Method Article

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

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DOI:

10.3791/57641

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March 27th, 2019

 ,  ,  , 
1Center for iPS Cell Research and Application (CiRA), Kyoto University

In This Article

Summary

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Abstract

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1β and HNF4α, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Introduction

The pancreas mainly consists of exocrine and endocrine cells, and its dysfunction or overload causes several diseases, such as pancreatitis, diabetes and pancreatic cancer. To elucidate the pathogeny of pancreatopathy, it is necessary to analyze the developmental process and function of pancreatic cells. In addition, a stable cell supply with robust quality is required to establish cell/tissue supplementation therapy. Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for these purposes, and the differentiation protocol toward pancreatic cells has been intensively studied1,2,3,4. Recent advances in the in vitro generation of pancreatic β cells mimic the generation of β cells in adult human, and these cells show therapeutic efficacy upon implantation into diabetic model mice2,3. In addition, the analysis of β cells generated from the induced pluripotent stem cells (iPSCs) of healthy and type 1 diabetes patient donors revealed no functional differences including when under stress5. Moreover, disease phenotypes have been partially reproduced in induced pancreatic cells with patient-derived iPSCs or hPSCs harboring genetic mutations in the same site as the patients6,7. 

To generate pancreatic cells from hPSCs, stepwise directed differentiation that recapitulates developmental processes is used. The pancreas is derived from the endoderm layer of the early embryo, which expresses sex determining region Y-box 17 (SOX17) and forkhead box A2 (FOXA2)8. Based on the mouse studies, the endodermal layer forms the primitive gut tube, which is marked by the expression of hepatocyte nuclear factor 1-beta (Hnf1β) and hepatocyte nuclear factor 4-alpha (Hnf4α). The primitive gut tube elongates and develops into the respiratory apparatus, digestive tract, and organs. After elongation, the posterior foregut area becomes the presumptive pancreatic region, as marked by the expression of the transcriptional factor pancreas/duodenum homeobox protein 1 (PDX1)8,9,10. The dorsal and ventral parts of the PDX1+ gut tube thicken to form pancreatic buds, which are marked by the co-expression of pancreas transcription factor 1 subunit alpha (PTF1A) and NK6 homeobox 1 (NKX6.1)8,11. This expression marks the morphological start of pancreatic organogenesis. Pancreatic endoderm cells, which are components of the pancreatic buds, form a branched tubular network of epithelial structures12 and eventually differentiate into exocrine and endocrine cells, including insulin-secreting β-cells and glucagon-secreting α-cells. Expression of PDX1 is detected first at the presumptive pancreatic region, which is then observed throughout the entire pancreatic development, and shows localization to β- and δ-cells9,13,14. Although the Pdx1+ cell area that does not express Ptf1a or Nkx6.1 differentiates into the gastric antrum, duodenum, extrahepatic bile duct and some intestinal cells at the middle to late stage of development in mice9, PDX1+ cells are considered the progenitors of the pancreas at the early developmental stage in humans. 

Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells for the generation of pancreatic lineages. The protocol initiates differentiation by seeding dissociated single cells15,16,17. Generally, undifferentiated hPSCs are maintained as colonies or cell aggregates in suspension or in adhesion. As a result, most protocols initiate the differentiation shortly after passaging. However, in the state of colonies or aggregates, cells are prone to spatial and transcriptional heterogeneities18,19,20,21,22, which might hamper the first differentiation step toward definitive endoderm followed by inefficient differentiation to PDX1+ cells. The present protocol may offer easy handling to improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently to endodermal lineages and PDX1+ cells23,24,25.

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Protocol

Experiments using hPSCs were approved by the ethics committee of the Department of Medicine and Graduate School of Medicine, Kyoto University.

1. Preparation of Materials

NOTE: Prepare all media and reagents for cell culture in a sterile environment. Warm up base culture media to room temperature (RT) before use. Medium for differentiation is used within 6 h at RT. Details of the reagents are listed in Table of Materials.

  1. Differentiation (Figure 1A)
    1. Stage 1A medium: Transfer RPMI 1640 medium into a tube using a pipette. Add the serum-free supplement, activin A, CHIR99021, and Y-27632 to a concentration of 1x, 100 ng/mL, 3 μM and 10 μM, respectively.
    2. Stage 1B medium: Transfer RPMI 1640 medium into a tube using a pipette. Add the serum-free supplement, activin A and CHIR99021 to a concentration of 1x, 100 ng/mL, ≤1 μM, respectively.
      NOTE: The concentration of CHIR99021 should be lower than in Stage 1A medium, and addition is not necessary, but it increases the cell number.
    3. Stage 2 medium: Transfer Improved MEM (iMEM) medium into a tube using a pipette. Add the serum-free supplement and keratinocyte growth factor (KGF) to a concentration of 0.5x and 50 ng/mL, respectively.
    4. Stage 3 medium: Transfer iMEM medium into a tube using a pipette. Add the serum-free supplement, KGF, NOGGIN, 3-Keto-N-aminoethyl-N’-aminocaproyldihydrocinnamoyl cyclopamine (CYC) and 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB) by pipette to concentrations of 0.5x, 50 ng/mL, 100 ng/mL, 0.5 μM and 10 nM, respectively.
  2. Flow cytometry (FCM)
    1. 1x Permeabilization/Wash buffer: Transfer water into a tube by a pipette. Add Permeabilization/Wash buffer by a pipette to a concentration of 1x.
    2. FCM blocking solution: Transfer 1x Permeabilization/Wash buffer into a tube by a pipette. Add donkey serum by a pipette to a concentration of 2% (vol/vol).
  3. Immunostaining
    1. Blocking solution: Transfer Dulbecco's Phosphate-Buffered Saline (DPBS) into a tube by a pipette. Add donkey serum and Triton X by pipette to concentrations of 5% (vol/vol) and 0.4% (vol/vol), respectively.

2. hPSC Differentiation to Posterior Foregut Cells/Pancreatic Progenitors (PDX1+ Cells)

NOTE: Conduct all procedures using sterile techniques. hPSCs are maintained on 6-well plates coated by a synthetic surface material for hPSCs with hPSC maintenance medium according to the manufacturer's instructions17,26. When cells reach 50-80% confluency (Stage 0) use them for differentiation.

  1. Prepare basement membrane matrix-coated plates.
    1. Transfer 6 mL of RPMI 1640 (4 °C) into a tube cooled to 4 °C on ice. Add 2 mg of basement membrane matrix (4 °C) with cooled 1 mL-pipette tips and mix well by gentle pipetting to make 0.33 mg/mL basement membrane matrix.
    2. Transfer 2 mL of diluted basement membrane matrix to each well of 6-well cell culture plates by a pipette. Place the plates at 37 °C for 60-90 min in an incubator. Afterward, keep the plates at RT until use (within 3 h).
  2. Seed the hPSCs and initiate differentiation to definitive endoderm (Stage 1A).
    1. Aspirate the used medium and add 2 mL of 0.5 mM ethylenediaminetetraacetic acid (EDTA) (RT) per well by pipette to wash the hPSCs cultured in the 6-well plates.
    2. Aspirate 0.5 mM EDTA and add 2 mL of 0.5 mM EDTA per well by pipette. Incubate the plates at 37 °C for 5 min.
    3. Aspirate 0.5 mM EDTA and add 1 mL of hPSC maintenance medium at RT supplemented with 10 μM Y-27632 per well by pipette. Pipette gently but quickly to blow off attached cells on the plates and to dissociate clumped cells into single cells. Do not bubble the cell suspension during pipetting.
    4. Transfer the cell suspension in a 50 mL centrifuge tube containing 4 mL of hPSC maintenance medium at RT supplemented with 10 μM Y-27632. Gently pipette the cell suspension. 
    5. Count the cell number in the cell suspension using the Trypan Blue stain exclusion procedure.
      1. Mix 15 μL of cell suspension and 15 μL of Trypan Blue in a tube.
      2. Transfer 10 μL of the cell suspension diluted with Trypan Blue onto cell counting slides in duplicate by a 10 μL pipette.
      3. Count the cell number with an automatic cell counter and calculate the cell density in the cell suspension. Viability is usually >98%.
    6. Aliquot the cell suspension in a 50 mL centrifuge tube at 1-1.5 x 106 cells per well of 6-well plates (1-1.5 x 105 cells/cm2).
    7. Centrifuge the 50 mL tube at 200 x g at RT for 5 min.
    8. Aspirate the supernatant using a pipette and resuspend the cells with 1 mL of Stage 1A medium (RT) per well. Gently pipette the cell suspension and add another 1 mL of Stage 1A medium (total, 2 mL per well).
    9. Aspirate the diluted basement membrane matrix in the wells of the cell culture plates prepared in step 2.1.2 by a 1,000 μL pipette. Proceed to the next step immediately.
    10. Gently pipette the cell suspension in step 2.2.8 again. Immediately after mixing, transfer the cell suspension (2 mL) into each well of a 6-well plate. Cover the plate with an aluminum foil to protect the plate from light. Place the plate in the clean bench at RT for 10-15 min.
      NOTE: Do not move the plate after seeding.
    11. Gently place the plate into a 37 °C, 5% CO2 incubator (humidified atmosphere) and culture for 24 h.
      NOTE: Do not shake the plate after seeding.
  3. Induce the differentiation into definitive endoderm (Stage 1B).
    1. Aspirate the used medium and add 2 mL of DPBS to the wells by pipette. Repeat the aspiration and addition of DPBS one time.
      NOTE: To remove any dead cells from the monolayer, gently shake the plate before each aspiration.
    2. Aspirate the used DPBS by a pipette and add 4 mL of Stage 1B medium (37 °C) per well. Gently place the plate into the 37 °C incubator (humidified atmosphere of 5% CO2) and culture for 48 h.
    3. Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time.
      NOTE: Remove the dead cells from the monolayer by gentle shaking before each aspiration.
    4. Aspirate the used DPBS by pipette and add 4 mL of Stage 1B medium (37 °C) per well. Gently place the plate into the incubator (37 °C, humidified atmosphere of 5% CO2) and culture for 24 h.
  4. Induce the differentiation into the primitive gut tube (Stage 2).
    1. Aspirate the used medium and add 2 mL of DPBS to the well by pipette.
      NOTE: Remove the dead cells from the monolayer by gentle shaking before each aspiration.
    2. Aspirate the used DPBS by pipette and add 4 mL of Stage 2 medium (37 °C) per well. Place the plate into the incubator (37 °C, humidified atmosphere of 5% CO2) and culture for 4 days.
  5. Induce the differentiation into PDX1+ cells (Stage 3).
    1. Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time.
    2. Aspirate the used DPBS by pipette and add 4 mL of Stage 3 medium (37 °C) per well. Place the plate into the incubator (37 °C, humidified atmosphere of 5% CO2) and culture for 3 days.
  6. Induce the differentiation into pancreatic endocrine cells.
    1. Perform NKX6.1+ cell induction (Stage 4, for 8 days) followed by endocrine cell induction (Stage 5, for 12 days) with previously described protocols3,16,26. Characterize and validate the endocrine cell differentiation by immunostaining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis.
      NOTE: Refer to Toyoda et al.16 and Kimura et al.26 for detailed experimental procedures. Refer Table 1 for primer sequences used for qRT-PCR analysis.

3. Flow Cytometry (FCM)

  1. Aspirate the used medium and add 2 mL of 0.5 mM EDTA to the well by pipette. Repeat the aspiration and addition of 0.5 mM EDTA one time.
    NOTE: Shake the plate gently to remove any dead cells from the monolayer cells before each aspiration.
  2. To dissociate the cells (approximately 3-6 × 106 cells per well), add 2 mL of 0.25% Trypsin containing 0.5 mM EDTA per well. Incubate the plates at 37 °C for 5-10 min.
  3. Pipette gently but quickly to dissociate clumped cells into single cells. Do not bubble the cell suspension during pipetting.
  4. Add 8-10 mL of base medium (RPMI 1640 or iMEM, RT) containing 0.5x serum-free supplement and 10 μM Y-27632 per well and mix the cell suspension. Transfer the cell suspension into a centrifuge tube.
  5. Centrifuge the tube at 300 x g at RT for 5 min.
  6. Aspirate the supernatant and resuspend the cells with 2 mL of 0.5 mM EDTA (RT). Gently pipette the cell suspension.
  7. Centrifuge the tube at 300 x g at RT for 5 min.
  8. Aspirate the supernatant and resuspend the cells with 100 μL of fixation and permeabilization buffer per 106 cells under a hood. Gently pipette the cell suspension under a hood. Keep the tube at RT for 30 min.
  9. Centrifuge the tube at 400 x g at RT for 5 min.
  10. Aspirate the supernatant under a hood and resuspend the cells with 500 μL FCM Blocking solution per 106 cells. Gently pipette the cell suspension. Keep the tube at RT for 30 min.
  11. Transfer 50 μL of the cell suspension to a tube (approximately 1 x 105 cells). Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 100 μL of diluted primary antibody (see Table of Materials) in FCM blocking solution and incubate at 4 °C for >16 h.
  12. Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 μL of 1x Perm/Wash buffer.
  13. Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 100 μL of diluted secondary antibody (see Table of Materials) in FCM blocking solution. Incubate the cells at RT for 60 min or at 4 °C for >16 h with protection from light.
  14. Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 μL of 1x Perm/Wash buffer.
  15. Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 μL of 2% donkey serum in DPBS.
  16. Filter the cell suspension by transferring on a 5 mL round bottom polystyrene tube with a cell strainer (35 µm nylon mesh) and keep at 4 °C with protection from light until the analysis.
  17. Analyze a proportion of positively stained cells by a flow cytometer27.

4. Immunostaining

  1. Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time.
    NOTE: Gently shake the plate to remove any dead cells from the monolayer cells before each aspiration.
  2. Add 2 mL of 4% paraformaldehyde (PFA) (4 °C) per well by pipette under a hood. Keep the plate at 4 °C for 20 min.
  3. Remove the PFA by pipette under a hood. Add 2 mL of DPBS at RT to the well by pipette under a hood. Repeat the aspiration and addition of DPBS one time.
  4. Add 2 mL of blocking solution per well and keep the plate at RT for 30 min.
  5. Aspirate the used solution and add 1 mL of primary antibody (see Table of Materials) in blocking solution per well. Incubate at RT for 60 min or at 4 °C for >16 h.
  6. Aspirate the used solution, add 2 mL of DPBS containing 0.4% Triton X-100 and incubate at RT for 10 min. Repeat the aspiration, addition of solution and incubation one time.
  7. Aspirate the used solution and add 1 mL of secondary antibody (see Table of Materials) in blocking solution per well. Incubate at RT for 60 min with protection from light.
  8. Aspirate the used solution, add 2 mL of DPBS containing 0.4% Triton X-100 (RT) and incubate at RT for 5 min with protection from light. Repeat the aspiration, addition of solution and incubation two times.
  9. Take micrographs to examine the differentiation efficiency under a fluorescence microscope28.

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Results

Propagating hiPSCs (585A129,30) are condensed and form a homogenous monolayer (Figure 1B) that is suitable for differentiation. Undifferentiated hiPSCs (Stage 0) are dissociated and re-seeded as single cells at low cell densities (1-1.5 x 105 cells/cm2). Within 1 h, the cells are attached to the plate and start to show protrusion. On day 1, the cells are proliferated and well distributed to cover 80-90% of the s...

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Discussion

The generation of PDX1+ cells is comprised of multiple steps; therefore, it is critical to treat cells at the appropriate time. Among the steps, the induction efficiency of definitive endoderm largely affects the final induction efficiency, possibly by interference from other contaminating lineage cells (i.e., mesoderm and ectoderm), which may proliferate and/or secrete factors that disrupt specific differentiation. If the proportion of SOX17+ cells is lower than 80% on day 4 (Stage 1B), an efficien...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported in part by funding from the Japan Society for the Promotion of Science (JSPS) through Scientific Research (C) (JSPS KAKENHI Grant Number15K09385 and 18K08510) to T.T., and Grant-in-Aid for JSPS Research Fellows (JSPS KAKENHI Grant Number 17J07622) to A.K., and the Japan Agency for Medical Research and Development (AMED) through its research grant “Core Center for iPS Cell Research, Research Center Network for Realization of Regenerative Medicine” to K.O. The authors thank Dr. Peter Karagiannis for reading the manuscript.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
3-Keto-N-aminoethyl-N′-aminocaproyldihydrocinnamoyl cyclopamineToronto Research ChemicalsK171000CYC
4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acidSanta Cruz BiotechnologySC-203303TTNPB
50 mL Conical Sterile Polypropylene Centrifuge TubesThermo Fisher Scientific339652
Anti-CDX2 antibody [EPR2764Y]AbcamAb76541Anti-CDX2, × 1/1000 dilution
B-27 Supplement (50 ×)Thermo Fisher Scientific17504-044Serum-free supplement
BD FACSAria II Cell SorterBD BiosciencesFor flow cytometry
Biomedical freezerSANYOMDF-U538For -30 °C storing
Cell Counting Slides for TC10/TC20 Cell Counter, Dual-ChamberBIO-RAD1450011Counting slide glass
CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEARGreiner bio-one657165For differentiation culture/6-well plate
CentrifugeTOMYAX-310For cell culturing
CentrifugeTOMYMX-305For RT-qPCR
CHIR99021Axon MedchemAxon 1386
CLEAN BENCHSHOWA KAGAKU S-1601PRVClean bench
Corning CellBIND 6-well plateCorning3335For feeder-free culture of hPSCs/6-well plate
Corning Matrigel Basement Membrane Matrix Growth Factor ReducedCorning354230Basement membrane matrix
Corning Synthemax II-SC SubstrateCorning3535For feeder-free culture of hPSCs/synthetic surface material for hPSCs
CryostatLeicaLeica CM1510 SFor immunostaining of aggregates.
Cytofix/Cytoperm KitBecton Dickinson554714Perm/Wash buffer is  Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.
Dako penDakoS2002For immunostaining of aggregates
dNTP mix (10 mM)Thermo Fisher Scientific18427-088For RT-qPCR
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher ScientificA11055Secondary antibody, × 1/500 dilution
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546Thermo Fisher ScientificA10036Secondary antibody, × 1/500 dilution
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546Thermo Fisher ScientificA10040Secondary antibody, × 1/500 dilution
Donkey SerumMerck MilliporeS30Donkey serum
D-PBS(-) without Ca or MgNacalai tesque14249-95DPBS
Essential 8 MediumThermo Fisher ScientificA1517001For feeder-free culture of hPSCs/hPSC maintenance medium
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap CapCorning3522355 mL round bottom polystyrene tube with cell strainer
Filter Tip, 1,000 µLWatoson124-1000SUse together with pipettes
Filter Tip, 20 µLWatoson124-P20SUse together with pipettes
Filter Tip, 200 µLWatoson124-P200SUse together with pipettes
Fluorescence MicroscopeKeyenceBZ-X700For immunostaining
Forma Steri-Cycle CO2 incubatorThermo Fisher Scientific370AIncubator
HNF-1β Antibody (C-20)Santa Cruz Biotechnologysc-7411Anti-HNF1β, × 1/200 dilution
HNF-4α Antibody (H-171)Santa Cruz Biotechnologysc-8987Anti-HNF4α, × 1/200 dilution
Hoechst 33342Thermo Fisher ScientificH3570For nucleus staining, × 1/200 dilution
Human Pancreas Total RNAAmbionAM7954For RT-qPCR
Human PDX-1/IPF1 AntibodyR&D SystemsAF2419Anti-PDX1, goat IgG, × 1/200 dilution
Human SOX17 AntibodyR&D SystemsAF1924Anti-SOX17, × 1/200 dilution
Improved MEM Zinc Option mediumThermo Fisher Scientific10373-017iMEM
Incubation chamberCosmo Bio10DOFor immunostaining of aggregates
Latex Examination GlovesAdachi
MAS coated slide glassMatsunami Glass83-1881For immunostaining of aggregates
MicroAmp Fast 96-well Reaction PlateApplied Biosystems/Thermo Fisher Scientific4346907For RT-qPCR
MicroscopeOlympusCKX41N-31PHPFor cell culturing
MicrotubeWatoson131-515CS
Monoclonal Anti-α-FetoproteinSIGMAA8452Anti-AFP, × 1/200 dilution
Nanodrop 8000Thermo Fisher ScientificFor RT-qPCR
Oligo dTFASMACCustom made Oligo For RT-qPCR of sequence is "TTTTTTTTTTTTTTTTTTTT"
Paraformaldehyde, powderNacalai tesque26126-54PFA, fixative, diluted in DPBS
Pharmaceutical refrigeratorSANYOMPR-514For 4 °C storing
PIPETMAN P GILSONPipette
Recombinant Human KGF/FGF-7R&D Systems251-KGKGF
Recombinant Human NogginPeproTech120-10CNOGGIN
Recombinant Human/Mouse/Rat Activin AR&D Systems338-ACActivin A
ReverTra Ace (100 U/μL)TOYOBOTRT-101For RT-qPCR
RNase-free DNase Set (50)QIAGEN79254For RT-qPCR
RNeasy Mini KitQIAGEN74104For RT-qPCR
RPMI 1640 with L-GlnNacalai tesque30264-85RPMI 1640
Sealing Film for Real TimeTakaraNJ500For RT-qPCR
Serological pipettes 10 mLCostar/Corning4488For cell culturing
Serological pipettes 25 mLCostar/Corning4489For cell culturing
Serological pipettes 5 mLCostar/Corning4487For cell culturing
Sox2 (D6D9) XP Rabbit mAbCell signaling3579SAnti-SOX2, × 1/200 dilution
StepOnePlusApplied Biosystems/Thermo Fisher ScientificFor RT-qPCR
SucroseNacalai tesque30406-25For immunostaining of aggregates
TB Green
Premix Ex Taq II 		TakaraRR820BFor RT-qPCR
TC20 Automated Cell CounterBIO-RAD1450101J1Automatic cell counter
Tissue-Tek OCT compound 4583 Sakura Finetechnical4583For immunostaining of aggregates
Tissue-Tek Cryomold Molds/AdaptersSakura Finetechnical4566For immunostaining of aggregates
Triton X-100Nacalai tesque35501-15
Trypan BlueBIO-RAD1450021
Ultracold freezerSANYOMDF-U33VFor -80 °C storing
UltraPure 0.5M EDTA, pH 8.0Thermo Fisher Scientific15575-038Dilute with DPBS to prepare 0.5 mM EDTA
Veriti Thermal CyclerApplied Biosystems/Thermo Fisher ScientificFor RT-qPCR
Y-27632Wako251-00514

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PDX1 Positive CellshPSC DifferentiationDefinitive EndodermStromal Vascular FractionFlow CytometryWestern BlotConfocal MicroscopyCell IsolationTissue DigestionProgenitor Cells

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