Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene.

Hyunah Lee,Chan Young Park,Hans R Schöler,Donggyu Nam,Taehee Lee,Jae-Kyung Choi,Hyun-Wook Kang,Holm Zaehres,Jeong Beom Kim,Soon-Yong Kwon,Marcos J Araúzo-Bravo

doi:10.1038/srep20708

Abstract

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Highlights

Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells, hold great potential for regenerative medicine[1,2]
Polycrystalline nickel (Ni) films were deposited onto the plates at room temperature, and the carbon (C) atoms dissociated from graphite powder were diffused through the grain boundaries (GBs) of Ni during the diffusion assisted synthesis (DAS) process[25]
Root-mean-square roughness (Rq) measured by atomic force microscopy (AFM) showed that DAS/GL (2.2 ± 0.35 nm) was 4 times higher than those of chemical vapor deposition (CVD) coated GL (CVD/GL) (0.65 ± 0.1 nm) and bare GL substrate (0.5 ± 0.05 nm), which indicates the presence of graphene ridges on diffusion assisted synthesis-grown nanocrystalline graphene (DAS-nanocrystalline graphene (NG)) layers (Fig. 1f and Fig. S1c)

Summary

Introduction

Human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), hold great potential for regenerative medicine[1,2]. Xenogeneic substrates including mouse embryonic fibroblast (MEF) feeders[1], or Matrigel and extracellular matrix (ECM) isolated from mouse sarcoma[3,5,7] must be avoided for generating clinical-grade hPSC due to the risk of xenogeneic contamination[10,11,12] To address this issue, feeder-dependent or feeder-free culture methods under xeno-free condition have been developed by employing allogeneic substrates such as human fibroblast feeder cells[4,9] or purified human ECMs (collagen, fibronectin, laminin or vitronectin)[6,8,13] respectively. We successfully established feeder-free and xeno-free culture system for long-term maintenance of hPSC in an undifferentiated state through employing DAS-NG This is the first report of hPSC maintenance on synthetic graphene surface without ECM coating, which allows the generation of clinical-grade hPSC at large-scale on a pathogen free culture platform

Methods

Results

Conclusion