Protocol to generate stem cell-derived alpha cells in 3D suspension culture

Summary

Pancreatic alpha cells represent a key islet cell type that acts in tandem with pancreatic beta cells to regulate blood glucose levels. Here, we present a protocol to generate stem cell-derived alpha (SC-α) cells using chemically defined small-molecule and growth factor induction in a scalable 3D suspension culture. We describe steps for embryonic stem cell culture and SC-α differentiation. We then detail procedures for quality control and the assessment of SC-α cell function.

For complete details on the use and execution of this protocol, please refer to Peterson et al.¹ and Shrestha et al.²

Graphical abstract

Highlights

• •Guidance on 3D ESC culture and stem cell-derived alpha (SC-α) cell differentiation
• •Instructions for SC-α differentiation factor preparation and media composition
• •Assessment of differentiation efficacy and functional glucagon secretion

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Pancreatic alpha cells represent a key islet cell type that acts in tandem with pancreatic beta cells to regulate blood glucose levels. Here, we present a protocol to generate stem cell-derived alpha (SC-α) cells using chemically defined small-molecule and growth factor induction in a scalable 3D suspension culture. We describe steps for embryonic stem cell culture and SC-α differentiation. We then detail procedures for quality control and the assessment of SC-α cell function.

Before you begin

Stem cell-derived therapies for the treatment of type 1 diabetes (T1D) are on the cusp of becoming a clinically viable treatment. Recent advancements have led to the development of stem cell-derived pancreatic beta cells, which are currently undergoing clinical trials to assess their therapeutic potential in individuals with diabetes.^3,4,5,6 A critical area of focus in cell therapy for T1D is the advancement of a single-cell approach such as beta-cells only to a multicellular approach that includes both beta and alpha cells and replicates the complete functionality of the pancreatic islet.^7,8 Additionally, studies have demonstrated important roles for alpha cells in both the pathophysiology of T1D and in glucose homeostasis.^9,10,11 Understanding the contribution of alpha cells in the development and management of T1D is critical for the advancement of comprehensive and robust treatment strategies for this disease. This first-of-its-kind protocol outlines a method for generating stem cell-derived alpha (SC-α) cells using a clinically scalable culture system. By employing chemically defined small molecules and growth factors, pluripotent stem cells can be differentiated into SC-α cells which are transcriptionally similar to human pancreatic alpha cells and secrete glucagon in similar quantities to human alpha cells.^1,2 These SC-α cells can be used to study the regulation of glucagon secretion in alpha cells or the interplay of alpha and beta cells in blood glucose homeostasis. SC-α cells may also be incorporated into next generation cellular therapies for T1D to provide key counter-regulatory signaling to SC-β cells for better blood glucose management. Furthermore, these SC-α cells can be used to model and study diabetic phenotypes, providing a key platform for the discovery and testing of therapeutic interventions, making them a promising tool for advancing our understanding and treatment of T1D.²

Innovation

The study of the pancreatic islets has largely focused on the generation of functional beta cells, with multiple protocols to generate stem cell-derived beta cells being reported.^{4,5,12,13,14,15} However, this has resulted in other islet cell types such as the pancreatic alpha cell to be underexplored. Previously, methods to study pancreatic alpha cells required the use of immortalized cell lines, often with limited transcriptional and functional similarity to human pancreatic alpha cells, or the use of cadaveric islets which complicates the study of alpha cells due to the presence of beta and delta cell populations also found in the pancreatic islets. Here we report on the first protocol for large scale production of SC-α cells from human pluripotent stem cells.¹ This protocol generates a primary population of monohormonal glucagon expressing SC-α cells with limited presence of beta or delta cells commonly found in pancreatic islets. As previous protocols predominantly generate stem cell-derived beta cells with only minor populations of glucagon expressing cells, this protocol represents a significant breakthrough in the ability to study and utilize pancreatic alpha cells.

Institutional permissions

Prior approval for the use of human pluripotent stem cells may be required by an institutional committee before starting this protocol. All data presented in this protocol were obtained with approval by the Mayo Clinic Stem Cell Research Oversight Committee.

Preparation of stock solutions

• 1.
  1.0 Molar D-Glucose Solution.

  • a.
    Dissolve 1.801 grams of D-Glucose into 10 mL of Ultrapure H₂O. MW: 180.16 g/mol.
• 2.
  2.56 Molar Sodium Chloride Solution.

  • a.
    Dissolve 74.803 grams of Sodium Chloride into 500 mL of Ultrapure H₂O. MW: 58.44 g/mol.
• 3.
  0.5 Molar Potassium Chloride Solution.

  • a.
    Dissolve 9.319 grams of Potassium Chloride into 250 mL of Ultrapure H₂O. MW: 74.55 g/mol.
• 4.
  2.0 Molar Potassium Chloride Solution.

  • a.
    Dissolve 37.276 grams of Potassium Chloride into 250 mL of Ultrapure H₂O. MW: 74.55 g/mol.
• 5.
  0.27 Molar Calcium Chloride Solution.

  • a.
    Dissolve 7.491 grams of Calcium Chloride into 250 mL of Ultrapure H₂O. MW: 110.98 g/mol.
• 6.
  0.12 Molar Magnesium Sulfate Solution.

  • a.
    Dissolve 3.611 grams of Magnesium Sulfate into 250 mL of Ultrapure H₂O. MW: 120.37 g/mol.
• 7.
  0.1 Molar Sodium Phosphate (Dibasic) Solution.

  • a.
    Dissolve 3.549 grams of Sodium Phosphate (Dibasic) into 250 mL of Ultrapure H₂O. MW: 141.96 g/mol.
• 8.
  0.12 Molar Potassium Phosphate Solution.

  • a.
    Dissolve 4.083 grams of Potassium Phosphate into 250 mL of Ultrapure H₂O. MW: 136.09 g/mol.
• 9.
  0.5 Molar Sodium Bicarbonate Solution.

  • a.
    Dissolve 10.501 grams of Sodium Bicarbonate into 250 mL of Ultrapure H₂O. MW: 84.01 g/mol.
• 10.
  4% Paraformaldehyde.

  • a.
    Mix 10 mL of 16% PFA (aqueous) with 30 mL of PBS.

Preparation of small molecules and growth factors

Note: Dissolve and aliquot all factors in a tissue culture hood under sterile conditions.

• 11.
  10 mM Rho Kinase inhibitor (Y-27632 or ROCK inhibitor) stock (working concentration = 10 μM).

  • a.
    Dissolve 500 mg of Y-27632 in 156 mL of sterile water.
  • b.
    Transfer 1 mL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 12 months.
• 12.
  15 mg/mL Activin A stock (working concentration = 100 ng/mL).

  • a.
    Dissolve 1 mg of Activin A with a total of 66 mL of 0.22 μm filtered 0.1% BSA Solution.
  • b.
    Aliquot 4.1 mL into sterile 15-mL conical tubes.
  • c.
    Store at −20°C for up to 6 months.
• 13.
  9.0 mM CHIR99021 stock (working concentration = 3 μM).

  • a.
    Dissolve 10 mg of CHIR99021 in a total of 2.388 mL of DMSO.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 12 months.
  • c.
    Protect from light during aliquot preparation, storage and use.
• 14.
  150 mg/mL Keratinocyte Growth Factor (KGF) stock (working concentration = 50 ng/mL).

  • a.
    Dissolve 1 mg of KGF in 6.67 mL of 0.22 μm filtered 0.1% BSA Solution.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 6 months.
• 15.
  6.0 mM Retinoic Acid (RA) stock (working concentration = 2 μM).

  • a.
    Dissolve 50 mg of RA in 27.7 mL of DMSO.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 6 months.
  • c.
    Protect from light during aliquot preparation, storage and use.
• 16.
  600 mM LDN 193189 stock (working concentration = 200 nM).

  • a.
    Dissolve 10 mg of LDN 193189 in 37.6 mL of DMSO.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 6 months.
• 17.
  30 mM RepSox (Alk5 Inhibitor II) stock (working concentration = 10 μM).

  • a.
    Dissolve 100 mg of RepSox in 11.6 mL of DMSO.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 12 months.
• 18.
  1.5 mM Phorbol 12,13-dibutyrate (PDBu) stock (working concentration = 500 nM).

  • a.
    Dissolve 5 mg of Phorbol 12,13-dibutyrate (PDBu) in 6.6 mL of DMSO.
  • b.
    Transfer 105 μL aliquots to sterile 1.5 mL Eppendorf tubes and store at −80°C for up to 12 months.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-OCT3/4 (use at 1:250 dilution)	Santa Cruz Biotechnology	Cat# sc-5279
Goat polyclonal anti-Sox17 (reconstitute at 0.2 μg/μL, use at 1:250 dilution)	R&D Systems	Cat# AF1924
Mouse monoclonal anti-HNF-1β (use at 1:100 dilution)	Santa Cruz Biotechnology	Cat# sc-130407
Goat polyclonal anti-PDX1 (reconstitute at 0.2 μg/μL, use at 1:250 dilution)	R&D Systems	Cat# AF2419
Mouse monoclonal anti-NKX6.1 (use at 1:100 dilution)	DSHB	Cat# F55A12-s
Rabbit polyclonal anti-Chromogranin A (use at 1:1,000 dilution)	Abcam	Cat# AB283265
Rat monoclonal anti-C-peptide (use at 1:250 dilution)	DSHB	Cat# GN-ID4-s
Mouse monoclonal anti-glucagon (use at 1:1,000 dilution)	Sigma	Cat# G2654
Donkey anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488	Invitrogen	Cat# A11055
Donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647	Invitrogen	Cat# A31571
Donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488	Invitrogen	Cat# A21206
Donkey anti-rat IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488	Invitrogen	Cat# A21208
Chemicals, peptides, and recombinant proteins
Sodium chloride	Sigma	Cat# S9625
Potassium chloride	Sigma	Cat# P5405
Calcium chloride	Sigma	Cat# 383147
Magnesium sulfate	Sigma	Cat# M7506
Sodium phosphate (dibasic)	Sigma	Cat# S7907
Potassium phosphate	Sigma	Cat# P9791
Sodium bicarbonate	Sigma	Cat# S5761
D-glucose	Sigma	Cat# G7021
HEPES Buffer (1 M)	Gibco	Cat# 15630-080
Fatty acid-free bovine serum albumin (FAF-BSA)	Proliant Biologicals	Cat# 68700
Sodium hydroxide (1N)	Fisher Scientific	Cat# SS266-1
Hydrochloride acid (1N)	Fisher Scientific	Cat# SA48-1
L-ascorbic acid (vitamin C)	Sigma	Cat# A4544-25G
Insulin Transferrin Selenium Ethanolamine (ITS-X)	Life Technologies	Cat# 51500-056
Glutagro, 200 mM solution, 100× [+] 8.5 g/L NaCl	Corning	Cat# 25-015-CI
Penicillin Streptomycin solution (100×)	Corning	Cat# 30-001-CI
Rho kinase inhibitor (Y-27632)	R&D Systems	Cat# 1254
Activin A	R&D Systems	Cat# 338-AC-01M
CHIR99021	R&D Systems	Cat# 4423
KGF (FGF7)	PeproTech	Cat# 100-19-1MG
Retinoic acid (RA)	R&D Systems	Cat# 0695
LDN 193189	R&D Systems	Cat# 6053
RepSox	R&D Systems	Cat# 3742
Phorbol 12,13-dibutyrate (PDBu)	R&D Systems	Cat# 4153
16% Paraformaldehyde (aqueous)	Thomas Scientific	Cat# C993M24
DMSO	Fisher Scientific	Cat# BP231-100
Triton X-100	Sigma	Cat# T9284
Critical commercial assays
DuoSet ELISA Ancillary Reagent Kit 2	R&D Systems	DY008B
Glucagon DuoSet ELISA	R&D Systems	DY1249
Experimental models: Cell lines
Human: HUES8 ESC – 3D culture adapted	HSCI iPS Core	NIHhESC-10-0021
Software and algorithms
FlowJo v.10.10.0	BD Biosciences	https://www.flowjo.com/flowjo/download
Attune Cytometric v.5.2	Thermo Fisher Scientific	Cat# A25554
SkanIt Software v.6.1.1	Thermo Fisher Scientific	N/A
Other
Countess 3 Automated cell counter	Thermo Fisher Scientific	Cat# AMQAX2000
Countess Cell counting chamber slides	Thermo Fisher Scientific	Cat# C10283
TrypLE	Gibco	Cat# 12604-013
Falcon Round-bottom polystyrene test tubes with cell strainer snap cap, 5 mL	Fisher Scientific	Cat# 352235
Falcon Round-bottom polypropylene test tubes with cap	Fisher Scientific	Cat# 352063
1.5 mL Eppendorf tubes	Thomas Scientific	Cat# 1148T71
Attune NxT Acoustic flow cytometer	Thermo Fisher Scientific	Cat# A28993
0.22 μm PES vacuum filter top	Sigma	Cat# S2GPT05RE
Sterile receiver bottle – 500 mL	Sigma	Cat# S200B05RE
Orion Star A111 pH meter	Thermo Fisher Scientific	Cat# STARA1110
Barnstead GenPure Pro Water Purification System	Thermo Fisher Scientific	Cat# 50131950
24-well Ultralow attachment culture plate	Corning	Cat# 3473
96-well Ultralow attachment culture plate	Corning	Cat# 3474
Millicell Transwell Inserts	Sigma	Cat# PIXP01250
Normal donkey serum	Jackson ImmunoResearch	Cat# 017-000-121
Cell culture grade H2O	Corning	Cat# 25-055-CV
mTeSR1 Complete Kit - GMP	STEMCELL Technologies	Cat# 85850
MCDB131	Corning	Cat# 15-100-CV
Accutase solution	Sigma	Cat# A6964-500ML
500 mL 3D Spinner flask	Corning	Cat# 3153
PluriStrainer 300 μm sterile filter	pluriSelect	Cat# 43-50300-03
Corning phosphate-buffered saline, 1X without calcium and magnesium, pH 7.4 ± 0.1	Corning	Cat# 21-040-CV
Magnetic stir plate	Chemglass Life Sciences	Cat# CLS-4100-09
Varioskan Lux microplate reader	Thermo Fisher Scientific	Cat# VL0000D0

Materials and equipment

Complete mTeSR for ESC maintenance

Reagent	Amount	Final concentration
mTeSR1 Basal Medium	400 mL	N/A
mTeSR1 5X Supplement	100 mL	1x

Store at 4°C for up to 1 week.

S1 Medium for stage 1 of the SC-α differentiation

Reagent	Amount	Final concentration
MCDB131	600 mL	N/A
FAF-BSA	12 g	0.3011 mM
Glucose	0.264 g	8.0 mM
NaHCO3	1.476 g	43.2857 mM
Vitamin C	0.0264 g	0.025 mM
Glutagro	6.0 mL	2.0 mM
Penicillin-Streptomycin (P/S) (100x)	6.0 mL	1x
ITS-X (100x)	12 mL	2x

Store at 4°C for up to 2 weeks.

Note: Allow the FAF-BSA to dissolve completely in MCDB131 (for 18–24 h at 4°C if needed) before adding all other components. Filter the final medium through a 0.22 μm filter into a sterile bottle.

S2 Medium for stage 2 of the SC-α differentiation

Reagent	Amount	Final concentration
MCDB131	300 mL	N/A
FAF-BSA	6.0 g	0.3011 mM
Glucose	0.132 g	8.0 mM
NaHCO3	0.369 g	28.6429 mM
Vitamin C	0.0132 g	0.025 mM
Glutagro	3.0 mL	2.0 mM
P/S (100x)	3.0 mL	1x
ITS-X (100x)	6.0 mL	2x

Store at 4°C for up to 2 weeks.

Note: Allow the FAF-BSA to dissolve completely in MCDB131 (for 18–24 h at 4°C if needed) before adding all other components. Filter the final medium through a 0.22 μm filter into a sterile bottle.

S3 Medium for stage 3 to 6 of the SC-α differentiation

Reagent	Amount	Final concentration
MCDB131	2.0 L	N/A
FAF-BSA	40 g	0.3011 mM
Glucose	0.88 g	8.0 mM
NaHCO3	2.46 g	28.6429 mM
Vitamin C	0.088 g	0.025 mM
Glutagro	20 mL	2.0 mM
P/S (100x)	20 mL	1x
ITS-X (100x)	10 mL	0.5x

Store at 4°C for up to 2 weeks.

Note: Allow the FAF-BSA to dissolve completely in MCDB131 (for 18–24 h at 4°C if needed) before adding all other components. Filter the final medium through a 0.22 μm filter into a sterile bottle.

Phosphate-buffered saline with Triton X-100 (PBST)

Reagent	Amount	Final concentration
Phosphate Buffered Saline (PBS)	1.0 L	N/A
Triton X-100	1.0 mL	0.1 %

This solution can be stored at 20–22°C for up to 6 months.

5% Donkey serum blocking buffer

Reagent	Amount	Final concentration
Freeze Dried Donkey Serum	10 mg	5.0 %
Ultrapure H2O	10 mL	N/A
PBST	190 mL	N/A

This solution can be stored at 4°C for up to 3 months.

3.3 mM Glucose krebs-ringer bicarbonate buffer solution (low glucose KRB)

Reagent	Amount	Final concentration
Ultrapure H2O	870 mL	N/A
Sodium Chloride [2.56 M]	50 mL	128 mM
Potassium Chloride [0.5 M]	10 mL	5.0 mM
Calcium Chloride [0.27 M]	10 mL	2.7 mM
Magnesium Sulfate [0.12 M]	10 mL	1.2 mM
Sodium Phosphate (Dibasic) [0.1 M]	10 mL	1 mM
Potassium Phosphate [0.12 M]	10 mL	1.2 mM
Sodium Bicarbonate [0.5 M]	10 mL	5.0 mM
D-Glucose [1.0 M]	3.3 mL	3.3 mM
HEPES [1.0 M]	10 mL	10 mM
FAF-BSA	1.0 g	0.1 %
Sodium Hydroxide [1.0 M]	N/A	pH 7.4

This solution should be filtered via 0.22 μm filter. Solution can be stored at 4°C for up to 2 weeks.

16.5 mM (High) Glucose KRB solution (high glucose KRB)

Reagent	Amount	Final concentration
3.3 mM Glucose KRB Solution	500 mL	N/A
D-Glucose [1.0 M]	6.6 mL	16.5 mM

This solution should be filtered via 0.22 μm filter. Solution can be stored at 4°C for up to 2 weeks.

30 mM KCl KRB solution (KCl KRB)

Reagent	Amount	Final concentration
3.3 mM Glucose KRB Solution	250 mL	N/A
Potassium Chloride [2.0 M]	3.125 mL	30 mM

This solution should be filtered via 0.22 μm filter. Solution can be stored at 4°C for up to 2 weeks.

Step-by-step method details

Thawing, seeding, and maintenance of ESCs

Timing: 10–15 days

This section describes the procedure for thawing, seeding, and maintaining 3D adapted human embryonic stem cells (ESCs) that will be differentiated into stem cell-derived alpha (SC-α) cells. These cells are cultured under feeder-free conditions and will need to be passaged every 72 h (see Part 2).

• 1.
  Prior to thawing frozen vials of ESCs prepare the 3D spinner flask as described below.

  • a.
    Add 293 mL of complete mTeSR through one of the side necks of the flask.
  • b.
    Add 300 μL of Y-27632 (ROCK inhibitor) solution to the flask.
  • c.
    Add 7 mL of complete mTeSR to a 15 mL conical tube and set aside.

CRITICAL: Adding ROCK inhibitor is necessary for the recovery and survival of single cell ESCs. Not including ROCK inhibitor at this step will result in excessive cell death.

• 2.
  Thaw 150 × 10⁶ ESCs and seed into 3D spinner flask.

  • a.
    Remove 6 frozen ESC cryotubes (25 × 10⁶ cells/tube) from the liquid nitrogen and place in a 37°C water bath for 2 min without submerging the tube cap until only a small sliver of ice remains.

    Note: Thawed ESCs should be from stocks earlier than passage 96 to ensure ESCs do not reach passage 100 prior to the start of the differentiation protocol. It is recommended to use stocks from passage 80 or less, especially if extended ESC maintenance is expected.

  • b.
    Carefully dry the tubes and spray with 70% ethanol, wipe and repeat, prior to placing the tubes in the tissue culture hood.
  • c.
    Using a 1000 μL pipette, gently and drop-wise add 1 mL of the mTeSR set aside at step 1c to each cryo tube and mix gently.
  • d.
    Using a new 1000 μL pipette tip, transfer the cell suspension of each tube into previously prepared flask (from Step 1a-b) and place on a magnetic stir plate at 70 rpm in the incubator (37°C and 5% CO₂) for 2 days.

    CRITICAL: Always confirm that the impeller of the flask rotates smoothly to ensure even and homogenous formation of clusters.

• 3.
  Feed the ESCs with 300 mL complete mTeSR 48 h after seeding.

  • a.
    Take the flask out of the incubator and place in the tissue culture hood.
  • b.
    Carefully turn the impeller several times using the tip of a 10 mL serological pipette to ensure the cells are resuspended evenly and remove 1 mL of cell suspension and place in one well of a 24-well plate for imaging.
  • c.
    Set a 5 min timer to allow the cells to settle to the bottom of the flask.

    Note: Cells may settle on the magnetic impeller in the flask. A gentle swirl of the flask after 2 min of settling time can help remove cells from the impeller.

  • d.
    In the meantime, using an inverted cell culture microscope, observe the morphology of the ESC clusters and take an image for future reference (see Figure 1).
  • e.
    After 5 min, carefully tilt the flask at a 45° angle and aspirate the media from the flask until only about 10 mL remain, take care not to aspirate the settled ESC clusters.
  • f.
    Using a 10 mL serological pipette, mix the ESC clusters by pipetting up and down forcefully 5 times.

    CRITICAL: Mixing the ESCs forcefully is important to prevent individual clusters from sticking to each other and creating large clusters prone to spontaneous differentiation. A Drummond Pipette-Aid should be used at full speed to mix clusters.

  • g.
    Add 290 mL fresh complete mTeSR (total volume in the flask is 300 mL) to the spinner flask and place the flask back on the stir plate in the incubator for 24 h until passaging.

    CRITICAL: Newly thawed ESCs should be passaged at least 3 times before starting new differentiations to ensure cells are adequately adapted to 3D culture.

Figure 1.

Morphology of pluripotent ESCs in 3D culture

(A) Bright-field images of 3-dimensional embryonic stem cell culture 2 days (top) and 3 days (bottom) after flask seeding/passaging. ESCs depicted here are from passage number 87. Representative flow cytometry demonstrates that ESCs are OCT4-positive.

(B) Representative images of good (1,2) and poor (3,4) ESC morphology. Good ESCs have uniform size, vacuoles, and are smaller than 300 μm. Poor ESC culture has variable cluster size and many clusters exceeding 300 μm. Scale bars = 500 μm.

Passaging 3D-adapted ESCs

Timing: 1 h

This section describes the passaging of human ESCs maintained in a 3D spinner flask. The ESCs need to be passaged every 72 h when maintained and not used for a differentiation.

• 4.
  Remove large ESC clusters from culture.

  • a.
    Take the flask out of the incubator and place in the tissue culture hood.
  • b.
    Carefully stir the impeller of the flask using a 10 mL serological pipette and remove 1 mL of media (with cells), and place in one well of a 24-well plate for imaging.
  • c.
    Set a 5 min timer to allow the cells to settle at the bottom of the flask.
  • d.
    In the meantime, using an inverted cell culture microscope, observe the morphology of the ESC clusters and take an image for future reference.

    Note: Cell clusters should be uniform in size and shape and should not be larger than 300 μm. Cell straining will be used to remove any clusters larger than this, but this should not represent more than 5% of your total clusters. Images of normal ESC morphology and poor ESC morphology can be found in Figure 1B.

  • e.
    After the 5 min timer is up, carefully tilt the flask at a 45° angle and aspirate the media through one of the side arms of the flask until only about 10 mL remain, take care not to aspirate the settled ESC clusters.
  • f.
    Place a 300 μm cell strainer in the mouth of a 50 mL conical tube and using a 25 mL serological pipette transfer the remaining media and cells through the cell strainer into the conical tube.
  • g.
    Rinse the spinner flask gently with 15-20 mL sterile PBS and transfer through the cell strainer.

    Note: This rinse should not be used to clean sides of flask or impeller from cell debris. Only easily detached and suspended cell clusters should be collected.

  • h.
    Allow the cells to settle in the tube for 5 min.
  • i.
    Once the clusters have settled, carefully aspirate the supernatant from the conical tube and add 35 mL sterile PBS to wash the cells.
  • j.
    Let the clusters settle for 5 min again.
• 5.
  Wash and prepare the spinner flask while the cells settle.

  • a.
    Add 12 mL sterile PBS through one of the side arms to the flask and remove all clusters and debris sticking to the sides of the flask using forceful pipetting.

    Note: Most debris is usually found at the top of the liquid interface and on the impeller. The longer the flask is in use the more difficult it will be to remove the debris, switch to a new flask if the flask sides are too sticky.

  • b.
    Aspirate the PBS, close the side arm that was used and repeat the wash step entering the flask from the other side arm to clean the other half of the flask.
  • c.
    Once clean, add 300 mL of fresh complete mTeSR media to the spinner flask.
  • d.
    Add 300 μL of Y-27632 (ROCK inhibitor) solution and set the flask aside to be reseeded later.

    CRITICAL: Do not forget to add ROCK inhibitor to the spinner flask that is to be seeded during passaging; it is necessary for the survival of single cell ESCs.

• 6.
  Dissociate the ESC clusters using Accutase and mechanical disruption.

  • a.
    Aspirate the PBS from the conical tube and add 7 mL fresh sterile PBS to the clusters.
  • b.
    Immediately add 7 mL Accutase to the clusters and set a timer for 6 min.
  • c.
    After 2 min, use a 10 mL serological pipette to aspirate the supernatant and dispense it vigorously back into the conical tube to mix the clusters once.

    CRITICAL: Be very cautious not to aspirate clusters into the pipette during this mixing procedure to prevent them from sticking to the pipette and dissociating too much. This mixing step only aims to keep cells from settling due to gravity.

  • d.
    Repeat after another 2 min (4 min since the start of the timer) to mix the cells a second time.
  • e.
    After the 6 min timer is up, inspect that all clusters have settled at the bottom of the conical tube.

    Note: If clusters are not completely settled, allow settling for an additional 1–2 min before aspiration. Excessive time in Accutase can lead to reduced cell viability. ESCs must be exposed to Accutase for at least 6 min to allow for sufficient dispersion but should not be exposed to Accutase for longer than 10 min to prevent excessive cell death.

  • f.
    Tilt the tube at a 45° angle and gently aspirate the supernatant while being cautious not to aspirate the clusters, then place the cap on the tube and tighten.
  • g.
    Disperse the clusters into single cells using mechanical disruption by dragging the tube across the grate in the front of the hood.

    • i.
      Hold the tube at a 45° angle and drag from side to side approximately 30 times.

      Note: This step may vary based on the tissue culture hood used; the goal is to use gentle vibration to disperse the cells. It is not recommended to vortex, sonicate or harshly pipette as these methods may cause too much cell death.

  • h.
    Inspect that no visible clusters remain; the cell suspension should appear milky at this point.
  • i.
    Add 35 mL mTeSR and 35 μL Y-27632 (ROCK inhibitor) solution to the single cell suspension.
  • j.
    Mix by inverting the tube 2-3 times.
• 7.
  Perform a cell count and seed the 300 mL spinner flask.

  • a.
    Dilute 100 μL of ESC suspension 1:5 with sterile PBS prior to cell count in a 1.5 mL Eppendorf tube by adding 100 μL cell solution to 400 μL PBS and mix well.

    • i.
      Note the total cell count, percentage of viable cells and the total viable cells.
  • b.
    Calculate the number of cells per mL and the volume needed to reseed a flask with 150 × 10⁶ viable ESC cells using the equations below.

    • i.
      Viable cells per mL.

      $$\left(viablecellcount\right)\times 5_{\left(Dilution\right)}=viablecells/mL$$
    • ii.
      Volume needed to seed 150 × 10⁶ ESCs.

      $$\frac{150\times {10}^6{}_{\left(Cellstobeseeded\right)}}{\left(viablecells/mL\right)}=volumeneededinmL$$
  • c.
    Seed 150 × 10⁶ ESCs into the prepared spinner flask by adding the calculated volume of single cells from the conical tube to the flask.
  • d.
    Place the flask back in the incubator onto the stir plate.

    Note: A total of 300–600 million viable ESCs are expected from a single passaged 300 mL flask. Seed additional flasks for culture expansion or differentiations as described above.

    Note: Maintained ESCs need to be fed with complete mTeSR 48 h after seeding and passaged every 72 h (split – rest – feed – split). ESCs can be maintained indefinitely with monthly karyotyping to ensure genetic stability. It is recommended that ESCs be discarded after passage 100 and replaced with a lower passage stock.

Stage 1: Differentiation to definitive endoderm

Timing: 6 days

This section describes the differentiation of human ESCs into definitive endoderm – the first stage in the differentiation towards SC-α cells.

Note: A complete schedule of media and factors for stage 1 through stage 6 of the SC-α differentiation can be found in Table 1. A schematic of the differentiation protocol can be found in Figures 2A and 3A.

Table 1.

SC-α differentiation schedule

Day	Stage	Media	Factor(s)
0	ESC Day 0	Complete mTeSR	Y-27632 (10 μM)
1	ESC Day 1	N/A	N/A
2	ESC Day 2	Complete mTeSR	N/A
3	Stage 1 Day 0	S1 Media	Activin A (100 ng/mL) CHIR 99021 (3 μM)
4	Stage 1 Day 1	S1 Media	Activin A (100 ng/mL)
5	Stage 1 Day 2	N/A	N/A
6	Stage 2 Day 0	Acquire Quality Control Sample: Stage 1 Complete
		S2 Media	KGF (50 ng/mL)
7	Stage 2 Day 1	N/A	N/A
8	Stage 3 Day 0	Acquire Quality Control Sample: Stage 2 Complete
		S3 Media	Retinoic Acid (2 μM)
9	Stage 3 Day 1	S3 Media	Retinoic Acid (2 μM) LDN 193189 (200 nM)
10	Stage 4 Day 0	Acquire Quality Control Sample: Stage 3 Complete
		S3 Media	LDN 193189 (200 nM)
11	Stage 4 Day 1	S3 Media	N/A
12	Stage 4 Day 2	N/A	N/A
13	Stage 4 Day 3	S3 Media	N/A
14	Stage 4 Day 4	N/A	N/A
15	Stage 5 Day 0	Acquire Quality Control Sample: Stage 4 Complete
		S3 Media	RepSox (10 μM)
16	Stage 5 Day 1	N/A	N/A
17	Stage 5 Day 2	S3 Media	RepSox (10 μM)
18	Stage 5 Day 3	N/A	N/A
19	Stage 5 Day 4	S3 Media	RepSox (10 μM)
20	Stage 5 Day 5	N/A	N/A
21	Stage 5 Day 6	S3 Media	RepSox (10 μM)
22	Stage 6 Day 0	Acquire Quality Control Sample: Stage 5 Complete
		S3 Media	PDBu (500 nM)
23	Stage 6 Day 1	N/A	N/A
24	Stage 6 Day 2	S3 Media	PDBu (500 nM)
25	Stage 6 Day 3	N/A	N/A
26	Stage 6 Day 4	S3 Media	PDBu (500 nM)
27	Stage 6 Day 5	N/A	N/A
28	Stage 6 Day 6	S3 Media	PDBu (500 nM)
29	Stage 6 Day 7	N/A	N/A
30	Stage 6 Day 8	S3 Media	PDBu (500 nM)
31	Stage 6 Day 9	N/A	N/A
32	Stage 6 Day 10	S3 Media	PDBu (500 nM)
33	Stage 6 Day 11	N/A	N/A
34	Stage 6 Day 12	S3 Media	PDBu (500 nM)
35	Stage 6 Day 13	N/A	N/A
36	Stage 6 Day 14	S3 Media	PDBu (500 nM)
37	Stage 6 Day 15	N/A	N/A
38	Stage 6 Day 16	S3 Media	PDBu (500 nM)
39	Stage 6 Day 17	N/A	N/A
40	Stage 6 Day 18	S3 Media	PDBu (500 nM)
41	Stage 6 Day 19	N/A	N/A
42	Stage 6 Day 20	S3 Media	PDBu (500 nM)
43	Stage 6 Day 21	N/A	N/A
44	Stage 6 Day 22	S3 Media	PDBu (500 nM)
45	Stage 6 Day 23	N/A	N/A
46	Stage 6 Day 24	S3 Media	PDBu (500 nM)
47	Stage 6 Day 25	N/A	N/A
48	Stage 6 Day 26	S3 Media	PDBu (500 nM)
49	Stage 6 Day 27	N/A	N/A
50	Stage 6 Day 28	Acquire Quality Control Sample: Stage 6 Complete
		S3 Media	PDBu (500 nM)

Figure 2.

Morphology and key cell populations of early SC-α differentiation

(A) Schematic representation of key cell populations generated in Stages 1-4 of the SC-α differentiation protocol. Bright-field images and flow cytometry characterization of SC-α differentiation progress at: (B) Stage 1 Complete, (C) Stage 2 Complete, (D) Stage 3 Complete, and E) Stage 4 Complete. The desired cell population specific to each stage is highlighted in the flow cytometry plots. Scale bars = 500 μm.

Figure 3.

SC-α cells are derived from polyhormonal pre-alpha cells and exhibit functional glucagon secretion

(A) Schematic representation of key cell populations generated in Stages 4-6 of the SC-α differentiation protocol. Bright-field images and flow cytometry characterization of SC-α differentiation progress at: (B) Stage 5 Complete, and (C) Stage 6 Complete. A population of Pre-Alpha cells will lose C-peptide expression and become GCG-monohormonal SC-α cells by the end of Stage 6.

(D) Schematic representation of plate preparation for glucose stimulated glucagon secretion assay.

(E) Glucagon secretion of SC-α cells exposed to 3.3 mM and 16.5 mM glucose challenges quantified by ELISA and normalized to 1000 cells. Data are represented as mean ± SEM (n = 5, p = 0.187).

(F) Stimulation index of unique SC-α differentiations (n = 5). Scale bars = 500 μm. Figures 3E and 3F were adapted and reprinted with permission from Shrestha et al.² Significance was calculated using a one-tailed, paired Student’s t test.

• 8.
  Prepare the human ESCs for differentiation.

  • a.
    Passage the ESCs according to schedule as described in Part 2 and seed a 3D spinner flask with 150 × 10⁶ ESCs in 300 mL complete mTeSR and ROCK inhibitor.
  • b.
    Allow the cells to rest for 48 h before feeding with 290 mL complete mTeSR as described in Part 1.
  • c.
    Start the differentiation 72 h after passaging.
• 9.
  Prepare all media and factors prior to starting the differentiation.

  • a.
    Prepare 600 mL of S1 media and store at 4°C when not in use.
  • b.
    Thaw one 4 mL aliquot of Activin A and one 100 μL aliquot of CHIR99021.

    • i.
      Thaw Activin A for 18–24 h at 4°C.
    • ii.
      Thaw CHIR99021 protected from light at 20°C–22°C and use within 30 min.
• 10.
  Feed 72 h post-passage ESCs with Stage 1 Day 0 media and factors.

  • a.
    Remove media and image cell clusters.

    • i.
      Take the spinner flask with the ESCs out of the incubator and place in the tissue culture hood.
    • ii.
      Using a 10 mL serological pipette, stir the impeller 10 times and take a 1 mL sample of media and cells to observe under an inverted cell culture microscope; take an image.
    • iii.
      Allow the cells to settle to the bottom of the flask for 5 min, then tilt the flask at a 45° angle and aspirate the media without disturbing the cells until only about 10 mL remain.

      CRITICAL: Perform this cell cluster imaging step at the start of the SC-α differentiation protocol and at the end of each differentiation stage (Stage 1 Day 0, Stage 2 Day 0, …, Stage 6 Day 28).

  • b.
    Add 290 mL of S1 media to the spinner flask.
  • c.
    Add 2 mL of thawed Activin A to the flask and store the remainder 2 mL at 4°C until use the next day.
  • d.
    Add 100 μL of thawed CHIR99021 to the flask.
  • e.
    Place the flask back in the incubator onto a stir plate set to 70 rpm for 24 h.
• 11.
  Feed the cells with Stage 1 Day 1 factors.

  • a.
    Remove media as mentioned in Step 10a (i and iii).
  • b.
    Add 290 mL of fresh S1 media to the spinner flask.
  • c.
    Using a 1000 μL pipette, add the remaining 2 mL of Activin A from previously thawed aliquot.
  • d.
    Place the flask back in the incubator onto a stir plate for 48 h.

Stage 2: Differentiation to gut tube endoderm

Timing: 2 days

This section describes the differentiation of definitive endoderm to gut tube endoderm – the second stage in the differentiation towards SC-α cells.

• 12.Collect a quality control sample, see section “stage complete: quality control sampling” (Steps 28–30).
• 13.
  Prepare all media and factors prior to starting Stage 2.

  • a.
    Prepare 300 mL of S2 media and store at 4°C when not in use.
  • b.
    Thaw one 100 μL aliquot of KGF at 20°C–22°C and use within 30 min.
• 14.
  Feed the cells with Stage 2 Day 0 media and factors to initiate the differentiation into gut tube endoderm.

  • a.
    Remove media and image cell clusters as mentioned in Step 10a (i-iii).

    Note: At this time, the clusters should be circular with dark cores (see Figure 2B). At this stage >70% of cells should be SOX17⁺/OCT4⁻, indicating successful definitive endoderm induction.

  • b.
    Add 290 mL of fresh S2 media to the spinner flask.
  • c.
    Using a 200 μL pipette, add the thawed 100 μL of KGF to the flask.
  • d.
    Place the flask back in the incubator onto a stir plate for 48 h.

Stage 3: Differentiation to pancreatic progenitors

Timing: 2 days

This section describes the differentiation of gut tube endoderm to pancreatic progenitors – the third stage in the differentiation towards SC-α cells.

• 15.Collect a quality control sample, see section “stage complete: quality control sampling” (Steps 28–30).
• 16.
  Prepare all media and factors immediately prior to starting Stage 3.

  • a.
    Prepare 2 L of S3 media and store at 4°C when not in use. This media type will be used for the rest of the differentiation.

    Note: Prepare S3 media in 2 L batches for remainder of differentiation protocol. Do not keep the media longer than 2 weeks at 4°C.

  • b.
    Thaw one 100 μL aliquot of retinoic acid (RA) at 20°C–22°C and protected from light. Use within 30 min.
• 17.
  Feed the cells with Stage 3 Day 0 media and factors to initiate the differentiation into pancreatic progenitors.

  • a.
    Remove media and image cell clusters as mentioned in Step 10a (i-iii).

    Note: At this time, the clusters will have changed dramatically in morphology and are referred to as “flowering.” The clusters should have a darker core and lighter “petal-like” outgrowths (see Figure 2C). At this stage >90% of cells should be SOX17⁺/HNF-1β⁺, indicating successful gut tube endoderm induction.

  • b.
    Add 290 mL of fresh S3 media to the spinner flask.
  • c.
    Using a 200 μL pipette, add the thawed 100 μL of RA to the flask.
  • d.
    Place the flask back in the incubator onto a stir plate for 24 h.
• 18.
  Feed the cells with Stage 3 Day 1 media and factors.

  • a.
    Thaw one 100 μL aliquot of RA and one 100 μL aliquot of LDN 193189 at 20°C–22°C and protected from light. Use within 30 min.
  • b.
    Remove media as mentioned in Step 10a (i and iii).
  • c.
    Add 290 mL of fresh S3 media to the spinner flask.
  • d.
    Using a 200 μL pipette, add the thawed 100 μL of RA and 100 μL of LDN 193189 to the flask.
  • e.
    Place the flask back in the incubator onto a stir plate for 24 h.

Stage 4: Differentiation into endocrine progenitors

Timing: 5 days

This section describes the differentiation of pancreatic progenitors to endocrine progenitors – the fourth stage in the differentiation towards SC-α cells.

• 19.Collect a quality control sample, see section “stage complete: quality control sampling” (Steps 28–30).
• 20.
  Feed the cells with Stage 4 Day 0 media and factors to initiate the differentiation into endocrine progenitors.

  • a.
    Thaw one 100 μL aliquot of LDN 193189 at 20°C–22°C and protected from light. Use within 30 min.
  • b.
    Remove media and image cell clusters as mentioned in Step 10a (i-iii).

    Note: At this time, the clusters will have become more spherical again but can be very heterogeneous in size and shape as they are still coming back together since “flowering” (Figure 2D). At this stage >90% of cells should be PDX1⁺/HNF-1β⁺, indicating successful pancreatic progenitor induction.

  • c.
    Add 290 mL of fresh S3 media to the spinner flask.
  • d.
    Using a 200 μL pipette, add the thawed 100 μL of LDN 193189 to the flask.
  • e.
    Place the flask back in the incubator onto a stir plate for 24 h.
• 21.
  On Day 1 and Day 3 of Stage 4, change the media in the spinner flask - no factors are added at this time – and let the cells rest for 48 h.

  • a.
    Remove media as mentioned in Step 10a (i and iii).
  • b.
    Add 290 mL of fresh S3 media to the spinner flask and place back in the incubator onto the stir plate for 48 h.

Stage 5: Differentiation into pre-alpha cells

Timing: 7 days

This section describes the differentiation of endocrine progenitors to Pre-Alpha cells – the fifth stage in the differentiation towards SC-α cells.

• 22.Collect a quality control sample, see section “stage complete: quality control sampling” (Steps 28–30).
• 23.
  Feed the cells with Stage 5 Day 0 media and factors to initiate the differentiation into Pre-Alpha cells.

  • a.
    Thaw one 100 μL aliquot of RepSox at 20°C–22°C and use within 30 min.
  • b.
    Remove media and image cell clusters as mentioned in Step 10a (i-iii).

    Note: At this time, the clusters should be very circular again with rough edges and a darker core and more homogeneous in size (see Figure 2E). At this stage approximately 50%–80% of cells should be CHGA⁺/NKX6.1⁻, indicating successful endocrine induction.

  • c.
    Add 290 mL of fresh S3 media to the spinner flask.
  • d.
    Using a 200 μL pipette, add the thawed 100 μL of RepSox to the flask.
  • e.
    Place the flask back in the incubator onto a stir plate for 48 h.
• 24.
  Repeat step 23 every 48 h on Stage 5 Day 2, 4, and 6.

  • a.
    After Stage 5 Day 6, place the flask back in the incubator for 24h, then proceed to Stage 6 without a rest day.

Note: During Stage 5, pancreatic endocrine cells will adopt expression of both C-peptide (C-PEP, an equimolar, more stable byproduct of insulin processing) and glucagon, a polyhormonal cell population known as a Pre-Alpha cell (see Figure 3B). For more information see Peterson et al.¹

Stage 6: Differentiation into SC-α cells

Timing: 28 days

This section describes the differentiation of Pre-Alpha cells into SC-α cells – the sixth and last stage in the differentiation towards SC-α cells.

• 25.Collect a quality control sample, see section “stage complete: quality control sampling” (Steps 28–30).
• 26.
  Feed the cells with Stage 6 Day 0 media and factors to initiate the differentiation into SC-α cells.

  • a.
    Thaw one 100 μL aliquot of PDBu at 20°C–22°C and use within 30 min.
  • b.
    Remove media and image as mentioned in Step 10a (i and iii).

    Note: At this time, the clusters are shaped like irregular spheroids with a dark main body and light colored “blebs” protruding from the surface (See Figure 3B). At this stage 30%–80% of the cells should be C-PEP⁺/GCG⁺, indicating successful Pre-Alpha cell induction.

  • c.
    Add 290 mL of fresh S3 media to the spinner flask.
  • d.
    Using a 200 μL pipette, add the thawed 100 μL of PDBu to the flask.
  • e.
    Place the flask back in the incubator onto a stir plate for 48 h.
• 27.
  Repeat step 26 every 48 h until Stage 6 Day 28, when the differentiation will be complete.

  • a.
    At stage 6 day 28, remove media and image as mentioned in Step 10a (i and iii).

Note: At this stage the clusters will look as they did at the end of Stage 5, with dark cores and lighter surface blebs (see Figure 3C). Approximately 15%–40% of cells should be C-PEP⁻/GCG⁺, indicating successful SC-α cell induction.

Note: If the cells are not used immediately, they may be kept in culture for up to 2 weeks. Cells should be fed with S3 media and PDBu every 48 h for as long as culture remains. Extending culture 2 weeks beyond Stage 6 Day 28 may cause decreases in function and viability.

Stage complete: Quality control sampling

Timing: 2 h

Following completion of each stage of the SC-α differentiation, cells are sampled and fixed for downstream analysis including flow cytometry and immunofluorescence staining.

• 28.
  Sampling SC-α differentiation.

  • a.
    Collect two unique 10 mL samples from the 300 mL SC-α differentiation immediately prior to the first feed of a new differentiation stage (Stage 2 Day 0, Stage 3 Day 0, etc.) and labels as “1” and “2”. See Table 1.

    Note: Cells at Stage 1 Day 0 are not collected as they represent ESCs. A sample can be collected if there are concerns about pluripotency of ESCs prior to differentiation.

  • b.
    Allow clusters in both tubes to gravity settle for 5 min, aspirate supernatant.
  • c.
    Wash clusters twice with 10 mL of PBS, allowing them to settle completely between washes.
  • d.
    Aspirate the final wash leaving just the cell pellet.
• 29.
  Immunofluorescence and frozen cluster sample preparation of tube 1.

  • a.
    Add 4 mL of PBS to tube 1 of the 10 mL cluster samples.
  • b.
    Distribute PBS evenly between four 1.5 mL Eppendorf tubes.
  • c.
    Pellet clusters from three 1.5 mL Eppendorf tubes using tabletop centrifuge and aspirate all supernatant. Store at −80°C for downstream protein/DNA/RNA analysis as needed.
  • d.
    Pellet cluster from the remaining 1.5 mL Eppendorf tube at 200 x g for 20 sec and resuspend clusters in 1 mL of 4 % PFA.
  • e.
    Incubate at 20°C–22°C for 1 h. Gently pellet and replace PFA with 1 mL PBS.
  • f.
    Store fixed cluster sample at 4°C for up to 6 months to use for sectioning and immunofluorescent staining as needed.
• 30.
  Flow cytometry sample fixation of tube 2.

  • a.
    Add 2 mL of TrypLE Express to tube 2 and incubate for 10 min in a 37°C water bath. Flick-mix once after 5 min.
  • b.
    Disperse cell clusters into a single cell suspension by gently pipetting up and down 20–25 times with a 1000 μL pipette.
  • c.
    Quench TrypLE reaction with 2 mL of stage-specific media (S1, S2, or S3 media).
  • d.
    Count single cell suspension using a hemocytometer or an automated cell counter.
  • e.
    Centrifuge single cell suspension at 200 x g for 5 min at 4°C using a swinging bucket centrifuge.
  • f.
    Aspirate supernatant and wash cell suspension with 2 mL of PBS.
  • g.
    Centrifuge single cell suspension at 200 x g for 5 min at 4°C using a swinging bucket centrifuge.
  • h.
    Aspirate supernatant and resuspend cell pellet in 1 mL of 4 % PFA.
  • i.
    Incubate at 20°C–22°C for 1 h. Centrifuge again at 200 x g for 5 min to pellet cells and replace PFA with 1 mL PBS using a swinging bucket centrifuge.
  • j.
    Store fixed single cell suspension at 4°C for up to 6 months.

Flow cytometry analysis of differentiation stages

Timing: 5 h

Fixed single cell suspensions are analyzed by flow cytometry using stage-specific antibodies to evaluate the progression of the differentiation.

Note: Performing flow cytometry in stage-batches can reduce hands-on time of flow cytometry analysis. It is recommended to run flow cytometry analysis following Stage 3 completion (S1C-S3C), Stage 5 completion (S4C-S5C), and Stage 6 completion (S6C).

Note: This protocol uses an Attune NxT flow cytometer. If using a different flow cytometer, adjust secondary antibodies, laser/filter configurations and software details as needed.

• 31.
  Blocking.

  • a.
    Pipette ∼400,000 cells from the fixed single cell suspension obtained from quality control sample collection through a 35 μm cell strainer cap into the attached round-bottom polystyrene test tube.

    Note: Pre-wetting the cell strainer filter with 1 mL of PBS can ease the passage of cells through the strainer.

  • b.
    Pass 1 mL of PBS through cell strainer cap to collect remaining cells.
  • c.
    Centrifuge cells at 1000 x g for 5 min at 4°C using a swinging bucket centrifuge.
  • d.
    Aspirate supernatant and resuspend cell pellet in 2 mL of PBST.
  • e.
    Centrifuge cells at 1000 x g for 5 min at 4°C using a swinging bucket centrifuge.
  • f.
    Prepare 5 % donkey serum blocking buffer while the cells spin down.
  • g.
    Aspirate supernatant and resuspend cell pellet in 1 mL of 5 % Donkey Serum Blocking Buffer.
  • h.
    Split each sample into 2x 500 μL samples for primary staining and a secondary antibody control in round-bottom polypropylene test tubes.
  • i.
    Incubate in blocking buffer at 20°C–22°C for 1 h.
• 32.
  Primary antibodies.

  • a.
    Centrifuge cells at 1000 x g for 5 min at 4°C using a swinging bucket centrifuge.
  • b.
    Prepare the appropriate primary antibody solutions for each sample by diluting in 500 μL of 5% donkey block.
  • c.
    Resuspend cells in 500 μL of primary antibody solution (primary staining samples) or 500 μL block (secondary antibody control).

    Note: Primary antibodies used for quality control are specific to each stage of SC-α differentiation sample. See Tables 2 and 3.

  • d.
    Incubate in primary antibody solution at 20°C–22°C for 1 h or at 4°C for 18–24 h, if desired.
• 33.
  Secondary antibodies.

  Note: The choice of secondary antibody may need to be adjusted based on the model of flow cytometer and laser/filter configuration available. The antibodies used in this protocol are for a 488 nm laser with a 530/30 bandpass filter and a 638 nm laser with a 670/14 bandpass filter.

  • a.
    Centrifuge cells at 1000 x g for 5 min at 4°C using a swinging bucket centrifuge and aspirate supernatant.
  • b.
    Wash the cells by resuspending the pellet in 1 mL PBST and centrifuge again at 1000 x g for 5 min at 4°C before aspirating the supernatant. Repeat one additional time.
  • c.
    Prepare the appropriate secondary antibody solutions for each sample by diluting in 500 μL of 5% donkey block.
  • d.
    Resuspend cell pellets in 500 μL of secondary antibody solutions.

    Note: Secondary antibodies used for quality control here are specific to each stage of SC-α differentiation sample. See Table 2.

  • e.
    Incubate in secondary antibody solution at 20°C–22°C for 1 h protected from light or at 4°C for 18–24 h, if desired.
  • f.
    Wash cells 3x with 2 mL PBST, resuspend final cell pellet in 500 μL mL of PBST.
• 34.
  Flow Cytometry.

  • a.
    Turn on flow cytometer, filling all solutions and installing the following filters if necessary.

    • i.
      530/30 bandpass filter for the 488 nm laser.
    • ii.
      670/14 bandpass filter for the 647 nm laser.
  • b.
    Open Attune NxT flow cytometry software and create a new workspace for samples to be analyzed.
  • c.
    Draw gates for the following cell populations (see Figure 4):

    • i.
      Cells vs. Debris – FSC-A (linear) vs. SSC-A (log).
    • ii.
      Single Cells vs. Doublets – FSC-H (linear) vs. FSC-W (log).
    • iii.
      488-antibody and 647-antibody Expression – 488-H (log) vs. 647-H (log).
  • d.
    Use secondary only control sample to adjust Photomultiplier Tube (PMT) values and flow rate.

    • i.
      Fluorescent values for both 488-H and 647-H should fall between 10² and 10³ in the secondary only control. This will place all measured cells in the bottom left quadrant of the 488-H vs. 647-H scatterplot (see Figure 4A).
  • e.
    Save secondary antibody data and collect remaining samples using the same PMT values and flow rate(s).
  • f.
    When complete, export FCS files for analysis using FlowJo software.

Table 2.

Antibodies for stage-specific quality control of SC-α differentiation

Differentiation Stage	Primary antibody 1:	Primary antibody 2:	Secondary antibody 1:	Secondary antibody 2:
Stage 1 Complete (Stage 2 Day 0)	Goat anti-SOX17 (1:250)	Mouse anti-OCT3/4 (1:250)	Donkey anti-Goat IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)
Stage 2 Complete (Stage 3 Day 0)	Goat anti-SOX17 (1:250)	Mouse anti-HNF-1β (1:100)	Donkey anti-Goat IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)
Stage 3 Complete (Stage 4 Day 0)	Goat anti-PDX1 (1:250)	Mouse anti-HNF-1β (1:100)	Donkey anti-Goat IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)
Stage 4 Complete (Stage 5 Day 0)	Rabbit anti-CHGA (1:1000)	Mouse anti-NKX6.1 (1:100)	Donkey anti-Rabbit IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)
Stage 5 Complete (Stage 6 Day 0)	Rat anti-C-PEP (1:250)	Mouse anti-GCG (1:1000)	Donkey anti-Rat IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)
Stage 6 Complete (Stage 6 Day 28)	Rat anti-C-PEP (1:250)	Mouse anti-GCG (1:1000)	Donkey anti-Rat IgG (1:1000)	Donkey anti-Mouse IgG (1:1000)

Table 3.

Stage-specific biomarkers of SC-α differentiation progress

Differentiation stage	Primary antibody 1 biomarker:	Primary antibody 2 biomarker:
Stage 1 Complete	SOX17 – Definitive Endoderm	OCT4 – Pluripotency Marker
Stage 2 Complete	SOX17 – Definitive Endoderm	HNF-1β – Gut Tube Endoderm
Stage 3 Complete	PDX1 – Pancreatic Lineage	HNF-1β – Gut Tube Endoderm
Stage 4 Complete	CHGA – Endocrine Cell	NKX6.1 – β Cell Lineage
Stage 5 Complete	C-PEP – Insulin Production	GCG – Glucagon Production
Stage 6 Complete	C-PEP – Insulin Production	GCG – Glucagon Production

Figure 4.

Flow cytometry gating strategy

Gating Strategy for flow cytometry analysis, applicable to all stages of the SC-α differentiation protocol.

(A) Secondary only control sample.

(B) Example gating of Stage 6 day 28 SC-α cells. Cellular debris and doublets are excluded before stage-specific Alexa Fluor-antibody expression is plotted and quantified.

Glucose-stimulated glucagon secretion

Timing: 2 days

Stage 6 complete SC-α cells are exposed to varied glucose concentrations to induce glucagon secretion. Intensity of SC-α cell secretion in response to low or high glucose conditions is a critical aspect of SC-α cell function.

• 35.
  KRB plate preparation (see Figure 3D).

  • a.
    Add 0.5 mL of High Glucose KRB Solution to Column 1 in each of two 24 well ultralow attachment plates. Place Millicell transwell inserts into each well.

    Note: Ensure transmembrane liquid has passed through the transwell membrane, allowing solution and solute transfer. Placing a small volume of high glucose KRB solution on top of the membrane can aid solution transfer across the membrane.

  • b.
    Add 1 mL of High Glucose KRB Solution to column 2 of both plates.
  • c.
    Add 1 mL of High Glucose KRB Solution to column 3 of one plate and 1 mL of Low Glucose KRB Solution to column 3 of the other plate.
  • d.
    Add 1 mL of KCl KRB Solution to column 4 of both plates.
  • e.
    Add 1 mL of TrypLE Express to column 5 of both plates.
  • f.
    Reserve 30 mL of High Glucose KRB Solution in a 50 mL conical tube.
  • g.
    Place both ultralow attachment plates and reserved KRB solution into an incubator for 1–2 h to allow for temperature equilibration.
• 36.
  “Fasting” SC-α cells in high glucose KRB.

  • a.
    Remove approximately 6 million cells from a Stage 6 complete SC-α differentiation.
  • b.
    Allow cells to gravity settle for 5 min and wash twice with 10 mL of pre-warmed High Glucose KRB Solution.
  • c.
    Resuspend cells in 4 mL of High Glucose KRB Solution.
  • d.
    Pipette 0.5 mL (0.75 million cells) into each Millicell transwell insert in prepared KRB Solution plates.

    Note: Optimal cell density for each transwell is between 0.5 and 1 million cells. Too many cells may lead to autocrine inhibition of glucagon secretion.

  • e.
    Incubate plates at 37°C for 1 h with gentle rocking.
• 37.
  SC-α cell glucose and KCl challenge.

  • a.
    Transfer transwell membranes in both 24 well plates to column 2.

    • i.
      Carefully tilt the transwell insert using forceps and pull out slowly, allowing the liquid in the transwell to wick into the main plate well.

      CRITICAL: Ensure less than 15 μL of solution is transferred between wells at every transfer during the GSGS. This will prevent the carryover of significant concentrations of solutes that may otherwise skew the glucagon secretion.

      Note: Maintaining contact between the membrane of the transwell and the main liquid interface in the plate well for 3–5 s will help in draining the transwell.

  • b.
    Immediately transfer transwell membranes from column 2 to column 3.
  • c.
    Incubate plates at 37°C for 1 h with gentle rocking.
  • d.
    Transfer transwell membranes in both 24 well plates from column 3 to column 4.
  • e.
    Immediately collect 200 μL of solution from each well in column 3 and store in a 96 well ultralow attachment plate. Store plate at −20°C immediately after collection.

    Note: Glucagon has a very short in vitro half-life. Rapid collection and storage at −20°C will help prevent degradation.

  • f.
    Incubate plates at 37°C for 1 h with gentle rocking.
  • g.
    Transfer transwell membranes in both 24 well plates from column 4 to column 5.
  • h.
    Immediately collect 200 μL of solution from each well in column 4 and store in a 96 well ultralow attachment plate. Store plate at −20°C immediately after collection.

    Note: Glucagon secretions can be stored at −20°C for up to 1 month. Storage at −80°C can extend storage to 6 months.

• 38.
  SC-α cell cluster dispersion and cell counting.

  • a.
    Incubate plates at 37°C for 15 min with gentle rocking.
  • b.
    Disperse cells in transwell inserts by pipetting up and down 15-20 times with a 200 μL pipette. Break transwell insert and mix with the entire 1 mL of TrypLE solution.
  • c.
    Count cells in TrypLE solution using a hemocytometer or automated cell counter.
  • d.
    Repeat for all transwell inserts.

Note: Single-cell suspensions have the tendency to aggregate on the transwell inserts. Samples should be dispersed one-by-one, and a counting sample should be collected immediately.

• 39.
  ELISA quantification of glucagon secretion.

  • a.
    Use a commercially available glucagon ELISA kit such as the R&D Systems DuoSet Glucagon ELISA to measure glucagon secretion in each KRB solution collected during GSGS.

    • i.
      Dilute glucagon secretion samples to linear detection range of ELISA kit before starting ELISA. A typical sample should be diluted 1:100 in high glucose KRB buffer.
  • b.
    Read out ELISA quantification on a microplate reader with absorbance reading capabilities such as the Thermo Fisher Varioskan Lux.
  • c.
    Glucagon concentration in each KRB condition will be normalized to 1000 cells using the counts obtained from each transwell during GSGS procedure.
  • d.
    Calculate the stimulation index (SI).

    • i.
      The stimulation index (SI) of SC-α cells is calculated as the ratio of glucagon secreted in the 3.3 mM glucose condition to the 16.5 mM glucose condition.

      $$SI=\frac{{\left[GCG\right]}_{3.3mM}}{{\left[GCG\right]}_{16.5mM}}$$
    • ii.
      A typical secretion index is between 1.0 and 2.0 (see Figures 3E and 3F).

Expected outcomes

Starting with 150 million ESCs at the time of flask seeding, this protocol is expected to produce 150-300 million total cells at Stage 6 day 28 (∼50 million SC-α cells). At the start of the differentiation (S1D0), approximately 300-600 million cells should be present in suspension culture. At the end of Stage 1, significant expansion will occur along with many dead cells and cellular debris present in the flask. The Stage 1 complete cell population should be >70% SOX17+/OCT4- when measured by flow cytometry (Figure 2B). By the end of Stage 2, a significant “flowering” should occur morphologically and >90% of cells should be SOX17+/HNF-1β+ (Figure 2C). With the establishment of the pancreatic progenitor cell fate during Stage 3, cell clusters will start to return to their round morphology (some oblong clusters will remain) and over 90% of cells should be PDX1+/ HNF-1β+ (Figure 2D). At the end of Stage 3, a flask should have approximately between 400-700 million cells. Establishment of the endocrine cell fate is less efficient than previous stages, with about 50-80% of cells expected to be CHGA+/NKX6.1- at the end of Stage 4 (Figure 2E). Some cell loss is expected at this stage, with typical total cell numbers between 200-400 million cells.

Pre-Alpha cell fate commitment should be approximately 30-80% GCG+/C-PEP+ at the end of Stage 5, with between 200-300 million cells remaining in the culture (Figure 3B). It is important to perform flow cytometry soon after the completion of Stage 5, as a low Pre-Alpha commitment is unlikely to yield necessary SC-α cell numbers at the end of the 28-day Stage 6. Establishing new differentiations may be a suitable alternative as opposed to extended 28-day culture in these instances.

The completion of the 28-day Stage 6 culture period should yield approximately 15-40% SC-α cells at the end of the differentiation process (Figure 3C). Total cell numbers should remain between 150-300 million cells (∼50 million total SC-α cells) by the end of the differentiation process. SC-α cells should display a stimulation index between 1.0-2.0 when measured by the glucose stimulated glucagon secretion assay (Figures 3E and 3F).

Limitations

The protocol here describes how to generate stem cell-derived alpha cells from 3D embryonic stem cell cultures using chemically defined small molecule and growth factor induction. This protocol, including ESC maintenance and SC-α differentiation has only been validated using culture materials described here. Changes to culture flask (impeller shape, glass vs. plastic casing), magnetic stir speed, and media composition require validation.

Additionally, the completed differentiation product contains approximately 15-40% of SC-α cells. Remaining cell populations need to be considered when designing and interpreting experiments using SC-α populations.

Isolated SC-α cells generated here may not respond to glucose stimulation in the same manner as isolated human islets due to the complexity of intra-islet interactions between alpha, beta, and delta cells, which are not present in the SC-α cell product.

This protocol describes a lengthy process with multiple complex steps that can cause significant opportunities for user error to occur. As a result, it is not uncommon for a differentiation to not meet the expected outcomes. Following this protocol as closely as possible can help reduce the risk of unsatisfactory differentiations but may not eliminate it.

Troubleshooting

Problem 1

Excessive cell death in ESC culture/passaging.

Potential solution

Check that ROCK Inhibitor is not expired and correctly reconstituted. Assure the cells are not handled harshly during passaging or lost during aspiration of supernatant while feeding. Ensure cell counting is accurate, as flask seeding density can significantly influence health of culture at 72 h post passage. High passage number can lead to unhealthy ESC culture. Cells beyond passage 100 should be discarded for more recent passages. Also, it is recommended to ensure karyotype stability of ESC culture approximately once per month. Finally, ensure that there is no contamination (bacterial, fungal or mycoplasma) leading to excess death.

Problem 2

Excessive cell loss over the course of SC-α differentiation.

Potential solution

There are two major reasons for cell loss during SC-α differentiation. The first is cell loss due to cellular aspiration during media changes. This occurs when cell clusters are not allowed to settle for a sufficient amount of time before removing spent media. Ensure that during media changes, cells are sufficiently settled and are not being aspirated with spent media. Additional settling time may be necessary at early stages (ESC, Stage 1, Stage 2), however settling will be faster at later differentiation stages. Another reason for cell loss is excessive cell death. The most common cause of cell death during differentiation is the improper placing of the spinner flask onto the magnetic stir plate, causing irregular and “jerky” rotations. During media changes, if spent media appears cloudy or opaque, cell death is likely occurring. If excessive cell death is expected, an image can be taken under a bright-field microscope (Step 10a) to observe for excessive cellular debris. Importantly, some cell death is expected during stages 1 and 2 of the differentiation due both high cell counts and the dynamic morphological process of “flowering”. Large scale cell death is not expected beyond stage 3 of the differentiation. At the end of each differentiation stage, compare cell counts obtained during quality control sampling to the expected cell counts described in the expected outcomes section to determine if cell loss is excessive. In the case of cell death, ensure the smooth rotation of the culture flask impeller each time flask is placed back into an incubator. Finally, ensure there are no cell death due to contamination issues (bacterial, fungal or mycoplasma).

Problem 3

Failure of cells to differentiate to definitive endoderm (Stage 2 Day 0).

Potential solution

• •In cases where cells are failing to proceed through the first differentiation stage, ensure that both Activin A and CHIR99021 are properly reconstituted and are not expired. Secondly, ensure that ESCs have not spontaneously differentiated by the start of the differentiation protocol (Stage 1 day 0). Collecting a culture sample and testing for OCT4+ expression (or other pluripotency markers if available) will ensure starting ESC material is suitable for the differentiation.
• •Additionally, it is normal for Stage 1 culture to have lots of cell debris associated with cell death. This excessive debris can confound flow cytometry results, making it appear as though differentiation to definitive endoderm has occurred at a lower efficiency. Continue culture to Stage 2 (gut tube endoderm, SOX17+/HNF-1β+) and assess cell populations by flow cytometry. The “cleaner” cell culture often reveals a >90% differentiation efficiency.

Problem 4

Differentiation stages do not meet expected cell proportion outcomes.

Potential solution

If SC-α differentiations are continuously not meeting expected outcomes at various stages, it is important to confirm that all factors are not expired and are being used at the appropriate concentrations. Additionally, it is important to ensure that media changes, especially those at the end of each differentiation stage, are thorough to ensure factors are not carried over from unchanged media. Maintaining consistent feed times (within ± 1 h time window every day) will ensure that cells are exposed to differentiation factors for the intended period of time, extended or shortened factor exposure time can significantly skew differentiation efficiency.

Problem 5

ES cell clusters are not forming or becoming too large (>300 μm) (See Figure 1B).

Potential solution

If ESC cell clusters are not forming following passage, ensure ROCK inhibitor is being added properly as previously described. Additionally, lowering the stir speed of the magnetic stir plate could promote cluster formation. Care should be taken that clusters are not routinely becoming larger than 300 μm in diameter, as spontaneous differentiation and necrosis can occur. If cell clusters are consistently larger than this, increasing magnetic stir speed can reduce cluster size. ES cells are also very sticky after seeding due to the addition of ROCK inhibitor, therefore ensuring media changes are thorough and the clusters are mixed vigorously with a serological pipette on the feed day, will prevent the formation of too large clusters.

Problem 6

High variability in glucagon secretion quantification.

Potential solution

In some instances, glucagon secretion from SC-α cells will not show glucose responsiveness or will display high variability in total glucagon secretion. Consistent quantification of glucagon from SC-α cells using ELISA technology is a difficult procedure with many variables influencing results. Firstly, the glucose stimulated secretion of SC-α cells in the absence of islet cell interactions (beta and delta cells) and other physiological stimulants (amino acids, hormones, etc.) is poorly understood and is not expected to match the more robust glucagon secretion response of alpha cells from intact isolated human or mouse islets. Exposing SC-α cells to other glucagon stimulants such as arginine could serve as an alternative method to assess alpha cell functionality.¹ To help ensure reproducible results, ensure that 0.5–2 million cells are present in each transwell insert during the GSGS procedure. Too few cells can lead to unquantifiable concentrations of glucagon to be secreted into the 1 mL solution volume, while too many cells could lead to self-inhibition of glucagon secretion due to the saturation of glucagon in the solution. Additionally, the extremely short half-life of glucagon can lead to variability in total measured glucagon if not properly stored. Ensure that glucagon secretion samples are not thawed more than 3 times and take care to keep time at 20°C–22°C to a minimum. Alternatively, the use of a dipeptidyl peptidase 4 (DPP4) inhibitor could help to prevent the enzymatic degradation of glucagon.

Problem 7

KRB buffer forms precipitates and/or solutes are not completely dissolved.

Potential solution

Confirm that all preparatory reagents are correctly made and not expired. Ensure the BSA is dissolved completely (this may take a few hours) before proceeding with pH adjustments as incorrect pH may cause certain solutes to precipitate out of solution. Ensure HEPES buffer is present to prevent pH changes upon incubation in 37°C incubator.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Quinn P. Peterson (peterson.quinn@mayo.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Quinn P. Peterson (peterson.quinn@mayo.edu).

Materials availability

This study did not generate any new or unique reagents.

Data and code availability

This study did not generate new or unique code and did not analyze new or unique datasets.

Acknowledgments

This work was funded by the Khalifa bin Zayed Al Nahyan Foundation and the Mayo Clinic. Parts of the graphical abstract and some figures were generated using BioRender.

Author contributions

T.S., K.R.K., and S.S. collected and analyzed the data used in this manuscript. T.S. and K.R.K. wrote and edited the protocol and prepared the manuscript. Q.P.P. was involved in all aspects of this work.

Declaration of interests

Q.P.P. serves on the scientific advisory board of MelliCell, Inc., and is listed as an inventor on intellectual property licensed by Vertex.