Developmental biology studies have revealed that pancreatic delta cells and beta cells share very similar developmental and signaling pathways. Both cell types originate from PDX1-, NGN3-, and NEUROD1-positive islet progenitors and subsequently express PAX4, NKX2.2, and PAX6 during the formation of endocrine progenitor cells (Chiang and Melton, 2003; Gu et al., 2002; Sosa-Pineda et al., 1997). Later, the delta cells specifically express the transcription factor HHEX and restrict the expression of NKX6.1 (Oster et al., 1998; Zhang et al., 2014). Based on these similarities, our strategy is to screen for small molecules that can specifically induce delta cell differentiation based upon the current beta cell differentiation method, thus to establish a method for the directed differentiation of pancreatic delta cells.

We performed a low-throughput screening of small molecular compounds/fibroblast growth factors (Ameri et al., 2010; Ye et al., 2005; Elghazi et al., 2002) in the established islet organoid differentiation system (Rezania et al., 2014) and determined the expression of various cell marker genes by qRT-PCR. We found that during primitive gut tube (S2) and posterior foregut (S3) induction, FGF7 with a concentration of 50 ng/mL could increase the expression of PDX1 and CHGA, the markers of pancreatic endoderm/endocrine precursors (Afelik et al., 2006). Notably during the following pancreatic endoderm (S4) and pancreatic endocrine precursor cell induction (S5), FGF2 with a concentration of 20 ng/mL was shown to significantly promote the expression of delta cell marker gene SST and the specific transcription factor HHEX, as well as their protein expression during endocrine cell induction (S6). Based upon this, we further explored the concentration and combination of FGF2 with FGF7 (FGF2/7), using qRT-PCR, Western blot, flow cytometric analysis, and immunocytochemistry (ICC) experiments. Although there was no significant difference between FGF7 and FGF2/7 combination during early stages, the combination of 20 ng/mL FGF2 with 50 ng/mL FGF7 during S4 and S5 was able to synergically increase the proportion of SST-positive delta cells, while decreasing alpha and beta cells at the end stage of differentiation, thus confirming the efficiency and robustness of our delta cell differentiation method (Chen et al., 2024).

The efficient differentiation protocol of human pancreatic delta cells can provide a stable source of delta cells, which will greatly facilitate the study of the physiological role of delta cells in the development of diabetes and provide systematic support for screening of delta cell targeting drugs. Moreover, the study of physiologically mature islet organoids containing delta cells will promote the development and refinement of cell therapeutic strategies and provide guidance for pre-clinical studies and clinical applications.

In contrast to established protocols for differentiating pluripotent stem cells into pancreatic alpha and beta cells, which report a fraction of delta cells as a by-product, our method significantly increases the proportion of pancreatic delta cells that are functional in vivo and in vitro. Here, our seven-step approach combining planar and suspension cultures provides stable and efficient production of delta cells with quality control to ensure differentiation efficiency and save differentiation costs.

In this SC-delta cell differentiation protocol, we used a source of hPSCs, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

• The H1 and H9 cell lines were obtained from WiCell Research Institute.

• mTeSR1 complete kit (Stemcell Technologies, cat. no. 85850)

• 500 mL MCDB 131 (Thermo Fisher, cat. no. 10372019)

• Activin A (StemCell Technologies, cat. no. 78001)

• Rabbit anti-FOXA2 (Abcam, cat. no. ab108422; RRID: AB_11157157)

• DAPI (Thermo Fisher Scientific, cat. no. 622492)

• Pipettors (Eppendorf, cat. no. 3120000216; 3120000232; 3120000259; 3120000267)

• Serological pipettes (NEST, cat. no. 326001, 327001; Corning, cat. no. 4489)

Here, we describe in detail a stepwise differentiation protocol for directed generating functional pancreatic delta cells from hPSCs as shown in Figure 1. The methodology consists of seven stages to activate or inhibit various signaling pathways using small molecules and growth factors (Figure 1A), dedicating to recreate the organogenesis of pancreatic islets in culture medium. The methods to assess the cells are also detailed including quality control at critical checkpoints as well as determination of cell composition at the endpoint.

hPSCs are seeded onto Matrigel-coated 6-well plates at a density of 0.2 × 10⁵ cells/cm² and cultured in mTeSR1 (Figure 2). When cells reach approximately 80% confluency after 6∼7 days, they are dispersed into a) single cells using TrypLE and seeded onto new Matrigel-coated 24-well plates at a density of 1.25 × 10⁵ cells/cm² for differentiation; or b) small cell colonies using gentle cell dissociation reagent and seeded onto new Matrigel-coated plates with mTeSR1 with 10 μM Y-27632 (Rho-kinase inhibitor) for passage.

The stem cells should be confluent 48 h after seeding onto 24-well plates, and mTeSR1 is replaced with stage 1 day 1 (S1D1) medium containing Activin A (TGF-β superfamily member) and CHIR99021 (Wnt agonist). 24 h later, the medium is replaced with S2D2 medium containing Activin A and a lower concentration of CHIR99021. The subsequent S1D3 medium used contains only Activin A. At the end of this stage, there should be more than 90% of the cells expressing the endoderm markers FOXA2 and SOX17 (Figure 3A). As a quality control checkpoint, high expression of these markers is critical to the success of the protocol, so it is important to optimize this step in each cell line prior to subsequent differentiation stages. All the markers for quality control can be checked by ICC visually or flow cytometry quantitatively.

During stage 2, the definitive endoderm cells are driven towards primitive gut tube with the keratinocyte growth factor (FGF7) treatment. At the end of S2D2, FOXA2 and SOX17 as reliable markers should be co-expressed in >85% of cells (Figure 3B) to distinguish the differentiation efficiency into SC-delta cells.

In the 2 days of stage 3, the cells are converted to posterior foregut with FGF7 accompanied by a high concentration of Retinoic Acid (RA), LDN193189 (BMP inhibitor), SANT1 (inhibitor of hedgehog signaling inhibitor), PdBU (protein kinase C activator) and Y-27632 by turning on the transcription factor PDX1. By the end of S3D2, >80% of the cells should be induced to express PDX1 (Figure 3C).

The pancreatic endoderm stage is induced in S4 medium for the next 3 days with same factors as S3 medium, exception of the innovative combination of FGF2 with FGF7 and drastically lower RA concentration. This stage aims to optimize PDX1+ cells. FGF2/7 synergically increase the HHEX expression whereas repress the beta-cell transcription factor NKX6.1 expression in the following stages. The end of this stage is an important quality-control checkpoint, where should generate >30% PDX1+/NKX6.1 + cells (Figure 3D) to ensure the ultimate expression of SST at the terminal of the SC-delta cell differentiation.

In stage 5, cells are induced to endocrine precursors with continuous FGF2/7 treatment to further increase SST and HHEX expression, XXI (γ-secretase inhibitor) to downregulate Notch signaling pathway, Betacellulin (member of epidermal growth factor), ALK5 inhibitor II, RA, SANT-1, LDN193189, Zinc Sulfate and heparin. Distinct with the methodology of beta-cell differentiation, thyroid hormone (T3) is depleted since stage 5, which has been reported to repress the expression of SST (Rezania et al., 2014).

To optimize the derivation of delta cells, at the beginning of this stage, the 2D-plated cells are dissociated into single cells by Accutase, and suspended with S6 medium in low-attachment 6-well plates at a density of 6 × 10⁶ cells/well for 3D cultivation (Figure 1B). The medium in this stage contains XXI, LDN193189, Zinc Sulfate, heparin, N-acetyl cysteine (NAC, promotes the proliferation), and R428 (AXL inhibitor) for 5 days of culture. 24 h after suspension, cells begin to aggregate to islet-like organoids.

The induced SC-delta cells in 3D culture need time to be mature, thus we develop the S7 medium with combination of Zinc Sulfate, heparin and α-Tocopherol (Vitamin E). After S7D6, >20% of SST+/CHGA+ and >25% SST+/PDX1 + cells are generated, which can be statistically analyzed by flow cytometry (Figures 3E, F) and ICC (Figures 3G, H).

1. Prepare 6-well plates for cell culture by coating with at least 700uL/well of diluted Matrigel for 2 h at 37°C. DMEM/F12 is used for dilution with the ratio described in the Matrigel product information. After coating, aspirate the DMEM/F12 and the plates are ready for use.

Caution: Do not start differentiation directly after thawing, but allowing at least one passage before seeding for differentiation, since cells need time to recover after thawing from the cryopreservation.

Critical Step: It is important to ascertain that the morphology and proliferation rate of the cells are consistent with those observed prior to cryopreservation. We recommend using qRT-PCR or immunostaining to check pluripotency markers such as OCT4 and NANOG.

Critical Step: It is recommended that an adequate number of cells be propagated to ensure the maintenance of a passage and differentiation. Therefore, it is advised that a propagation plan be developed for each experiment.

a) For cell passage and propagation:

6. Before passing the hPSCs, prepare a new Matrigel-coated 6-well plate as described in step 1 to continue the propagation.

b) For differentiation:

10. Prepare Matrigel coated 24-well plates before seeding.

Caution: Cell colonies rather single cells during passage can properly avoid karyotypic abnormalities, due to excessive passaging of stem cells. We also remind you to initially expand and cryofrozen hPSCs to build a cell bank which provides new cells after long passages.

14. 48 h after seeding for differentiation, cells should reach 100% confluence and form a monolayer (Figure 2). Add Activin A (100 ng/mL) and CHIR99021 (3 µM) to pre-warmed S1 basal medium to prepare S1D1 medium. Table 2 shows the composition of the basal medium for the different steps, and Table 3 summarizes the stored and diluted concentrations of the small molecules used in the different stages. Table 1 will facilitate the calculation of the amount of medium required for each feed throughout the differentiation process.

Caution: If the cells are not 100% confluence after 2 days, extending the incubation time or directly initiation of differentiation would affect the differentiation efficiency. It is essential to ascertain whether any issues have arisen during the plate seeding phase. Potential complications may include inaccurate cell counting or excessive manipulation, which could compromise cell viability.

Critical Step: To achieve optimum efficiency, differentiation factors must be added freshly before each day’s feeding throughout the entirety of the protocol. It is important to note that there is a two-cycle limit for freeze-thaw cycles. Furthermore, it is advisable to avoid maintaining the medium containing differentiation factors in a water bath for an extended period.

15. Rinse cells once with PBS and add S1D1 medium.

Caution: It is not uncommon for some cell death to occur during stage 1.

Caution: It is important to note that when replacing the medium, the cells should be washed to remove any residual CHIR from the previous medium. Otherwise, the differentiation efficiency may be adversely affected.

Critical Step: Effective endoderm induction at the end of stage 1 is important to the success of the protocol. It is therefore beneficial to check the proportion of FOXA2+ and SOX17+ cells at the end of this stage through ICC (Figure 3A) to ensure that >90% of cells express these markers.

18. Prepare S2 medium by adding FGF7 (50 ng/mL) to pre-warmed S2 basal medium.

Critical Step: The percentage of FOXA2+ and SOX17+ cells at the completion of this stage can be checked by ICC (Figure 3B). We suggest that typically >85% expression is advantageous for the induction of delta cells.

Caution: On the first day of a new stage, wash the cells with PBS prior to the addition of the new period medium. This procedure will ensure the complete removal of differentiation factors from the previous medium, thus facilitating the differentiation efficiency.

21. At the beginning of this stage, as some cells congregate while the majority remain in a monolayer configuration, they typically show a “valley-like” morphology (Figure 2). Prepare S3 medium by adding FGF7 (50 ng/mL), RA (1 μM), SANT-1 (0.25 μM), LDN193189 (100 nM), PdBU (500 nM) and Y27632 (10 μM) to the S3 basal medium to obtain the S3 medium and warm in a 37°C water bath before use.

Critical step: Pancreatic transcription factor PDX1 is turned on at stage 3, and should be quantified at the completion of this stage by ICC (Figure 3C) to ensure that >80% of the cells express PDX1.

24. At the beginning of this stage, although some cell clusters from the previous stage may persist, most cells should exhibit a confluent monolayer (Figure 2). Prepare S4 medium by adding FGF7 (50 ng/mL), FGF2 (20 ng/mL), RA (0.1 μM), LDN193189 (100 nM), SANT-1 (0.25 μM), PdBU (100 nM), Y27632 (10 μM) and ALK5i II (10 μM) to the S4 basal medium and warm in a 37°C water bath before use.

Caution: S4 medium is almost the same as S3 medium, except for the addition of FGF2 and a much lower concentration of RA.

Critical Step: The addition of a combination of FGF2 and FGF7 from S4 onwards will significantly promote the expression of HHEX and SST, which represents a key aspect of this protocol for the induction of delta cells.

25. Aspirate S3 medium, gently rinse cells once with PBS and add S4 medium.

Critical Step: It is recommended to perform quality control at this stage by checking the percentage of PDX1+ and NKX6-1+ cells by ICC (Figure 3D). At the end of this stage, cells expressing both PDX1 and NKX6-1 should be >30%.

27. At the beginning of this stage, the cells should still be in a monolayer, often with some clusters and sometimes small holes (Figure 2). Prepare S5 medium by adding FGF7 (50 ng/mL), FGF2 (20 ng/mL), RA (0.05 μM), LDN193189 (100 nM), SANT-1 (0.25 μM), Zinc Sulfate (10 μM), XXI (1 μM), ALK5i II (10 μM), Betacellulin (20 ng/mL) and heparin (10 μg/mL) to the S5 basal medium and warm in a 37°C water bath before use.

Critical Step: During stage 5, pancreatic progenitor cells convert into endocrine cells. The continuous addition of FGF2 and FGF7 to the S5 medium is crucial for effective induction of delta cells.

28. Aspirate the S4 medium, gently rinse the cells once with PBS and add S5 medium.

30. Prepare the S6 medium by adding LDN193189 (100 nM), Zinc Sulfate (10 μM), XXI (0.1 μM), R428 (2 μM), NAC (1 mM) and heparin (10 μg/mL) to the S6 basal medium and warm in a 37°C water bath.

Critical Step: After suspending, cells can be lost up to 50% during aggregation, depending on the differentiation efficiency. Using this method, most endocrine cells tend to aggregate into clusters, while any remaining progenitors and other cell types are susceptible to death during this process. Hence, this aggregation step is prone to purify the endocrine cell population, therefore increasing the proportion of delta cells obtained by induction.

37. Prepare S7 medium by adding Zinc Sulfate (10 μM), α-Tocopherol (10 μM), NAC (1 mM) and heparin (10 μg/mL) to the S7 basal medium and warm at 37°C water bath.

Critical Step: We have improved the S5-S7 medium by removing T3, which has been reported to decrease the expression of SST.

38. Aspirate S6 medium with the method described in step 35 and add S7 medium.

Caution: Remove S6 medium as much as possible and wash the cell aggregates once with S7 medium.

39. Culture the cells on the orbital shaker. Feed cells every other day with S7 medium until the end of differentiation when cells are to be assessed.

40. At the end of the desired stage, aspirate the medium in plates, wash with PBS, and add 0.5 mL TrypLE/well. For aggregated cell spheroids after stage 5, pipette them into a microcentrifuge tube using a P1000 pipettor, wash with PBS, and add 1 mL TrypLE. Incubate at 37°C for 5–10 min.

Caution: We recommend using 4% PFA in a chemical fume hood.

44. Centrifuge at 300 g at RT for 5 min.

Pause point: The fixed cells can be stored at 4°C for up to 2 months.

46. Centrifuge at 300 g for 5 min at RT.

Critical Step: Stem cells should be stained at the same time with samples, as it is often necessary to use stem cells as a negative biological control when running samples on a flow cytometer, as well as a secondary antibody only control (Figures 3E, F).

58. At desired stage for detecting, aspirate the medium in plates, wash with PBS, and add 0.5 mL TrypLE/well.

74. To immunostain S6 or S7 aggregated spheroids, first, remove the cell clusters into a microcentrifuge tube using a P1000 pipettor. Wait for a few minutes until cell clusters settle down, then remove the medium from the top. Add 1 mL PBS and gently invert the centrifuge tube to clean the cell clusters.

Pause Point: Sectioned slides can be stored at room temperature for at least 1 year.

81. Circle the location of cell clusters with an immunohistochemistry pen prior to staining.

Caution: Be careful to avoid air bubbles when place the coverslip, especially where the organoids locate.

85. Store samples in a slide box at 4°C before imaging with a confocal or widefield fluorescence microscope.

86. Aspirate the medium in the plates needed to be detected, wash with PBS, and add 0.5 mL TrypLE/well. For aggregated cell clusters, pipette them into a microcentrifuge tube using a P1000 pipettor, wash with PBS, and add 1 mL TrypLE. Incubate at 37°C for 5–10 min.

Pause Point: Lysed cells can be stored in RLT buffer at −80°C for several months.

89. According to the manufacturer’s instructions, proceed mRNA extraction, reverse transcription, as well as PCR reactions. Sequences of primers are listed in Table 5.

The protocol takes approximately 4-5 weeks to induce the differentiation of delta cells, plus 1-2 weeks for stem cell expansion and differentiation efficiency evaluation. The time needed for culturing cells each day will depend on plate configurations.

Steps 1–13: stem cell culture and seeding differentiations (stage 0), 7 days (0.5–2 h/d hands-on)