Reduction of N-Glycolylneuraminic Acid in Human Induced Pluripotent Stem Cells Generated or Cultured under Feeder- and Serum-Free Defined Conditions

Abstract

BackgroundThe successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells.Methodology/Principal FindingsHere we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative.Conclusion/SignificanceThis study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells

“…Finally, another important challenge before attempting clinical application pertains to the use of agents and cells derived from xenogeneic sources. Currently, animal-derived serum (bovine serum) is used for establishing iPS cells and inducing neural differentiation; however, a method to establish iPS cells without using such serum has also been reported [50], although these iPS cells will have to be characterized from the beginning. To accelerate these preclinical studies in the future, traceability of animal-derived agents should be ensured and methods of clean-up at the stage of the final product, namely, iPS-NS/PCs, according to GMP should be established as a practical strategy.…”

Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples

“…23 Imatinib (LC Laboratories) was added to the culture medium at the various concentrations (1-10M). U0126 and LY294004 (LC Laboratories) were used to inhibit ERK and AKT, respectively.…”

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

“…iPS cells are usually maintained on feeder cells that are derived from mouse embryonic fibroblasts (MEFs) (Takahashi and Yamanaka 2006;Takahashi et al 2007;Yu et al 2007). However, several feederfree conditions were developed recently especially for human embryonic stem (ES) cells and iPS cells to ensure biosafe human clinical applications (Xu et al 2001;Amit et al 2004;Rosler et al 2004;Lu et al 2006;Navarro-Alvarez et al 2008;Miyazaki et al 2008;Sun et al 2009;Kitajima and Niwa 2010;Rodin et al 2010;Hayashi et al 2010;Fukusumi et al 2013). Animal-derived components are relatively high risk for potential infections in clinical regenerative medicine.…”