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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Abstract

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling, however, with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell's physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

Introduction

Diabetes is a complex metabolic disorder affecting millions of people globally. Supplementation of insulin is considered the only treatment option for diabetes. More advanced cases are treated with beta cell replacement therapy, achieved through transplantation of either whole cadaveric pancreas or islets1,2. Several issues surround transplantation therapy, such as limitation with the availability and quality of the tissue, invasiveness of transplantation procedures in addition to the continuous need for immunosuppressants. This necessitates the need for discovering novel and alternative options for beta cell replacement therapy2,3. Human pluripotent stem cells (hPSCs) have recently emerged as a promising tool for understanding human pancreas biology and as a non-exhaustive and potentially a more personalized source for transplantation therapy4,5,6,7. hPSCs, including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), have a high self-renewal capacity and give rise to any tissue type of the human body. hESCs are derived from the embryo's inner cell mass, and hiPSCs are reprogrammed from any somatic cell4,8.

Directed differentiation protocols are optimized to generate pancreatic beta cells from hPSCs that sequentially direct hPSCs through pancreatic developmental stages invitro. These protocols generate hPSC-derived islet organoids. While they have greatly improved at increasing the proportion of pancreatic beta cells therein, the efficiency of protocols is highly variable. It does not increase to more than ~40% of NKX6.1+/INSULIN+ or C-PEPTIDE + cells5,9,10,11,12,13. However, the generated beta cells are not entirely identical to the adult human beta cells in terms of their transcriptional and metabolic profiles and their response to glucose4,5,14. The hPSC-derived beta cells lack gene expression of key beta cell markers such as PCSK2, PAX6, UCN3, MAFA, G6PC2, and KCNK3 compared to adult humans islets5. Additionally, the hPSC-derived beta cells have diminished calcium signaling in response to glucose. They are contaminated with the co-generated polyhormonal cells that do not secrete appropriate amounts of insulin in response to increasing glucose levels5. On the other hand, hPSC-derived pancreatic progenitors, which are islet precursors, could be generated more efficiently in vitro compared to beta cells and, when transplanted in vivo, could mature into functional, insulin-secreting beta cells15,16. Clinical trials are currently focused on demonstrating their safety and efficacy upon transplantation in T1D subjects.

Notably, expression of the transcription factors PDX1 (Pancreatic and Duodenal Homeobox 1) and NKX6.1 (NKX6 Homeobox 1) within the same pancreatic progenitor cell is crucial for commitment towards a beta cell lineage5. Pancreatic progenitors that fail to express NKX6.1 give rise to polyhormonal endocrine cells or non-functional beta cells17,18. Therefore, a high co-expression of PDX1 and NKX6.1 in the pancreatic progenitor stage is essential for ultimately generating a large number of functional beta cells. Studies have demonstrated that an embryoid body or 3D culture enhances PDX1 and NKX6.1 in pancreatic progenitors where the differentiating cells are aggregated, varying between 40%-80% of the PDX1+/NKX6.1+ population12,19. However, compared to suspension cultures, 2D differentiation cultures are more cost-effective, feasible, and convenient for application on multiple cell lines5. We recently showed that monolayer differentiation cultures yield more than up to 90% of PDX1+/NKX6.1+ co-expressing hPSC-derived pancreatic progenitors20,21,22. The reported method conferred a high replicating capacity to the generated pancreatic progenitors and prevented alternate fate specifications such as hepatic lineage21. Therefore, herein, this protocol demonstrates a highly efficient method for the differentiation of hPSCs to pancreatic beta-cell precursors co-expressing PDX1 and NKX6.1. This method utilizes the technique of dissociating hPSC-derived endoderm and manipulating the cell density, followed by an extended FGF and Retinoid signaling as well as Hedgehog inhibition to promote PDX1 and NKX6.1 co-expression (Figure 1). This method can facilitate a scalable generation of hPSC-derived pancreatic beta-cell precursors for transplantation therapy and disease modeling.

Protocol

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and Qatar Biomedical Research Institute (QBRI) (no. 2016-003). This work is optimized for hESCs such as H1, H9, and HUES8. Blood samples were obtained from healthy individuals from Hamad Medical Corporation (HMC) hospital with full informed consent. The iPSCs are generated from peripheral blood mononuclear cells (PBMCs) of control, healthy individual23.

1. Preparation of the culture media

  1. Prepare human pluripotent stem cells (hPSC) culture media
    1. Prepare hPSC culture media from the commercially available medium for maintaining and expanding human embryonic stem cells by supplementing 100 units/mL of Penicillin and 100 ug/mL of Streptomycin (see Table of Materials). Aliquot and store the complete media at -20 °C for the long term or 4 °C for immediate use.
  2. Prepare Stage 1 differentiation media (for Definitive Endoderm (DE)).
    1. Prepare Basal media from MCDB 131 media by supplementing with 0.5% of fatty acid-free Bovine serum albumin (FFA-BSA), 1.5 g/L of Sodium bicarbonate (NaHCO3), 10 mM of glucose, 2 mM of Glutamax, 100 units/mL of Penicillin, and 100 µg/mL of Streptomycin (see Table of Materials). Filter the prepared media using a 0.2 µm filter and store at 4 °C.
    2. Prepare Stage 1 media containing CHIR99021 from the Basal media (prepared in step 1.2.1) by warming the basal media at 37 °C and then supplementing with 2 µM of CHIR99021, 100 ng/mL of Activin A, 10 µM of Rock inhibitor (Y-27632), and 0.25 mM of Vitamin C (see Table of Materials). Mix the supplemented media well and cover it with aluminum foil to protect it from light.
      NOTE: This media will be used only on the first day of differentiation.
    3. Prepare Stage 1 media without CHIR99021 from the Basal media by warming it at 37 °C and supplementing it with 100 ng/mL of Activin A and 0.25 mM of Vitamin C and mix well.
      NOTE: 5 ng/mL of basic FGF or FGF2 can be optionally added to this media. This media will be used for the remaining days of Stage 1.
  3. Prepare Stage 2 differentiation media (for Primitive Gut Tube; PGT).
    1. Prepare Stage 2 differentiation media containing Rock inhibitor from the Basal media by warming it at 37 °C and supplementing with 50 ng/mL of FGF10, 50 ng/mL of NOGGIN, 0.25 µM of CHIR99021, 10 µM of Y-27632 (Rock inhibitor), and 0.25 mM of Vitamin C.
      NOTE: This media is used on the day of dissociation of endodermal cells.
    2. Prepare Stage 2 differentiation media without Rock inhibitor following the same procedure for the Media prepared in step 1.3.1, excluding the Rock inhibitor.
      NOTE: This media is used on the second day of Stage 2.
  4. Prepare Stage 3 differentiation media (for Posterior Foregut).
    1. Prepare DMEM media containing 4.5 g/L of Glucose, then supplement with 1% of Penicillin-streptomycin and 2 mM of Glutamax and use as basal media for Stages 3 and 4.
    2. Warm the DMEM media (prepared in step 1.4.1) and supplement with 2 µM of Retinoic acid, 0.25 µM of SANT-1, 50 ng/mL of FGF10, 50 ng/mL of NOGGIN, 0.25 mM of Vitamin C, and 1% B27 supplement without vitamin A (see Table of Materials).
      NOTE: This media is used for 4 days of Stage 3 for P2-D (Protocol 2, optimized, dissociated) and 2 days of Stage 3 for P1-ND (Protocol 1, non-optimized, non-dissociated).
  5. Prepare Stage 4 differentiation media (for Pancreatic Progenitors).
    1. Add DMEM containing 4.5 g/L of Glucose with 100 ng/mL of EGF, 10 mM of Nicotinamide, 50 ng/mL of NOGGIN, 0.25 mM of Vitamin C, and 1% of B27 supplement without Vitamin A (see Table of Materials). Use this media for all days of Stage 4.
      NOTE: Maintain all reagents at appropriate temperatures, and all cytokine and sensitive reagents need to be aliquoted. Thaw the aliquoted reagents and add to the basal media at the time of changing the differentiation media. The compositions of the different differentiation media are provided in Table 1.

2. Preparation of basement membrane matrix coated dishes

  1. Thaw commercially available basement matrix (see Table of Materials) and aliquot on ice; freeze the aliquots at -20 °C.
  2. Before coating the tissue culture dishes, thaw the frozen aliquots on ice. Add appropriate volume of the membrane matrix solution to the chilled KO-DMEM/F-12 media (KnockOut DMEM/F-12) (see Table of Materials) to achieve the desired diluted concentration and mix well. Store the diluted matrix solution at 4 °C for immediate use.
  3. Cover the surface of the tissue culture-treated plates (see Table of Materials) with diluted membrane matrix solution and place the plates at 37 °C for at least 60 min before plating the cells. Use a dilution of 1:50 of the membrane matrix solution in KO-DMEM/F-12 for plating cells for pancreatic differentiation experiment and 1:80 for expansion of undifferentiated hPSCs.

3. Culture of undifferentiated hPSCs

  1. Passage the hPSCs when the colonies reach a 70%-80% confluency.
  2. To passage, wash the hPSCs once with warm PBS, aspirate using a portable vacuum aspirator inside the tissue culture hood, and add 0.5 mM of EDTA solution in PBS to cover the colony surface. Incubate at 37 °C, 5% CO2 for 1 min or until the colonies' borders detach from the plate surface.
  3. Remove EDTA solution and collect the detaching colonies with hPSC culture medium using a P1000 micropipette. Centrifuge the collected cells at 128 x g for 4 min at room temperature. Discard the supernatant and supplement the cells with hPSC culture media containing 10 µM of Y-27632 (Rock inhibitor)23,24.
    ​NOTE: Passage the hPSCs in at least a 1:3 ratio on 1:80 membrane matrix-coated dishes. However, for differentiation experiments, plate the hPSCs on 1:50 coated dishes.

4. Induction of Definitive Endoderm (DE) differentiation in hPSCs (Stage 1)

  1. When the hPSC colonies reach a confluency of 70%-80%, wash them twice with warm PBS and aspirate using a portable vacuum aspirator inside the tissue culture hood to begin Stage 1 differentiation.
  2. Add Stage 1 differentiation medium containing CHIR99021 (from step 1.2.2) to the colonies, 2 mL per 6-well plate, and incubate at 37 °C for 24 h.
  3. The next day, replace the spent media with Stage 1 differentiation medium without CHIR99021 (step 1.2.3).
  4. Every 24 h, aspirate the spent media using a portable vacuum aspirator inside the tissue culture hood and replace it with a fresh Stage 1 differentiation medium without CHIR99021.
    ​NOTE: Stage 1 can be extended up to 4 days. The length of Stage 1 is dependent on the hPSC line being used and should be optimized accordingly.

5. Immunofluorescence analysis of hPSC-derived DE (Stage 1)

  1. For immunofluorescence, aspirate the spent media using a portable vacuum aspirator inside the tissue culture hood from the wells and wash twice with warm PBS. Swirl the plate to get rid of any cell debris.
  2. Cover the surface of the wells with 4% of paraformaldehyde (PFA) to fix the DE cells; for example, add 250 uL of PFA per well of a 24-well plate. Place the plate on a 2D shaker at 20 x g for 20 min.
  3. After fixation, wash the DE cells with tris-buffered saline with 0.5% of Tween (TBST) (see Table of Materials) and place the plate on the shaker at 20 x g for 10 min. Repeat this step once more.
  4. Permeabilize the fixed cells by adding a generous volume of phosphate-buffered saline with 0.5% of Triton X-100 (PBST); for example, add 1 mL of PBST per well of a 24-well plate and place the plate back on the shaker at 20 x g for 20 min.
  5. Freshly prepare 5%-6% of BSA in PBST as blocking buffer and add it to the permeabilized cells. Incubate the plate for at least 1 h in the blocking solution on the shaker.
  6. Dilute the primary antibodies against SOX17 and FOXA2 together (see Table of Materials) in 2%-3% BSA in PBST solution. Add the combined antibodies to blocked cells and place the plate on the shaker at 4 °C overnight at a low speed with gentle shaking.
    NOTE: SOX17 and FOXA2 are well-established DE markers25,26.
  7. The next day, aspirate the primary antibodies using a portable vacuum filter and wash the wells with TBST three times, each wash for 10 mins on the shaker.
  8. Prepare 1:500 dilution of Alexa fluor 488- and 568- conjugated secondary antibodies (see Table of Materials) against the species the primary antibodies were raised in.
  9. Add the secondary antibody combination to the stained well and cover the plate with aluminum foil to protect from light. Place the plate on the shaker for 1 h at room temperature.
  10. Aspirate the secondary antibody solution using a portable vacuum filter and wash the stained wells with TBST on the shaker for 10 min, covering the plate with foil. Repeat the wash step a total of three times.
  11. Prepare a 1 µg/mL of Hoechst 33342 dilution in PBS to stain the nuclei. Add the Hoechst solution to the wells and place the plate on the shaker for 2-3 min.
  12. Aspirate the Hoechst solution using a portable vacuum filter and rinse the wells with PBS twice.
  13. Finally, add PBS to the stained cells and image them using an inverted fluorescence microscope (see Table of Materials) in the dark. Keep the plate covered with foil when not imaging to minimize the fluorophore bleaching.
    ​NOTE: Alternatively, flow-cytometry can be used to assess DE efficiency as described in step 9.2.

6. Generation of the primitive gut tube (PGT) from hPSCs (Stage 2)

NOTE: If the immunofluorescence analysis in step 5.13 is determined to be a SOX17-FOXA2 co-expression of 80% and above, the experiment proceeds to Stage 2. If the efficiency is <80%, extend the duration of Stage 1 to 4 days.

  1. On day 1 of Stage 2, dissociate the hPSC-derived endodermal cells using TrypLE or Accutase (see Table of Materials) for the optimized P2-D protocol. Wash the adherent cells with warm PBS and add warm 1 mL of TrypLE or Accutase solution per well of a 6-well plate for 3-5 min at 37 °C, 5% CO2, or until the cells begin to detach from one another.
  2. Dissociate the detached sheets or monolayer of cells in the wells and then collect them together in a 15 mL polypropylene tube using basal Stage 1/2 media without cytokines containing at least 0.5% of either fetal bovine serum (FBS) or KnockOut serum (KOSR) (see Table of Materials).
  3. Spin down the cells at 800 x g for 5 min at 4 °C and discard the supernatant. Add 1 mL of sterile PBS and resuspend the pellet into single cells.
  4. Count the cells using an automated counter (see Table of Materials) by loading the recommended volume of cells in the chamber slide. Spin the resuspended cells at 800 x g for 5 min at 4 °C and discard the supernatant.
  5. Resuspend the pellet in the appropriate volume of Stage 2 differentiation medium containing Rock inhibitor at a density of 2.5-3.5 x 105 cells/cm2.
    NOTE: This count may come down to a 1:2 splitting ratio for most cell lines, dependent on the proliferation rate. The total volume will be 2 mL of media with resuspended cells in 1 a well of 6-well plate.
  6. Plate the resuspended cells on 1:50 membrane matrix-coated plates (prepared in step 2) and incubate them at 37 °C, 5% CO2 in the incubator.
  7. 24 h later, replace the media with Stage 2 differentiation medium without Rock inhibitor (prepared in step 1.3.2).

7. Generation of posterior foregut from hPSCs (Stage 3)

  1. Aspirate the spent Stage 2 media using a portable vacuum aspirator inside the tissue culture hood and wash the cells with warm PBS.
  2. Add Stage 3 differentiation media from step 1.4 to the cells and incubate at 37 °C, 5% CO2.
  3. After 24 h, replace the spent media with freshly prepared Stage 3 differentiation media. Repeat this for a total of 4 days for P2-D protocol (optimized) and only 2 days for the non-dissociated P1-ND.

8. Generation of Pancreatic Progenitors from hPSCs (Stage 4)

  1. After 4 days of Stage 3 treatment, wash the cells with warm PBS, gently swirl the plate, and aspirate using a portable vacuum aspirator. Then, add Stage 4 differentiation media from step 1.5 to the cells.
  2. After 24 h, replace the spent media with freshly prepared Stage 4 media. Repeat this for a total of 4 days.

9. Assessment of differentiation efficiency of generating pancreatic progenitors from hPSCs

  1. Perform the immunofluorescence analysis of the hPSC-derived pancreatic progenitors (Stage 4) for expression of PDX1 and NKX6.1.
    NOTE: PDX1 and NKX6.1 are well-established pancreatic progenitor markers17,18.
    1. Perform fixation, permeabilization, blocking, and antibody incubation and washes according to step 5.
    2. Stain the hPSC-derived pancreatic progenitors with a combination of PDX1 and NKX6.1 antibodies (see Table of Materials) diluted in 2%-3% BSA in PBST.
    3. Use 1:500 dilutions of appropriate Alexa fluor 488- and 568- conjugated secondary antibodies (see Table of Materials).
  2. Perform flow-cytometry analysis of hPSC-derived pancreatic progenitors for expression of PDX1 and NKX6.1.
    NOTE: Flow-cytometry analysis of pancreatic markers in the generated hPSC-derived pancreatic progenitors provides a way to quantify the PDX1 and NKX6.1 co-expressing cells.
    1. At the end of Stage 4, wash the cells twice with warm PBS and add enough TrypLE or Accutase to cover the surface of the wells, for example, 1 mL of TrypLE or Accutase per well of 6-well plate. Place the plate in the incubator for 5-7 min or until the cells detach from the surface.
    2. Dissociate the adherent sheets of cells within the well using a P1000 micropipette before collecting them in a 15 mL polypropylene tube.
    3. Spin down the cells in hPSC-derived pancreatic progenitors at 800 x g for 5 min at 4 °C, then discard the supernatant. Wash the cells with PBS by dissociating them into single cells. Count the cells using an automated counter (see Table of Materials) and note the concentration in the number of cells per mL.
    4. Spin at 800 x g for 5 min at 4 °C, and discard the supernatant. Add 200 µL of chilled PBS to the pellet and dissociate.
    5. Add 2 mL of chilled 80% ethanol dropwise, with the tube on a vortex at low-medium speed (400 x g at room temperature). Close the caps tightly and place the tubes slightly tilted on the shaker at 4 °C overnight.
    6. Spin down the cells at 800 x g for 5 min at 4 °C, and wash with PBS to dissociate any clumps of fixed cells.
    7. Block the fixed cells with 5%-6% BSA solution in PBST for at least 1 h at room temperature or 4 °C overnight on the shaker.
    8. Stain for the pancreatic progenitor markers following the steps below.
      1. Distribute 2,00,000 cells per condition, including appropriate isotype controls dependent on the IgG subclass of the host species of the primary antibody, unstained and secondary antibody controls in a 96-well V bottom plate or 1.5 mL centrifuge tubes.
      2. Spin down the plate at 800 x g for 5 min at 4 °C and flip the plate with a swift motion to discard the supernatant without losing the pellets. Prepare primary antibody dilutions in 3% BSA solution (see Table of Materials).
        NOTE: The concentration of primary antibodies can be between 1:50 to 1:200.
      3. Incubate the stained cells for at least 2 h at room temperature or overnight at 4 °C on a shaker with gentle shaking at low speed.
      4. Wash the stained cells with TBST thrice by pipetting the cells up and down in the wells. Spin and discard the supernatant as in step 9.2.8.2.
      5. Add 1:500 dilution of secondary antibodies (Alexa fluor 488- and 647-conjugated antibodies) prepared in PBS (see Table of Materials). Incubate for 30 min at room temperature.
      6. Wash the stained cells with TBST at least twice by pipetting the cells up and down. Spin the plate and discard the supernatant as in step 9.2.8.2.
      7. Collect the stained cells in at least 100 µL of PBS and transfer them to light-protected FACS tubes (see Table of Materials). Run the samples on a flow-cytometry machine.

Results

The results show that optimized protocol P2-D (Figures 1A) enhanced pancreatic progenitor differentiation efficiency by upregulating PDX1 and NKX6.1 co-expression (Figure 2A,B, and Figure 3A). In particular, the results showed that dissociation of endodermal cells and their replating on fresh membrane matrix along with a longer duration of Stage 3 enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors (op...

Discussion

This work describes an enhanced protocol for generating pancreatic progenitors from hPSCs with a high co-expression of PDX1 and NKX6.1. Dissociation and replating of the hPSC-derived endoderm at half density on fresh matrix resulted in higher PDX1 and NKX6.1 in hPSC-derived pancreatic progenitors.

Although the growth factor cocktail for each stage is highly similar to P1-ND27, it has been shown that a more extended Stage 3 treatment including FGF and retinoid signaling ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).

Materials

NameCompanyCatalog NumberComments
15 mL, conical, centrifuge tubesThermo Scientific339651
20X TBS Tween 20Thermo Scientific28360
24-well culture plates, flat bottom with lidCostar3524
50 mL, conical, centrifuge tubesThermo Scientific339652
6- well culture plates, multidishThermo Scientific140685
AccutaseStem Cell Technologies0-7920
Activin AR&D338-ACReconstituted in 4 mM HCl
Anti NKX6.1 antibody, mouse monoclonalDSHBF55A12-CDiluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
Anti-PDX1 antibody, guinea pig polyclonalAbcamab47308Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining
B27 minus Vit AThermoFisher12587010
Bovine serum albumin, heat shock fraction, fatty acid freeSigmaA7030
CHIR 99021Tocris4423Reconstituted in DMSO
DMEM, high glucoseThermoFisher41965047
Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568InvitrogenA10037
Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488A-21206
DPBS 1XThermoFisher14190144
EGFThermoFisherPHG0313Reconstituted in 0.1% BSA in PBS
FGF10R&D345-FGReconstituted in PBS
GlucoseSigma AldrichG8644
Hoechst 33258Sigma23491-45-4
Inverted microscopeOlympusIX73
KnockOut DMEM/F-12 (1X)Gibco12660-012
KnockOut SR serum replacementGibco10828-028
L-Ascorbic acid (vitamin C)SigmaA92902Reconstituted in distilled water
Matrigel Growth Factor Reduced (GFR) Basement Membrane MatrixCorning354230Aliquot the thawed stock and freeze at -20C.
MCDB131ThermoFisher10372019
Mouse anti-SOX17ORIGENECF500096Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining
mTeSR PlusStem Cell Technologies85850Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use
Nalgene filter units, 0.2 µm PESThermoFisher566-0020
NicotinamideSigma72340Reconstituted in distilled water
NOGGINR&D6057-NGReconstituted in 0.1% BSA in PBS
Paraformaldehyde solution 4% in PBSChemCruzsc-281692
Penicillin-Streptomycin (10,000 U/mL)ThermoFisher15140122
Portable vacuum aspirator
Rabbit anti-FOXA2Cell signaling technology3143Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining
Retinoic AcidSigma AldrichR2625Reconstituted in DMSO
Rock inhibitor (Y-27632)ReproCell04-0012-02Reconstituted in DMSO
Round Bottom Polystyrene FACS Tubes with Caps, STERILEStellar ScientificFSC-9010
SANT-1Sigma AldrichS4572Reconstituted in DMSO
Sodium bicarbonateSigmaS5761-500G
StemFlexThermoFisherA3349401Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use
TALI Cellular Analysis SlideInvitrogenT10794
Tali image-based cytometer automated cell counterInvitrogenT10796
Triton X-100Sigma9002-93-1
TrypLE 100 mLThermoFisher12563011
Tween 20SigmaP2287
UltraPure 0.5 M EDTA, pH 8.0Invitrogen15575-038

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