Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Research Base for Cell Manufacturability, Osaka University, Suita, Osaka, Japan.
Biotechnol J. 2024 Jan;19(1):e2300364. doi: 10.1002/biot.202300364. Epub 2023 Nov 20.
Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1 and NKX6.1 pancreatic progenitor cells in high yields. The PDX1 and PDX1 /NKX6.1 cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.
高效地将人诱导多能干细胞（hiPSCs）分化为功能性胰腺细胞，为糖尿病的研究和治疗带来了巨大的希望。然而，缺乏一种能够产生高均一性胰腺祖细胞的稳健培养策略。在这里，我们建立了一种简单的分化策略，通过两步法（使用肉毒杆菌神经毒素 HA 进行顺序细胞同步化）来产生同步的 iPSC 衍生胰腺祖细胞。在测试的各种方法中，HA 暴露的第一步同步方法通过空间控制异质细胞分布，诱导从 E-到 N-钙黏蛋白和从 N-到 E-钙黏蛋白表达的同步转换，从而提高其向 iPSC 定向分化为确定内胚层细胞的能力。iPSC 衍生的确定内胚层细胞可以高效地产生高产量的 PDX1 和 NKX6.1 胰腺祖细胞。与未同步培养相比，PDX1 和 PDX1/NKX6.1 细胞密度分别增加了 1.6 倍和 2.2 倍。同一运行和不同运行之间的变异系数低于 10%，表明在不同的培养和运行中具有稳定而强大的分化能力。我们的方法是一种简单而高效的策略，可以在多阶段胰腺祖细胞分化过程中产生具有最高均一性的大量分化细胞，为指导 iPSC 分化为功能性胰岛素产生细胞提供了一种潜在的工具。
