Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.
Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.
复制标题
增强了人多能干细胞分化为胰腺祖细胞共表达PDX1和NKX6.1。
DOI：
10.1186/s13287-017-0759-z
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发表时间：
2018-01-23
影响因子：
7.5
通讯作者：
Abdelalim EM
中科院分区：
文献类型：
--
作者：
Memon B;Karam M;Al-Khawaga S;Abdelalim EM
胰腺祖细胞（Pancreatic progenitors，PP）共表达两种转录因子PDX1和NKX6.1，是功能性胰腺β细胞不可或缺的前体细胞。在这里，我们的目的是建立一种有效的方案，用于最大限度地从人多能干细胞（hPSC）中产生PDX1 +/NKX6.1 + PP。为了增强PDX1 +/NKX6.1+群体，我们通过解离密集形成的内胚层细胞并以不同密度重新铺板来操纵分化期间的体外培养条件。使这些解离的细胞经受类视色素和成纤维细胞生长因子（FGF）10信号传导的增加的持续时间以诱导更高的PDX1和NKX6.1表达。我们的优化方案显著增加了NKX6.1的表达，导致单层中PDX1 +/NKX6.1+祖细胞的比例增加（约90%），高于先前发表的方案，以及控制胰腺发育的关键TF上调。胰腺分化效率的提高得到了肝脏特异性抑制和NKX6.1+细胞增殖增加的补充。有趣的是，我们能够通过操纵重新铺板密度来富集新的PDX1-/NKX6.1+群体;这些群体在三维簇中定向。进一步分化验证了我们的PDX1 +/NKX6.1+祖细胞产生NGN3+内分泌祖细胞的能力。我们提供了一种新的技术，该技术促进单层培养中的适当细胞重排，以产生高比例的具有提高的自我复制能力的PDX1 +/NKX6.1 + PP，从而有助于在体外从hPSC可规模化地生产功能性β细胞。我们的创新方法还丰富了一种新的NKX6.1 +/PDX1-群体，具有拟议的内分泌前体的特征，允许进一步研究破译β细胞发育的途径。本文的在线版本（doi：10.1186/s13287 - 017 - 0759-z）包含补充材料，可供授权用户使用。
Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1–/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1– population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development. The online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users.
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影响因子：
11
作者：
Lin HY;Tsai CC;Chen LL;Chiou SH;Wang YJ;Hung SC
通讯作者：
Hung SC
影响因子：
5.4
作者：
Kimmel RA;Onder L;Wilfinger A;Ellertsdottir E;Meyer D
通讯作者：
Meyer D
影响因子：
8.2
作者：
Lyttle, B. M.;Krishnamurthy, J. Li M.;Wang, R.
通讯作者：
Wang, R.
影响因子：
4.6
作者：
Nostro, M. Cristina;Sarangi, Farida;Keller, Gordon
通讯作者：
Keller, Gordon
DOI：
10.1016/j.bbrc.2012.03.041
发表时间：
2012-04-13
影响因子：
3.1
作者：
Abdelalim, Essam Mohamed;Tooyama, Ikuo
通讯作者：
Tooyama, Ikuo