Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Summary

This article presents a protocol for directed differentiation and functional analysis of β-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional β-cell like cells secreting insulin in response to glucose.

Abstract

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic β-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for β-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic β-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic β-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

Introduction

Human pluripotent stem cells (hPSCs) have the unique ability to differentiate into various cell types, making them a viable alternative to human pancreatic β-cells¹. These hPSCs are categorized into two types: embryonic stem cells (hESCs), derived from the blastocyst², and induced pluripotent cells (hiPSCs), generated by reprogramming somatic cells directly³. The development of techniques to differentiate hPSCs into β-cells, has important implications for both fundamental research and clinical practice^1,4. Diabetes mellitus is a chronic disease affecting >400 million people worldwide and results from the inability of the body to regulate glycemia due to malfunction or loss of pancreatic β-cells⁵. The limited availability of pancreatic islet cells for transplantation has hindered the development of cell replacement therapies for diabetes^2,4,6,7. The ability to generate glucose-responsive insulin-secreting cells using hPSCs serves as a useful cellular model for studying human islet development and function. It can also be used to test potential therapeutic candidates for diabetes treatment in a controlled environment. Moreover, hPSCs have the potential to produce pancreatic islet cells that are genetically identical to the patient, reducing the risk of immune rejection after transplantation^2,4,7.

In recent years, there have been significant advancements in the refinement of hPSC culture and differentiation protocols, resulting in increased efficiency and reproducibility of the differentiation process toward generating pancreatic β-cells^8,9.

The following protocol outlines the essential stages of directed differentiation of pancreatic β-cells. It involves the regulation of specific cell signaling pathways at distinct time points. It is based on the protocol developed by Sui L. et al.¹⁰ (2018) for the generation of hPSCs into pancreatic β-cells. The protocol was adjusted to recent updates from Sui L. et al.¹¹ (2021), as the latest research emphasizes the significance of using aphidicolin (APH) treatment to enhance the differentiation of β-cells. The current protocol includes the addition of APH to the medium during the later stages of the process. Furthermore, modifications have been made to the composition of the medium during the early stages of differentiation compared to the initial protocol. A notable change is the addition of Keratinocyte Growth Factor (KGF) on Day 6 and continuing until Day 8. The keratinocyte growth factor (KGF) is introduced from day 6 to day 8, which slightly differs from the initial protocol¹⁰, where KGF was not included in the stage 4 medium.

The first and essential step in the generation of β-cell-like cells is the directed differentiation of hPSCs into definitive endoderm (DE), a primitive germ layer that gives rise to the epithelial lining of various organs, including the pancreas. After the formation of DE, the cells undergo differentiation into the primitive gut tube, which is followed by the specification of the posterior foregut fate. The posterior foregut then develops into pancreatic progenitor cells, which have the potential to differentiate into all cell types of the pancreas, including the endocrine and exocrine cells. The subsequent stage in the process involves pancreatic endocrine progenitors giving rise to the hormone-secreting cells found in the islets of Langerhans. In the end, the differentiation process reaches its final stage by producing fully functional pancreatic β-cell like cells^9,10. It is important to note that this process is complex and often requires optimization of the culture conditions, such as specific growth factors and extracellular matrix components, to improve the efficiency and specificity of differentiation^9,10. Furthermore, generating functional β-cell like cells from hPSCs in vitro is still a major challenge. Ongoing research focuses on improving differentiation protocols and enhancing the maturation and function of the resulting β-cells⁹.

In this protocol, the use of gentle cell dissociation during the culture and passage of hPSCs is essential to maintain cell viability and pluripotency, significantly improving the efficiency of differentiation into pancreatic β-cells. Additionally, each stage-specific medium has been meticulously optimized following the protocol developed by Sui L. et al.¹⁰ to promote a high yield of insulin-secreting cells in clusters that closely resemble the human islet.

Protocol

Prior to initiating differentiation, it is recommended to determine the required number of islet-like organoids for experimental purposes. In a 6 well plate, a single well with over 80% confluency typically consists of 2-2.3 million hPSCs. While an accurate prediction is challenging due to variations in hPSC lines and differentiation efficiency, a rough estimate is 1.5 times the number of initial wells. An effectively directed differentiation usually yields 1.6 to 2 million cells per well in six-well plates, encompassing all cells within the clusters rather than exclusively insulin-producing cells. For a 50 µm cluster, it can be approximated to contain around 10,000 cells. Table 1 provides a summary of the media composition used for each day/stage of directed differentiation on top of stem cell matrix and medium, along with the glucose-stimulated insulin secretion buffer.

1. Passaging human pluripotent stem cells prior differentiation in 6 well plates

NOTE: The appropriate passaging of human stem cells prior to differentiating into β-cell-like cells is a crucial step in establishing the experimental process. Incorrect passage dilution or attachment cell number can compromise differentiation efficiency and fidelity.

1. Prepare stem cell culture medium, coating, and dissociation solutions (as specified in Table 1).
2. 1-2 h before passaging, coat a six-well plate with cold coating solution (1 mL per well) and incubate at 37 °C, 5% CO₂.
3. Warm an aliquot of stem cell culture medium at room temperature (15 -25 °C) for 20 min.
4. Aspirate the stem cell medium and add 1 mL of calcium/magnesium free D-PBS.
5. Repeat step 1.4. twice.
   NOTE: D-PBS is added to wash the cells and remove the previous medium and compounds. It is important to note that no specific time frame is required for the washing steps, as exposure to D-PBS does not harm the hPSCs, preventing cell rupture or shrinkage caused by osmosis.
6. Aspirate D-PBS, add 500 µL of dissociation solution for each well, and let it sit for 2-5 min at room temperature (15 - 25 °C).
7. While cells are dissociating, aspirate the coating solution of the new 6 well plate and add 1-2 mL of calcium/magnesium-free D-PBS to each well.
8. Regularly check the dissociation process under the inverted microscope.
9. Aspirate the dissociation solution when 80 - 90% of the cells have rounded but are still adherent.
10. Add 1 mL of the stem cell medium containing 10 µM ROCK inhibitor.
11. Collect the dissociated cells into a 15 mL conical tube using 1,000 µL sterile filtered pipette tips and a P1000 pipette.
12. Slowly triturate the cell suspension up and down in the pipette with a 1,000 µL sterile pipette filtered tip to break the colonies, but do not exceed 10 times.
13. Add 1 mL of the stem cell medium containing ROCK inhibitor to help detach the remaining hPSCs and transfer the cell suspension to the same 15 mL conical tube (1.10).
14. Aspirate the D-PBS from the newly coated plate and immediately add the cell suspension from the 15 mL conical tube. To ensure optimal cell growth, seed a range of 2 x 10⁵- 1 x 10⁶ viable cells per well when using a 6 well plate (1 in 10 to 1 in 50 splits).
    NOTE: For the volume calculation, each well of a 6-well plate must contain 2 mL of stem cell medium with ROCK inhibitor.
15. Move the plate quickly back and forth, and side to side 5 - 10 times.
16. Place the plate at 37 °C, 5% CO₂ incubator for 24 h.
17. Replace with fresh stem cell medium without ROCK inhibitor the next day, and then every 2 days until the confluency of hPSCs has reached 80 - 95%.

2. Human stem cell-derived β-cells directed differentiation

NOTE: The hPSCs can be used for the direct differentiation process into pancreatic β-cells when 80-95% confluence is achieved.

1. Day -1: Passaging of the cells Day -1 of differentiation:
   1. 1-2 h before passage, coat a 6 well plate with cold coating solution in a 37 °C, 5% CO₂ incubator for 1 h.
   2. Follow steps 1.1 to 1.10 and proceed to counting cells.
   3. Load 10 µL of the cell mixture, add an equal volume of Trypan Blue, and gently mix.
   4. Calculate the cell concentration. Use 0.8 × 10⁶ cells/mL - 1.0 x 10⁶ cells/mL of stem cell medium containing 10 µM ROCK inhibitor to plate the coated 6 well plate with 2 mL of media per well.
   5. Move the plate quickly back and forth, side to side three times.
   6. Place the plate in a 37 °C, 5% CO₂ incubator. Quickly move the plate back and forth, and side to side again 10 times. Then incubate the plate for 4 h.
      NOTE: Ensure that the maximum attachment and the even distribution of cells do not disturb the cells by moving the plate once placed in the incubator. Open and close the incubator very carefully.
   7. Place the bottle of basal medium for Days 0 to 4 at 4 °C to thaw it overnight.
2. Day 0
   NOTE: The hPSCs must be 80 - 95% confluent, do not start the differentiation process to definitive endoderm under that confluency.
   1. On ice, thaw both the basal medium for Days 0 to 4 and supplements (see Table of Materials).
   2. Prepare in two conical tubes only the necessary volume to be used on Day 1 (2 mL per well of a 6-well plate), and in a separate tube, double the volume for Day 2.5 to 4 (2 x 2 mL per well of a 6-well plate). Store the Day 2.5 to Day 4 medium at 4 °C.
   3. Warm an aliquot of the necessary volume of the Day 1 medium and washing medium 1 in a 37 °C water bath for 5 min.
   4. Aspirate the stem cell medium and add 2 mL of washing medium 1.
      NOTE: The washing medium used in the protocol consists of the basal medium specific to each day/stage, supplemented with 1% antibiotics. The washing steps of the direct differentiation protocol only involve the addition of the designated washing medium (referred to as "washing medium 1 or 2", Table of Materials) and subsequent aspiration, following the described procedure 2.2.4. It is important to mention that there is no specific time frame indicated for these washing steps.
   5. Aspirate washing medium 1 and immediately add 2 mL of the Day 1 medium to the side of the well.
   6. Incubate the cells at 37 °C and 5% CO₂ for 24 h.
3. Day 1
   1. Pre-warm an aliquot of the necessary volume of Days 2.5 to 4 medium in a 37 °C water bath for 5 min.
   2. Aspirate the Day 1 medium and immediately add 2 mL of the Day 2.5 to 4 medium to the side of the well. Do not wash the cells.
   3. Place the plate back at 37 °C and 5% CO₂ for 36 h.
4. Days 2.5 to 4
   1. Pre-warm an aliquot of the remaining volume of the Day 2.5 to 4 medium in 37 °C water bath for 5 min.
   2. Aspirate the medium and immediately add 2 mL of freshly prepared Days 2.5 to 4 medium to the side of the well.
   3. Place the plate back at 37 °C and 5% CO₂ for 36 h.
5. Day 4
   NOTE: At this stage, the expression of the definitive endoderm markers is at its maximum, and cells can initiate primitive gut tube differentiation.
   1. Prepare an aliquot of the necessary volume of the Day 4 primitive gut stage medium (2 mL per well of a six-well plate) and warm it at room temperature (15 °C -25 °C) for 20 min.
   2. Aspirate Days 2.5 to 4 medium and add 2 mL of washing medium 1.
   3. Aspirate the washing medium 1, and immediately add 2 mL of Day 4 primitive gut stage to the side of the well.
   4. Place the plate back at 37 °C and 5% CO₂ for 48 h.
6. Days 6 to 8
   NOTE: At this stage, the cells initiate further differentiation onto posterior foregut fate.
   1. Prepare the necessary volume of Days 6 to 8 posterior foregut medium (2 mL per well of a 6 well plate) and warm the medium at room temperature (15 °C -25 °C) for 20 min.
   2. Aspirate the Day 4 medium and immediately add 2 mL of Days 6 to 8 posterior foregut medium to the side of the well.
   3. Place the plate at 37 °C incubator containing 5% CO₂ for 48 h.
7. Days 8 to 12
   NOTE: The cells are ready to undergo pancreatic progenitors' differentiation.
   1. Prepare the necessary volume of Days 8 to 12 pancreatic progenitors stage medium (2 mL per well of a 6 well plate) and warm the medium at room temperature (15 °C -25 °C) for 20 min.
   2. Aspirate Days 6 to 8 medium and immediately add 2 mL of Days 6 to 8 posterior foregut medium to the side of the well.
   3. Place the plate in an incubator at 37 °C containing 5% CO₂ for 48 h.
8. Day 12: Performing the clustering step
   NOTE: At this stage, the cells form a dense monolayer, and will be transitioned to 3D cell culture to form clusters. This step is crucial for the differentiation to enhance the structural resemblance of β-like cells to native human islets but also improves their functional resemblance at both the cellular and cluster levels¹¹(Figure 1).
   1. Treat a 6 well plate containing microwells that are 400 µm (as specified in the Table of materials: Human Stem Cell-Derived β-cells Directed Differentiation, Day 12) with 2 mL of anti-adherence rinsing solution at room temperature (15 °C - 25 °C).
      NOTE: The microwell prevents cells from attaching to the well bottom and facilitates the mechanical formation of 3D clusters.
   2. Centrifuge plate at 1,300 x g for 5 min.
   3. Check for the presence of any bubbles under an inverted microscope. If no bubbles are observed, aspirate anti-adherence rinsing solution and immediately add 2 mL of washing medium 2 (Table of materials).
   4. Repeat step 1.8.3. two times.
   5. Prepare the necessary volume of the cluster medium and warm it at room temperature (15 -25 °C) for 20 min. Ensure that two wells of a 6 well plate go into one well of the 400 µm microwells 6 well plate with 4 mL of the cluster medium.
   6. Aspirate pancreatic progenitor medium and add dissociation buffer (0.5 mL per well).
   7. Incubate at room temperature (15 °C - 25 °C) for 2-5 min. Regularly check the dissociation process under an inverted microscope and aspirate dissociation buffer when most of the cells have rounded but are still adherent.
   8. Aspirate the dissociation buffer and immediately add 1 mL cluster medium to each well.
   9. Dissociate cells by gently pipetting up and down six times using a P1000 pipette and 1,000 µL filter tips.
   10. Transfer the cells and the cluster medium mixture into a 50 mL conical tube.
   11. Add 1 mL of the cluster medium to collect all remaining cells.
   12. Add the cluster medium to the total volume needed so each well of the microwells plate has 4 mL of mixture cluster medium/cells.
   13. Aspirate the washing medium 2 from microwells 6-well plate and transfer the cell suspension. Incubate the plate at 37 °C for 24 h.
9. Day 13: Handling the cells post-clustering and changing their media from Day 13 onward.
   NOTE: Clusters are highly sensitive and require careful handling to ensure their integrity and viability in the following days. Therefore, it is crucial to closely monitor the clusters during the post-clustering period after Day 12. Regular evaluation and adjustments are essential to promote successful outcomes in the experimental process.
   1. Prepare the necessary volume of Days 13 medium (2 mL per well of a 6 well plate), and in a separate conical tube of 50 mL for Day 15 to 20 pancreatic endocrine progenitor medium.
   2. Slowly collect clusters from the microwells 6 well plate into a 50 mL conical tube using a p1000 pipette and 1,000 µL filtered tips.
   3. Add 1 mL of Day 13 medium to collect the remaining clusters and transfer the clusters into the tube.
   4. Let the cells set naturally under gravity to the bottom of the conical tube for 5 min.
   5. Aspirate as much supernatant as possible from the tube without disturbing the cell clusters. The pipette tips should not touch nor aspirate the clusters but the supernatant only.
   6. Add the necessary volume of Day 13 medium according to the following: 1 well of microwells 6 well plate goes into three wells of a low-attachment 6 well plate (2 mL of media per well).
   7. Gently pipette up and down, avoiding aspirating clusters.
   8. Transfer the suspension to a very low-adherent 6 well plate.
   9. Move the plate quickly back and forth, side to side six times.
   10. Incubate the plate at 37 °C for 48 h.
   11. Perform the medium changes in all the next steps until the end of the differentiation as described in this section 2.9.
10. Days 15 to 20
    1. Change to pancreatic endocrine progenitor stage medium every 48 h until day 21 of differentiation by collecting the cell suspension into a 50 mL conical tube and following steps 2.9.4 to 2.9.10 for Day 13.
11. Days 21 to 27
    NOTE: At this stage, clusters differentiate onto pancreatic β-cells.
    1. Prepare pancreatic β-cell stage medium.
    2. Change the medium every other day as described for days 15 to 21.
       ​NOTE: After Day 25, the islet-like organoids can undergo analysis to confirm they are fully functional.

3. Staining of pancreatic β-cell clusters

NOTE: Perform this step to study the functional assessment of clusters post-differentiation

1. Collect five to ten clusters in a 1.5 mL microcentrifuge tube at the end of differentiation.
2. Fix the cell clusters with 200 µL of 4% PFA for 15 min at room temperature.
3. Add 1 mL of PBS to the clusters and remove the PBS with a 1,000 µL pipette tip without disturbing the clusters.
4. Add 200 µL of 30% sucrose to the clusters and incubate overnight at 4 °C.
5. Transfer the clusters to a cryomold, remove extra sucrose, add a drop of O.C.T. medium, and mix the clusters with O.C.T. medium.
6. Freeze the cryomold on dry ice and store at -80 °C.
7. Cut 5 µm sections from the frozen block on microscope slides using a microtome/or cryostat.
8. Store slides at -80 °C freezer until staining.
9. On the day of staining, take the slides out of the -80 °C freezer.
10. Carefully remove the ice around the cryomold's sections.
11. Circle cell clusters on slides with a hydrophobic pen.
12. Rehydrate slides in a slide staining jar containing PBS for 15 min.
13. Add cold methanol to the slide staining jar and place the jar with slides at -20 °C for 10 min.
14. Immerse slides in a PBS jar.
15. Repeat step 3.14 three times.
    NOTE: Use this method for all the following washing steps.
16. Remove PBS from the slides and cover immediately with 100 µL of blocking solution with 2-5% BSA in PBS-T.
17. Add blocking solution to each slide and cover with paraffin film.
18. Incubate for 1 h at room temperature (15 -25 °C).
19. Dilute the primary antibodies in the blocking solution using the ratios provided in the Reagents and Solutions section.
20. Remove the blocking solution from the slides.
21. Immediately add diluted antibodies and cover slides with parafilm to prevent the slide from getting dry.
22. Incubate overnight at 4 °C.
23. The following day wash the slides 3 - 5 times with PBS-T.
24. Prepare diluted secondary antibodies and DNA stain.
25. Remove excess PBS-T from the slides.
26. Add diluted secondary antibodies and DNA stain. Incubate for 45 min at room temperature (15 -25 °C).
27. Wash the slides 3 - 5 times with PBS-T.
28. Remove any liquid from the slides.
29. Add a drop of histology mounting medium to each section.
30. Cover the slide with a glass cover.
31. Take pictures of stained slides using a fluorescent microscope.

4. GSIS (glucose-stimulated insulin secretion assay)

1. Prepare three different solutions: low glucose KREBS solution, high glucose KREBS solution, and low glucose KREBS solution with KCl.
2. Warm all the solutions at 37 °C water bath.
3. Carefully select around 10 - 15 clusters of similar size using a 1000 µL pipette tip and transfer them into a 1.5 mL tube along with a small amount of differentiated medium to avoid drying out the clusters.
   NOTE: The clusters should be visible to the naked eye, but if not, place the plate containing differentiated clusters under an inverted microscope to select the clusters and transfer them to a 1.5 mL tube.
4. Place the tube with clusters and medium on a tube rack and wait for the clusters to sink to the bottom of the tube.
5. Slowly aspirate the supernatant using a 200 µL pipette and add 200 µL of low glucose KREBS solution.
6. Incubate the islets in the low glucose solution for 1 h at 37 °C.
   NOTE: To ensure the accurate assessment of cluster numbers and consistency in experiments, it is advisable to check for the presence of all clusters in the solution during transfer, as they tend to adhere to the tube.
7. Remove the tube from the incubator and slowly aspirate 200 µL of KREBS solution without aspirating any clusters.
8. Add 200 µL of low glucose KREBS solution and incubate for 30 min at 37 °C.
9. Aspirate the volume of 200 µL of low glucose KREBS solution into a 500 µL tube and immediately store it at -80 °C for insulin measurements. Repeat for separate treatments.
10. To wash the clusters, add 200 µL of KREBS solution without glucose to the tube containing the clusters. Allow the clusters to settle to the bottom of the tube, and carefully remove the supernatant without disturbing the clusters.
11. Add 200 µL of high glucose KREBS solution and incubate for 30 min at 37 °C.
12. Aspirate 200 µL of high glucose KREBS into a 500 µL tube and immediately store it at -80 °C for insulin measurement. Repeat for separate treatments.
13. To wash the clusters, add 200 µL of KREBS solution without glucose to the tube containing the clusters. Allow the clusters to settle to the bottom of the tube, and carefully remove the supernatant without disturbing the clusters.
14. Add 200 µL of KCl KREBS solution and repeat for separate treatments.
15. Incubate for 30 min at 37 °C.
16. Aspirate 200 µL of KCl KREBS solution into a 500 µL tube and immediately store it at -80 °C for insulin measurements. Repeat for other treatments.
17. Add 50 µL of ultrapure water and proceed to measure total insulin content.
18. Add 150 µL of acid ethanol solution to the tube with islets and ultrapure water.
19. Store samples at 4 °C overnight (12 - 15 h).
20. The following day, vortex each sample.
21. Perform sonication with an electric sonicator set to 20% power for 1 s to ensure complete lysis of the islet tissue.
    NOTE: Repeat step 4.21 until there are no remaining clusters visible.
22. Centrifuge all samples at 10,000 × g for 10 min and keep the supernatant.
23. Measure DNA concentration using a nanodrop spectrophotometer, which can then be used to normalize insulin measurements.

Results

The protocol described in this paper offers a highly efficient approach for differentiating β-like cells from hPSCs¹⁰. This process utilizes a 2D culture system that is easily scalable, enabling its use in various experimental settings, such as learning differentiation, smaller projects and laboratories, and pilot tests to assess the potential of an iPSC line for differentiation.

It is essential to characterize the functional properties of differentiated β-cel...

Discussion

The successful differentiation of hPSCs into pancreatic β-cells depends on optimizing all aspects of routine culturing and passage of the selected hPSCs. This includes ensuring that the cell line has a normal karyotype, is negative for mycoplasma infection, and is free of plasmid or viral vector genomes. Furthermore, when using hiPSCs, it is important to avoid using the earliest passage which are still undergoing reprogramming, for pilot experiments. These experiments should be conducted on a small scale to identify...

Materials

Name	Company	Catalog Number	Comments
1.5 mL TubeOne Microcentrifuge Tube	Starlabs	S1615-5500
6-well Cell culture plate	ThermoFisher Scientific	165218
AggreWell 400 6-well plate	STEMCELL Technologies	34425
Anti-Glucagon	Sigma-aldrich	G2654-100UL
Anti-Insulin	Dako	A0564
Anti-NKX6.1	Novus Biologicals	NBP1-49672SS
Anti-PDX1	Abcam	ab84987
Aphidicolin	Sigma-Aldrich	A4487
B-27 Supplement (50X), serum free	Thermo Fisher Scientific	17504044
Bovine Serum Albumin, fatty acid free	Sigma-Aldrich	A3803-100G
Calcium chloride dihydrate	Sigma-Aldrich	C3306
Calcium/Magnesium free D-PBS	Thermo Fisher Scientific	14190144
Cyclopamine-KAAD	Calbiochem	239804
D-(+)-Glucose,BioXtra	Sigma-Aldrich	G7528
Disodium hydrogen phosphate, anhydrous	Sigma-Aldrich	94046-100ML-
DMEM plus GlutaMAX	Thermo Fisher Scientific	10566016	For Washing Medium 2: DMEM plus GlutaMAX 1% PS.
DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)	Thermo Fisher Scientific	10565-018
Epredi SuperFrost Plus Adhesion slides	Thermo Fisher Scientific	10149870
Ethanol	VWR	20821.33
Fetal Bovine Serum	Thermo Fisher Scientific	10270098
Gamma-Secretase Inhibitor XX	Thermo Fisher Scientific	J64904
Geltrex LDEV-Free Reduced Growth Factor Basement	Thermo Fisher Scientific	A1413302	Geltrex 1:1 into cold DMEM/F-12 medium to provide a final dilution of 1:100.
Goat Anti-Guinea pig, Alexa Fluor 555	Thermo Fisher Scientific	A-21435
Goat Anti-Guinea pig, Alexa Fluor 647	Abcam	ab150187
Goat anti-Mouse Secondary Antibody, Alexa Fluor 633	Thermo Fisher Scientific	A-21052
Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 568	Thermo Fisher Scientific	A-11011
Heparin	Sigma-Aldrich	H3149
HEPES buffer	Sigma-Aldrich	H3375-500G
Hoechst 33342, Trihydrochloride	Thermo Fisher Scientific	H1399
Human FGF-7 (KGF) Recombinant Protein	Thermo Fisher Scientific	PHG0094
Hydrogen chloride	Sigma-Aldrich	295426
ImmEdge Hydrophobic Barrier PAP Pen	Agar Scientific	AGG4582
LDN193189	Sigma-Aldrich	SML0559-5MG
Magnesium chloride hexahydrate	Sigma-Aldrich	M9272-500G
OCT Compound 118 mL	Agar Scientific	AGR1180
PBS Tablets, Phosphate Buffered Saline, Fisher BioReagents	Thermo Fisher Scientific	7647-14-5
Penicillin-Streptomycin (PS)	Thermo Fisher Scientific,	15070-063
Potassium chloride	Sigma-Aldrich	7447-40-7
Recombinant Human EGF Protein	R&D Systems	236-EG-200
Rectangular cover glasses, 22×50 mm	VWR	631-0137
RepSox (Hydrochloride)	STEMCELL Technologies	72394
RPMI 1640 Medium, GlutaMAX Supplement	Thermo Fisher Scientific	61870036	For Washing Medium 1: RPMI 1640 plus GlutaMAX 1% PS.
Shandon Immu-mount	Thermo Fisher Scientific	9990402
Sodium bicarbonate	Sigma-Aldrich	S6014-500G
Sodium chloride	Sigma-Aldrich	S3014
Sodium dihydrogen phosphate anhydrous	Sigma-Aldrich	7558-80-7
STEMdiff Endoderm	STEMCELL Technologies	5110
StemFlex Medium	Thermo Fisher Scientific	A3349401	Thaw the StemFlex Supplement overnight at 4°C, transfer any residual liquid of the supplement bottle to StemFlex Basal Medium.
Stemolecule All-Trans Retinoic Acid	Reprocell	04-0021
Thyroid Tormone 3 (T3)	Sigma-Aldrich	T6397
Trypan Blue Solution, 0.4%	ThermoFisher Scientific	15250061
TrypL Express Enzyme (1X)	Thermo Fisher Scientific	12604013
TWEEN 20	Sigma-Aldrich	P2287-500ML
Ultra-Low Adherent Plate for Suspension Culture	Thermo Fisher Scientific	38071
UltraPure DNase/RNase-Free Distilled Water	Thermo Fisher Scientific	10977015
Y-27632 (Dihydrochloride)	STEMCELL Technologies	72302
Zinc Sulfate	Sigma-Aldrich	Z4750