Davenport Claudia, Diekmann Ulf, Naujok Ortwin
Institute of Clinical Biochemistry, Hannover Medical School.
Institute of Clinical Biochemistry, Hannover Medical School;
J Vis Exp. 2016 Mar 3(109):53655. doi: 10.3791/53655.
The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.
多能干细胞（如胚胎干细胞，ESCs）的分化能力为细胞替代疗法提供了潜在的治疗应用。终末分化的细胞类型可用于治疗各种退行性疾病。这些细胞在体外向肺、肝和胰腺组织分化，第一步需要生成确定的内胚层细胞。这一步骤是进一步分化为终末成熟细胞类型（如产生胰岛素的β细胞、肝细胞或其他内胚层衍生细胞类型）的限速步骤。向内胚层谱系定向的细胞高度表达多种转录因子，如FOXA2、SOX17、HNF1B、GATA家族成员以及表面受体CXCR4。然而，分化方案很少能达到100%的效率。在这里，我们描述了一种通过使用磁性微珠在分化为确定内胚层后纯化CXCR4+细胞群体的方法。这种纯化还能去除不需要的谱系的细胞。这种温和的纯化方法快速且可靠，可用于改善下游应用和分化。
