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.
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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
- Prepare human pluripotent stem cells (hPSC) culture media
- 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.
- Prepare Stage 1 differentiation media (for Definitive Endoderm (DE)).
- 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.
- 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. - 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.
- Prepare Stage 2 differentiation media (for Primitive Gut Tube; PGT).
- 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. - 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.
- 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.
- Prepare Stage 3 differentiation media (for Posterior Foregut).
- 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.
- 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).
- Prepare Stage 4 differentiation media (for Pancreatic Progenitors).
- 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.
- 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.
2. Preparation of basement membrane matrix coated dishes
- Thaw commercially available basement matrix (see Table of Materials) and aliquot on ice; freeze the aliquots at -20 °C.
- 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.
- 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
- Passage the hPSCs when the colonies reach a 70%-80% confluency.
- 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.
- 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)
- 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.
- 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.
- The next day, replace the spent media with Stage 1 differentiation medium without CHIR99021 (step 1.2.3).
- 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)
- 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.
- 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.
- 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.
- 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.
- 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.
- 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. - 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.
- 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.
- 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.
- 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.
- 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.
- Aspirate the Hoechst solution using a portable vacuum filter and rinse the wells with PBS twice.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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. - 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.
- 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)
- Aspirate the spent Stage 2 media using a portable vacuum aspirator inside the tissue culture hood and wash the cells with warm PBS.
- Add Stage 3 differentiation media from step 1.4 to the cells and incubate at 37 °C, 5% CO2.
- 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)
- 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.
- 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
- 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.- Perform fixation, permeabilization, blocking, and antibody incubation and washes according to step 5.
- 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.
- Use 1:500 dilutions of appropriate Alexa fluor 488- and 568- conjugated secondary antibodies (see Table of Materials).
- 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.- 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.
- Dissociate the adherent sheets of cells within the well using a P1000 micropipette before collecting them in a 15 mL polypropylene tube.
- 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.
- 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.
- 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.
- 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.
- 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.
- Stain for the pancreatic progenitor markers following the steps below.
- 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.
- 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. - 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.
- 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.
- 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.
- 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.
- 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.
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Representative 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 (optimized protocol, P2-D) (Figure 2 and Figure 3A), in comparison to the non-dissociated protocol (P1-ND) that was modified from a previously published study27. The present enhanced protocol also generated the highest proportion of PDX1+/NKX6.1+ progenitors compared to "P1-D" (Protocol 1, S3 = 2 days, dissociated) and "P2-ND" (Protocol 2, S3 = 4 days, non-dissociated), and the detailed results have been previously published21. Pancreatic progenitors generated using P2-D also have increased numbers of SOX9+ cells compared to the non-dissociated P1-ND (Figure 3B). The optimized method also generated a higher proportion of proliferative NKX6.1+ cells that co-express the proliferation marker Ki67 (Figure 3C).
The representative results presented here are from iPSCs generated from peripheral blood mononuclear cells (PBMCs) of control, healthy individual. However, we have reproducibly applied the current enhanced protocol on multiple hPSC lines such as H1-hESCs, H9-hESCs, HUES8-hESCs, and several other control iPSC lines to yield ~90% PDX1 and NKX6.1 co-expressing pancreatic progenitors20,21,28.
Following DE induction in hPSCs, mild levels of cell death are expected. However, if a high cell death rate was observed, the experiment was stopped and started again with a fresh batch of cells. The efficiency of DE induction should be higher than 70%-80% to ensure a high proportion of DE cells in the culture that will serve as an ideal starting source for pancreatic progenitor induction (Figure 1C).
The density of replating endodermal cells following dissociation can be manipulated based on the growth rate of that particular cell. For example, slow-growing cells may be replated at a higher density than recommended. This will minimize the chances of obtaining irrelevant pancreatic populations at Stage 4. A high co-expression of PDX1 and NKX6.1 can be obtained following dissociation after Stage 1 and replating at half density (Figure 1A, Figure 2A,B, and Figure 3A). If a high expression of PDX1 is observed but only a moderate expression of NKX6.1, Stage 4 can be extended by 2 days to enhance NKX6.1 expression in PDX1-expressing cells.
Figure 1: Timeline for the addition of cytokines and growth factors during pancreatic progenitor differentiation. (A) Schematic representation of the optimized protocol (P1-D) for generation of PDX1 and NKX6.1 from hPSCs. hPSC-derived definitive endoderm (DE) is dissociated and replated at half density and then sequentially directed towards a pancreatic progenitor fate. (B) Images of hPSCs in brightfield before starting differentiation at Day 0. (C) Immunostaining analysis for expression of endodermal markers SOX17 and FOXA2 in hPSC-derived endoderm (Stage 1) cells. SOX17, green; FOXA2, red. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 2: Generation of hPSC-derived pancreatic progenitors, co-expressing PDX1 and NKX6.1. Immunostaining analysis for comparison of PDX1 and NKX6.1 expression in hPSC-derived pancreatic progenitors using the enhanced protocol (P2-D) (A) and a non-optimized, previously published protocol (P1-ND) (B). The enhanced protocol achieved the highest co-expression of PDX1 and NKX6.1. PDX1, green; NKX6.1, red. Magnified images are provided in the second panel for each protocol. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Quantification of pancreatic markers in pancreatic progenitors generated using the enhanced protocol. Flow-cytometry analysis in hPSC-derived pancreatic progenitors yielded using P2-D in comparison to the non-dissociated P1-ND. (A) Histograms for PDX1 and NKX6.1 expression and double-positive staining graphs. (B) The histograms for SOX9 expression and (C) co-expression of NKX6.1 with the proliferation marker Ki67. Please click here to view a larger version of this figure.
| Stage | Media | Cytokines | Days | ||||||
| 1 | MCDB 131 media + 0.5% fatty acid-free bovine serum albumin (FFA-BSA), 1.5 g/L sodium bicarbonate (NaHCO3), 10 mM of glucose | Day 1: 2 μM CHIR99021, 100 ng/mL Activin A, 10 μM Rock inhibitor (Y-27632), 0.25 mM Vitamin C. Day 2 onwards: 100 ng/mL Activin A, 0.25 mM Vitamin C | 3 or 4 | ||||||
| 2 | MCDB 131 media + 0.5% fatty acid-free bovine serum albumin (FFA-BSA), 1.5 g/L sodium bicarbonate (NaHCO3), 10 mM glucose | Day 1 (Dissociation): 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 μM CHIR99021, 10 μM Y-27632 (Rock inhibitor) , 0.25 mM Vitamin C. Day 2: 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 μM CHIR99021, 0.25 mM Vitamin C. | 2 | ||||||
| 3 | DMEM + 4.5 g/L glucose | 2 μM Retinoic acid, 0.25 μM SANT-1, 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 mM Vitamin C, 1% B27 supplement without vitamin A | 4 | ||||||
| 4 | DMEM + 4.5 g/L glucose | 100 ng/mL EGF, 10 mM Nicotinamide, 50 ng/mL NOGGIN, 0.25 mM Vitamin C, 1% B27 supplement without vitamin A. | 4 | ||||||
Table 1: The compositions of the different differentiation media used in the study.
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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 and BMP and hedgehog inhibition increases NKX6.1 expression, which is in contrast to Nostro et al.'s findings27. While PDX1 expression in the pancreas during embryonic development is positively regulated by FGF and retinoid signaling and BMP and hedgehog inhibition27,29,30, the present results demonstrated the extension of this signaling cocktail also upregulates NKX6.1. Nevertheless, the effect of an extended Stage 3 treatment was aided by dissociation and replating of endodermal cells that led to increased PDX1 and NKX6.1 expression. Furthermore, this method (P2-D) also increased SOX9-expressing cells in hPSC-derived pancreatic progenitors, in addition to the proportion of proliferative NKX6.1+ cells that co-express the proliferation marker Ki67.
Another crucial factor affecting the efficiency of differentiation was the availability of fresh membrane matrix to the dissociated cells. The extracellular matrix components have been previously demonstrated to regulate stem cell fate specification31,32. Overall, the favorable effects of extracellular matrix components on pancreatic development have been recorded33,34,35, particularly for the membrane matrix, which, along with laminin, was shown to have a pro-endocrine effect on pancreatic lineage cells36. Therefore, replating endodermal cells on fresh membrane matrix may have enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors.
The cell density of differentiating cells also controls gene expression. Multiple studies have demonstrated the significance of cell-cell contact in regulating pancreatic development37,38,39. Cellular aggregation or embryoid body formation has been shown to induce higher pancreatic endocrine gene expression than 2D cultures19. However, the results demonstrate that higher pancreatic expression, especially NKX6.1, can be obtained by culturing cells in a 2D monolayer at half the endodermal density using our optimized protocol21. Interestingly, this method also inhibited hepatic cell fate (the alternative fate of hPSC-derived DE) as noticed by the decreased expression of hepatic genes AFP (Alpha-fetoprotein) and ALB (Albumin)21, indicating that physical factors play a role in lineage specification of hPSCs.
Overall, the results demonstrate that modulating the physical environment of the differentiating cells can enhance pancreatic gene expression21. Specifically, the dissociation of hPSC-derived endoderm and replating on fresh membrane matrix with a longer FGF and retinoid signaling and hedgehog and BMP inhibition can enhance the PDX1 and NKX6.1 co-expression in hPSC-derived pancreatic progenitors. However, the optimized protocol is at least 2 days longer than previously published protocols. Also, the length of Stage 4 may be extended beyond the minimum recommended, i.e., 4 days, based on the cell line being used. Multiple recombinant human growth factors are employed in the optimized protocol that can be substituted by their less expensive, small molecule compounds that perform the same action. Nevertheless, this optimized protocol can facilitate the scalable generation of pancreatic progenitors from hPSCs for cell therapy and disease modeling.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).
Materials
| Name | Company | Catalog Number | Comments |
| 15 mL, conical, centrifuge tubes | Thermo Scientific | 339651 | |
| 20X TBS Tween 20 | Thermo Scientific | 28360 | |
| 24-well culture plates, flat bottom with lid | Costar | 3524 | |
| 50 mL, conical, centrifuge tubes | Thermo Scientific | 339652 | |
| 6- well culture plates, multidish | Thermo Scientific | 140685 | |
| Accutase | Stem Cell Technologies | 0-7920 | |
| Activin A | R&D | 338-AC | Reconstituted in 4 mM HCl |
| Anti NKX6.1 antibody, mouse monoclonal | DSHB | F55A12-C | Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining |
| Anti-PDX1 antibody, guinea pig polyclonal | Abcam | ab47308 | Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining |
| B27 minus Vit A | ThermoFisher | 12587010 | |
| Bovine serum albumin, heat shock fraction, fatty acid free | Sigma | A7030 | |
| CHIR 99021 | Tocris | 4423 | Reconstituted in DMSO |
| DMEM, high glucose | ThermoFisher | 41965047 | |
| Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Invitrogen | A10037 | |
| Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | A-21206 | ||
| DPBS 1X | ThermoFisher | 14190144 | |
| EGF | ThermoFisher | PHG0313 | Reconstituted in 0.1% BSA in PBS |
| FGF10 | R&D | 345-FG | Reconstituted in PBS |
| Glucose | Sigma Aldrich | G8644 | |
| Hoechst 33258 | Sigma | 23491-45-4 | |
| Inverted microscope | Olympus | IX73 | |
| KnockOut DMEM/F-12 (1X) | Gibco | 12660-012 | |
| KnockOut SR serum replacement | Gibco | 10828-028 | |
| L-Ascorbic acid (vitamin C) | Sigma | A92902 | Reconstituted in distilled water |
| Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 354230 | Aliquot the thawed stock and freeze at -20C. |
| MCDB131 | ThermoFisher | 10372019 | |
| Mouse anti-SOX17 | ORIGENE | CF500096 | Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining |
| mTeSR Plus | Stem Cell Technologies | 85850 | Mix 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 PES | ThermoFisher | 566-0020 | |
| Nicotinamide | Sigma | 72340 | Reconstituted in distilled water |
| NOGGIN | R&D | 6057-NG | Reconstituted in 0.1% BSA in PBS |
| Paraformaldehyde solution 4% in PBS | ChemCruz | sc-281692 | |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140122 | |
| Portable vacuum aspirator | |||
| Rabbit anti-FOXA2 | Cell signaling technology | 3143 | Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining |
| Retinoic Acid | Sigma Aldrich | R2625 | Reconstituted in DMSO |
| Rock inhibitor (Y-27632) | ReproCell | 04-0012-02 | Reconstituted in DMSO |
| Round Bottom Polystyrene FACS Tubes with Caps, STERILE | Stellar Scientific | FSC-9010 | |
| SANT-1 | Sigma Aldrich | S4572 | Reconstituted in DMSO |
| Sodium bicarbonate | Sigma | S5761-500G | |
| StemFlex | ThermoFisher | A3349401 | Mix the basal media with supplement. Aliquot and store at -20 °C for longer time or at 4 °C for instant use |
| TALI Cellular Analysis Slide | Invitrogen | T10794 | |
| Tali image-based cytometer automated cell counter | Invitrogen | T10796 | |
| Triton X-100 | Sigma | 9002-93-1 | |
| TrypLE 100 mL | ThermoFisher | 12563011 | |
| Tween 20 | Sigma | P2287 | |
| UltraPure 0.5 M EDTA, pH 8.0 | Invitrogen | 15575-038 |
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