Kamishibahara Yu, Okamoto Satoshi, Ohkuma Takuya, Taniguchi Hideki
Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, Japan.
Biol Methods Protoc. 2022 Dec 10;8(1):bpac034. doi: 10.1093/biomethods/bpac034. eCollection 2023.
Human-induced pluripotent stem cell (hiPSC)-derived hepatic cells are useful tools for regenerative medicine, and various culture substrates are currently used for their differentiation. We differentiated hiPSC-derived hepatic endoderm (HE), endothelial cells (ECs), and mesenchymal cells (MCs) using Laminin-511 (LN) coating to generate liver organoids, hiPSC-liver buds (hiPSC-LBs), which exhibited therapeutic effects when transplanted into disease model animals. Stably producing significant amounts of hiPSC-LBs is necessary for sufficient therapeutic effects. However, general precoating (standard coating) requires quick manipulation, often causing failure for inexperienced cell cultures, we thus tested direct LN addition to the culture medium (Direct coating). Using quantitative gene expression, flow cytometry, albumin secretion, and ammonia metabolism, we demonstrated that Standard and Direct coating similarly induce hiPSC-derived hepatocyte, mesodermal cell, EC, and MC differentiation. Standard and Direct coating-differentiated cells generated iPSC-LBs with equivalent hepatic functions. Furthermore, Direct coating enabled stable induction of differentiation independent of individual culture skills and reduced total amount of LN use as the same differentiated cell quality can be obtained upon LN supplementation at lower concentrations. In summary, the results of this study suggest that Direct coating could enable stable hiPSC-LB production at a low cost, thereby yielding mass cell production using hiPSCs.
人诱导多能干细胞（hiPSC）来源的肝细胞是再生医学的有用工具，目前各种培养底物被用于其分化。我们使用层粘连蛋白-511（LN）包被来分化hiPSC来源的肝内胚层（HE）、内皮细胞（EC）和间充质细胞（MC），以生成肝类器官，即hiPSC肝芽（hiPSC-LB），将其移植到疾病模型动物中时显示出治疗效果。为获得足够的治疗效果，稳定大量生产hiPSC-LB是必要的。然而，一般的预包被（标准包被）需要快速操作，对于缺乏经验的细胞培养人员来说常常导致失败，因此我们测试了将LN直接添加到培养基中（直接包被）。通过定量基因表达、流式细胞术、白蛋白分泌和氨代谢，我们证明标准包被和直接包被同样能诱导hiPSC来源的肝细胞、中胚层细胞、EC和MC分化。标准包被和直接包被分化的细胞产生了具有同等肝功能的iPSC-LB。此外，直接包被能够独立于个人培养技能稳定诱导分化，并减少LN的总用量，因为在较低浓度下补充LN就能获得相同质量的分化细胞。总之，本研究结果表明，直接包被能够以低成本稳定生产hiPSC-LB，从而利用hiPSC实现大规模细胞生产。
