Diekmann Ulf, Davenport Claudia, Kresse Jasmin, Naujok Ortwin
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Curr Protoc Stem Cell Biol. 2017 Feb 2;40:1D.9.1-1D.9.17. doi: 10.1002/cpsc.22.
Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is required. Upon commitment into this lineage, the cells express transcription factors such as FOXA2, SOX17, HNF1B; GATA family members; and the surface protein CXCR4. Unfortunately, some pluripotent stem cells resist the differentiation and contaminate the culture. Thus, we describe here an endoderm differentiation protocol, which yields endoderm-committed cells in high numbers in a 4-day treatment protocol. Second, a method for the purification of CXCR4-positive endoderm cells by magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) is described. The purification by MACS is quick and reliable and can be used to obtain pure endoderm cells either meant for downstream analysis such as omics or further differentiation experiments into endoderm-derived somatic cells. © 2017 by John Wiley & Sons, Inc.
多能干细胞具有分化为人体任何体细胞类型的能力。生成用于治疗肝脏、肺部和胰腺疾病的替代细胞具有重大医学意义。首先，需要在体外将其诱导形成定形内胚层细胞。一旦确定进入该谱系，细胞会表达转录因子，如叉头框蛋白A2（FOXA2）、SRY-盒转录因子17（SOX17）、肝细胞核因子1β（HNF1B）；GATA家族成员；以及表面蛋白CXC趋化因子受体4（CXCR4）。不幸的是，一些多能干细胞会抵抗分化并污染培养物。因此，我们在此描述一种内胚层分化方案，该方案在4天的处理方案中能大量产生定形内胚层细胞。其次，描述了一种通过磁珠分选（MACS）和荧光激活细胞分选（FACS）纯化CXCR4阳性内胚层细胞的方法。通过MACS进行的纯化快速且可靠，可用于获得用于下游分析（如组学）或进一步分化为内胚层来源体细胞的进一步分化实验的纯内胚层细胞。© 2017约翰威立国际出版公司
