Decellularized Feeders: An Optimized Method for Culturing Pluripotent Cells
Decellularized Feeders: An Optimized Method for Culturing Pluripotent Cells
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DOI：
10.5966/sctm.2013-0077
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发表时间：
2013-12-01
影响因子：
6
通讯作者：
Macchiarini, Paolo
中科院分区：
文献类型：
--
作者：
Lim, Mei Ling;Jungebluth, Philipp;Macchiarini, Paolo
多能细胞如人胚胎干细胞和人诱导多能干细胞可用于再生医学领域，因为它们可以无限增殖并分化成所有细胞类型。然而，维持和繁殖干细胞的限制因素是需要灭活的成纤维细胞作为生长基质，因为这些可能潜在地导致交叉污染。在这项研究中，我们的目的是保持干细胞的细胞外基质（ECM）的未照射或π照射的成纤维细胞。已经证明，ECM含有对多能细胞的粘附、增殖和分化至关重要的因子和蛋白质。为了保存ECM，通过用0.05%十二烷基硫酸钠（SDS）处理使成纤维细胞的细胞层脱细胞，这导致与常规饲养层培养物相比不存在DNA。然而，SDS处理没有引起ECM结构和完整性的可检测的变化。此外，免疫组化表明，主要ECM蛋白，如纤连蛋白，胶原蛋白和层粘连蛋白的表达，保持不变。在该脱细胞基质上培养的人多能细胞保持多能性标志物NANOG和OCT 4的基因表达，并且具有分化为三个胚层的潜能。这里所示的体外培养系统具有极好的潜力，因为主要的同种异体组分（即，饲养细胞的DNA）。这也是一种技术上简单、快速、安全和廉价的方法，用于维持精细的无饲养层干细胞培养，以用于进一步的细胞分化研究。
Pluripotent cells such as human embryonic stem cells and human induced pluripotent stem cells are useful in the field of regenerative medicine because they can proliferate indefinitely and differentiate into all cell types. However, a limiting factor for maintaining and propagating stem cells is the need for inactivated fibroblasts as a growth matrix, since these may potentially cause cross-contamination. In this study, we aimed to maintain stem cells on the extracellular matrix (ECM) of either nonirradiated or pi-irradiated fibroblasts. It has been demonstrated that the ECM contains factors and proteins vital for the adhesion, proliferation, and differentiation of pluripotent cells. In order to preserve the ECM, the cell layers of the fibroblasts were decellularized by treatment with 0.05% sodium dodecyl sulfate (SDS), which resulted in an absence of DNA as compared with conventional feeder culture. However, SDS treatment did not cause a detectable change in the ECM architecture and integrity. Furthermore, immunohistochemistry demonstrated that expressions of major ECM proteins, such as fibronectin, collagen, and laminin, remained unaltered. The human pluripotent cells cultured on this decellularized matrix maintained gene expression of the pluripotency markers NANOG and OCT4 and had the potency to differentiate to three germ layers. The in vitro culture system shown here has an excellent potential since the main allogeneic components (i.e., DNA of the feeder cells) are removed. It is also a technically easy, fast, safe, and cheap method for maintaining a refined feeder-free stem cell culture for further cell differentiation studies.