Abstract
Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.
Highlights
Human pluripotent stem cell lines were grown in a coculture system using a layer of mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells.[1,2,3]
We suggest that choosing the components of the culture medium and the scaffolding materials can ensure maintenance of pluripotency and cell survival through PI3K/AKT and Fyn-Ras homolog gene family member A (RhoA)-ROCK signaling pathways in humaninduced pluripotent stem cells (hiPSCs) culture
This review highlights the components that are suitable for serum- and xeno-free media and feeder cell-free culture systems that use laminin-511 as the scaffold
Summary
Human pluripotent stem cell (hPSC) lines were grown in a coculture system using a layer of mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells.[1,2,3] to be clinically compliant, humaninduced pluripotent stem cells (hiPSCs) need to be cultured in a medium that is serum free, chemically defined, and free of animal products (xeno free). These proteins and glycans are necessary for hiPSC attachment, proliferation, and self-renewal[36] (Fig. 1). Matrigel, which includes laminin-111, collagen IV, fibronectin, and vitronectin, can be used to replace the MEF feeder layer and support robust hPSC growth.[12,13,14] induces transcription of TGF-b-dependent genes that regulate signaling pathways involved in pluripotency and self-renewal, such as the MAPK and PI3K/ AKT pathways Signaling through these pathways supports proliferation and continued pluripotency in hiPSCs44 (Fig. 2). TGF-b is an essential additive in culture medium using a laminin-511 scaffold (Fig. 3)
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