iPSC-Derived Pancreatic Islet Cell (iPIC) Therapy Department, Orizuru Therapeutics Inc., Fujisawa, Kanagawa, 251-8555, Japan.
Takeda-CiRA Joint Program for iPS Cell Applications (T-CiRA), Fujisawa, Kanagawa, 251-8555, Japan.
Stem Cell Res Ther. 2023 Jan 5;14(1):1. doi: 10.1186/s13287-022-03220-4.
Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs.
We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose-response assays, single-cell RNA-sequencing and in vivo efficacy study.
We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII.
Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism.
源自人诱导多能干细胞（hiPSC）的分化细胞移植为临床治疗带来了巨大的希望。消除恶性细胞转化的风险因素对于确保此类细胞的安全性至关重要。本研究旨在评估和减轻 hiPSC 衍生的胰岛样细胞（iPIC）分化方案中细胞培养过程中可能出现的致突变性。
我们评估了用于 hiPSC 衍生的胰岛样细胞（iPICs）分化的分化因子的致突变性。我们采用了艾姆斯致突变性检测、流式细胞术分析、免疫染色、基于时间分辨荧光共振能量转移（TR-FRET）的无细胞剂量反应测定、单细胞 RNA 测序和体内功效研究。
我们观察到激活素受体样激酶 5 抑制剂 II（ALK5iII）具有致突变作用。ALK5iII 是一种广泛使用的β细胞诱导剂，但没有其他测试的 ALK5 抑制剂能诱导β细胞。我们获得了激酶抑制谱，并发现只有 ALK5iII 抑制了所有测试的 ALK5 抑制剂中的细胞周期蛋白依赖性激酶 8 和 19（CDK8/19）。一致地，CDK8/19 抑制剂在没有 ALK5iII 的情况下有效地诱导了β细胞。非致突变的 ALK5 抑制剂 SB431542 和 CDK8/19 抑制剂 senexin B 的联合治疗提供了具有体外细胞组成和体内功效的 iPICs，与使用 ALK5iII 观察到的相当。
我们的发现为细胞治疗提供了一种新的风险缓解方法，并推进了我们对β细胞分化机制的理解。
