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Abstract：
Mouse embryonic stem cells (mESCs) have been widely used as a model system to study the basic biology of pluripotency and to develop cell-based therapies. Traditionally, mESCs have been cultured in a medium supplemented with fetal bovine serum (FBS). However, serum with its inconsistent chemical composition has been problematic for reproducibility and for studying the role of specific components. While some serum-free media have been reported, these media contain commercial additives whose detailed components have not been disclosed. Recently, we developed a serum-free medium, DA-X medium, which can maintain a wide variety of adherent cancer lines. In this study, we modified the DA-X medium and established a novel serum-free condition for both naïve mESCs in which all components are chemically defined and disclosed (DA-X-modified medium for robust growth of pluripotent stem cells: DARP medium). The DARP medium fully supports the normal transcriptome and differentiation potential in teratoma and the establishment of mESCs from blastocysts that retain the developmental potential in all three germ layers, including germ cells in chimeric embryos. Utility of chemically defined DA-X medium for primed mouse epiblast stem cells (mEpiSCs) revealed that an optimal amount of cholesterol is required for the robust growth of naïve-state mESCs, but is dispensable for the maintenance of primed-state mEpiSCs. Thus, this study provides reliable and reproducible culture methods to investigate the role of specific components regulating self-renewal and pluripotency in a wide range of pluripotent states.
摘要：
小鼠胚胎干细胞（mESCs）已被广泛用作模型系统，以研究多能性的基础生物学并开发基于细胞的疗法。传统上,mESC已在补充有胎牛血清(FBS)的培养基中培养。然而,血清的化学成分不一致，在重现性和研究特定成分的作用方面存在问题。虽然已经报道了一些无血清培养基，这些介质含有商业添加剂，其详细成分尚未公开。最近,我们开发了一种无血清培养基,DA-X培养基,可以维持各种各样的粘附癌细胞系。在这项研究中,我们对DA-X培养基进行了改良,并为两种初始mESC建立了一种新的无血清条件,其中所有组分均在化学上进行了定义和公开(DA-X改良的多能干细胞稳健生长培养基:DARP培养基).DARP培养基完全支持畸胎瘤的正常转录组和分化潜能，以及从胚泡中建立mESC，这些胚泡保留了所有三个胚层的发育潜能，包括嵌合胚胎中的生殖细胞。化学定义的DA-X培养基用于引发小鼠表皮干细胞（mEpiSCs）的效用表明，原始状态mESCs的稳健生长需要最佳量的胆固醇。但对于维持启动状态的mEpiSCs是可有可无的。因此，这项研究提供了可靠和可重复的培养方法，以研究特定成分在多种多能状态下调节自我更新和多能性的作用。
