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Introduction
The increasing number of patients with pancreatic disorders, such as diabetes mellites, pancreatic cancer and pancreatitis, is alarming in many countries (1-3). It has been suggested that genetic variants, which cause hereditary disorders, can serve as risk factors for idiopathic dis-eases. For example, variants of certain genes involved in monogenic diabetes, such as neonatal diabetes and maturity-onset diabetes of the young (MODY) also increase the risk of type 2 diabetes (T2D) (4). Moreover, there has been growing interest in the cause of pancreatic diseases, as the significant increase in pancreatic complications was observed even in healthy individuals who were administered glucagon-like peptide 1 (GLP-1) receptor agonists for weight loss purposes (5, 6). The recent study has revealed an increased risk of gastrointestinal adverse events, such as biliary disease, pancreatitis, bowel obstruction, and gastroparesis, among diabetic patients taking these drugs (5). Especially, the risk of pancreatitis was ten times higher with GLP-1 agonists, including semaglutide or liraglutide, than with other weight loss agent like bupropion-naltrexone (5). Therefore, it is important to understand the genetic etiology of inheritable pancreatic diseases to pave a way to develop the better therapy for common pancreatic diseases.
Although there is a much need for studying pancreatic disorders in-depth, the modeling system for pancreatic diseases is limited. The anatomy and physiology of pancreas is significantly different between mouse and human, making it unfeasible to use mouse model to dissect the genetic etiology of pancreatic diseases (7, 8). For example, contrast to human, which have a pancreas as a solid organ, mouse pancreas disperses in abdominal cavity spanning from spleen to duodenum (7, 8). Also, in mouse, insulin-producing β-cells are positioned in the middle and glucagon-producing α-cells and somatostatin-producing δ-cells are marginated in the islet of Langerhans (7, 8). In contrast, human islet does not exhibit the distinct compartmentalization: β-cells are interspersed among other endocrine cell types, establishing extensive direct cell-to-cell contacts. This implies that intercellular interactions among endocrine cells would be much frequent and complex in humans compared to mice (7, 8). These differences also exist at the molecular level leading different responses from mouse and human pancreas for the same chemical treatment. Streptozotocin (STZ) is commonly used to cause β-cell death which result in diabetes in rodent model (9). However, human pancreas is extensively insensitive to pancreatic cytotoxicity caused by STZ via DNA alkylation and poly ADP-ribose polymerase activation followed by lethal nicotinamide adenine dinucleotide depletion (10). Moreover, the genetic structure of mouse is substantially different from human genome, making it difficult to investigate the genetic requirement of human disease in mice. For instance, mice have a total of 20 trypsinogen genes, while humans have only one cationic trypsinogen gene, recapitulating human herediatary pancreatitis using mouse models has many obstacles (11). Thus, pancreatic disease modeling should be performed in a human context. In this regard, stem cell-derived modeling is the most ideal approach to recapitulate human physiological condition in vitro.
Recently, the three-dimensional (3D) organoid culture system, which best imitates physiological conditions of human body by providing cell orientation and polarity for proliferation, growth, and differentiation, has been established for both of pancreatic endocrine and exocrine cells (12). As the 3D organoid culture system reproduce the interactions between cells, it has become the finest platform to examine the impact of other lineages for disease pathogenesis, such as the impact of pancreatic ductal cells for type 1 diabetes (T1D) (13). Due to the lack of adult pancreatic stem cells, pancreatic organoids are originated to pancreatic progenitors (PPs) derived from human pluripotent stem cells (hPSCs) (14) or pancreatic cancer, which produces patient-derived tumor organoids (15-17). An effort to generate pancreatic organoids directly from human adult pancreas generated organoids which are primarily composed by progenitor-like pancreatic ductal epithelial cells, with a low presence of endocrine cells (18). The limited number of endocrine cells in organoids became mature only after in vivo transplantation (18). Pancreatic organoids derived from human fetal pancreas also were comprised of epithelial cells, implying clear limitations to establish pancreatic organoids directly from pancreatic tissue (19).
Furthermore, the advancement of clustered regularly interspaced short palindromic repeats (CRISPRs) gene editing technology has revolutionized disease modeling using stem cells. By introducing genetic mutations found in human patients using CRISPR techniques and comparing normal cells with genetically mutated cells during pancreatic differentiation, it is possible to investigate the molecular mechanisms that the genetic mutation can cause within pancreatic cells. This review will provide the details regarding genetic risk factors of pancreatic diseases, which can be examined by hPSCs combined with the CRISPR genome editing and 3D organoids culture technology.
Anatomy of Pancreas
The pancreas is composed of two compartments: endocrine, which produce hormones, and exocrine, which secrete digestive enzymes (20, 21). The pancreas is located behind the stomach and is embedded in the curve of duodenum (20, 22). The pancreas can be divided into the head, body, and tail (Fig. 1A) (22).
Endocrine
The endocrine portion of the pancreas, which comprises 2% of the pancreatic tissue consists of scattered clusters of cells called islets of Langerhans which secrete hormones directly into the bloodstream (Fig. 1A) (22). These islets contain five types of hormone-producing cells: α-cells account for 15%∼20% of islet cells, β-cells account for 65%∼80%, γ-cells for 3%∼5%, δ-cells for 3%∼10%, and ε-cells for less than 1% (Table 1) (22, 23). The α-cells within the islets secrete the hormone glucagon, which raises blood glucose levels by promoting glycogenolysis, the breakdown of glycogen into glucose, and gluconeogenesis, the production of new glucose in the liver, muscle, kidney, and intestine. In contrast, the β-cells produce insulin, a hormone that lowers blood glucose levels by facilitating the uptake of glucose into cells and promoting glycogenesis, the conversion of glucose to glycogen for storage in the liver and muscles. The δ-cells secrete somatostatin, which inhibits the release of both insulin and glucagon. The pancreatic polypeptide cells, formerly known as γ-cells, or F cells, release pancreatic polypeptide, a hormone involved in regulating the secretory activity of both the exocrine and endocrine components, and slowing digestion by decreasing gastrointestinal motility. ε-Cells produce ghrelin, which plays a pivotal role in regulating appetite by acting on the hypothalamus region of the brain to stimulate feelings of hunger and promote food intake. Beyond its appetite-stimulating effects, ghrelin influences energy utilization by promoting the breakdown of adipose tissue via lipolysis and stimulating gluconeogenesis in the liver, thereby producing more glucose (20, 22).
Disruptions in the function of these endocrine cells can lead to a range of disorders. The most well-known pancreatic endocrine disorder is diabetes mellitus, which occurs due to either the autoimmune destruction of β-cells (T1D) or the development of insulin resistance and eventual β-cell dysfunction (T2D). In both cases, the lack of insulin or the body’s inability to respond to it properly leads to hyperglycemia, which can cause serious complications such as cardiovascular disease, kidney damage, and neuropathy if left untreated. In addition to insulin-related disorders, imbalances in glucagon secretion can lead to difficulty in maintaining stable blood sugar levels. Excessive glucagon production can manifest as hyperglycemia, and insufficient glucagon secretion can result in hypoglycemia. Disor-ders related to pancreatic polypeptide and ghrelin are less common and not as well understood. Through this finely tuned system of hormone secretion, the endocrine pancreas plays a vital role in maintaining blood glucose homeostasis, energy metabolism, and proper nutrient processing within the body.
Exocrine
The exocrine part of the pancreas, which composes 98% of the pancreatic tissue, is responsible for producing and secreting digestive enzymes into the small intestine to help break down and absorb nutrients from the food (Fig. 1A) (20-22). This part consists of acinar cells, which secrete digestive enzymes, and ductal cells, which form a network of ducts that transport these enzymes into the duodenum. The acinar cells synthesize and secrete digestive enzymes, including amylase for carbohydrate digestion, lipase for fat digestion, and proteases such as trypsinogen, and chymotrypsin for protein breakdown. As the enzymes are produced, they travel through a network of tiny ducts within the pancreas, converging into larger ducts that eventually merge to form the main pancreatic duct. This main duct traverses through the center of the pancreas, collecting the secretions from the acinar cells, and ultimately joins the common bile duct, which carries bile from the liver and gallbladder (Fig. 1A). Together, the pancreatic duct and common bile duct form the hepatopancreatic ampulla (or ampulla of Vater), which empties into the duodenum at the major duodenal papilla. The pancreatic juice, which contains these enzymes, is alkaline due to the bicarbonate (HCO3−) secreted by the pancreatic ductal epithelial cells, helping to neutralize the acidic chyme that enters the duodenum from the stomach (Fig. 1A) (20, 22, 24). This process ensures optimal conditions for the enzymes to function and protects the duodenum from the acidic environment. When the exocrine function of the pancreas is compromised, one of the most common disorders is exocrine pancreatic insufficiency (EPI), where the pancreas fails to produce enough digestive enzymes and HCO3− to achieve normal digestion and absorption (Table 1). Without sufficient enzymes, proper digestion cannot occur, leading to malabsorption of nutrients, particularly fats. This can result in symptoms such as steatorrhea (fatty stools), weight loss, and nutritional deficiencies especially in fat-soluble vita-mins. Pancreatic enzyme replacement therapy (PERT) is the primary management strategy for EPI patients, which supplements the insufficient pancreatic enzymes with oral preparations containing lipase, amylase, and protease. Many PERT preparations are the most commonly used preparations are enteric-coated microspheres, which protect the enzymes from the acidic environment of the stomach and release them in the alkaline milieu of the duodenum, where they can effectively mix with the chyme and perform their digestive functions (25).
Pancreatic Cancer
Another serious condition related to the pancreas is pancreatic cancer (26). Pancreatic neuroendocrine tumors (PNETs) can arise from the endocrine cells of the pancreas, often referred as “islet cell tumors.” These tumors can be functional, secreting hormones, or non-functional, halting hormone secretion. Functional PNETs occurred in β-cells is insulinomas causing hypoglycemia and in α-cells causes glucagonomas resulting diabetes and necrolytic migratory erythema. Less than 5% of PNET occurs in δ-cells resulting somatostatinoma, which can cause diabetes mellitus, cholelithiasis, and steatorrhea due to the inhibitory effects of somatostatin on other hormones like insulin and glucagon. PNETs can be a part of multiple endocrine neoplasia type-1 (MEN1) which causes tumors in the multiple endocrine organs including parathyroid and pituitary glands, and the pancreas.
Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, originating from the ductal epithelial cells lining the pancreatic ducts. Pancreatic cancer is often asymptomatic in its early stages and diagnosed when it has advanced. Symptoms include abdominal pain, jaundice, weight loss, digestive problems, and loss of appetite appearing as the tumor grows. Although the treatment options vary from surgery, chemotherapy, to radiation therapy, the prognosis for pancreatic cancer remains poor, with a low five-year survival rate of around 12%, which makes it one of deadliest cancers (26, 27).
Risk factors for PDAC include smoking, obesity, chronic pancreatitis (CP), inherited genetic syndromes, and advanced age (28). PDAC arises from precursor lesions, namely pancreatic intraepithelial neoplasias (PanINs) and intraductal papillary mucinous neoplasms (IPMNs). These lesions undergo a multistep progression, acquiring genetic alterations and ultimately developing into invasive PDAC. The most common genetic driver of PDAC is activating mutations in the KRAS gene, which are found in over 90% of cases. Additionally, loss-of-function mutations in tumor suppressor genes, such as TP53, SMAD4, and CDKN2A, are frequently observed (28). The immune landscape of PDAC is predominantly immunosuppressive, with a paucity of cytotoxic CD8 T cells and an abundance of suppressive myeloid cells, such as tumor-associated macrophages and myeloid-derived suppressor cells (29). This poor immune responsiveness was the reason that the administration of immune oncotherapy, such as checkpoint inhibitor, exhibit minimum benefits among PDAC patients.
Pancreatitis
Another common pancreatic disorder is pancreatitis, which is the inflammation of the pancreas (30). Acute pancreatitis (AP) occurs suddenly and can cause severe abdominal pain, nausea, vomiting, and fever. It is often caused by gallstones blocking the pancreatic duct or excessive alcohol consumption. If left untreated, AP can cause complications such as pancreatic necrosis, where portions of the pancreatic tissue die, resulting in organ failure. Recurrent episodes of AP can lead to the development of CP in affected individuals, a long-term condition characterized by persistent inflammation and fibrosis of the pancreas leading to permanent damage to both exocrine and endocrine parts (Fig. 1B) (31). This damage can result in EPI and endocrine dysfunction (32). As a consequence of endocrine dysfunction, 25%∼48% of CP patients experience adult-onset diabetes (33). The diagnosis of CP is challenging due to the initial lack of clear symptoms and overlapping symptoms with other conditions (34). As CP serves as a strong risk factor for pancreatic cancer, individuals with CP have at least 15-fold increased risk of developing pancreatic cancer with earlier onset (35).
Hereditary Pancreatic Disorders
Several studies indicate that genetic variants associated with hereditary pancreatic disorders could also act as risk factors, increasing the likelihood of developing sporadic pancreatic diseases (4). By dissecting the genetic components of inheritable pancreatic diseases, we can achieve better understanding of disease mechanism which causes common pancreatic diseases. The most common non-neoplastic pancreatic diseases from the endocrine and exocrine lineages are diabetes and pancreatitis, respectively. Rare forms of diabetes, neonatal diabetes and MODY, are strongly influenced by genetic mutations. Neonatal diabetes is caused by defective pancreatic development, with the approximate incidence of 1：90,000 live births. It occurs mainly before 6 months of age and requires immediate insulin injection to treat transient or permanent hyperglycemia and PERT to resolve EPI (36). MODY, which accounts for approximately 1%∼5% of all diabetes cases, is a group of hereditary diabetes characterized by early onset, usually before the age of 25 years, and caused by genetic defects in insulin secretion (37-39). Unlike T1D, MODY patients do not have autoantibodies, and unlike T2D, they are not usually obese and are not insulin-resistant (37-39). MODY is inherited in an autosomal dominant manner, meaning that a single defective gene inherited from one parent is sufficient to cause the disorder (37-39). In the case of some transcription factors (TFs), including pancreatic and duodenal homeobox 1 (PDX1) and neurogenic differentiation 1 (NEUROD1), which play critical roles during pancreatic development, their heterozygous mutations causes MODY, while their homozygous mutations causes neonatal diabetes (40, 41). This implies that the insufficient gene dosage during pancreatic development can predispose individuals to diabetes in their later lifetime.
Genetics of MODY
To date, at least 14 different MODY genes have been identified, each causing a specific subtype of MODY (Table 2). The most common forms are HNF4A-MODY (MODY1), GCK-MODY (MODY2), and HNF1A-MODY (MODY3), which together account for approximately 70%∼80% of all MODY cases (Fig. 2A, 2B) (37).
HNF4A-MODY (MODY1)
Hepatocyte nuclear factor 4 alpha (HNF4α) is a TF that regulates the expression of genes involved in glucose and lipid metabolism in the liver and pancreatic β-cells (42). Mutations in HNF4A lead to a progressive decrease in insulin secretion, resulting in hyperglycemia (37). HNF4A-MODY accounts for 5%∼10% of MODY cases and typically presents in adolescence or early adulthood (37). Patients may initially respond to sulfonylureas but often require insulin treatment as the disease progresses (43). Additionally, HNF4A-MODY is associated with fetal macrosomia, which the size of fetus is much larger than average, and transient neonatal hyperinsulinemic hypoglycemia (Fig. 2A) (43).
GCK-MODY (MODY2)
Glucokinase (GCK) is a key enzyme in the pancreatic β-cells that acts as a glucose sensor, regulating insulin secretion in response to changes in blood glucose levels (44). GCK-MODY, which accounts for 30%∼50% of MODY cases, is often asymptomatic, rarely requiring treatment as it causes a mild, stable hyperglycemia from birth (37, 43). However, during pregnancy, women with GCK-MODY may require insulin therapy to prevent fetal macrosomia (43, 44).
HNF1A-MODY (MODY3)
HNF1α is a TF that regulates the expression of genes involved in glucose metabolism and insulin secretion in the pancreatic β-cells (45). Mutations in HNF1A lead to a progressive decrease in insulin secretion, resulting in hyperglycemia (46). HNF1A-MODY is the most common form of MODY, accounting for 30%∼65% of cases (37). Patients typically present with symptomatic diabetes in adolescence or early adulthood and may initially respond to sulfonylureas, but often require insulin treatment as the disease progresses (Fig. 2B) (43).
PDX1-MODY (MODY4)
PDX1 is a TF that plays a crucial role in pancreatic development and the regulation of insulin gene expression (40). Mutations in PDX1 are rare and lead to a variable clinical presentation, ranging from mild hyperglycemia to severe diabetes requiring insulin treatment (Fig. 2C) (43). PDX1-MODY accounts for less than 1% of MODY cases (37).
HNF1B-MODY (MODY5)
HNF1β is a TF that regulates the development and function of the pancreas, kidney, liver, and genital tract (47). Mutations in HNF1B lead to a variable clinical presen-tation, including diabetes, renal cysts and malformations, genital tract abnormalities, liver dysfunction, and pancreatic hypoplasia (Fig. 2D). HNF1B-MODY accounts for approximately 5% of MODY cases and typically presents in adolescence or early adulthood (37). Patients often require insulin treatment and may develop end-stage renal disease requiring renal replacement therapy (43).
NEUROD1-MODY (MODY6)
NEUROD1 is a TF that plays a role in the development and maintenance of pancreatic β-cells and the regulation of insulin gene expression (41). Mutations in NEUROD1 are rare and lead to a variable clinical presentation, ranging from mild hyperglycemia to severe diabetes requiring insulin treatment (43). NEUROD1-MODY accounts for less than 1% of MODY cases (37).
KLF11-MODY (MODY7)
Kruppel-like factor 11 (KLF11) is a TF that regulates the expression of genes involved in insulin secretion and β-cell function (48). Mutations in KLF11 are rare and have been reported in a small number of families with early-onset diabetes (37). The clinical presentation and management of KLF11-MODY are not well-characterized due to its rarity.
CEL-MODY (MODY8)
Carboxyl ester lipase (CEL) is a digestive enzyme secreted by the acinar cells that plays a role in the hydrolysis of dietary fat and fat-soluble vitamins (49). Mutations in CEL lead to a syndrome characterized by diabetes, pancreatic exocrine dysfunction, and lipomatosis (49). As CEL-MODY is a rare and their clinical presentation are not well-characterized (37). Genetic mutations in CEL are found in not only MODY but also T1D and T2D patients, implying that the crosstalk between acinar cells and β-cells plays such a critical role for diabetes initiation (48).
PAX4-MODY (MODY9)
Paired box 4 (PAX4) is a TF that plays a role in the development and maintenance of pancreatic β-cells. Mutations in PAX4 are rare and have been reported in a small number of families with early-onset diabetes (37). The clinical presentation and management of PAX4-MODY are not well-characterized due to its rarity (38, 39).
INS-MODY (MODY10)
Insulin (INS) is the gene that encodes the insulin protein. Mutations in INS lead to a spectrum of disorders, including neonatal diabetes, familial hyperinsulinemia, and early-onset diabetes. INS-MODY is a rare subtype, and the clinical presentation and management depend on the specific mutation and its effect on insulin structure and function (37-39).
BLK-MODY (MODY11)
B lymphoid tyrosine kinase (BLK) is a non-receptor tyrosine kinase that is expressed in pancreatic β-cells and plays a role in insulin secretion. Mutations in BLK have been reported in a small number of families with early-onset diabetes (37). The clinical presentation and management of BLK-MODY are not well-characterized due to its rarity (38, 39).
ABCC8-MODY (MODY12)
ATP binding cassette subfamily C member 8 (ABCC8) encodes the sulfonylurea receptor 1 (SUR1) subunit of the ATP-sensitive potassium (K-ATP) channel in pancreatic β-cells, which regulates insulin secretion. ABCC8-MODY is a rare subtype, and the clinical presentation and management depend on the specific mutation and its effect on K-ATP channel function (37-39).
KCNJ11-MODY (MODY13)
Potassium inwardly rectifying channel subfamily J member 11 (KCNJ11) encodes the Kir6.2 subunit of the K-ATP channel in pancreatic β-cells, which regulates insulin secretion. KCNJ11-MODY is a rare subtype, and the clinical presentation and management depend on the specific mutation and its effect on K-ATP channel function (37-39).
APPL1-MODY (MODY14)
Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) is a multifunctional adaptor protein that regulates various cellular processes, including insulin signaling and glucose metabolism. Mutations in APPL1 have been reported in a small number of families with early-onset diabetes (37). The clinical presentation and management of APPL1-MODY are not well-characterized due to its rarity (38, 39).
Genetics of Pancreatitis
A rare and inherited form of CP is hereditary pancreatitis (HP), in which CP symptoms pass from generation to generation in a family, occurring in 3∼6 individuals per million (50, 51). Inheritance pattern of HP is an autosomal dominant manner, meaning that a single copy of the mutated gene is sufficient to cause the disorder. The age of onset for HP is typically early, with many patients experiencing their first episode of acute pancreatitis during childhood or adolescence. As a result, HP patients face a higher risk of mortality due to early-onset pancreatic cancer and T1D (52). Patients with HP have mutations in one of the following genes: serine protease 1 (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC), carboxypeptidase A1 (CPA1), or cystic fibrosis transmembrane conductance regulator (CFTR) (Fig. 2) (53). As HP-causing genetic variants are also present in idiopathic chronic pancreatitis (ICP) patients who did not exhibit a family history, understanding the genetic etiology of HP is important.
PRSS1
The pancreatic acinar cells secrete digestive proteases in the form of inactive precursors. The precursor of trypsin, known as trypsinogen, is secreted by the pancreas and activated by the serine protease enteropeptidase in the small intestine (Fig. 1B) (54). Premature activation of trypsinogen inside the pancreatic acinar cells, prior to its release into the small intestine, results in elevated trypsin activity. This enhanced trypsin activity damages the acinar cells and triggers inflammatory responses. About 80% of HP cases are caused by heterozygous gain-of-function mutations in the human PRSS1 gene, which encodes human cationic trypsinogen, leading to the overactivity of trypsin and other proteases in the pancreas (50). HP caused by PRSS1 mutations follows an autosomal dominant inheritance pattern, with a strong familial clustering (55-58). The p.Arg122His mutation is the most common variant of PRSS1 associated with HP (59), followed by the p.Asn29Ile mutation (60, 61). These two heterozygous mutations are found in 90% of HP patients, with 65% harboring the p.Arg122His variant and 25% carrying the p.Asn29Ile variant. The remaining 10% patients have other heterozygous mutations, including p.Lys23Arg, p.Arg116Cys, and p.Arg122Cys (57). Approximately 80%∼90% of individuals with the p.Arg122His genetic variant in the PRSS1 gene develop pancreatitis (51, 62-64). Over 40 PRSS1 gene mutations contribute to the development of HP (Supplementary Table S1) (57).
Trypsinogen can be activated by trypsin through a process known as autoactivation (54). Mutations in trypsino-gen that enhance autoactivation are the risk factors for CP and are associated with HP (55). Two main mechanisms prevent intrapancreatic trypsinogen activation: trypsin inhibition by the lysosomal protease cathepsin L, and trypsinogen degradation by SPINK1 and CTRC (55, 65, 66). PRSS1 variants promote trypsinogen autoactivation or hinder CTRC-mediated trypsinogen degradation, leading to increased trypsin activity in the pancreas (57). Trypsin triggers trypsinogen autoactivation by cleaving the activation peptide at the Lys23-Ile24 site in trypsinogen, which occurs more vigorously under certain conditions. For example, CTRC cleaves the Phe18-Asp19 peptide bond within the activation peptide, generating a shorter form of trypsinogen that undergoes more active autoactivation. Whereas, the cleavage of the Leu81-Glu82 peptide bond by CTRC and the cleavages of the Arg122-Val123 peptide bond by trypsin induce irreversible degradation of trypsinogen preventing its conversion to trypsin, thereby reducing the concentration of trypsin in the pancreas (Fig. 1C) (65, 67, 68). Therefore, p.Arg122His mutation of PRSS1 gene interfere with this process of trypsinogen inactivation, prolonging its activity and contributing to the development of pancreatitis. The p.Asn29Ile mutation both promotes trypsinogen autoactivation and inhibits its degradation by CTRC. Mutations occurred in activation peptide from position 16 to 22, such as p.Ala16Val, stimulate trypsinogen autoactivation independently of CTRC (Fig. 1C) (69-72). Other genetic variants in the PRSS1 gene also are predicted to be pathogenic from several cohort studies, which need more validation (Fig. 2E, Supplementary Table S1). Many HP families are found not to have any PRSS1 mutations, leading to the identification of mutations in genes that can regulate the activation and degradation of trypsinogen and trypsin.
SPINK1
The SPINK1 gene encodes a trypsin-regulating molecule in acinar cells. As an inhibitor of both cationic and anionic trypsin, SPINK1 functions to inhibit trypsin activity, thereby mitigating the acute inflammatory response and preventing recurrent acute pancreatitis (73-77). In the pancreas, SPINK1 inhibits approximately 20% of trypsin activity, thus preventing premature trypsinogen activation (78-83). SPINK1 mutations do not cause disease by themselves but play a role in lowering the threshold of pancreatitis caused by other genetic or environmental factors (76). Studies have shown that SPINK1 mutations are more significant risk factors for ICP than for pancreatitis induced by environmental factors such as alcohol, suggesting that ICP develops through the trypsin activation mechanism (84). More genetic variants of SPINK1 from several cohort studies are listed, which are predicted to be pathogenic (Fig. 2F, Supplementary Table S1).
CTRC
CTRC is a digestive protease that is synthesized and secreted by pancreatic acinar cells. CTRC is produced as an inactive proenzyme, chymotrypsinogen C, and is activated later in the duodenum by tryptic cleavage (85). Loss-of-function variants in CTRC have been reported as a risk factor for CP because CTRC can protect the pancreas from trypsin-related damages by degrading trypsin (86, 87). Although many variants were found pancreatitis patients, functional study of CTRC variants demonstrated that only a portion of variants have a reduced enzyme activity, emphasizing the performing functional evaluation of variants (Fig. 2G, Supplementary Table S1) (88).
CPA1
The CPA1 gene encodes the enzyme carboxypeptidase A1, which are produced by acinar cells and play a role in digestion by hydrolyzing the C-terminal peptide bonds in dietary polypeptide chains. Most of CPA1 genetic variants found in CP patients were located from exons 7 to exon 10 including one of most commonly found variants in CP patients, c.768C>G, p.Asn256Lys (Fig. 2H, Supplementary Table S1) (89). The mechanism how CPA1 mutations increase the risk of pancreatitis is believed to be associated with endoplasmic reticulum stress induced by misfolding rather than increased trypsin activity (89, 90).
CFTR
The CFTR gene encodes an anion channel that transports a chloride (Cl−) and HCO3− across epithelial cell membranes, responsible for normal physiological fluid secretion functions in the pancreas (91). Anion transportation by CFTR is the rate-limiting step for Cl− and HCO3− secretion to epithelial luminal surfaces, which is followed by H2O transport. As a result, CFTR plays a critical role in maintaining the thin, watery mucus. Decreased CFTR expression results in abnormal Cl− and HCO3− secretion, leading to increased sodium (Na＋) absorption and sticky mucus secretion (92-95). As trypsin remains inactive under high pH conditions even if the activation peptide is cleaved within the pancreas, the high pH environment maintained by CFTR plays a critical role to suppresses pancreatitis (73). Thus, CFTR gene mutations ultimately lead cystic fibrosis (CF), which is characterized by the production of thick mucus in the lungs, pancreas, and other organs and 85% of people with CF experience CP early in their lives (96).
Among more than 2,000 mutations found in CFTR gene, pancreatic insufficiency develops when both CFTR alleles harbor severe loss-of-function mutations (Table 1) (97). When a severe mutation on one CFTR allele and a milder mutation on the other allele with some residual CFTR function, the patients develop in CF with CP. Heterozygous carriers of CFTR mutations do not develop CF but have an increased risk of developing CP later in their lives (97). Studies have shown that the CFTR p.Arg75Gln variant increases the risk of CP but not the risk of CF by decreasing permeability of HCO3− without compromising Cl− transport, especially when it occurs together with the SPINK1 p.Asn34Ser mutation (91, 98). However, CFTR p.Arg75Gln mutation alone is not sufficient to cause CP (99).
Disease Modeling of Pancreatitis Using hPSCs
Although many genetic variants are found in pancreatic disease patients, it is difficult to speculate the disease-causation based on genetic study without functional vali-dation. Therefore, disease modeling using hPSCs is an ideal platform to examine the genetic factors for disease mechanism. hPSCs, which include human embryonic stem cells (hESCs) derived from the inner cell mass of blastocysts and induced pluripotent stem cells (iPSCs) reprogrammed from adult cells, possess the ability to form all three germ layers: ectoderm, mesoderm, and endoderm (Fig. 3A) (100). To generate pancreatic cells, hPSCs are first directed towards a definitive endoderm (DE) (101). This is followed by the formation of the primitive gut tube (PGT), which is sometimes referred to as the gut tube endoderm (GTE). PGT gives rise to the foregut, which subsequently forms the stomach, liver, and pancreas, and the midgut and hindgut, which develops into the small and large intestines, respectively. The pancreatic specification TF, PDX1, induces the emerge of PP cells from the posterior portion of the foregut, forming dorsal and ventral pancreas. PDX1-expressing dorsal and ventral PP cells start to express pancreas associated transcription factor 1a (PTF1A) and NK6 homeobox 1 (NKX6-1), eventually fuse to create a multi-layered epithelial structure with a lumen (Fig. 3A). This pancreatic epithelium then expands and branches, forming the tip domain, which expresses PTF1A without NKX6-1, and the trunk domain, which expresses NKX6-1 and SRY-box transcription factor 9 (SOX9) without PTF1A (102). The PTF1A segregation at tip domain initiates the acinar cell differentiation, while the NKX6-1 expression at trunk domain induces rise of endocrine and pancreatic ductal cells (Fig. 3A) (103). The pancreatic differentiation from hPSCs recapitulates in vivo pancreatic development, involving the sequential activation or inhibition of growth factors and signaling pathways using various molecules (Fig. 3B, 3C). The initial step in hPSC differentiation towards DE involves activating the transforming growth factor-beta (TGF-β) pathways by the treatment of Activin A or growth differentiation factor-8 (GDF-8) and WNT pathways by adding CHIR99021 (Fig. 3B, 3C). PDX1 mediates pancreatic lineage commitment from the foregut, which is induced by inhibition of sonic hedgehog and bone morphogenetic protein (BMP) signaling and activating retinoic acid and fibroblast growth factor (FGF) pathway.
The differentiation of endocrine progenitors from the bipotent trunk epithelium is driven by the activation of the pro-endocrine transcription factor neurogenin-3, which can be induced by suppressing Notch, BMP, and TGF-β signaling. Maturation of pancreatic β-cells after birth by expressing insulin secretion-related genes enables β-cells to respond to high glucose levels. TGF-β signaling, thyroid hormone, and gamma-secretase inhibition are involved in the process (Fig. 3B). Pancreatic organoids are made from PP cells via cell aggregation and orbital shaker to induce maturation of cells facilitating the differentiation process (104-107).
As both of exocrine and endocrine cells develop from the PP, the knowledge obtained for endocrine cells also paved a way to study exocrine cells. Several groups have established the differentiation for exocrine lineages by urging PP cells into ductal or acinar lineage after aggregation (16, 108-110). However, the differentiation methods and efficiency significantly vary among groups contrast to endocrine differentiation. Ductal differentiation was established to study CF and PDAC, which are originated from ductal cells. Pancreatic ductal epithelial cell (PDEC) generated by Simsek et al. (109) reached 98% after purification and iPSC derived from CF patient produced PDEC expressed reduced CFTR levels compared to control. Pancreatic duct-like organoids (PDLOs) were also generated to perform PDAC modeling (16, 108, 110). Breunig et al. (110) tested 30 compounds to generate pancreatic trunk-like organoids (PTrLOs) expressing SOX9 and PDX1 which will give rise to ductal cells later. After treating PTrLO with nicotinamide, ZnSO4, epidermal growth factor, and FGF10, carbonic anhydrase II, Keratin 19 and HNF1B expression was confirmed establishing PDLO (Fig. 3). However, CFTR expression was confirmed only in part of organoids implying the maturity of organoids occurred partially (110). Huang et al. (108) generated not only pancreatic ductal cells but also acinar cells expressing PTF1A and CTRC with amylase and lipase activity, promising the hPSC as a platform to study pancreatic exocrine disorder (Fig. 3C).
Discussion
Hereditary pancreatic diseases are good candidates for hPSC-based modeling due to their strong genetic influence. hPSC-derived cells offer a powerful tool to investigate disease mechanisms and test potential therapeutic interventions. For example, iPSCs derived from patients with HNF4A-MODY or HNF1A-MODY have successfully elucidated the underlying disease mechanism by utilizing pancreatic β-cell differentiation protocol from hPSCs (111, 112). Similarly, hPSC-derived pancreatic ductal cells have been utilized to model CFTR (109) and PDAC (16, 108, 110, 113, 114). However, pancreatitis, which need an immune cell involvement, has not yet been examined using hPSC-based models, emphasizing the need for further advancements in this field.
One of the major challenges in the disease modeling using hPSC-derived pancreatic organoids is the lack of robust differentiation method for lineages other than endocrine cells. While endocrine differentiation can generate upto 90% of glucose-responsive insulin-producing β-cells and has progressed to clinical trials for T1D patients (115, 116), the differentiation efficiency of exocrine lineages, such as acinar or ductal cells, remains variable ranging from 5% to 30% depending on the differentiation methods. This low efficiency hinders the ability to detect the subtle phenotypic difference in vitro, often necessitating in vivo transplantation to evaluate their disease phenotypes (16, 108, 110, 113, 114). Moreover, the hPSC-derived pancreatic β-cells frequently exhibit transcriptional and functional immaturity compared to primary β-cells, emphasizing the need for further optimization to achieve fully mature and functional pancreatic cells in vitro.
Another limitation of the current hPSC-derived pancreatic disease modeling is the absence of immune cells, which play a central role in the pathogenesis of inflammation and fibrosis. To address this, an effort has been made to co-culture immune cells with pancreatic cells. While one study was able to recapitulate some aspects of T1D using this approach, its application is limited, as the authors isolated T cells from peripheral blood mononuclear cells of the same human donor who provided cells for iPSCs derivation (117). As most of researchers do not have access to blood of the iPSCs donor, this strategy can be used by a small numbers of scientists. Differentiating T cells and pancreatic cells from the same hPSCs theoretically possible; however, many immunological obstacles must be overcome to establish such a model, including matching the major histocompatibility complex among cells to prevent immune rejection and facilitating appropriate T-cell receptor-antigen interactions.
In conclusion, the disease modeling using hPSCs is a complex process the requires the generation of multiple cell types and the formation of 3D organoids to enable physiologically relevant cell-cell interactions. Despite significant progress in generating insulin-secreting β-cells, current protocols require further optimization to achieve fully mature and functional pancreatic endocrine and exocrine cells in vitro. Therefore, continued research into the mechanisms governing pancreatic development and efforts to incorporate immune cells will be crucial for advancing hPSC-based pancreatic disease modeling.
Supplementary Materials
Supplementary data including one table can be found with this article online at https://doi.org/10.15283/ijsc24036
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