Type 1 Diabetes is an autoimmune disease following the destruction of pancreatic β cells. Although replacing the β cells is an effective therapy, it is not a widely applied strategy. Nonsufficient cadaveric donors and low yield of transplantable islets besides chronic immunosuppression provide the necessity of generating renewable sources of insulin-expressing β cells with clinical utility. Patient-specific human-induced pluripotent stem cells (hiPSCs) are the most promising cells for cell-based therapies in regenerative medicine without the concern of immunological incompatibility or controversial issues. Developing novel approaches to maintain the function and survival of β cells should be considered as well.
The development of pancreas is regulated by a set of regulatory transcription factors. Pancreatic and duodenal homeobox 1 (PDX1) is a master regulatory transcription factor that involves in pancreatic development and glucose-dependent regulation of insulin gene expression. Sustaining expression of PDX1 in insulin-producing cells (IPCs) causes maintaining the β-cell phenotype by inducing the insulin expression.1 In addition, insulin is the main regulator of islet survival, mediated by a signaling target of PDX1. Through antiapoptotic effects of the PDX1, insulin prevents apoptosis in primary islets. Translocation of PDX1 to the nucleus triggers antiapoptotic insulin signaling by the adequate expression of antiapoptotic proteins, BCl-2 and BClXL, and loss of PDX1 stimulates caspase-3 activity. Also, oxidative stress suppression and mitochondrial metabolism are the pathways regulated by insulin to protect β cells through targeting the PDX1 promoter.2–4 Notably, defects in PDX1 gene are a cause of pancreatic agenesis, early-onset insulin-dependent diabetes mellitus, and maturity-onset diabetes of the young type 4.5
So, the strategy of induced PDX1 overexpression is well beneficial to enhance β-cell production, maturation, function, and survival. On the other hand, because the PDX1 is a transcriptional activator of several genes, including insulin, islet amyloid polypeptide, glucokinase, somatostatin, and glucose transporter type 2,1 an efficient β-cell differentiation protocol is needed, after transducing the hiPSCs with PDX1, to direct the cells toward functional pancreatic β cells.
In this study, through an efficient step-wise protocol, designed in our laboratory, the established patient-specific PDX1-overexpressing hiPSC lines were directed differentiation toward mature functional β-cell populations with the ability to release insulin in response to elevated glucose.
Materials and Methods
Culture of hiPSCs and PDX1-Overexpressing hiPSCs
The hiPSCs were maintained on mouse embryonic fibroblast (MEF) feeder layers in DMEM/F12 (Bioidea, Tehran, Iran) supplemented with 20% (vol/vol) KnockOut serum replacement (Invitrogen, Carlsbad, CA), 1 mM nonessential amino acids (Invitrogen), 2 mM Glutamax (Invitrogen), 50 U/ml penicillin, 50 μg/ml streptomycin (Bioidea), 0.55 mM 2-mercaptoethanol (Invitrogen), 10 ng/ml recombinant human bFGF (Peprotech, Rocky Hill, NJ). Cultures were passaged enzymatically using collagenase type IV (Bioidea) at a 1:4–1:8 split ratio every 5–7 days.6
Lentivirus Production and Transduction
The HEK 293T cells were plated at 6 × 106 cells per 100 mm dish and incubated overnight. Cells were cotransfected with 8 µg of pMD2G encoding the vesicular stomatitis virus glycoprotein (VSV-G) envelope and 15 µg of psPax2 packaging plasmid along with 23 µg of a plasmid harboring PDX1 gene, using CaPO4 precipitation as previously described.7,8 After transfection of 48 and 72 hours, the supernatant of transfectant was collected and filtered through a 0.45 mm pore-size cellulose acetate filter (Whatman, Maidstone, UK). Lentiviral titer was calculated by Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA). The hiPSCs were seeded at 1 × 105 cells per 100 mm dish one day before transduction and infected with PDX1-harboring virus supplemented with 4 µg/ml polybrene (Nacalai Tesque, Kyoto, Japan) with multiplicity of infection (MOI) of five, three times by 12 hours intervals. Immediately after third transduction, the cells were treated with primate embryonic stem (ES) cell medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF) as well as puromycin to a final concentration of 2 µg/ml during 10 days for stable transfection and clone selection.
Differentiation of hiPSCs and PDX1-Overexpressing hiPSCs
Differentiation was carried out according to the novel protocol established in our laboratory (Figure 1A),9 on MEF feeder layer in the dishes precoated by collagen (R & D System), laminin (R & D System), fibronectin (R & D System). Differentiation initiated on days 4–6 after passage, and the media were changed every day throughout the differentiation protocol. The cells were cultured in Roswell Park Memorial Institute (RPMI) Medium (without fetal bovine serum [FBS]) supplemented with the activin A (100 ng/ml) and Wnt3a (25 ng/ml) for the first day. The next two days, the cells were cultured in RPMI with 0.2% vol/vol FBS and activin A (100 ng/ml). Next, the cells were washed with phosphate-buffered saline (PBS) (with Mg2+/Ca2+) and then cultured in RPMI with 2% vol/vol FBS and keratinocyte growth factor (KGF) (50 ng/ml) as well as SB431542 (2.5 µM) for 3 days. The medium was changed to DMEM with 1% vol/vol B27 supplement, KAAD cyclopamine (0.25 µM), all-trans retinoic acid (RA, 2 µM), and Noggin (50 ng/ml) for another 3 days. The medium was changed to Dulbecco’s Modified Eagle’s Medium (DMEM) with 1% vol/vol B27 supplement, Noggin (50 ng/ml), KGF (50 ng/ml), epidermal growth factor (EGF) (50 ng/ml) for the next 3 days. Finally, the cells were cultured in DMEM supplemented with 1% (vol/vol) B27, Noggin (50 ng/ml), KGF (50 ng/ml), EGF (50 ng/ml), and 3-isobutyl-1-methylxanthine (IBMX) (100 µM) for the last 3 days.
Supplements were obtained from Sigma, St. Louis, MO (RA); Invitrogen, Carlsbad, CA (B27); Stemgent, Cambridge, Massachusetts, MA (KAAD-cyclopamine); R & D Systems, Minneapolis, MN (Activin A, Wnt3a); Peprotech, Rocky Hill, NJ (KGF, EGF, and Noggin); Stemcell Technologies, Vancouver, Canada (SB431542); Wako (IBMX, CA).
Real-Time Quantitative PCR
Small scrapings of cells were harvested, and total RNA was isolated with high pure RNA isolation kit (Roche). Total RNA of healthy human islet was purified as a positive control as well. RNA (5 µg) was used for reverse transcription with AccuPower RocketScript RT PreMixkit (Bioneer). PCR reactions were run in quadruplicate using 100 ng of the cDNA per reaction and 400 nM forward and reverse primers with SYBR Premix Ex Taq II (Tli RNaseH Plus), Bulk (Takara, RR820L). Real-time polymerase chain reaction (PCR) was performed using the Applied Biosystems StepOne instrument. The threshold cycle (Ct) for each gene of interest and GAPDH as housekeeping gene were determined, and data were analyzed by Relative Expression Software Tool (REST; Qiagen, Hilden, Germany). Changes in the expression ratio were tested for significance by a Pair Wise Fixed Reallocation Randomization Test and plotted using standard error (SE) estimation via a complex Taylor algorithm. Primer sequences were reported elsewhere.9
Cultures were fixed for 30 minutes in room temperature (RT) in 4% (wt/vol) paraformaldehyde in PBS, washed several times in PBS followed by adding 0.2% Triton X-100 in PBS for 15 minute in RT and blocked for 30–45 minutes in RT in PBST [PBS/0.1% (wt/vol) Triton X-100 (Sigma)] containing 1% (vol/vol) bovine serum albumin (BSA) (Sigma-Aldrich). Primary antibodies were diluted in 1% (vol/vol) BSA and secondary antibodies in PBS as 1:250 to 1:500 in dark condition. Primary antibodies were incubated for 24 hours at 4 °C, and secondary antibodies were incubated for 2 hours in RT. Followed by several wash, nuclei were stained with DAPI solution (1:1000 in double distilled water) for 5 minutes in dark condition.9 The antibodies and dilutions used in this study were as follow: goat anti-SOX17 (R & D System), 5 µg/ml; rabbit anti-FOXA2 (Chemicon, Billerica, MA), 1:200; goat anti-GSC (Santa Cruz Biotechnology, Dallas, TX), 1:50; goat anti-PDX1 (Santa Cruz Biotechnology, Dallas, TX), 1:50; rabbit anti-C-Peptide (abcam, Cambridge, UK), 1:100.
For intracellular staining, after fixation by 4% (wt/v) paraformaldehyde, 1–2 × 106 single cells were permeabilized with Perm buffer [PBS, 0.25% (v/v) Triton X-100, 0.01% (wt/v) NaN3] for 5 minutes in RT and then washed with IC buffer [PBS, 1% (wt/v) BSA, 0.01% (wt/v) NaN3]. Cells were incubated with primary antibodies (0.5–1 µg) diluted with blocking buffer [PBS, 0.1% (v/v) Triton X-100, 1% (v/v) bovine serum albumin, 0.01% (wt/v) NaN3] overnight at 4°C. Cells were washed in IC buffer and then incubated with appropriate secondary antibodies at a final 1:100 dilution for 60 minutes in RT. Cells were washed with IC buffer and then in FACS buffer [PBS, 0.1% (wt/v) BSA, 0.01% (wt/v) NaN3]. Cells were resuspended in FACS buffer for flow acquisition. Flow cytometry data were acquired with a FACSCalibur™ (BD Biosciences, San Jose, CA), using Alexa Fluor 488 with the excitation line at 499 nm and detecting fluorescence at 519 nm as well as Alexa Fluor 594 with the excitation line at 591 nm and detecting fluorescence at 618 nm. Data were analyzed using FloMax software. The background was estimated using unstained control and isotype immunoglobulin.10
C-Peptide Release Assay
C-peptide level in culture supernatants was measured using the C-peptide chemiluminescent immunoassay (CLIA) technology (Liaison, Alpharetta, GA). Briefly, differentiated cells at the end of stage 5 were preincubated at 37°C for 30 minutes with DMEM containing a minimal essential medium, 1% B27 supplement, and 2.5 mM glucose. The culture medium was collected, and the same cells were further incubated with DMEM containing 20 mM glucose without any nutrient secretagogues or secretory stimuli at 37°C for another 60 minutes. Then, the culture media were collected every 15 minutes, and C-peptide secretion into the culture media was measured.11
The differentiated cells were prepared for transmission electron microscopy according to the routine transmission electron microscopy staining protocol. The differentiated cells were fixed in 4% (wt/vol) formaldehyde and 1% (wt/vol) glutaraldehyde in 0.1 M PB (pH, 7.4) overnight. The fixative was replaced with 8% (wt/vol) (0.2 M) sucrose in 0.1 M PB at 4°C overnight and then, washed and osmicated with 1% (vol/vol) OsO4 in 0.1 M PB. In continue, cells were washed and dehydrated through a graded series of ethanol solutions and embedded in beam capsules and baked in 60°C oven for 48 hours. Sectioned semithin (thick section, 0.5–1 µm) was stained with toluidine blue. Lastly, they were ultrathin sectioned and collected on grids. Grids were stained with 5% (wt/vol) uranyl acetate for 15 minutes and then lead citrate for 5 minutes. Imaging was performed using a Philips 420 TEM at 80 kV.9
Induction of PDX1 Overexpression in hiPSCs
The patient-specific hiPSCs were produced by lentiviral transfer of four transcription factors, OCT4-SOX2-KLF4-C-MYC, to the fibroblast of a diabetic patient. The hiPSCs clones were picked up 16 days after transduction and undergone the characterization tests to demonstrate the pluripotency features of the established hiPSCs, reported in our previous article.9 Then the hiPSC-characterized cell was induced to overexpress PDX1 by a lentivirus-carrying PDX1 gene. The PDX1 overexpression was verified by Immunofluorescence assay in the PDX1-overexpressing hiPSCs, after puromycin selection, 10 days after transfection (Figure 2B).
The mRNA Profile of Directed Pancreatic Differentiation Pathway of PDX1-Overexpressing hiPSCs
The PDX1-overexpressing hiPSC line was undergone the sequential generation of various endoderms according to the new pancreatic differentiation protocol developed in our laboratory (Figure 1A).9 Each endodermal stage was verified by monitoring the expression level of stage-specific gene markers.
At the first day of differentiation, the cells were transitioned into the mesendoderm stage, verified by the upregulation of BRACHYURY gene expression (Figure 3).
In stage 1, at the third day of differentiation, SOX17, FOXA2, and GSC genes were upregulated. This expression profile confirmed the formation of definitive endoderm at the third day of differentiation (Figure 3).
The upregulation of HNF4A expression on day 6 demonstrated the primitive gut tube formation in the second stage of differentiation (Figure 3).
The expression of PDX1 and HNF6 enhanced during third stage of differentiation and reached to their maximum level on day 9, indicated the appearance of the posterior foregut endoderm (Figure 3). The PDX1 maintained its expression during the course of differentiation. While, the expression level of HNF6 was dropped through transition toward the other stages. The upregulation of HNF6 in stage 3 was in favor of posterior foregut formation, but sustained expression of HNF6 may lead to pancreatic duct formation.
The pancreatic endoderm and endocrine precursors appeared by the upregulation of NKX2.2, NKX6.1, PTF1A, and NGN3 markers (Figure 3). Interestingly, the NKX2.2 and NKX6.1 genes began to express sooner than what expected, from the sixth day of differentiation. The NGN3 and PTF1A were transiently expressed in stage 4, and their expression levels dropped as the differentiation progressed. The decrease in PTF1A in the latter days of differentiation prevented acinar formation.
At the end of stage 5, by about 15 days of differentiation, islet-like cluster structures expressed high level of insulin against the low level of glucagon and somatostatin (Figure 3).
Surprisingly, the expression of INS triggered from the day of 12 of pancreatic differentiation and PDX1 overexpression induced higher expression level of insulin in PDX1-overexpressing hiPSC-derived β-like cells in comparison to the nontransduced counterpart cells (Figure 3). Also, the expression level of insulin in the produced β-like cells was similar to the insulin expression level in the control positive islet. Indeed, PDX1 overexpression optimized and expedited the pathway of β-cell production.
In contrast, the SST and the GCG genes were expressed in lower level in the differentiated cells derived from PDX1-overexpressing hiPSC compared with the nontransduced counterparts, and their expression levels were extremely lower than the expression levels of these hormones in the control positive islet (Figure 3).
Interestingly, the AMY gene was not significantly expressed in the end-stage cells indicating that the exocrine cells were not existed in the cell populations produced in our culture (Figure 3).
Immunocytochemical Analysis of Stage-Specific Markers
During the 15 day pancreatic differentiation pathway, expression of stage-specific markers was monitored in the stage 1, 3, and 5 by immunocytochemical (ICC) analysis. The overexpression of FOXA2, SOX17 and GSC confirmed the definitive endoderm formation. While PDX1 overexpression indicated the derivation of posterior foregutendoderm. At the last stage of differentiation, the formation of β-like cells was demonstrated by C-peptide marker (Figure 2A).
Flow Cytometry Analysis of Stage-Specific Markers
According to flow cytometry results, approximately 47.67% FOXA2-expressing cells, 42.95% SOX17-expressing cells, and 21.54% GSC-expressing cells were detected in stage 1 (Figure 4A). Furthermore, 70.23% of the cells in stage 3 expressed PDX1 marker (Figure 4B). Interestingly, 85.61% of the end cells were C-peptide–positive cells (Figure 4C).
Insulin Release from PDX1-Overexpressing hiPSC-Derived β Cells
The differentiated cells secreted C-peptide in a glucose-dependent manner with rapid release kinetics in response to glucose. The insulin release level reached to its highest level 30 minutes after exposure to glucose (Figure 1C). The amount of secreted C-peptide 30 minutes after 20 mM glucose treatment was approximately more than seven times higher than the basal level (Figure 1B).
Electron microscopy images illustrated the ultrastructure of insulin granule, a crystalline core, and a clear halo surrounding, observed in the β cells in our culture (Figure 2C).
In this research, the strategy of induced PDX1 overexpression besides an efficient β-cell differentiation protocol led to high-efficient in vitro generation of ectopic IPCs with the effectively reduced number of polyhormonal cells and increased number of insulin single-positive cells.
PDX1 is a master regulatory transcription factor that involves in pancreatic development and glucose-dependent regulation of insulin gene expression. Sustaining expression of PDX1 in IPCs causes maintaining the β-cell phenotype by inducing insulin expression.1 On the other hand, PDX1 is a regional endoderm marker whose expression marks the dorsal and ventral pancreatic buds, as well as a part of the stomach and duodenal endoderm.12 Therefore, we applied an efficient β-cell differentiation protocol besides the PDX1-overexpression strategy to uniquely direct the cells toward β-cell production and maturation.
In this attempt, inhibition of the bone morphogenetic proteins (BMP) signaling pathway by Noggin and induction of pancreas progenitors by retinoid signaling in addition to blocking the Sonic hedgehog (Shh) signaling by KAAD cyclopamine led to the cells to the destination of foregut endoderm formation. Notably, passing through the foregut endoderm stage was facilitated by induced PDX1 overexpression. This strategy yielded 85.61% glucose-responsive insulin-secreting cells in vitro.
The C-peptide secretion in a glucose-dependent manner is a characteristic of a mature β cell, whereas response to secretory stimuli or nutrient secretagogues is indicative of immature fetal β cell.13 Remarkably, the kinetics of C-peptide release in our produced mature β cells was approximately seven times higher than the basal level (Figure 1B). While, other study exhibited a limited capacity of in vitro glucose-dependent C-peptide secretion in the β-like cells derived from the PDX1-overexpressed human embryonic stem cells.14 On the other hand, it was indicated in our previous report9 that the amount of secreted C-peptide in the nontransduced hiPSC-derived β-like cells was about five times higher than the basal level. While, other reports indicated 0- to 3.2-fold increase of glucose-responsive C-peptide secretion, observed in hES/hiPSC-derived insulin-positive cells.13,15–22 Indeed, PDX1 overexpression raised the insulin secretion in β-like cells derived from PDX1-overexpressing hiPSC in comparison to the nontransduced counterparts. This is certified by the fact that the PDX1 promotes proliferation of pancreatic progenitor cells and regulates the biosynthesis of insulin. Whenever the cells expose to high glucose, PDX1 protein translocates from the cytoplasm or nuclear periphery to the nuclei and induces the high expression of insulin. While, during hypoglycemia, PDX1 protein transports out of the nuclei of β cell, leads to avoid excessive insulin production.1 Furthermore, sustained expression of PDX1 in IPCs caused maintaining the β-cell phenotype by inducing the insulin expression and repressing glucagon expression and α-cell differentiation (Figure 3). Therefore, induced PDX1-overexpression strategy reduced the rate of polyhormonal cells generation.
Moreover, PDX1 regulates the expression of NKX6.1, which is exclusively restricted to β cells to induce their maturation.1 Surprisingly, NKX2.2 and NKX6.1 genes began to express earlier than what expected in the cells derived from the PDX1-overexpressing hiPSC line, indicated the faster formation of pancreatic endoderm and endocrine precursors (Figure 3).
Also, the expression of INS gene triggered from the day of 12 of pancreatic differentiation and PDX1 overexpression induced higher expression level of insulin in PDX1-overexpressing hiPSC-derived β-like cells in comparison to the nontransduced counterpart cells (Figure 3), and the expression level of insulin was similar to the control positive islet. Indeed, PDX1 overexpression optimized and expedited the pathway of β-cell production. In contrast, the SST and the GCG genes expressed in lower level in the differentiated cells derived from PDX1-overexpressing hiPSC compared with nontransduced counterparts, and these expression levels were extremely lower than the expression levels of these hormones in the control positive islet (Figure 3). Interestingly, the AMY gene was not significantly expressed in the end-stage cells, indicating that the exocrine cells were not existed in the cell populations produced in our culture (Figure 3).
Moreover, PDX1 regulates the formation and maturation of insulin granules.1 Transmission electron microscopy images illustrated the insulin granules in the produced β cells (Figure 2C), which confirms the generation of insulin-expressing cells.
In addition to what discussed above, regarding the fact that insulin regulates the islet survival through a signaling target of PDX1,4 hence the strategy of induced PDX1 overexpression is well beneficial to enhance β-cell production, maturation, function, and survival.
In spite of that, some believe that induction of the overexpression of NGN3 together with PDX1 dramatically increases the level and timing of maximal INS1 mRNA expression.23 However, a comprehensive study is required to identify the more precise transcriptional network of pancreatic progenitor cell to design an efficient strategy to develop the mature and functional β cells.
On the other hand, although an efficient differentiation protocol with the potency to overexpress key marker genes in the pathway leads to enhanced expression of pancreatic enriched genes, transplantation of the β-like cells promotes the maturation of IPCs, because insulin expression may require additional signals, present in vivo.14 Accordingly, optimizing an efficient differentiation protocol yields the more mature β cells and finally better function of transplants.
Moreover, it is required to assess the functionality of produced β cells by evaluation of the glucose-responsive insulin secretion and the potency of the cells to remove the hyperglycemia in animal models. Of interest in this regard is the report, indicated the reversal of hyperglycemia in SCID-beige mice with STZ-induced diabetes after only 6 weeks of β-cell transplantation.24
Most notably, an efficient and safe diabetes cell therapy will require reconstituted β cells not only to release insulin in response to enhanced glucose level but also to appropriately suppress insulin secretion in response to hypoglycemia.25
Finally, an effective strategy should be provided to protect the β-cell survival and long-term performance as well as to prevent the loss of insulin function till reaching the clinical applications. Further, cell dose requirements for the treatment of diabetic patients and graft longevity should be determined.
In sum, although guided differentiation toward functional β cells presents substantial challenges, it is possible to produce glucose-responsive IPCs by activating the relevant signaling pathways at different developmental stages in vitro. Moreover, the strategy of PDX1 overexpression enhances β-cell production, maturation, function, and survival. These cells can be a renewable source of patient-specific induced pluripotent stem cell (iPSC)-derived β-like cells, a promising approach to develop diabetes cell therapy, disease modeling, and drug screening.
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