A Reliable and Efficient Protocol for Human Pluripotent Stem Cell Differentiation into the Definitive Endoderm Based on Dispersed Single Cells
A Reliable and Efficient Protocol for Human Pluripotent Stem Cell Differentiation into the Definitive Endoderm Based on Dispersed Single Cells
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DOI：
10.1089/scd.2014.0143
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发表时间：
2015-01-15
影响因子：
4
通讯作者：
Naujok, Ortwin
中科院分区：
文献类型：
--
作者：
Diekmann, Ulf;Lenzen, Sigurd;Naujok, Ortwin
多能细胞分化为内胚层相关细胞类型最初需要体外原肠胚形成为定形内胚层（DE）。大多数分化方案从多能细胞集落开始，由于起始条件定义不充分，使其适应复杂化。本文所述的方案从确定的细胞数量的分散的单细胞开始，并在三种不同的人胚胎干细胞系和一种人诱导多能干细胞系上进行测试。激活素A/Nodal信号传导和GSK 3抑制的组合激活持续前24小时，随后激活素A/Nodal信号传导有效地诱导DE状态。单独激活ActivinA/Nodal信号传导并不有效。有效的GSK 3抑制允许在整个方案期间降低激活素A浓度。多能细胞的非饲养层依赖性培养是优选的，以实现高效率和稳健性，因为饲养层细胞阻碍分化过程。此外，抑制磷脂酰肌醇3-激酶（PI 3 K）信号通路是不需要的，但产生高细胞数有效地致力于DE。最后，产生的内胚层可以进一步分化成PDX 1阳性的泛胰腺细胞和NGN 3阳性的内分泌祖细胞。因此，由于基于来自无饲养层培养的人多能细胞的分散单细胞的明确定义的条件，这种有效且稳健的DE分化方案是朝着更好的再现性前进的一步。
Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h, followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally, inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required, nonetheless yielding high cell numbers efficiently committed toward the DE. Finally, the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus, this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells.