Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells / Journal of Rural Medicine

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

Engineering Biomaterials for Feeder-Free Maintenance of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) are capable of differentiating into any type of somatic cell, a characteristic that imparts significant therapeutic potential. Human embryonic stem cells and induced pluripotent stem cells are types of hPSCs. Although hPSCs have high therapeutic potential, their clinical relevance is limited by the requirement for animal feeder layers, which maintain their pluripotency and self-renewal. hPSCs grown on animal feeder cells are at high risk for pathogen contamination and can be affected by the immunogenicity of the feeder layer. The presence of animal feeder cells also limits the scalability of hPSCs in culture because of the high cost of culturing and batch-to-batch variations. Therefore, development of feeder-free systems is imperative for robust, lower-cost, xeno-free, scalable culture of hPSCs. Biomaterials engineered with bioactive molecules such as adhesion proteins and extracellular matrix proteins, or synthetic materials such as peptides and polymers, may provide alternative substrates to animal feeder cells. This article reviews biomaterial-based, feeder-free systems for hPSC growth and maintenance, which provide clinically relevant alternatives to feeder cell systems.