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

Taro Toyoda; Azuma Kimura; Hiromi Tanaka; Kenji Osafune

doi:10.3791/57641

JoVE Journal > Developmental Biology

Biologia dello sviluppo

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

Published: March 27, 2019

doi:

10.3791/57641

Taro Toyoda¹, Azuma Kimura¹, Hiromi Tanaka¹, Kenji Osafune¹

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

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.

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.

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.
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).
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.

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).
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% CO₂incubator (humidified atmosphere) and culture for 24 h.
  NOTE: Do not shake the plate after seeding.
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.
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.
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.
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)

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.
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.
Pipette gently but quickly to dissociate clumped cells into single cells. Do not bubble the cell suspension during pipetting.
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.
Centrifuge the tube at 300 x g at RT for 5 min.
Aspirate the supernatant and resuspend the cells with 2 mL of 0.5 mM EDTA (RT). Gently pipette the cell suspension.
Centrifuge the tube at 300 x g at RT for 5 min.
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.
Centrifuge the tube at 400 x g at RT for 5 min.
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.
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.
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.
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.
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.
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.
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.
Analyze a proportion of positively stained cells by a flow cytometer²⁷.

4. Immunostaining

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.
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.
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.
Add 2 mL of blocking solution per well and keep the plate at RT for 30 min.
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.
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.
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.
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.
Take micrographs to examine the differentiation efficiency under a fluorescence microscope²⁸.

Representative 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 surface area. During Stage 1B, the media appear cloudy due to dead cells. The removal of dead cells is critical for highly efficient differentiation since dead cells likely disturb the survival and differentiation of cells lying underneath. On days 3-4, cells form a homogenous monolayer sheet that can be described as a cobblestone appearance. At this point, most cells stop expressing sex determining region Y-box 2 (SOX2), a marker for undifferentiated cells, and instead express a definitive endoderm marker, SOX17, at more than 90% (Figure 2A and B). Most SOX17⁺ cells express FOXA2 (Figure 2A). Starting the differentiation with an inappropriate cell density compromises the differentiation efficiency at this step (Figure 3). At Stages 2 and 3, cell death relaxes, and the used medium is not as cloudy as it is in Stage 1B. Cells express the primitive gut tube markers HNF1β and HNF4α (Figure 2C) and eventually express a posterior foregut/pancreatic progenitor marker, PDX1, at more than 90% (Figure 2D and E). The PDX1⁺ cell induction is reproducible in another hiPSC line, 1231A3 ³¹, and an hESC line, KhES-3 ³² (Figure 4). qRT-PCR results of the mRNA expression of stage markers were consistent with immunostaining (Figure 5A). The mRNA expression of PDX1 is evident at Stage 3 and substantially increases afterword.

PDX1⁺ cells at early developmental stages have the potential to differentiate into not only pancreatic cells but also gastric antrum, duodenum, extrahepatic bile duct and a part of the intestine ⁹. The differentiation potential of in vitro generated PDX1⁺ cells to pancreatic cells can be assessed by extended culture with reported protocols for pancreatic endoderm and pancreatic endocrine cells³^,¹⁶^,²⁶. The expressions of a pancreatic endoderm marker, NKX6.1, and two pancreatic endocrine markers, INSULIN, and GLUCAGON, were observed on days 19 (Stage 4) and 31 (Stage 5), respectively (Figure 5).

Figure 1: Representative appearance of cells under stepwise differentiation. (A) A scheme of directed differentiation from hPSCs to pancreatic lineages. Numbers in parentheses indicate concentrations (units are written below). (B) Representative bright field micrographs of hiPSCs at key steps of the differentiation culture. 585A1 hiPSCs were dissociated as single cells and induced to differentiate into definitive endoderm, primitive gut tube and posterior foregut/pancreatic progenitor. The lower panels are enlarged views of the upper panels. RPMI, RPMI 1640; AA, activin A (ng/mL); CH, CHIR99021 (μM); KGF (ng/mL); NOG, NOGGIN (ng/mL); CYC, 3-Keto-N-aminoethyl-N’-aminocaproyldihydrocinnamoyl cyclopamine (μM); TTNPB, 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (nM). Scale bars = 300 μm. Please click here to view a larger version of this figure.

Figure 2: Representative induction toward pancreatic lineages from hiPSCs. (A) The proportion of SOX2^–SOX17⁺ cells analyzed by flow cytometry before differentiation (Stage 0) and on day 4 (Stage 1B). Undifferentiated hiPSCs (SOX2⁺SOX17^–) were differentiated into definitive endoderm (SOX2^–SOX17⁺). Most SOX17⁺ cells co-expressed FOXA2. (B) Representative immunofluorescent micrographs on day 4 (Stage 1B). The fixed cells were stained for SOX17 (green), SOX2 (red), and nuclei (blue). (C) Representative immunofluorescent micrographs on days 4 (Stage 1B) and 8 (Stage 2). The fixed cells were stained for HNF1β (green), HNF4α (red) and nuclei (blue). (D) The proportion of cells positive for PDX1, as analyzed by flow cytometry on days 4 (Stage 1B) and 11 (Stage 3). (E) Representative immunofluorescent micrographs of PDX1 (green) and nuclei (blue) on day 11 (Stage 3). Scale bars = 100 μm. Please click here to view a larger version of this figure.

Figure 3: Representative induction toward definitive endoderm initiated from different cell densities. (A) Representative bright field micrographs of cells 1 h after differentiation. hiPSCs were dissociated as single cells and induced to differentiate into definitive endoderm at different cell densities (1-50 x 10⁴/cm²). The lower panels are enlarged views of the upper panels. (B) The proportion of SOX2^–SOX17⁺ cells analyzed by flow cytometry before differentiation (Stage 0) and on day 4 (Stage 1B). (C) The proportion of PDX1⁺ cells analyzed by flow cytometry on days 8 (Stage 2) and 11 (Stage 3). Scale bars = 300 μm. Please click here to view a larger version of this figure.

Figure 4: Representative induction toward PDX1⁺ cells in hiPCSs and hESCs. An hiPSC line, 1231A3 (A), and an hESC line, KhES-3 (B), were differentiated to definitive endoderm and PDX1⁺ cells. The cell composition was analyzed by flow cytometry. The proportion of SOX2^–SOX17⁺ cells was analyzed before differentiation (Stage 0) and on day 4 (Stage 1B). The proportion of PDX1⁺ cells was analyzed on days 8 (Stage 2) and 11 (Stage 3). Please click here to view a larger version of this figure.

Figure 5: Representative induction toward pancreatic endoderm and endocrine cells. hiPSCs (585A1) were differentiated into PDX1⁺ cells. The cells were further differentiated into pancreatic endoderm and pancreatic endocrine cells with reported protocols³^,¹⁶^,²⁶. (A) mRNA expressions of stage markers were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized to GAPDH expression and presented as the fold-change in gene expression relative to the peak value. Note that PDX1 expression was increased at >20-fold from days 8 (Stage 2) to 11 (Stage 3) and at >100-fold from days 11 (Stage 3) to 19 (Stage 4). The expression in the adult human pancreas is shown as Panc. SOX2, black; SOX17, purple; HNF1β, brown; HNF4α, orange; PDX1, light green; NKX6.1, green; INSULIN, blue; GLUCAGON, red. (B) Representative immunofluorescent micrographs of a pancreatic endoderm marker, NKX6.1 (red), on days 11 (Stage 3) and 19 (Stage 4). PDX1 (green) and nuclei (blue) were co-stained. (C) Representative immunofluorescent micrographs of two pancreatic endocrine makers, insulin (INS, green) and glucagon (GCG, red), on days 19 (Stage 4) and 31 (Stage 5). Nuclei (blue) were co-stained. Please click here to view a larger version of this figure.

Gene name	Gene symbol	Forward primer	Reverse primer
glyceraldehyde-3-phospha te dehydrogenase	GAPDH	GAAGGTGAAGGTCGGAGTC	GAAGATGGTGATGGGATTTC
SRY-box 2	SOX2	AGTCTCCAAGCGACGAAAAA	TTTCACGTTTGCAACTGTCC
SRY-box 17	SOX17	CGCACGGAATTTGAACAGTA	TTAGCTCCTCCAGGAAGTGTG
HNF1 homeobox B	HNF1β	CCTCTCCTCCAAACAAGCTG	TGTTGCCATGGTGACTGATT
hepatocyte nuclear factor 4 alpha	HNF4α	GAGCTGCAGATCGATGACAA	TACTGGCGGTCGTTGATGTA
pancreatic and duodenal homeobox 1	PDX1	AGCAGTGCAAGAGTCCCTGT	CACAGCCTCTACCTCGGAAC
NK6 homeobox 1	NKX6.1	ATTCGTTGGGGATGACAGAG	TGGGATCCAGAGGCTTATTG
glucagon	GCG	GAATTCATTGCTTGGCTGGT	CGGCCAAGTTCTTCAACAAT
insulin	INS	CTACCTAGTGTGCGGGGAAC	GCTGGTAGAGGGAGCAGATG

Table 1: Primers for qPCR.

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 efficient induction to PDX1⁺ cells is likely to be compromised.

Undifferentiated states of hPSCs are maintained as colonies or aggregates of compacted cells. However, methods that start the differentiation from colonies or aggregates may suffer from heterogeneity, because of different cell adhesion and density in the colony, such as in the center and periphery²², and because cells are at different stages in the cell cycle²⁵. On the other hand, our method starts with dissociated single cells, which enables a relatively homogenous state of cell adhesion of single cells or cell density in every single cell. In terms of homogenous handling for each cell, our methods might be easier than others that start with colony or aggregation cultures.

Although our protocol can be used to induce PDX1⁺ cells from multiple hPSC lines, the differentiation could still be inefficient. In such cases, differences in adhesion state right after seeding among the hPSC lines could be the cause, and modification of the seeding density could be a solution³³. Indeed, Figure 3 shows that inappropriate seeding cell density compromises differentiation into definitive endoderm and PDX1⁺ cells. Interestingly, the optimal cell density for PDX1⁺ cell induction was different among the cell populations that achieved >90% definitive endoderm. Another possibility for inefficient differentiation is the poor maintenance condition of the undifferentiated hPSCs, which compromises the quality of pluripotency despite the expression of markers for the undifferentiated state. In this case, the hPSC expansion culture should be recommenced from early passage frozen stocks or sub cloning should be performed to obtain hPSCs in suitable conditions. In the case of inefficient differentiation on days 8 (Stage 2) or 11 (Stage 3), the duration of these steps should be optimized. Supporting this idea, the duration of Stage 3 has been shown critical for acquiring later stage cell characteristics and is cell line-dependent in pancreatic lineage³⁴.

The generation of PDX1⁺ cells is crucial for the in vitro generation of pancreatic cells. PDX1 is functionally essential for pancreatic development based on knowledge from Pdx1 null mice, which are apancreatic³⁵. Consistently, in vitro and in vivo implantation studies showed hPSC-derived PDX1⁺ cells have the potential to develop into all pancreatic components, including exocrine and endocrine cells such as pancreatic β cells³^,¹⁶^,³⁶^,³⁷. Thus, the efficient generation of PDX1⁺ cells from hPSCs leads to a stable pancreatic cell supply for the establishment of β cell therapy against diabetes and the understanding of human pancreas development and pancreatic diseases.

The limitations of this method are related to the two-dimensional (2D) monolayer culture format, which is not suitable for some cell types and cell processing. In recent years, three-dimensional (3D) cultures have been shown to promote the generation of mature cells and tissues, possibly due to mimicking the in vivo microenvironment. For example, β cells generated in 3D cultures but not 2D monolayer cultures could attain the ability to secrete insulin in response to extracellular glucose levels³⁸.

To use PDX1⁺ cells for the generation of developmentally later cell types, it is important to shift to 3D cultures, such as suspension cultures of aggregates embedded in an extracellular matrix and aggregate cultures on an air-liquid interface³^,¹⁶^,³⁷. In addition, 2D monolayer cultures require more surface area for culturing than suspension cultures, limiting scalability. The processing of large amounts of cells for commercial use requires modifications such as the use of microbeads. At the same time, the present method is suitable for the screening of differentiation-inducing factors and the exploration of molecular mechanisms by gene transfer.

Divulgazioni

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.

Materials

3-Keto-N-aminoethyl-N′-aminocaproyldihydrocinnamoyl cyclopamine	Toronto Research Chemicals	K171000	CYC
4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid	Santa Cruz Biotechnology	SC-203303	TTNPB
50 mL Conical Sterile Polypropylene Centrifuge Tubes	Thermo Fisher Scientific	339652
Anti-CDX2 antibody [EPR2764Y]	Abcam	Ab76541	Anti-CDX2, × 1/1000 dilution
B-27 Supplement (50 ×)	Thermo Fisher Scientific	17504-044	Serum-free supplement
BD FACSAria II Cell Sorter	BD Biosciences		For flow cytometry
Biomedical freezer	SANYO	MDF-U538	For -30 °C storing
Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber	BIO-RAD	1450011	Counting slide glass
CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR	Greiner bio-one	657165	For differentiation culture/6-well plate
Centrifuge	TOMY	AX-310	For cell culturing
Centrifuge	TOMY	MX-305	For RT-qPCR
CHIR99021	Axon Medchem	Axon 1386
CLEAN BENCH	SHOWA KAGAKU	S-1601PRV	Clean bench
Corning CellBIND 6-well plate	Corning	3335	For feeder-free culture of hPSCs/6-well plate
Corning Matrigel Basement Membrane Matrix Growth Factor Reduced	Corning	354230	Basement membrane matrix
Corning Synthemax II-SC Substrate	Corning	3535	For feeder-free culture of hPSCs/synthetic surface material for hPSCs
Cryostat	Leica	Leica CM1510 S	For immunostaining of aggregates.
Cytofix/Cytoperm Kit	Becton Dickinson	554714	Perm/Wash buffer is Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.
Dako pen	Dako	S2002	For immunostaining of aggregates
dNTP mix (10 mM)	Thermo Fisher Scientific	18427-088	For RT-qPCR
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Fisher Scientific	A11055	Secondary antibody, × 1/500 dilution
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546	Thermo Fisher Scientific	A10036	Secondary antibody, × 1/500 dilution
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546	Thermo Fisher Scientific	A10040	Secondary antibody, × 1/500 dilution
Donkey Serum	Merck Millipore	S30	Donkey serum
D-PBS(-) without Ca or Mg	Nacalai tesque	14249-95	DPBS
Essential 8 Medium	Thermo Fisher Scientific	A1517001	For feeder-free culture of hPSCs/hPSC maintenance medium
Falcon 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Corning	352235	5 mL round bottom polystyrene tube with cell strainer
Filter Tip, 1000 µL	Watoson	124-1000S	Use together with pipettes
Filter Tip, 20 µL	Watoson	124-P20S	Use together with pipettes
Filter Tip, 200 µL	Watoson	124-P200S	Use together with pipettes
Fluorescence Microscope	Keyence	BZ-X700	For immunostaining
Forma Steri-Cycle CO₂ incubator	Thermo Fisher Scientific	370A	Incubator
HNF-1β Antibody (C-20)	Santa Cruz Biotechnology	sc-7411	Anti-HNF1β, × 1/200 dilution
HNF-4α Antibody (H-171)	Santa Cruz Biotechnology	sc-8987	Anti-HNF4α, × 1/200 dilution
Hoechst 33342	Thermo Fisher Scientific	H3570	For nucleus staining, × 1/200 dilution
Human Pancreas Total RNA	Ambion	AM7954	For RT-qPCR
Human PDX-1/IPF1 Antibody	R&D Systems	AF2419	Anti-PDX1, goat IgG, × 1/200 dilution
Human SOX17 Antibody	R&D Systems	AF1924	Anti-SOX17, × 1/200 dilution
Improved MEM Zinc Option medium	Thermo Fisher Scientific	10373-017	iMEM
Incubation chamber	Cosmo Bio	10DO	For immunostaining of aggregates
Latex Examination Gloves	Adachi
MAS coated slide glass	Matsunami Glass	83-1881	For immunostaining of aggregates
MicroAmp Fast 96-well Reaction Plate	Applied Biosystems/Thermo Fisher Scientific	4346907	For RT-qPCR
Microscope	Olympus	CKX41N-31PHP	For cell culturing
Microtube	Watoson	131-515CS
Monoclonal Anti-α-Fetoprotein	SIGMA	A8452	Anti-AFP, × 1/200 dilution
Nanodeop 8000	Thermo Fisher Scientific		For RT-qPCR
Oligo dT	FASMAC	Custom made Oligo	For RT-qPCR of sequence is "TTTTTTTTTTTTTTTTTTTT"
Paraformaldehyde, powder	Nacalai tesque	26126-54	PFA, fixative, diluted in DPBS
Pharmaceutical refrigerator	SANYO	MPR-514	For 4 °C storing
PIPETMAN P	GILSON		Pipette
Recombinant Human KGF/FGF-7	R&D Systems	251-KG	KGF
Recombinant Human Noggin	PeproTech	120-10C	NOGGIN
Recombinant Human/Mouse/Rat Activin A	R&D Systems	338-AC	Activin A
ReverTra Ace (100 U/μL)	TOYOBO	TRT-101	For RT-qPCR
Rnase-Free Dnase Set (50)	QIAGEN	79254	For RT-qPCR
Rneasy Mini Kit	QIAGEN	74104	For RT-qPCR
RPMI 1640 with L-Gln	Nacalai tesque	30264-85	RPMI 1640
Sealing Film for Real Time	Takara	NJ500	For RT-qPCR
Serological pipettes 10 mL	Costar/Corning	4488	For cell culturing
Serological pipettes 25 mL	Costar/Corning	4489	For cell culturing
Serological pipettes 5 mL	Costar/Corning	4487	For cell culturing
Sox2 (D6D9) XP Rabbit mAb	Cell signaling	3579S	Anti-SOX2, × 1/200 dilution
StepOnePlus	Applied Biosystems/Thermo Fisher Scientific		For RT-qPCR
Sucrose	Nacalai tesque	30406-25	For immunostaining of aggregates
TB Green Premix Ex Taq II	Takara	RR820B	For RT-qPCR
TC20 Automated Cell Counter	BIO-RAD	1450101J1	Automatic cell counter
Tissue-Tek OCT compound 4583	Sakura Finetechnical	4583	For immunostaining of aggregates
Tissue-Tek Cryomold Molds/Adapters	Sakura Finetechnical	4566	For immunostaining of aggregates
Triton X-100	Nacalai tesque	35501-15
Trypan Blue	BIO-RAD	1450021
Ultracold freezer	SANYO	MDF-U33V	For -80 °C storing
UltraPure 0.5M EDTA, pH 8.0	Thermo Fisher Scientific	15575-038	Dilute with DPBS to prepare 0.5 mM EDTA
Veriti Thermal Cycler	Applied Biosystems/Thermo Fisher Scientific		For RT-qPCR
Y-27632	Wako	251-00514

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Toyoda, T., Kimura, A., Tanaka, H., Osafune, K. Efficient Generation of Pancreas/Duodenum Homeobox Protein 1⁺ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures. J. Vis. Exp. (145), e57641, doi:10.3791/57641 (2019).