Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar.
Cancer Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar.
Stem Cell Res Ther. 2018 Jan 23;9(1):15. doi: 10.1186/s13287-017-0759-z.
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.
同时表达转录因子 PDX1 和 NKX6.1 的胰腺祖细胞（PPs）被认为是功能性胰腺β细胞的不可或缺前体细胞。在这里，我们旨在建立一种从人多能干细胞（hPSC）中高效生成 PDX1/NKX6.1 PPs 的方案。
为了增强 PDX1/NKX6.1 群体，我们通过在不同密度下重新铺板来操纵分化过程中的体外培养条件，以解离密集形成的内胚层细胞。这些分离的细胞接受延长的视黄酸和成纤维细胞生长因子（FGF）10 信号，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（~90%）的比例增加，高于以前发表的方案，以及上调了控制胰腺发育的关键 TFs。胰腺分化效率的提高伴随着肝特异性的抑制和 NKX6.1 细胞的增殖增加。有趣的是，我们能够通过操纵重新铺板密度来富集一种新型的 PDX1/NKX6.1 群体；这些细胞在三维培养物中自我定向成簇。进一步的分化验证了我们的 PDX1/NKX6.1 祖细胞生成 NGN3 内分泌祖细胞的能力。
我们提供了一种新技术，可促进单层培养中适当的细胞重排，以产生具有高比例的 PDX1/NKX6.1 PPs 和提高的自我复制能力，从而有助于从 hPSC 体外规模化生产功能性β细胞。我们的创新方法还富集了一种新型的 NKX6.1/PDX1 群体，具有拟议的内分泌前体细胞的特征，允许进一步研究β细胞发育的途径。
