessed for each measurement.

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Myotube diameter was measured at the widest point

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Flow cytometry.

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For flow cytometry analysis, cells were detached from substrates with Versene (Gibco) and

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pelleted; the cellular membrane was permeabilized with 0.25 v% Triton X-100 (Sigma-Aldrich)

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in PBS for 10 min. After incubated with 10% fetal calf serum (Sigma-Aldrich) in PBS for 30 min

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to block non-specific protein adsorption, the samples were incubated with primary mouse anti

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human antibodies for Myogenin, Myf5, Myf6, and MHC (Abcam) in PBS at a 1:100 dilution for

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1 hr at room temperature. The cells were rinsed 3 times with PBS for each step. Fluorescently

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labeled cells were analyzed with a FACSCanto flow cytometer (BD Biosciences). 11


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Statistical Methods. The data are expressed as mean ±s.d. Statistical significance was determined using one-

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way analysis of variance (ANOVA), followed by pair-wise comparison via Student’s t-test.

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Values of p < 0.05 were considered statistically significant.

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RESULTS

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Screening myogenic micro-environment conditions.

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To determine the appropriate in vitro micro-environment for effective myogenic

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differentiation, hESCs were cultured in combined conditions of ECM-like substrates and

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signaling molecules. The expression of myogenic marker protein MyoD was quantified using

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flow cytometry 48 hours after hESCs were cultured in specific micro-environments under

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various culture conditions. MyoD, a myogenic regulatory factor in myogenic differentiation, is

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commonly used to identify myogenic progenitors.32-35 25 different micro-environments

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comprised of 5 different substrates in combination with 5 different media were screened (Fig. 1).

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The substrates include randomly-oriented PCL nanofibers (PCL random), Collagen I films

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(collagen I), randomly-oriented C-PCL nanofibers (C-PCL random), C-PCL films, and aligned

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C-PCL nanofibers (C-PCL aligned). Culture media include the standard culture medium 12


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containing 4 ng/ml bFGF and five differentiation media without bFGF: serum-free medium,

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serum-free medium containing Wnt3a (Wnt3a+), serum-free medium containing retinoic acid

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(RA+), and serum-free medium containing Pax3 (Pax3+). Serum-free conditions have been

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explored for the successful differentiation of hESCs into skeletal myoblasts14 and

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cardiomyocytes.36 Wnt3a protein is known to activate the Wnt pathway and also induce

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activation of myogenic differentiation genes.6 Retinoid acid (RA), a derivative of vitamin A,

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plays a crucial role in a wide variety of embryonic developmental process37 and was reported to

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upregulate Pax3 and MyoD expressions in mESC.12 Pax3, a transcription factor in muscle

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development, has successfully induced hESCs differentiation into myogenic lineage.9,

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As

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shown in Fig. 1a, hESCs on all C-PCL substrates (film, random and aligned nanofibers) had

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higher MyoD expression than those on PCL random nanofiber and collagen I film when cultured

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in Wnt3a+ medium. hESCs showed higher MyoD expression when cultured in Wnt3a+ medium

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than in any media without Wnt3a on all the substrates (p < 0.05). For the media containing

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Wnt3a+, hESCs showed greater MyoD expressions on the C-PCL substrates than on PCL random

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nanofibers or collagen I film (Fig. 1b). For the aligned C-PCL nanofibers, hESCs cultured in

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Wnt3a+ medium showed the greatest MyoD expression (with ~22% of the cell population

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expressing MyoD) among all the media including standard medium, serum-free medium, and

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media containing RA or Pax3 (Fig. 1c). From this screening, the combination of C-PCL 13


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substrates and Wnt3a+ was identified as the most effective micro-environment for myogenic

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differentiation of hESCs. Thus, the subsequent investigation focused on the combinations of C-

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PCL substrates and Wnt3a+.

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Effects of micro-environment conditions on gene expression.

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The change in myogenic gene transcription is indicative of myogenic differentiation. Real-

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time PCR was performed to examine the myogenic gene expressions of hESCs cultured in

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controlled micro-environments (Fig. 2). Seven mark genes (Pax3, Pax7, My5, My6, MyoD,

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myogenin, and MHC) associated with myogenic differentiation were examined. Pax 7, a paired

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box transcription factor, is essential for satellite cell formation and particularly important for

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satellite cell function.13 Myf5 is the earliest marker of myogenic activity, followed by Myf6 and

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MyoD.39 MyoD signaling upregulates myogenin activity, while activities of both MyoD and

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myogenin genes are upregulated by Myf5 and Myf6 activities.39 Myogenin activity is a later

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regulator of the myogenic program and is necessary for the expression of muscle structural

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protein MHC.39 When hESCs were cultured on collagen I film in Wnt3a+ medium, only three of

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the examined genes (Pax3, Pax7 and MyoD) were elevated as compared to those cultured in

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standard and serum free media (Fig. 2a). hESCs cultured on PCL random nanofibers displayed

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myogenic gene expressions similar to those on collagen I film (Fig. 2b). On C-PCL films, hESCs 14


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showed significant up-regulation of all myogenic gene expressions except MHC (Fig. 2c).

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hESCs on C-PCL random nanofibers display increased myogenic gene expressions except Myf6

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as compared to standard and serum-free media when cultured in Wnt3a+ medium (Fig. 2d).

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Notably, on C-PCL aligned nanofibers, hESCs showed significant increases in the expressions in

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all myogenic genes in both serum-free and Wnt3a+ media as compared to hESCs cultured in

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standard (Fig. 2e).

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Effects of micro-environment conditions on cell morphology and alignment.

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SEM images were acquired to examine hESC morphology in controlled micro-

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environments (Fig. 3). After 48 hrs of culture in standard media, hESCs retained a spherical

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morphology on all substrates (Collagen I film, C-PCL film, and C-PCL aligned nanofiber).

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Specifically, hESCs displayed stacked cell colonies on collagen I film and C-PCL aligned

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nanofibers (Fig. 3a), but loosely dispersed as single cells on C-PCL films and C-PCL random

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nanofibers indicating reduced initial adhesion and limited colony formation on these materials

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(Fig. 3a). hESCs cultured on all substrates in Wnt3a+ medium displayed significantly different

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morphologies (Fig. 3b) than in standard media. On collagen I film, cells displayed mixed

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spherical and spindle morphologies. On C-PCL films, hESCs exhibited a more spread and

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flattened morphology. On C-PCL random nanofibers, the cells maintained mixed elongated and 15


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spherical morphologies. Notably on C-PCL aligned nanofibers in Wnt3a+ medium, hESCs

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displayed an elongated morphology along the orientation of the aligned nanofibers (Fig. 3b and

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Supplementary Fig. 1).

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To further illustrate the ability of ECM topology to direct hESC alignment, cellular

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alignment was quantified by SEM image analysis. The direction of each hESC was defined as an

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angle value relative to the orientation of the aligned nanofibers. Quantitative morphometric

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analysis confirmed that ECM topology can direct hESC alignment (Fig. 3c). Long range

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alignment of hESCs was observed on C-PCL aligned nanofibers. In contrast, no long range order

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was observed in hESCs cultured on collagen I film, C-PCL film, or C-PCL random nanofibers.

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Effects of micro-environment conditions on protein expression.

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After gene transcription into mRNA, depending on downstream processing, the mRNA may

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translate into functional proteins. To examine the distribution of myogenic marker proteins in

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different culture micro-environments, hESCs were immunofluorescently stained for Myf5, Myf6,

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MyoD, myogenin and MHC (FITC, green), and counterstained for cell nuclei (DAPI, blue). As

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expected, fluorescence images of hESCs cultured on both collagen I film (Fig. 4a) and C-PCL

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aligned nanofibers (Fig. 4b) for 48 hrs showed weak protein expressions of myogenic marker

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genes. These results suggest that nanofibers alone could not enhance myogenic differentiation. 16


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When cultured in Wnt3a+ medium, the myogenic marker proteins of hESCs highly

2

expressed on both collagen I film (Fig. 5a) and C-PCL aligned nanofibers (Fig. 5b) after 48 hrs.

3

In contrast, hESCs cultured on C-PCL films displayed no expression of myogenic proteins

4

(absence of green color) in either standard medium or Wnt3a+ medium (Supplementary Fig. 2).

5

In addition, MHC expression was only faintly visible in hESCs cultured on collagen I film (Fig.

6

5a), but was highly expressed in hESCs cultured on C-PCL aligned nanofibers (Fig. 5b).

7

Morphological analysis of hESCs undergoing myogenic differentiation was performed (Table

8

1.). hESCs undergoing myogenic differentiation on C-PCL aligned nanofibers were greater in

9

diameter and length than hESCs cultured on collagen I film. Additionally, nuclear elongation,

10

another hallmark of myogenic differentiation,30, 31 was greater in the C-PCL aligned nanofibers

11

condition than in collagen I film. Furthermore, cell counting of MHC positive cells determined a

12

62% frequency of myotubes among hESCs cultured on C-PCL aligned nanofibers versus just a

13

2.8% frequency of myotubes among hESCs cultured collagen I.

14

Flow cytometry was then used to quantify the myogenic protein expression of hESCs (Fig.

15

6). Myf5 expression was detected in 63% of hESCs cultured on C-PCL aligned nanofibers, while

16

only in 18% of hESCs on collagen I. Myf6 expression was also observed in 91% of hESCs

17

cultured on C-PCL aligned, compared to 21% on collagen I film. Myogenin expression was

18

detected in 81% of hESCs cultured on C-PCL aligned nanofibers, compared to 24% on collagen 17


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I film. Finally, MHC expression was detected in 63% of hESCs cultured on C-PCL aligned

2

nanofibers, compared to 4% on collagen I film, consistent with cell counting of MHC positive

3

cells. Together, these results indicate that culture on aligned nanofibers in the presence of Wnt3a

4

promotes the directed myogenic differentiation of hESCs.

5 6

Effects of micro-environment conditions on pluripotency and the Wnt signaling pathway.

7

The mRNA expression of genes associated with pluripotency and Wnt signaling pathway of

8

hESCs was also evaluated by real-time PCR (Fig. 7). OCT4 is a hallmark gene of hESC

9

pluripotency, and the decreased OCT4 expression indicates differentiation.40, 41 Wnt1, Wnt3a and

10

Wnt7a are known as gatekeepers of the myogenic program, and their elevated expression

11

indicates myogenic differentiation.6,

12

substrates showed a small, but not statistically significant, decrease in OCT4 expression

13

compared to hESCs cultured in standard and serum free media. For hESCs cultured on collagen

14

I (Fig. 7a) or PCL randomly-oriented nanofibers (Fig. 7b) in the Wnt3a+ medium, an elevated

15

expression of Wnt3a was observed compared to cells cultured in standard or serum-free media.

16

When cultured on C-PCL films in Wnt3a+ media (Fig. 7c), hESCs showed increased expressions

17

of both Wnt3a and Wnt7a compared to those cultured in both standard and serum-free, indicating

18

the occurrence of myogenesis.42 hESCs on C-PCL random nanofibers induced statistically

34, 42

When cultured in Wnt3a+ medium, hESCs on all

18


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significant increases in expressions of Wnt1, Wnt3a and Wnt7a genes when cultured in Wnt3a+

2

medium (Fig. 7d), which is expected at the onset of myogenic differentiation.6,

3

aligned nanofibers were uniquely able to induce increased expressions of Wnt related genes of

4

hESCs when they are cultured in both serum-free and Wnt3a+ media than those cultured in

5

standard media (Fig. 7e).

42-44

C-PCL

6 7

DISCUSSION

8

The preliminary screening for appropriate myogenic micro-environments was based on the

9

early 48 hour expression levels of MyoD protein (Fig. 1), an early marker for myogenic

10

commitment.35 All the hESCs on C-PCL substrates showed better MyoD expression than those

11

on collagen I or PCL in Wnt3a+ medium. Among C-PCL substrates, hESCs on C-PCL aligned

12

nanofibers showed the greatest MyoD expression. This result suggests that the ECM topology is

13

important in directing myogenic differentiation of hESCs, which is consistent with previous

14

findings that the micro-environment affects cell behavior and fate.45-47

15

The use of serum-free media resulted in notable increased MyoD expression in hESCs

16

compared to the standard media, especially on C-PCL films and C-PCL aligned nanofibers. This

17

result is consistent with previous studies where serum-free media was found to induce myogenic

18

differentiation.14 The addition of Wnt3a substantially elevated MyoD transcription level for all 19


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substrates after 48 hrs of cell culture. Wnt3a has been repeatedly observed to play a key role in

2

regulating myogenesis.6, 23, 35, 43 Wnt3a is also reported to induce epaxial myotome myogenesis

3

which results in skeletal muscle formation during embryonic myogenesis.39 Wnt signaling is

4

considered as a molecular switch that regulates the cell transition from proliferation to myogenic

5

differentiation via β-catenin pathway activation.42 Our findings further suggest that the myogenic

6

response is highly dependent on the combination of topological and chemical cues of substrates

7

and signaling molecules. The combination of C-PCL materials and Wnt3a are shown to induce

8

greater increases in MyoD activity than singling molecule alone, or any other tested

9

combinations of materials and singling molecules.

10

The analysis of myogenic gene expressions of hESCs cultured in different controlled

11

conditions highlight the significance of appropriate micro-environment towards myogenic

12

differentiation. The use of C-PCL aligned nanofibers increased myogenic activity (Fig. 2).

13

Interestingly, hESCs on the PCL random expressed a similar level of mRNA expression of

14

myogenic genes in both serum-free and Wnt3a+ media, suggesting the dominant effect in

15

regulating myogenic activity was the ECM rather than the soluble growth factors. When hESCs

16

were cultured on C-PCL aligned fibers, MHC gene expression was significantly upregulated in

17

Wnt3a+ medium as compared to both standard and serum-free media (Fig. 2e). These gene

20


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activity assessments suggest that an optimized differentiation environment may improve the

2

likelihood of advanced myogenic differentiation.

3

Analysis of hESCs morphology further revealed the importance of controlled ECM micro-

4

environment (Fig. 3). The collagen I film represents a common myogenic and tissue engineering

5

substrate.16, 48-50 C-PCL film represents a flat two-dimensional C-PCL surface that was selected

6

to examine the cellular response to the C-PCL material devoid of cell scale topology. C-PCL

7

random-nanofibers create ECM-scale topology while maintaining overall isotropy. C-PCL

8

aligned-nanofiber represents both ECM-scale topology and long-range anisotropy. After 48

9

hours in standard media, hESCs in all substrates except for C-PCL random exhibited spherical

10

morphology, which is typical of undifferentiated state.51 The strong matrix-cell interactions

11

might have limited hESC spreading on the C-PCL random material. However, the adhesion of

12

hESCs to C-PCL film was poor, with minimal cell spreading. The topology-induced guidance for

13

hESCs was more evident 48 hours after culturing in Wnt3a+ medium. Only a very few elongated

14

hESCs were observed on collagen I, C-PCL films, and C-PCL random, and moreover, the

15

orientation of the cell elongation is random on these materials (Fig. 3c), indicating that myogenic

16

differentiation is very limited. In contrast, the hESC population on C-PCL aligned nanofibers

17

was nearly entirely elongated and aligned parallel to the direction of fiber alignment, with high

18

cell density (Fig. 3c). This study further indicates that the hESCs cultured on the C-PCL aligned 21


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nanofibers differentiate more rapidly under the morphogenetic guidance of the fibers, as has been

2

similarly observed in other studies of cell differentiation on fibers.2, 19, 22, 52 These anisotropic

3

nanofibrous substrates more closely replicated the native myofibrillar differentiation

4

microenvironment than traditional two-dimensional culture substrates.

5

The expression of myogenic marker proteins by hESCs was significantly different in

6

standard media (Fig. 4) than Wnt3a+ media (Fig. 5). Similar to the trends observed from

7

morphological analysis, the only minimal expression of myogenic protein markers in standard

8

medium was observed in both collagen I and C-PCL aligned nanofibers. Collectively, expression

9

levels of Myf5, Myf6, MyoD and myogenin were consistently elevated for cells on C-PCL

10

aligned nanofibers compared to hESCs cultured on collagen I nanofibers in Wnt3a+ medium.

11

Quantitative assessment of myogenic protein expressions in differentiating conditions by flow

12

cytometry (Fig. 6) corroborated immunofluorescent observations (Fig. 5 and Table 1).

13

Importantly, MHC expression was detected only in hESCs cultured on C-PCL aligned nanofibers

14

in Wnt3a+ medium, suggesting that terminal myogenic differentiation into mature myoblasts was

15

achieved only in the aligned C-PCL nanofibers supplemented with Wnt3a growth factor. A

16

combination of Wnt3a media condition and aligned ECM-like material induced 63% MHC-

17

expression cells in 2 days (Fig. 6), 16 times higher and 5 times faster than the highest reported

18

MHC expression by MSCs cultured in Wnt3a+ medium for 10 days.6 The protein expression 22


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patterns support that the underlying substrate material and morphology can be tuned to

2

significantly improve efficacy and expediency of myogenic differentiation from hESCs in vitro.

3

When examining genes relating to pluripotency and embryogenesis, expression of the

4

pluripotency marker OCT4 declined slightly in Wnt3a+ conditions, and the expressions of genes

5

related to Wnt signaling pathways are elevated in all C-PCL substrates (Fig. 7).

6 7

CONCLUSIONS

8

We presented a promising ECM-like micro-environment comprised of topology cue and

9

signaling function for embryonic myogenesis. Using C-PCL random nanofibers and C-PCL films

10

as topographical controls, and PCL and C-PCL random nanofibers and collagen I films as

11

material controls, the topological cues provided by C-PCL aligned nanofibers were shown to

12

play a key role in inducing hESCs myogenic differentiation. The unique topology of the aligned

13

C-PCL nanofibers significantly increased expressions of myogenic marker genes and proteins.

14

Addition of the growth factor Wnt3a further enhanced the hESC myogenic differentiation. To the

15

best of our knowledge, our culture system demonstrated the highest and most fast myogenic

16

differentiation from stem cells that have been studied to date. Significantly, the high myogenic

17

differentiation was achieved under a controlled culture condition without the support of feeder

18

layers. This feeder-free cell culture system would facilitate studies of myogenesis by shortening 23


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Page 24 of 38

1

experimental times, and serving as a consistent model of embryonic myogenesis. Further, the

2

system can be used to quickly generate a large population of highly committed myogenic

3

progenitors, which are necessary for therapeutic potential of hESCs in muscle repair.

4 5

ACKNOWLEDGEMENTS

6

This work was supported in part by the Kyocera Professor Endowment and the Washington

7

Research Foundation.

8 9

Supporting Information Available:

10

Primer sequences used for quantitative RT-PCR; confocal microscopy of hESCs cultured for 48

11

hours in standard media and Wnt3a+ media on C-PCL films. This material is available free of

12

charge via the Internet at http://pubs.acs.org.

13

24


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FIGURES

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Figure 1. Myogenic micro-environments screening by flow cytometry. (a) Populations of hESCs

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that express MyoD after 48 hrs of culture in various substrates and media containing various

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growth factors. (b) The populations of hESCs express MyoD after cultured on various material

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substrates in medium containing Wnt3a. (c) The populations of hESCs express MyoD after

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cultured on C-PCL aligned nanofibers in media containing various growth factors. Results are

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presented as mean ± s.d., and * indicates that each of the means in that group is statistically

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different from all means in the other group, p < 0.05, n = 3.

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Biomacromolecules

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Figure 2. Myogenic gene expressions of hESCs cultured for 48 hrs on (a) collagen I, (b) PCL

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randomly-oriented fibers, (c) C-PCL films, (d) C-PCL random nanofibers, and (e) C-PCL

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aligned nanofibers in standard medium, serum-free medium, and Wnt3a+ medium. The values

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are presented as relative to the expression of GAPDH. All the results are expressed as the mean ±

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s.d. * indicates the value is statistically different from the other in the same group, ** indicates

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the value is statistically different from the standard media value in that group p < 0.05, n = 3.

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Figure 3. SEM images showing the morphology of hESCs cultured in different controlled

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conditions. hESCs were cultured on either collagen I, C-PCL films, C-PCL random or C-PCL

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aligned nanofibers for 48 hrs in (a) standard medium, or (b) Wnt3a+ medium. The scale bar

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represents 25 µm. (c) Normalized histograms of hESC orientation after 48 hrs of culture in

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Wnt3a+ medium. The scale bar represents 10 µm.

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Biomacromolecules

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Figure 4. Confocal immunofluorescent images of hESCs cultured for 48 hrs in standard

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conditions on (a) collagen I film and (b) C-PCL aligned nanofibers. Cells were stained with

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DAPI to identify nuclei (blue, column 1), and specific antibodies to the proteins of interest

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(green, column 2). Co-localization of target protein and nuclei is also illustrated (merged, column

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3). The scale bars represent 20 µm.

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Page 34 of 38

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Figure 5. Confocal immunofluorescent images of hESCs cultured for 48 hrs in Wnt3a+ medium

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on (a) collagen I film and (b) C-PCL aligned nanofibers. Cells were stained with DAPI to

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identify nuclei (blue, column 1), and specific antibodies to the proteins of interest (green, column

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2). Co-localization of target proteins and nuclei is also illustrated (merged, column 3). The scale

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bars represent 20 µm.

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Biomacromolecules

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Figure 6. Myogenic protein expressions of hESCs cultured for 48 hrs in Wnt3a+ medium on

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collagen I films (top row) and C-PCL aligned nanofibers (middle row) as assessed by flow

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cytometry. hESCs were stained against Myf5, Myf6, myogenin and MHC (red peaks), compared

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with hESCs stained with isotype-IgG as negative controls (black peaks). Population percentage

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of hESCs stained positive by specific proteins are presented graphically (bottom row). 35


Biomacromolecules

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Page 36 of 38

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Figure 7. Pluripotency and Wnt signaling pathway assessment of hESCs cultured for 48 hrs on

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(a) collagen I film, (b) PCL random nanofibers, (c) C-PCL films, (d) C-PCL random nanofibers,

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and (e) C-PCL aligned nanofibers in either standard medium, serum-free medium, or Wnt3a+

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medium. The values are presented relative to the expressions of GAPDH. All the results are

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expressed as mean ± s.d. * indicated the value is statistically different from the all other values in

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the same treatment group, ** indicates the value is statistically different from the standard media

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value only in that treatment group, p < 0.05, n = 3.

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TABLES

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Table 1. Morphological analysis of MHC expressing cells undergoing myogenic differentiation

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after 48 hrs of culture in Wnt3a+ medium. Myotube diameter and length are means ± s.d.

Myotube Diameter Myotube Length Circularity of Nucleus Myotube Frequency (% of total cells)

Collagen I Substrate

C-PCL Aligned Substrate

6.7 ± 0.38 µm 10.8 ± 2.4 µm 0.74 ± 0.086 2.8 %

7.3 ± 1.5 µm 44 ± 12 µm 0.46 ± 0.18 62 %

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FOR TABLE OF CONTENTS USE ONLY

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Nanofiber-based In Vitro System for High Myogenic Differentiation of Human Embryonic Stem Cells

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Matthew Leung, Ashleigh Cooper, Soumen Jana, Ching-Ting Tsao, Timothy Petrie, and Miqin Zhang

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Nanofiber-Based in Vitro System for High Myogenic Differentiation of

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