Silk-Carbon Nanotube Composite for Stem Cell Neuronal Differentiation

Chi-Shuo Chen*, Catherine Le*, Sushant Soni, Eric Y-T Chen, Wei-Chun Chin

Bioengineering, University of California, Merced 5200 North Lake Road

Merced, California, USA Phone: (209) 228-8668, Fax: (209) 228-4047, E-mail: wchin2@ucmerced.edu

*these authors contributed equally to this work

Abstract This study aims to create a new paradigm for building 3D silk-carbon nanotube (CNT) based composite scaffolds in order to acquire better neuron differentiation efficiency from human embryonic stem cells (hESCs). CNT and silk have been successfully used for neuronal differentiation and tissue engineering applications. The silk-CNT composite scaffolds can serve as efficient support matrices for stem cell derived neuronal transplants that offer a promising opportunity for nerve injury treatments in spinal cord injury (SCI) patients. Keywords: stem cell, carbon nanotube, silk, neuron and differentiation.

Introduction

There are about 250,000 to 400,000 patients in the US suffer from spinal cord injury (SCI) [1]. Human embryonic stem cells (hESCs) can differentiate into specific lineages responding to regulated spatial and temporal signals, holding great promise for many SCI patients. The unique appeal of ESCs-based transplantation for SCI is the possibility of those transplanted cells to repair damaged tissues. However, the harsh harmful microenvironment and the lack of supportive substrate during transplantation result in only a small fraction of transplanted cells survive and diminish the potential of stem cell-related cell therapy [2]. CNTs with sizes comparable to ECM molecules such as collagens and laminins, which have been reported to favor neuron growth. CNTs have excellent mechanical strength while flexible. Therefore, CNTs are able to maintain scaffolds’ structural integrity during cell growth. It has been reported that substrates prepared using CNTs are biocompatible and can support neuron growth and differentiation[3] . It has also been proposed that neurons grown on CNT meshwork displayed better signal transmission, possibly because that CNTs may form tight contacts with neuron membranes to favor electrical shortcuts [4]. All of these characteristics make CNTs a promising material to repair damaged neurons. Silk from commercial silk worms (Bombyx mori) has been used as building material for scaffolds for many stem cell and tissue engineering applications [5,6,7]. Our preliminary results also reveal that silk-based and CNT-based biomaterials are able to promote neuronal differentiation from hESCs. This paper provide a new paradigm for building 3D silk-CNT scaffolds in order to acquire better neuron differentiation efficiency from hESCs and more effective neuronal cell transplantation.

Experiment

A. Silk Fibroin Preparation Based on the protocol published by Kaplan et al. [7,8], Bombyx mori silk pre-washed in boiling 0.02 M Na2CO3 (Sigma Aldrich, St. Louis, MO) for 1 h and then rinsed thoroughly with deionized water to remove the sericin protein from the fibroin. The remaining fibroin was then dissolved in 9.3M LiBr (Fisher Scientific) for 3 h at 60˚C. The fibroin solution was then dialyzed (MWCO 1,000) in DI water for 48 h. Following which the silk solution was centrifuged at 800 g and the supernatant was collected [8]. B. Fibroin Solution/CNT Coating MWCNT (Nano-Lab, Waltham, MA) were dispersed in DI water following which they were sonicated to help disperse the MWCNT throughout the solution more evenly. Glass micro coverslips were boiled in a mild surfactant for 30 mins and cleaned thoroughly by DI water. The coverslips were then washed with 2N HCl overnight and cleaned with DI water. The coverslips were then dried in an oven at 80˚C for 2 h. The MWCNT was further diluted to achieve a final concentration of 1 mg/ml by adding silk fibroin solution and DI water respectively. Furthermore, for the coating the silk fibroin solution was further diluted to 2% wt solution. The cover slips were then coated with the two solutions –– MWCNT with Silk and Silk with DI water on the coverslips three times at 60˚C. E. Maintenance and differentiation of human embryonic stem cells hESC lines H9 from Wicell (Madison, WI, passage 32-55) were cultured on feeder layers of mitomycin C treated mouse embryonic fibroblasts (MEF) as described in our previous study [9]. Medium were changed daily and differentiated cells were moved manually after 7 days. The EB cell colonies were detached from the MEF feeder layer with dispase (1 Uml-1) and transferred to ultra-low contact wells. The suspended EB cell colonies are allowed to grow for 4-6 days before plating on substrates. The EB cell aggregations were then seeded, about 7-10 colonies, onto each silk and silk/CNT substrates, with neuron induction medium consisting of F12/DMEM, N2 supplement, and FGF2 (20 ng/ml). Medium were once changed daily for the first 2 days and then once every other day. Cells were fixed with 4% paraformaldehyde at the 7th day.

C. Immunocytochemistry and fluorescence measurements We stained cells using β–tubulin III as a marker for neuronal

differentiation with a ratio of 1:500, Nestin (1:500) as markers for motor neuron progenitor [3], and DAPI (1:5,000) as nuclei markers. Images were taken with a Nikon Eclipse TE2000-U fluorescent microscope (Nikon Eclipse TE2000-U, Tokyo, Japan). Fluorescence intensities and axon lengths were quantified, using the image analysis software, simplePCI (Compix Inc., Sewickley, PA, USA).

Results The β–tubulin III and Nestin were used to determine the hESCs differentiation. The image analysis showed the neuron markers, β–tubulin III and Nestin, were highly up-regulated in hESCs grow on silk/CNT substrate (Fig. 2). Distinct neural somas and more noticeable axon shootings on silk/CNT substrates were observed (Fig. 1). However, less mature neural cell bodies and little to no axon shootings were seen on substrates derived from silk alone compared to cells on silk/CNT substrate. The axon length was measured using image analysis (Fig 2). We also noticed that both silk and silk/CNT substrates began to degrade and become more brittle after 14 days of incubation. This feature will be essential to the natural biodegradation of the silk/CNT substrates as a vehicle for potential neural implantation.

Fig. 1 Fluorescence image of β-tubulin III expression on silk/CNT substrates. Scale bar equals to 100 μm.

β-III Nestin0.0

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Fig. 2 Quantative measurements of β–tubulin III and Nestin expression in hESCs on silk and silk/CNT substrates.

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Fig. 3 Axon lengths as measured by β-tubulin III expression on silk and silk/CNT substrates.

Discussion CNT has been used to promoted hESCs into neuron lineage [9]. However, the difficulty of CNT transplantation limited its applications. The amphiphilic natural copolymer, fibrin, dispersed MWCNT in water and formed a polymer film to hold CNT within the biodegradable matrix. Integrating silk and MWCNT, our results demonstrated the potential of silk/CNT composite as scaffolds for neuron regenerative medicine.

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