Interdepartmental Stem Cell Institute Leuven, Catholic University Leuven, Leuven, Belgium.
Nat Protoc. 2010 Jul;5(7):1324-36. doi: 10.1038/nprot.2010.80. Epub 2010 Jun 24.
Differentiation of stem cells to hepatocytes has industrial applications, as well as the potential to develop new therapeutic strategies for liver disease. The protocol described here, sequentially using cytokines that are known to have a role in liver embryonic development, efficiently differentiates rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells by directing them through defined embryonic intermediates, namely, primitive streak/mesendoderm/definitive endoderm, hepatoblast and hepatocyte-like phenotype. After 20 days, the final differentiated multipotent adult progenitor cell progeny is a mixture of cells, comprising cells with the characteristics of hepatoblasts and a smaller cell fraction with the morphological and phenotypical features of mature hepatocytes, as well as other mesodermal cells and some persistent undifferentiated rMAPCs. A detailed functional characterization of the stem cell progeny is also described; this should be used to confirm that differentiated cells display the functional characteristics of mature hepatocytes, including albumin secretion, glycogen storage and several detoxifying functions such as urea production, bilirubin conjugation, glutathione S-transferase activity and cytochrome activity.
干细胞向肝细胞的分化具有工业应用价值，并有潜力为肝脏疾病开发新的治疗策略。本文描述了一种使用细胞因子的方法，这些细胞因子已知在肝胚胎发育中起作用，可通过定向诱导其经历特定的胚胎中间阶段（原始条纹/中胚层/确定内胚层、肝前体细胞和肝样细胞表型），将大鼠多能成体祖细胞（rMAPC）高效地分化为肝样细胞。20 天后，最终分化的多能成体祖细胞的后代是一种混合细胞，包含具有肝前体细胞特征的细胞和具有成熟肝细胞形态和表型特征的较小细胞群，以及其他中胚层细胞和一些持续未分化的 rMAPC。本文还描述了对干细胞后代的详细功能特征分析；这应用于确认分化细胞显示成熟肝细胞的功能特征，包括白蛋白分泌、糖原储存和几种解毒功能，如尿素生成、胆红素结合、谷胱甘肽 S-转移酶活性和细胞色素活性。
