The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters
* These authors contributed equally
In This Article
Summary
Abstract
Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.
Introduction
Pancreatic beta cells secrete insulin responding to rises in blood glucose levels. Patients lacking sufficient insulin production due to the autoimmune destruction of beta cells in type 1 diabetes (T1D)1, or due to beta cell dysfunction in type 2 diabetes (T2D)2, are typically treated with the administration of exogenous insulin. Despite this life-saving therapy, it cannot precisely match the exquisite control of blood glucose as achieved by dynamic insulin secretion from bona fide beta cells. As such, patients often suffer the consequences of life-threatening hypoglycemic episodes and other complications resulting from ....
Access restricted. Please log in or start a trial to view this content.
Protocol
This protocol is based on work with hPSC lines, including H1, HUES4 PDXeG, and Mel1 INSGFP/W, in feeder-free conditions. A step-by-step procedure is detailed in this section, with supporting data from the differentiation of Mel1 INSGFP/W in the representative results section. We recommend that further optimization is needed when working with other hPSC lines that are not stated here. See the Table of Materials for details related to all reagents and solutions used in this protocol.
1. Preparation of differentiation media and solutions
NOTE: See Table 1....
Access restricted. Please log in or start a trial to view this content.
Results
We developed a hybrid strategy to differentiate stem cells into insulin-secreting hPSC-islets in seven steps, which utilizes a pancreatic progenitor kit for the first four stages in planar culture, followed by a modified protocol built upon a previously reported method6 in a static suspension culture for the last three stages (Figure 1). With this protocol, ensuring a near confluency (90%-100%) culture at 24 h after cell seeding (Stage 0) is critical for initiating an.......
Access restricted. Please log in or start a trial to view this content.
Discussion
This paper describes a seven-stage hybrid protocol that allows for the generation of hPSC islets capable of secreting insulin upon glucose challenge within 40 days of culture in vitro. Among these multiple steps, efficient induction of definitive endoderm is believed to set an important starting point for the final differentiation outcomes18,27,28. In the manufacturer's protocol, a seeding density at 2.6 × 10
Access restricted. Please log in or start a trial to view this content.
Disclosures
Timothy J. Kieffer was an employee of ViaCyte during the preparation of this manuscript. The other authors declare no competing interests.
Acknowledgements
We gratefully acknowledge the support from STEMCELL Technologies, Michael Smith Health Research BC, Stem Cell Network, JDRF, and the Canadian Institutes of Health Research. Jia Zhao and Shenghui Liang are recipients of the Michael Smith Health Research BC Trainee Award. Mitchell J.S. Braam is a recipient of the Mitacs Accelerate Fellowship. Diepiriye G. Iworima is a recipient of the Alexander Graham Bell Canada Graduate Scholarship and the CFUW 1989 Ecole Polytechnique Commemorative Award. We sincerely thank Dr. Edouard G. Stanley from MCRI and Monash University for sharing the Mel1 INS GFP/W line and the Alberta Diabetes Institute Islet Core for isolating ....
Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 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 |
References
- Atkinson, M. A., Eisenbarth, G. S., Michels, A. W. Type 1 diabetes. Lancet. 383 (9911), 69-82 (2014).
- Petersen, M. C., Shulman, G. I. Mechanisms of insulin action and insulin resistance. Physiological Reviews. 98 (4), 2133-2223 (2018).
- Shapiro, A. M., et al.
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
