A graphene-based platform for induced pluripotent stem cells culture and differentiation

doi:10.1016/j.biomaterials.2011.09.071

Biomaterials

Volume 33, Issue 2, January 2012, Pages 418-427

https://doi.org/10.1016/j.biomaterials.2011.09.071 Get rights and content

Abstract

Induced pluripotent stem cells (iPSCs) hold great promise as a cell source for regenerative medicine yet its culture, maintenance of pluripotency and induction of differentiation remain challenging. Conversely, graphene (G) and graphene oxide (GO) have captured tremendous interests in the fields of materials science, physics, chemistry and nanotechnology. Here we report on that G and GO can support the mouse iPSCs culture and allow for spontaneous differentiation. Intriguingly, G and GO surfaces led to distinct cell proliferation and differentiation characteristics. In comparison with the glass surface, iPSCs cultured on the G surface exhibited similar degrees of cell adhesion and proliferation while iPSCs on the GO surface adhered and proliferated at a faster rate. Moreover, G favorably maintained the iPSCs in the undifferentiated state while GO expedited the differentiation. The iPSCs cultured on both G and GO surfaces spontaneously differentiated into ectodermal and mesodermal lineages without significant disparity, but G suppressed the iPSCs differentiation towards the endodermal lineage whereas GO augmented the endodermal differentiation. These data collectively demonstrated that the different surface properties of G and GO governed the iPSCs behavior and implicate the potentials of graphene-based materials as a platform for iPSCs culture and diverse applications.

Introduction

Graphene is an atomic-thick sheet of carbon atoms arranged in two-dimensional (2D) honeycomb structure with unique physical, chemical and mechanical properties [1], [2]. The capability of biofunctionalization of graphene and its derivative, graphene oxide (GO), has brought these nanomaterials under spotlight and has drawn intense attention for a plethora of applications in biotechnology including bioassays [3], biosensors [4], photothermal anticancer therapy [5] and electrical stimulation of cells [6]. Thanks to the biocompatibility at low concentration [7] and 2D nature with ultra-large surface area, graphene and GO have recently captured interests as cell culture substrates. Substrates coated with graphene or GO have enabled the culture of several mammalian cells including NIH-3T3 fibroblast [8] and A549 [9], but the widespread use of graphene and GO for cell culture necessitates more intensive research.

Induced pluripotent stem cells (iPSCs) are pluripotent cells that can be derived from somatic cells by introducing a cocktail of reprogramming factors while obviating the need to destroy embryos [10], [11]. Similar to embryonic stem (ES) cells, iPSCs can be maintained in the undifferentiated state indefinitely but can differentiate into cells belonging to all 3 germ layers: endoderm (e.g. hepatocytes, insulin-producing β cells and lung epithelium), mesoderm (e.g. osteoblast and chondrocytes) and ectoderm (e.g. neural cells). The pluripotency and avoidance of ethical issues render iPSCs a promising cell source for the regeneration of virtually all tissues/organs and their potentials in disease modeling [12] as well as the treatment of various diseases including Parkinson’s diseases [13], hemophilia [14] and sickle cell anemia [15] have been implicated. To maintain the pluripotency, iPSCs typically need to be cultured on the feeder layer cells (e.g. mouse embryonic fibroblasts) with appropriate cytokines such as leukemia inhibitory factor (LIF) [16]. Without these supporting cell/matrix and anti-differentiation factors, iPSCs in 2D or in suspension cultures spontaneously differentiate in vitro and form 3D aggregates known as embryoid bodies (EBs) which encompass cells of endodermal, mesodermal and ectodermal lineages. As differentiation continues, a variety of cell types are developed within the EBs environment.

Given the promise of iPSCs in regenerative medicine and graphene-based materials as biomaterials, the overriding objectives of this study were to evaluate the feasibility of culturing iPSCs on the surface of G- and GO-coated substrates, and to assess how the surface properties dictated the iPSCs proliferation and differentiation.

Section snippets

Preparation of graphene oxide (GO)

All of the chemicals were used as received. GO was prepared by oxidation and exfoliation of commercially available graphite by Hummer’s method. Briefly, 0.5 g of natural graphite (Bay Carbon, SP-1) and 0.5 g of sodium nitrate (NaNO₃, J. T. Baker) was mixed in a 500 ml round bottom flask, followed by the addition of concentrated sulfuric acid (95–97%, Sigma–Aldrich) and stirring on ice. When the temperature dropped to 0 °C, 3 g of potassium permanganate (KMnO₄, J. T. Baker) was slowly added to

Preparation and characterization of G- and GO-coated substrates

GO sheets were prepared by Hummer’s method and the presence of epoxide, hydroxyl, carbonyl and carboxyl groups was confirmed by FTIR and XPS (Fig. 1a–c). AFM images revealed that they were mostly single-layered with lateral size ranging from 2 to 6 μm (Fig. 2a,b). For iPSCs culture, GO was tightly immobilized onto clean glass coverslips. G-coated coverslips were obtained via direct hydrazine reduction of GO sheets. SEM (Fig. 3) and AFM (Fig. 4) images of G and GO sheets immobilized on the

Discussion

In this study we unveiled that G- and GO-coated substrates are biocompatible with iPSCs and enable the cell adherence and proliferation, which supports the notions that GO exerts low cytotoxicity to mammalian cells including A549, NIH-3T3 and human fibroblasts [9], [28], [29] and G is biocompatible to human mesenchymal stem cells [30]. However, GO enables more favorable iPSCs adherence and proliferation than G, probably because the more abundant oxide groups on the GO surface (Fig. 1) conferred

Conclusions

In summary, hereby we developed G- and GO-coated biomaterials, which allow for attachment, proliferation and differential differentiation of iPSCs and hold great promise for iPSCs culture.

Acknowledgements

The authors acknowledge the generous gift of mouse iPSCs from Dr. Shinya Yamanaka (Center for iPS Cell Research and Application, Kyoto University) and the financial support from the National Tsing Hua University (Booster Program 99N2544E1 and Toward World-Class University Project 100N2050E1) and National Science Council (99-2221-E-007-025-MY3, 99-2221-E-007-096), Taiwan.

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¹: These two authors contributed equally to this work.

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A graphene-based platform for induced pluripotent stem cells culture and differentiation

Abstract

Introduction

Section snippets

Preparation of graphene oxide (GO)

Preparation and characterization of G- and GO-coated substrates

Discussion

Conclusions

Acknowledgements

Biomaterials

Biomaterials

Biomaterials

Biomaterials

Cell

Mol Ther

Mol Ther

J Genet Genomics

Biochem Biophys Res Commun

Mol Ther

Biomaterials

Carbon

Biochem Biophys Res Commun

Graphene: the new two-dimensional nanomaterial

Angew Chem Int Ed

Electric field effect in atomically thin carbon films

Science

Biocompatibility of graphene oxide

Nanoscale Res Lett

Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies

ACS Nano

In vitro toxicity evaluation of graphene oxide on A549 cells

Toxicol Lett