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.
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.
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 chronic hyperglycemic excursions. Transplantation of human cadaveric islets successfully restores tight glycemic control in T1D patients but is limited by the availability of islet donors and difficulties in purifying healthy islets for transplantation3,4. This challenge can, in principle, be solved by using hPSCs as an alternative starting material.
Current strategies for generating insulin-secreting islets from hPSCs in vitro often aim to mimic the process of embryonic pancreas development in vivo5,6. This requires knowledge of the responsible signaling pathways and timed addition of corresponding soluble factors to mimic critical stages of the developing embryonic pancreas. The pancreatic program initiates with the commitment into definitive endoderm, which is marked by transcription factors forkhead box A2 (FOXA2) and sex-determining region Y-box 17 (SOX17)7. Successive differentiation of definitive endoderm involves the formation of a primitive gut tube, patterning into a posterior foregut that expresses the pancreatic and duodenal homeobox 1 (PDX1)7,8,9, and epithelial expansion into pancreatic progenitors that co-express PDX1 and NK6 homeobox 1 (NKX6.1)10,11.
Further commitment to endocrine islet cells is accompanied by the transient expression of pro-endocrine master regulator neurogenin-3 (NGN3)12 and stable induction of key transcription factors neuronal differentiation 1 (NEUROD1) and NK2 homeobox 2 (NKX2.2)13. The major hormone-expressing cells, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells, and pancreatic polypeptide-producing PPY cells, are subsequently programmed. With this knowledge, as well as discoveries from extensive, high-throughput drug screening studies, recent advancements have enabled the generation of hPSC-islets with cells resembling beta cells capable of insulin secretion14,15,16,17,18,19.
Step-wise protocols have been reported for generating glucose-responsive beta cells6,14,18,19. Built upon these studies, the present protocol involves the use of a pancreatic progenitor kit for generating PDX1+/NKX6.1+ pancreatic progenitor cells in a planar culture, followed by microwell plate aggregation into uniform-sized clusters and further differentiation toward insulin-secreting hPSC-islets with the R-protocol in a static 3D suspension culture. Quality control analyses, including flow cytometry, immunostaining, and functional assessment, are performed for rigorous characterization of the differentiating cells. This paper provides a detailed description of each step of the directed differentiation and outlines the in vitro characterization approaches.
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 for all solutions to be prepared and Table 2 for the differentiation media.
2. Differentiation of hPSCs into pancreatic progenitor cells
3. Aggregation into pancreatic progenitor clusters using 400 µm diameter microwell plates
4. Differentiation of kit-derived pancreatic progenitor clusters into hPSC-islets
5. Flow cytometric analysis
6. Whole mount immunostaining
7. Static GSIS assay
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 efficient differentiation for most hPSC lines (Figure 2A). Testing different seeding densities is strongly recommended to determine the optimal condition for a particular cell line. During Stage 1 culture, the media will likely appear cloudy due to floating dead cells, which are commonly observed at this stage. This does not compromise the differentiation efficiency of the kit as long as the attached cells fully cover the vessel surface. During Stages 2-4, as cells proliferate and cell death diminishes, the monolayers become more compacted and the spent media are not as cloudy as during Stage 1 (Figure 2A). As demonstrated with the insulin reporter Mel1 INSGFP/W hESCs, at least 95% FOXA2+/SOX17+ definitive endoderm cells at the end of Stage 1 and 80% PDX1+/NKX6.1+ pancreatic progenitor cells at the end of Stage 4 can be achieved routinely (Figure 2B–E). The transition of pancreatic progenitor cells from planar culture to suspension culture has been shown to promote subsequent endocrine cell induction23,24. In this updated protocol, we use microwell plates and allow for 24 h of cell aggregation. This generates thousands of pancreatic progenitor clusters with uniform size and morphology at a time (Figure 3A, B), and the average aggregation efficiency (by calculating the percentage of cells that are incorporated into aggregates) is 74.1% ± 4.4% (Figure 3C). Importantly, expression of PDX1 and NKX6.1 is maintained at high levels in these clusters post aggregation (Figure 3D, E), suggesting an efficient production of pancreatic progenitors in vitro with the differentiation kit and microwell plate.
We next use R-protocol (a method modified from Rezania et al.6) for the last 3-stage differentiation in a static suspension culture system using ULA flat bottom 96-well plates to further differentiate these kit-derived pancreatic progenitors toward hPSC-islets (Figure 1). Insulin-expressing cells are gradually induced within endocrine clusters, as indicated by the increased INS-GFP signals in living cultures over time (Figure 4A, B). Quantification of cluster size reveals that the average diameter of Stage 5 clusters is 150 µm and by Stage 7, the average diameter increases to 220 µm (Figure 4C). The clusters predominantly maintain their smooth, spherical appearance as they grow between Stage 5 and Stage 7, suggesting that the static culture method limits cluster clumping compared to orbital shaker-based suspension cultures (which typically generate some oversized clusters)25. Maintenance of compacted clusters with smooth spherical morphology is an indicator of good viability of the differentiating cells, which is important for a successful differentiation toward functional hPSC-islets in the end.
Characterization of cell composition shows that Stage 7 hPSC-islets generated by the hybrid protocol are primarily endocrine and comprised of four major islet cell types, including insulin-positive beta cells, glucagon-positive alpha cells, somatostatin-positive delta cells, and pancreatic polypeptide-positive PPY cells, while enterochromaffin cells (derived from an off-target lineage26) are also present (Figure 5A–C). Notably, this hybrid protocol generates largely monohormonal islet cells (~50% CPEP+/GCG–/SST– cells, ~17% GCG+/CPEP– cells, and ~12% SST+/CPEP– cells) and only a minority of bi-hormonal cells (CPEP+/GCG+ or CPEP+/SST+) in Stage 7 cultures (Figure 5B-D). Examination of several key transcription factors and mature beta cell markers in Stage 7 hPSC-islets reveals that differentiating beta cells express PDX1, NKX6.1, NEUROD1, MAFA, and glucose transporter GLUT1 (Figure 6A). Furthermore, ~60% INS+/PDX1+ cells (Figure 6B) and 50% CPEP+/NKX6.1+ cells (Figure 6C) are induced in the Stage 7 cultures, suggesting an efficient generation of functional beta cells using the hybrid strategy as presented here. Indeed, in vitro static glucose-stimulated insulin secretion (GSIS) assays demonstrate that Stage 7 hPSC-islets can secrete high levels of insulin responding to both high glucose and direct depolarization (Figure 6D). While this function is encouraging, the in vitro glucose responsiveness from hPSC-islets is still not equivalent to that of cadaveric islets in terms of the magnitude of insulin secretion (Figure 6D) and the total insulin content of hPSC-islets is approximately half the amount of that in cadaveric islets (Figure 6E). Continued optimization is required to achieve hPSC-islets with more mature beta-cell phenotypes.
Figure 1: Overview of differentiation scheme for generating functional hPSC-islets in vitro. The workflow diagram provides an overview of the seven-step procedure involved in the differentiation of hPSCs into pancreatic progenitor cells in a planar culture using the pancreatic progenitor kit, aggregation into pancreatic progenitor clusters using microwell plates, and transition toward hPSC-islets in a static suspension culture. An insulin reporter hESC line, Mel1 INSGFP/W, is used throughout this study as an example to demonstrate the efficacy of this hybrid method. Abbreviations: hPSCs = human pluripotent stem cells; GSIS = glucose-stimulated insulin secretion. Please click here to view a larger version of this figure.
Figure 2: Directed differentiation of hPSCs into pancreatic progenitor cells using the pancreatic progenitor kit. (A) Representative phase contrast images showing cell morphology at the indicated stages. Scale bars = 750 µm. (B, C) Representative flow cytometry plots for key markers of (B) Stage 1 definitive endoderm cells (FOXA2+/SOX17+) and (C) Stage 4 pancreatic progenitor cells (PDX1+/NKX6.1+). (D) Representative immunostaining images of pancreatic progenitor cells for PDX1 (red) and NKX6.1 (green). Nuclei were stained with DAPI (blue). Scale bar = 200 µm. (E) Flow cytometric quantification of PDX1+/NKX6.1+ cells in Stage 4 Day 4 pancreatic progenitor monolayer cells (n = 5 biological replicates). Abbreviations: hPSCs = human pluripotent stem cells; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 3: Aggregation into uniform-sized pancreatic progenitor clusters using 400 µm diameter microwell plates. (A) Representative phase contrast images at the end of Stage 4 showing cluster morphology in the microwell and after retrieval from the microwell. Scale bars = 300 µm. (B) Quantification of cluster diameter at the end of Stage 4 (n = 60 clusters from 3 biological replicates). (C) Quantification of aggregation efficiency at the end of Stage 4 (n = 5 biological replicates). (D) Representative immunostaining images of pancreatic progenitor clusters for PDX1 (red) and NKX6.1 (green). Nuclei were stained with DAPI (blue). Scale bar = 100 µm. (E) Flow cytometric quantification of PDX1+/NKX6.1+ cells in Stage 4 Day 5 pancreatic progenitor aggregates (n = 4 biological replicates). Abbreviation: DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 4: Differentiation of pancreatic progenitors into hPSC-islets in a static suspension culture system. (A) Representative phase contrast images (top panel) and fluorescent images (bottom panel) showing cluster morphology and insulin expression pattern at indicated stages. Scale bars = 300 µm. (B) Quantification of mean INS-GFP fluorescent intensities per cluster at indicated stages (n = 30 clusters from two biological replicates). (C) Quantification of cluster diameter at indicated stages (n = 60 clusters from three biological replicates). Abbreviations: hPSC = human pluripotent stem cell; INS = insulin; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
Figure 5: Characterization of cell composition in Stage 7 hPSC-islets. (A) Representative immunostaining images of Stage 7 hPSC-islets showing different cell types (SYN, pan-endocrine marker; CK19, ductal marker; INS-GFP, insulin is indicated by reporter GFP signals; GCG; SST; PPY; ENC was stained by SLC18A1 antibody). Nuclei were stained with DAPI (blue). Scale bars = 100 µm. (B,C) Representative flow cytometry plots of the (B) percentage of CPEP+ and GCG+ cells in Stage 7 hPSC-islets and (C) the percentage of CPEP+ and SST+ cells in Stage 7 hPSC-islets. (D) Flow cytometry quantification of indicated cell markers in Stage 7 hPSC-islets (n = 4 biological replicates). Abbreviations: hPSC = human pluripotent stem cell; INS= insulin; GFP = green fluorescent protein; SYN = synaptophysin; GCG = glucagon; SST = somatostatin; PPY = pancreatic polypeptide; ENC = enterochromaffin; CPEP = C-peptide; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 6: In vitro characterization and functional assessment of Stage 7 hPSC-islets. (A) Representative immunostaining images of Stage 7 hPSC-islets showing co-expression of insulin (indicated by reporter INS-GFP signals) with several key beta cell markers, such as NKX6.1, MAFA, NEUROD1, PDX1, and glucose transporter GLUT1. Nuclei were stained with DAPI (blue). Scale bars = 100 µm. (B,C) Representative flow cytometry plots of the (B) percentage of INS+/PDX1+ cells and the (C) percentage of CPEP+/NKX6.1+ cells. (D) Static GSIS assays showing insulin release from Stage 7 hPSC-islets (n = 6 including 3 biological and 2 technical replicates) and human islets (n = 6 including 3 biological and 2 technical replicates) in response to high glucose (16.7G, 16.7 mM glucose) and 30 mM KCl depolarization. Data are presented as mean ± SEM. An unpaired two-tailed t-test (**p < 0.01) was used to compare between two indicated groups. (E) Total insulin content of Stage 7 hPSC-islets (n = 6 including 3 biological and 2 technical replicates) and human islets (n = 6 including 3 biological and 2 technical replicates). An unpaired two-tailed t-test (*p < 0.05) was used to compare the two groups. Abbreviations: hPSC = human pluripotent stem cell; INS = insulin; GFP = green fluorescent protein; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Table 1: Formulation of all solutions and media used in this protocol. Please click here to download this Table.
Table 2: Formulation of Stages 1-7 differentiation media. The table provides the recipe to prepare the Stages 1-7 differentiation media. Please click here to download this Table.
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 × 105/cm2 is recommended to initiate differentiation, and cells are exposed to Stage 1 media for 2 days. To ensure that cells reach near confluency on Day 1 (90%-100%), we highly recommend testing seeding densities when working on a new hPSC line as the optimal seeding density may vary. For example, with all three hPSC lines tested in our hands, 1.6-1.80 × 105/cm2, rather than 2.60 × 105/cm2, was found to be an optimal seeding density. Extending the culture duration of Stage 1 from 2 days to 3 days with one additional day in Stage 1B medium increases the purity of FOXA2+/SOX17+ cells from 80% to > 93% (data not shown). If the proportion of FOXA2+/SOX17+ cells is lower than 70% at the end of Stage 1, induction of PDX1+ cells and PDX1+/NKX6.1+ cells will likely be compromised.
It is important to note that some cell death may be observed during the entire Stage 1 culture while the attached cells are required to cover 100% of the surface area. During Stages 2-4, the decrease in cell death concurrent with cell proliferation results in compacted and multilayered cells, which has been reported to be critical for the efficient induction of pancreatic progenitor markers PDX1 and NKX6.1 and their nuclear localization29,30. On the last day of Stage 4, the cells are dissociated and aggregated into clusters using the AggreWell 400 µm diameter microwell plates. Compared to other approaches such as suspended aggregation, manual aggregation, or using U-bottom 96-well plates, AggreWell aggregation generates thousands of spherical uniform-sized clusters, illustrating the robustness of this method.
For endocrine induction during Stages 5-7 in a static 3D culture, we transfer an average number of ~20 kit-derived pancreatic progenitor clusters into each well of ULA flat bottom 96-well plates. This method greatly reduces the formation of oversized clusters and improves cell viability, possibly by limiting cluster fusion and/or lowering shear stress-mediated cell loss during orbital shaker culture. To remove floating dead cells in spent media while reducing the risk of losing clusters during media change, we recommend refreshing about two-thirds of the total medium volume. We note that this partial medium change does not hamper endocrine differentiation and instead may offer a gentle transition when switching between different stages. Nevertheless, some cell death may still be observed during Stage 5 and early Stage 6 with this hybrid protocol when transitioning from planar culture to static suspension culture. Reporter hPSC lines (for instance, Mel1 INSGFP/W hESCs) are useful for monitoring late-stage differentiation in living cultures. Running flow cytometry will help examine NKX6.1+/NEUROD1+ at the end of Stage 5, INS+/NKX6.1+ at the end of Stage 6, and CPEP+/NKX6.1+ at the end of Stage 7. Efficient induction of CPEP+ populations co-expressed with NKX6.1 is critical for achieving glucose responsiveness in beta cells31,32. In this regard, this protocol consistently generates >50% CPEP+/NKX6.1+ cells in the resulting Stage 7 hPSC-islets that can secrete insulin in response to a high glucose challenge in vitro.
Although the present protocol has demonstrated an efficient generation of insulin-producing hPSC islets, there are limitations to this method. Differentiation efficiencies at the end of Stage 4 from various hPSC lines, particularly iPSCs, with this pancreatic progenitor kit could still be variable (data not shown). Optimization should be performed on a case-by-case basis. This method for the last three stages of differentiation relies on the use of static suspension culture in 96-well plates, which is labor-intensive and not scalable, thus limiting its use to research only. While it is encouraging to obtain insulin-secreting capability, the in vitro glucose responsiveness and insulin content of hPSC islets are significantly lower than those of cadaveric islets. Future efforts are still required for continued protocol optimization. Despite these limitations, we believe that the protocol presented here provides hPSC-derived materials suitable for screening differentiation-inducing factors and regulators of islet hormone secretion, as well as for modeling islet cell dysfunction.
The authors have nothing to disclose.
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 and distributing human islets. We also acknowledge the support from the Life Sciences Institute Imaging and Flow Cytometry facilities at the University of British Columbia. Figure 1 was created with BioRender.com.
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 |