干细胞分化为胰岛细胞为传统的糖尿病治疗和疾病建模提供了另一种解决方案。我们描述了一种详细的干细胞培养方案,该方案将商业分化试剂盒与先前验证的方法相结合,以帮助在培养皿中产生分泌胰岛素的干细胞衍生胰岛。
人类多能干细胞 (hPSC) 分化为分泌胰岛素的 β 细胞为研究 β 细胞功能和糖尿病治疗提供了材料。然而,在获得充分模拟天然人类β细胞的干细胞衍生的β细胞方面仍然存在挑战。在先前研究的基础上,已经生成了 hPSC 衍生的胰岛细胞,以创建具有改进的分化结果和一致性的方案。这里描述的方案在第 1-4 阶段使用胰腺祖细胞试剂盒,然后在第 5-7 阶段使用根据 2014 年发表的论文修改的方案(以下简称“R 协议”)。包括使用胰祖细胞试剂盒和 400 μm 直径微孔板生成胰祖细胞簇的详细程序、用于 96 孔静态悬浮形式内分泌分化的 R 方案,以及 hPSC 衍生胰岛的 体外 表征和功能评估。完整的方案需要 1 周进行初始 hPSC 扩增,然后需要 ~5 周才能获得产生胰岛素的 hPSC 胰岛。具有基本干细胞培养技术和生物测定培训的人员可以重现该协议。
胰腺β细胞分泌胰岛素,对血糖水平的升高做出反应。由于 1 型糖尿病 (T1D)1 中 β 细胞的自身免疫性破坏或 2 型糖尿病 (T2D)2 中 β 细胞功能障碍而缺乏足够胰岛素分泌的患者通常使用外源性胰岛素进行治疗。尽管这种疗法可以挽救生命,但它无法精确地与真正的β细胞动态分泌胰岛素所实现的血糖精确控制相媲美。因此,患者经常遭受危及生命的低血糖发作和慢性高血糖波动引起的其他并发症的后果。人类尸体胰岛的移植成功地恢复了 T1D 患者的严格血糖控制,但受到胰岛供体可用性和纯化健康胰岛进行移植的困难的限制 3,4。原则上,这一挑战可以通过使用hPSC作为替代起始材料来解决。
目前在体外从 hPSC 产生胰岛素分泌胰岛的策略通常旨在模拟体内胚胎胰腺发育的过程 5,6。这需要了解负责任的信号通路,并定时添加相应的可溶性因子,以模拟发育中的胚胎胰腺的关键阶段。胰腺程序从确定的内胚层开始,其标志是转录因子叉头盒 A2 (FOXA2) 和性别决定区 Y-box 17 (SOX17)7。最终内胚层的连续分化包括形成原始肠管,形成表达胰腺和十二指肠同源框 1 (PDX1)7,8,9 的后前肠,以及上皮扩张为共表达 PDX1 和 NK6 同源框 1 (NKX6.1)10,11 的胰腺祖细胞。
对内分泌胰岛细胞的进一步承诺伴随着促内分泌主调节因子神经原蛋白-3 (NGN3)12 的瞬时表达以及关键转录因子神经元分化 1 (NEUROD1) 和 NK2 同源框 2 (NKX2.2) 13 的稳定诱导。随后对主要的激素表达细胞进行编程,例如产生胰岛素的 β 细胞、产生胰高血糖素的 α 细胞、产生生长抑素的 delta 细胞和产生胰腺多肽的 PPY 细胞。凭借这些知识,以及广泛的高通量药物筛选研究的发现,最近的进展使 hPSC 胰岛能够产生类似于 β 细胞的细胞,能够分泌胰岛素 14,15,16,17,18,19。
已经报道了产生葡萄糖反应性 β 细胞的分步方案 6,14,18,19。基于这些研究,本方案涉及使用胰腺祖细胞试剂盒在平面培养物中产生 PDX1+/NKX6.1+ 胰腺祖细胞,然后将微孔板聚集成大小均匀的簇,并在静态 3D 悬浮培养物中使用 R 方案进一步分化为分泌胰岛素的 hPSC 胰岛。进行质量控制分析,包括流式细胞术、免疫染色和功能评估,以严格表征分化细胞。本文详细介绍了定向分化的每个步骤,并概述了体外表征方法。
本文描述了一种七阶段混合方案,该方案允许在体外培养后 40 天内产生能够在葡萄糖激发时分泌胰岛素的 hPSC 胰岛。在这些多个步骤中,有效诱导最终内胚层被认为为最终分化结果设定了重要的起点 18,27,28。在制造商的方案中,建议接种密度为2.6×105 / cm 2以启动分化,并将细胞暴露于第1阶段培养基中2?…
The authors have nothing to disclose.
我们非常感谢 STEMCELL Technologies、Michael Smith Health Research BC、Stem Cell Network、JDRF 和加拿大卫生研究院的支持。Jia Zhao 和 Shenghui Liang 是 Michael Smith Health Research BC 实习生奖的获得者。Mitchell J.S. Braam 是 Mitacs Accelerate Fellowship 的获得者。Diepiriye G. Iworima 是 Alexander Graham Bell 加拿大研究生奖学金和 CFUW 1989 年巴黎综合理工学院纪念奖的获得者。我们衷心感谢来自MCRI和莫纳什大学的Edouard G. Stanley博士分享Mel1 INS GFP / W 系和阿尔伯塔省糖尿病研究所胰岛核心分离和分布人类胰岛。我们还要感谢不列颠哥伦比亚大学生命科学研究所成像和流式细胞术设施的支持。 图 1 是使用 BioRender.com 创建的。
3,3’,5-Triiodo-L-thyronine (T3) | Sigma | T6397 | Thyroid hormone |
4% PFA solution | Santa Cruz Biotechnology | sc-281692 | Should be handled in fume hood |
96-Well, Ultralow Attachment, flat bottom | Corning Costar (VWR) | CLS3474 | Flat bottom; for static suspension culture in the last three stages |
Accutase | STEMCELL Technologies | 07920 | Dissociation reagent for Stage 4 cells |
Aggrewell400 plates | STEMCELL Technologies | 34415 | 400 µm diameter microwell plates |
Aggrewell800 plates | STEMCELL Technologies | 34815 | 800 µm diameter microwell plates |
Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG) | R&D Systems | IC2400G | 1:100 in flow cytometry; used for assaying Stage 1 cells |
Alexa Fluor 488 Goat IgG Isotype Control | R&D Systems | IC108G | 1:100 in flow cytometry |
Alexa Fluor 488 Mouse anti-Human SST (mouse IgG2B) | BD Sciences | 566032 | 1:250 in flow cytometry; used for assaying Stage 7 cells |
Alexa Fluor 488 Mouse IgG2B Isotype Control | R&D Systems | IC0041G | 1:500 in flow cytometry |
Alexa Fluor 647 Mouse anti-Human C-peptide (mouse IgG1κ) | BD Pharmingen | 565831 | 1:2,000 in flow cytometry; used for assaying Stage 7 cells |
Alexa Fluor 647 Mouse anti-Human INS (mouse IgG1κ) | BD Sciences | 565689 | 1:2,000 in flow cytometry |
Alexa Fluor 647 Mouse anti-Human NKX6.1 (mouse IgG1κ) | BD Sciences | 563338 | 1:33 in flow cytometry; used for assaying Stage 4 cells |
Alexa Fluor 647 Mouse anti-Human SOX17 (mouse IgG1κ) | BD Sciences | 562594 | 1:50 in flow cytometry; used for assaying Stage 1 cells |
Alexa Fluor 647 Mouse IgG1κ Isotype Control | BD Sciences | 557714 | 1:50 in flow cytometry |
ALK5i II | Cayman Chemicals | 14794 | TGF-beta signaling inhibitor |
Anti-Adherence Rinsing Solution | STEMCELL Technologies | 7010 | Microwell Rinsing Solution |
Assay chamber | Cellvis | D35-10-1-N | For static GSIS and confocal imaging purposes |
Bovine serum albumin (BSA) | Thermo Fisher Scientific | BP1600-100 | For immunostaining procedure |
CK19 antibody | DAKO | M0888 | 1:50 in whole mount immunofluorescence |
D-glucose | Sigma | G8769 | Medium supplement |
DAPI | Sigma | D9542 | For nuclear counterstaining |
DMEM/F12, HEPES | Thermo Fisher Scientific | 11330032 | Matrix diluting solution |
Donkey anti-goat Alexa Fluor 555 | Life technologies | A21432 | 1:500 in whole mount immunofluorescence |
Donkey anti-goat Alexa Fluor 647 | Life technologies | A21447 | 1:500 in whole mount immunofluorescence |
Donkey anti-mouse Alexa Fluor 555 | Life technologies | A31570 | 1:500 in whole mount immunofluorescence |
Donkey anti-mouse Alexa Fluor 647 | Life technologies | A31571 | 1:500 in whole mount immunofluorescence |
Donkey anti-rabbit Alexa Fluor 555 | Life technologies | A31572 | 1:500 in whole mount immunofluorescence |
Donkey anti-rabbit Alexa Fluor 647 | Life technologies | A31573 | 1:500 in whole mount immunofluorescence |
Donkey anti-sheep Alexa Fluor 647 | Life technologies | A21448 | 1:500 in whole mount immunofluorescence |
DPBS | Sigma | D8537 | Without Ca2+ and Mg2+ |
ELISA, insulin, human | Alpco | 80-INSHU-E01.1 | For human insulin measurement |
Fatty acid-free BSA | Proliant | 68700 | Medium supplement |
Fixation and Permeabilization Solution Kit | BD Sciences | 554714 | Fix/Perm and 10x Perm/Wash solutions included |
Gentle Cell Dissociation Reagent | STEMCELL Technologies | 7174 | For clump passaging hPSCs during maintenance culture |
Glucagon antibody | Sigma | G2654 | 1:400 in whole mount immunofluorescence |
GLUT1 antibody | Thermo Fisher Scientific | PA1-37782 | 1:200 in whole mount immunofluorescence |
GlutaMAX-I (100x) | Gibco | 35050061 | L-glutamine supplement |
Glycerol | Thermo Fisher Scientific | G33-4 | For tissue clearing and mounting |
GSi XX | Sigma Millipore | 565789 | Notch inhibitor |
Heparin | Sigma | H3149 | Medium supplement |
ITS-X (100x) | Thermo Fisher Scientific | 51500056 | Insulin-Transferrin-Selenium-Ethanolamine; medium supplement |
LDN193189 | STEMCELL Technologies | 72147 | BMP antagonist |
MAFA antibody | Abcam | ab26405 | 1:200 in whole mount immunofluorescence |
Matrigel, hESC-qualified | Thermo Fisher Scientific | 08-774-552 | Extracellular matrix for vessel surface coating |
MCDB131 medium | Life technologies | 10372019 | Base medium |
mTeSR1 Complete Kit | STEMCELL Technologies | 85850 | stem cell medium and 5x supplement included |
N-Cys (N-acetyl cysteine) | Sigma | A9165 | Antioxidant |
NaHCO3 | Sigma | S6297 | Medium supplement |
NEUROD1 antibody | R&D Systems | AF2746 | 1:20 in whole mount immunofluorescence |
NKX6.1 antibody | DSHB | F55A12-c | 1:50 in whole mount immunofluorescence |
Pancreatic polypeptide antibody | R&D Systems | AF6297 | 1:200 in whole mount immunofluorescence |
PBS | Sigma | D8662 | With Ca2+ and Mg2+ |
PDX1 antibody | Abcam | ab47267 | 1:200 in whole mount immunofluorescence |
PE Mouse anti-Human GCG (mouse IgG1κ) | BD Sciences | 565860 | 1:2,000 in flow cytometry; used for assaying Stage 7 cells |
PE Mouse anti-Human NKX6.1 (mouse IgG1k) | BD Sciences | 563023 | 1:250 in flow cytometry |
PE Mouse anti-Human PDX1 (mouse IgG1k) | BD Sciences | 562161 | 1:200 in flow cytometry; used for assaying Stage 4 cells |
PE Mouse IgG1κ Isotype Control | BD Sciences | 554680 | 1:2,000 in flow cytometry |
PE Mouse-Human Chromogranin A (CHGA, mouse IgG1k) | BD Sciences | 564563 | 1:200 in flow cytometry |
R428 | Cayman Chemicals | 21523 | AXL tyrosine kinase inhibitor |
Retinoid acid, all-trans | Sigma | R2625 | Light-sensitive |
RIPA lysis buffer, 10x | Sigma | 20-188 | For hormone extraction |
SANT-1 | Sigma | S4572 | SHH inhibitor |
SLC18A1 antibody | Sigma | HPA063797 | 1:200 in whole mount immunofluorescence |
Somatostatin antibody | Sigma | HPA019472 | 1:100 in whole mount immunofluorescence |
STEMdiff Pancreatic Progenitor Kit | STEMCELL Technologies | 05120 | Basal media and supplements included |
Synaptophysin antibody | Novus | NB120-16659 | 1:25 in whole mount immunofluorescence |
Triton X-100 | Sigma | X100 | For permeabilization |
Trolox | Sigma Millipore | 648471 | Vitamin E analog |
TrypLE Enzyme Express | Life technologies | 12604-021 | cell dissociation enzyme reagent for single cell passaging hPSCs |
Trypsin1/2/3 antibody | R&D Systems | AF3586 | 1:25 in whole mount immunofluorescence |
Y-27632 | STEMCELL Technologies | 72304 | ROCK inhibitor |
Zinc sulfate | Sigma | Z0251 | Medium supplement |