DOI QR코드

DOI QR Code

Pancreatic Diseases: Genetics and Modeling Using Human Pluripotent Stem Cells

  • Yuri Lee (Graduate School of Pharmaceutical Sciences, Ewha Womans University) ;
  • Kihyun Lee (Graduate School of Pharmaceutical Sciences, Ewha Womans University)
  • Received : 2024.03.29
  • Accepted : 2024.04.01
  • Published : 2024.08.30

Abstract

Pancreas serves endocrine and exocrine functions in the body; thus, their pathology can cause a broad range of irreparable consequences. Endocrine functions include the production of hormones such as insulin and glucagon, while exocrine functions involve the secretion of digestive enzymes. Disruption of these functions can lead to conditions like diabetes mellitus and exocrine pancreatic insufficiency. Also, the symptoms and causality of pancreatic cancer very greatly depends on their origin: pancreatic ductal adenocarcinoma is one of the most fatal cancer; however, most of tumor derived from endocrine part of pancreas are benign. Pancreatitis, an inflammation of the pancreatic tissues, is caused by excessive alcohol consumption, the bile duct obstruction by gallstones, and the premature activation of digestive enzymes in the pancreas. Hereditary pancreatic diseases, such as maturity-onset diabetes of the young and hereditary pancreatitis, can be a candidate for disease modeling using human pluripotent stem cells (hPSCs), due to their strong genetic influence. hPSC-derived pancreatic differentiation has been established for cell replacement therapy for diabetic patients and is robustly used for disease modeling. The disease modeling platform that allows interactions between immune cells and pancreatic cells is necessary to perform in-depth investigation of disease pathogenesis.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIT) (RS-2023-00261247, RS-2023-00223069) and the Ewha Womans University Research Grant of 2022.

References

  1. Saeedi P, Petersohn I, Salpea P, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract 2019;157:107843 
  2. Iannuzzi JP, King JA, Leong JH, et al. Global incidence of acute pancreatitis is increasing over time: a systematic review and meta-analysis. Gastroenterology 2022;162:122-134 
  3. Park W, Chawla A, O'Reilly EM. Pancreatic cancer: a review. JAMA 2021;326:851-862 
  4. Redondo MJ, Balasubramanyam A. Toward an improved classification of type 2 diabetes: lessons from research into the heterogeneity of a complex disease. J Clin Endocrinol Metab 2021;106:e4822-e4833 
  5. Sodhi M, Rezaeianzadeh R, Kezouh A, Etminan M. Risk of gastrointestinal adverse events associated with glucagon-like peptide-1 receptor agonists for weight loss. JAMA 2023;330:1795-1797 
  6. Patel F, Gan A, Chang K, Vega KJ. Acute pancreatitis in a patient taking semaglutide. Cureus 2023;15:e43773 
  7. Abdulreda MH, Caicedo A, Berggren PO. A natural body window to study human pancreatic islet cell function and survival. CellR4 Repair Replace Regen Reprogram 2013;1:111-122 
  8. Dolensek J, Rupnik MS, Stozer A. Structural similarities and differences between the human and the mouse pancreas. Islets 2015;7:e1024405 
  9. Furman BL. Streptozotocin-induced diabetic models in mice and rats. Curr Protoc 2021;1:e78 
  10. Yang H, Wright JR Jr. Human beta cells are exceedingly resistant to streptozotocin in vivo. Endocrinology 2002;143:2491-2495 
  11. Nemeth BC, Wartmann T, Halangk W, Sahin-Toth M. Autoactivation of mouse trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop. J Biol Chem 2013;288:24049-24062 
  12. Cala G, Sina B, De Coppi P, Giobbe GG, Gerli MFM. Primary human organoids models: current progress and key milestones. Front Bioeng Biotechnol 2023;11:1058970 
  13. Fasolino M, Schwartz GW, Patil AR, et al. Single-cell multi-omics analysis of human pancreatic islets reveals novel cellular states in type 1 diabetes. Nat Metab 2022;4:284-299 
  14. Millman JR, Xie C, Van Dervort A, Gurtler M, Pagliuca FW, Melton DA. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nat Commun 2016;7:11463 
  15. Boj SF, Hwang CI, Baker LA, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 2015;160:324-338 
  16. Huang L, Holtzinger A, Jagan I, et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nat Med 2015;21:1364-1371 
  17. Seino T, Kawasaki S, Shimokawa M, et al. Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression. Cell Stem Cell 2018;22:454-467.e6 
  18. Loomans CJM, Williams Giuliani N, Balak J, et al. Expansion of adult human pancreatic tissue yields organoids harboring progenitor cells with endocrine differentiation potential. Stem Cell Reports 2018;10:712-724 
  19. Bonfanti P, Nobecourt E, Oshima M, et al. Ex vivo expansion and differentiation of human and mouse fetal pancreatic progenitors are modulated by epidermal growth factor. Stem Cells Dev 2015;24:1766-1778 
  20. Adolph TE, Mayr L, Grabherr F, Schwarzler J, Tilg H. Pancreas-microbiota cross talk in health and disease. Annu Rev Nutr 2019;39:249-266 
  21. Puri S, Folias AE, Hebrok M. Plasticity and dedifferentiation within the pancreas: development, homeostasis, and disease. Cell Stem Cell 2015;16:18-31 
  22. Roder PV, Wu B, Liu Y, Han W. Pancreatic regulation of glucose homeostasis. Exp Mol Med 2016;48:e219 
  23. Brissova M, Fowler MJ, Nicholson WE, et al. Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J Histochem Cytochem 2005;53:1087-1097 
  24. Keller J, Layer P. Human pancreatic exocrine response to nutrients in health and disease. Gut 2005;54(Suppl 6):vi1-vi28 
  25. Dominguez-Munoz JE. Pancreatic enzyme therapy for pancreatic exocrine insufficiency. Curr Gastroenterol Rep 2007;9:116-122 
  26. Halbrook CJ, Lyssiotis CA, Pasca di Magliano M, Maitra A. Pancreatic cancer: advances and challenges. Cell 2023;186:1729-1754 
  27. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023;73:17-48 
  28. Pantaleo A, Forte G, Fasano C, et al. Understanding the genetic landscape of pancreatic ductal adenocarcinoma to support personalized medicine: a systematic review. Cancers (Basel) 2023;16:56 
  29. Ying H, Dey P, Yao W, et al. Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev 2016;30:355-385 
  30. Leung PS. Common pancreatic disease. Adv Exp Med Biol 2010;690:29-51 
  31. Weiss FU, Laemmerhirt F, Lerch MM. Etiology and risk factors of acute and chronic pancreatitis. Visc Med 2019;35:73-81 
  32. Mitra V, Munnelly S, Grammatikopoulos T, et al. The top 10 research priorities for pancreatitis: findings from a James Lind Alliance priority setting partnership. Lancet Gastroenterol Hepatol 2023;8:780-782 
  33. Bellin MD, Whitcomb DC, Abberbock J, et al. Patient and disease characteristics associated with the presence of diabetes mellitus in adults with chronic pancreatitis in the United States. Am J Gastroenterol 2017;112:1457-1465 
  34. Kleeff J, Whitcomb DC, Shimosegawa T, et al. Chronic pancreatitis. Nat Rev Dis Primers 2017;3:17060 
  35. Ma DM, Dong XW, Han X, et al. Pancreatitis and pancreatic cancer risk. Technol Cancer Res Treat 2023;22:15330338231164875 
  36. Beltrand J, Busiah K, Vaivre-Douret L, et al. Neonatal diabetes mellitus. Front Pediatr 2020;8:540718 
  37. Urakami T. Maturity-onset diabetes of the young (MODY): current perspectives on diagnosis and treatment. Diabetes Metab Syndr Obes 2019;12:1047-1056 
  38. Tshivhase A, Matsha T, Raghubeer S. Diagnosis and treatment of MODY: an updated mini review. Appl Sci 2021;11:9436 
  39. Nkonge KM, Nkonge DK, Nkonge TN. The epidemiology, molecular pathogenesis, diagnosis, and treatment of maturity-onset diabetes of the young (MODY). Clin Diabetes Endocrinol 2020;6:20 
  40. Ebrahim N, Shakirova K, Dashinimaev E. PDX1 is the cornerstone of pancreatic β-cell functions and identity. Front Mol Biosci 2022;9:1091757 
  41. Rubio-Cabezas O, Minton JA, Kantor I, Williams D, Ellard S, Hattersley AT. Homozygous mutations in NEUROD1 are responsible for a novel syndrome of permanent neonatal diabetes and neurological abnormalities. Diabetes 2010;59:2326-2331 
  42. Beinsteiner B, Billas IML, Moras D. Structural insights into the HNF4 biology. Front Endocrinol (Lausanne) 2023;14:1197063 
  43. Delvecchio M, Pastore C, Giordano P. Treatment options for MODY patients: a systematic review of literature. Diabetes Ther 2020;11:1667-1685 
  44. Hulin J, Skopkova M, Valkovicova T, et al. Clinical implications of the glucokinase impaired function - GCK MODY today. Physiol Res 2020;69:995-1011 
  45. Zhao Q, Ding L, Yang Y, et al. Clinical characteristics of patients with HNF1-alpha MODY: a literature review and retrospective chart review. Front Endocrinol (Lausanne) 2022;13:900489 
  46. Shepherd M, Ellis I, Ahmad AM, et al. Predictive genetic testing in maturity-onset diabetes of the young (MODY). Diabet Med 2001;18:417-421 
  47. Francis Y, Tiercelin C, Alexandre-Heyman L, Larger E, Dubois-Laforgue D. HNF1B-MODY masquerading as type 1 diabetes: a pitfall in the etiological diagnosis of diabetes. J Endocr Soc 2022;6:bvac087 
  48. Neve B, Fernandez-Zapico ME, Ashkenazi-Katalan V, et al. Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function. Proc Natl Acad Sci USA 2005;102:4807-4812 
  49. Johansson BB, Fjeld K, El Jellas K, et al. The role of the carboxyl ester lipase (CEL) gene in pancreatic disease. Pancreatology. 2018;18:12-19 
  50. Nelson HA, Johnson LM. Hereditary pancreatitis in a young adult: acute to chronic. Clin Biochem 2021;98:78-80 
  51. Rebours V, Boutron-Ruault MC, Schnee M, et al. The natural history of hereditary pancreatitis: a national series. Gut 2009;58:97-103 
  52. Rebours V, Boutron-Ruault MC, Schnee M, et al. Risk of pancreatic adenocarcinoma in patients with hereditary pancreatitis: a national exhaustive series. Am J Gastroenterol 2008;103:111-119 
  53. Piseddu I, Vielhauer J, Mayerle J. Genetic testing in acute and chronic pancreatitis. Curr Treat Options Gastro 2022;20:429-444 
  54. Rinderknecht H. Activation of pancreatic zymogens. Normal activation, premature intrapancreatic activation, protective mechanisms against inappropriate activation. Dig Dis Sci 1986;31:314-321 
  55. Hegyi E, Sahin-Toth M. Genetic risk in chronic pancreatitis: the trypsin-dependent pathway. Dig Dis Sci 2017;62:1692-1701 
  56. Girodon E, Rebours V, Chen JM, et al. Clinical interpretation of PRSS1 variants in patients with pancreatitis. Clin Res Hepatol Gastroenterol 2021;45:101497 
  57. Nemeth BC, Sahin-Toth M. Human cationic trypsinogen (PRSS1) variants and chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol 2014;306:G466-G473 
  58. Rowen L, Koop BF, Hood L. The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 1996;272:1755-1762 
  59. Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141-145 
  60. Gorry MC, Gabbaizedeh D, Furey W, et al. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997;113:1063-1068 
  61. Teich N, Mossner J, Keim V. Mutations of the cationic trypsinogen in hereditary pancreatitis. Hum Mutat 1998;12:39-43 
  62. Howes N, Lerch MM, Greenhalf W, et al. Clinical and genetic characteristics of hereditary pancreatitis in Europe. Clin Gastroenterol Hepatol 2004;2:252-261 
  63. Whitcomb DC, Preston RA, Aston CE, et al. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 1996;110:1975-1980 
  64. Solomon S, Gelrud A, Whitcomb DC. Low penetrance pancreatitis phenotype in a Venezuelan kindred with a PRSS1 R122H mutation. JOP 2013;14:187-189 
  65. Szmola R, Sahin-Toth M. Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: identity with Rinderknecht's enzyme Y. Proc Natl Acad Sci USA 2007;104:11227-11232 
  66. Wartmann T, Mayerle J, Kahne T, et al. Cathepsin L inactivates human trypsinogen, whereas cathepsin L-deletion reduces the severity of pancreatitis in mice. Gastroenterology 2010;138:726-737 
  67. Szabo A, Sahin-Toth M. Determinants of chymotrypsin C cleavage specificity in the calcium-binding loop of human cationic trypsinogen. FEBS J 2012;279:4283-4292 
  68. Szabo A, Sahin-Toth M. Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C. J Biol Chem 2012;287:20701-20710 
  69. Geisz A, Hegyi P, Sahin-Toth M. Robust autoactivation, chymotrypsin C independence and diminished secretion define a subset of hereditary pancreatitis-associated cationic trypsinogen mutants. FEBS J 2013;280:2888-2899 
  70. Joergensen MT, Geisz A, Brusgaard K, et al. Intragenic duplication: a novel mutational mechanism in hereditary pancreatitis. Pancreas 2011;40:540-546 
  71. Chen JM, Kukor Z, Le Marechal C, et al. Evolution of trypsinogen activation peptides. Mol Biol Evol 2003;20:1767-1777 
  72. Nemoda Z, Sahin-Toth M. The tetra-aspartate motif in the activation peptide of human cationic trypsinogen is essential for autoactivation control but not for enteropeptidase recognition. J Biol Chem 2005;280:29645-29652 
  73. LaRusch J, Whitcomb DC. Genetics of pancreatitis. Curr Opin Gastroenterol 2011;27:467-474 
  74. Lasson A, Borgstrom A, Ohlsson K. Elevated pancreatic secretory trypsin inhibitor levels during severe inflammatory disease, renal insufficiency, and after various surgical procedures. Scand J Gastroenterol 1986;21:1275-1280 
  75. Ogawa M. Pancreatic secretory trypsin inhibitor as an acute phase reactant. Clin Biochem 1988;21:19-25 
  76. Pfutzer RH, Barmada MM, Brunskill AP, et al. SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis. Gastroenterology 2000;119:615-623 
  77. Aoun E, Muddana V, Papachristou GI, Whitcomb DC. SPINK1 N34S is strongly associated with recurrent acute pancreatitis but is not a risk factor for the first or sentinel acute pancreatitis event. Am J Gastroenterol 2010;105:446-451 
  78. Kume K, Masamune A, Kikuta K, Shimosegawa T. [-215 G>A; IVS3+2T>C] mutation in the SPINK1 gene causes exon 3 skipping and loss of the trypsin binding site. Gut 2006;55:1214 
  79. Kiraly O, Wartmann T, Sahin-Toth M. Missense mutations in pancreatic secretory trypsin inhibitor (SPINK1) cause intracellular retention and degradation. Gut 2007;56:1433-1438 
  80. Kiraly O, Boulling A, Witt H, et al. Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor (SPINK1) cause autosomal dominant hereditary pancreatitis. Hum Mutat 2007;28:469-476 
  81. Kereszturi E, Kiraly O, Sahin-Toth M. Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis. Gut 2009;58:545-549 
  82. Boulling A, Le Marechal C, Trouve P, Raguenes O, Chen JM, Ferec C. Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene. Eur J Hum Genet 2007;15:936-942 
  83. Threadgold J, Greenhalf W, Ellis I, et al. The N34S mutation of SPINK1 (PSTI) is associated with a familial pattern of idiopathic chronic pancreatitis but does not cause the disease. Gut 2002;50:675-681 
  84. Aoun E, Chang CC, Greer JB, Papachristou GI, Barmada MM, Whitcomb DC. Pathways to injury in chronic pancreatitis: decoding the role of the high-risk SPINK1 N34S haplotype using meta-analysis. PLoS One 2008;3:e2003 
  85. Zhou J, Sahin-Toth M. Chymotrypsin C mutations in chronic pancreatitis. J Gastroenterol Hepatol 2011;26:1238-1246 
  86. Rosendahl J, Witt H, Szmola R, et al. Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis. Nat Genet 2008;40:78-82 
  87. Masson E, Chen JM, Scotet V, Le Marechal C, Ferec C. Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis. Hum Genet 2008;123:83-91 
  88. Beer S, Zhou J, Szabo A, et al. Comprehensive functional analysis of chymotrypsin C (CTRC) variants reveals distinct loss-of-function mechanisms associated with pancreatitis risk. Gut 2013;62:1616-1624 
  89. Witt H, Beer S, Rosendahl J, et al. Variants in CPA1 are strongly associated with early onset chronic pancreatitis. Nat Genet 2013;45:1216-1220 
  90. Nemeth BC, Orekhova A, Zhang W, et al. Novel p.K374E variant of CPA1 causes misfolding-induced hereditary pancreatitis with autosomal dominant inheritance. Gut 2020;69:790-792 
  91. Schneider A, Larusch J, Sun X, et al. Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis. Gastroenterology 2011;140:162-171 
  92. Pezzulo AA, Tang XX, Hoegger MJ, et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 2012;487:109-113 
  93. McMaster P. Bile studies after liver transplantation. Ann R Coll Surg Engl 1979;61:435-440 
  94. Derichs N, Jin BJ, Song Y, Finkbeiner WE, Verkman AS. Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching. FASEB J 2011;25:2325-2332 
  95. Chen JH, Stoltz DA, Karp PH, et al. Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia. Cell 2010;143:911-923 
  96. Stallings VA, Stark LJ, Robinson KA, Feranchak AP, Quinton H. Evidence-based practice recommendations for nutrition-related management of children and adults with cystic fibrosis and pancreatic insufficiency: results of a systematic review. J Am Diet Assoc 2008;108:832-839 
  97. Hegyi P, Wilschanski M, Muallem S, et al. CFTR: a new horizon in the pathomechanism and treatment of pancreatitis. Rev Physiol Biochem Pharmacol 2016;170:37-66 
  98. Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR, Cohn JA. Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology 2001;121:1310-1319 
  99. Berke G, Gede N, Szadai L, et al. Bicarbonate defective CFTR variants increase risk for chronic pancreatitis: a meta-analysis. PLoS One 2022;17:e0276397 
  100. Liang G, Zhang Y. Embryonic stem cell and induced pluripotent stem cell: an epigenetic perspective. Cell Res 2013;23:49-69 
  101. Jin W, Jiang W. Stepwise differentiation of functional pancreatic β cells from human pluripotent stem cells. Cell Regen 2022;11:24 
  102. Duque M, Amorim JP, Bessa J. Ptf1a function and transcriptional cis-regulation, a cornerstone in vertebrate pancreas development. FEBS J 2022;289:5121-5136 
  103. Schaffer AE, Freude KK, Nelson SB, Sander M. Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors. Dev Cell 2010;18:1022-1029 
  104. Hogrebe NJ, Maxwell KG, Augsornworawat P, Millman JR. Generation of insulin-producing pancreatic β cells from multiple human stem cell lines. Nat Protoc 2021;16:4109-4143 
  105. Pagliuca FW, Millman JR, Gurtler M, et al. Generation of functional human pancreatic β cells in vitro. Cell 2014;159:428-439 
  106. Liang S, Zhao J, Baker RK, Tran E, Zhan L, Kieffer TJ. Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system. Cell Rep Methods 2023;3:100466 
  107. Veres A, Faust AL, Bushnell HL, et al. Charting cellular identity during human in vitro β-cell differentiation. Nature 2019;569:368-373 
  108. Huang L, Desai R, Conrad DN, et al. Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids. Cell Stem Cell 2021;28:1090-1104.e6 
  109. Simsek S, Zhou T, Robinson CL, et al. Modeling cystic fibrosis using pluripotent stem cell-derived human pancreatic ductal epithelial cells. Stem Cells Transl Med 2016;5:572-579 
  110. Breunig M, Merkle J, Wagner M, et al. Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell 2021;28:1105-1124.e19 
  111. Ng NHJ, Jasmen JB, Lim CS, et al. HNF4A haploinsufficiency in MODY1 abrogates liver and pancreas differentiation from patient-derived induced pluripotent stem cells. iScience 2019;16:192-205 
  112. Cujba AM, Alvarez-Fallas ME, Pedraza-Arevalo S, et al. An HNF1α truncation associated with maturity-onset diabetes of the young impairs pancreatic progenitor differentiation by antagonizing HNF1β function. Cell Rep 2022;38:110425 
  113. Kim J, Hoffman JP, Alpaugh RK, et al. An iPSC line from human pancreatic ductal adenocarcinoma undergoes early to invasive stages of pancreatic cancer progression. Cell Rep 2013;3:2088-2099 
  114. Hohwieler M, Illing A, Hermann PC, et al. Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut 2017;66:473-486 
  115. Shapiro AMJ, Thompson D, Donner TW, et al. Insulin expression and C-peptide in type 1 diabetes subjects implanted with stem cell-derived pancreatic endoderm cells in an encapsulation device. Cell Rep Med 2021;2:100466 
  116. Ramzy A, Thompson DM, Ward-Hartstonge KA, et al. Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes. Cell Stem Cell 2021;28:2047-2061.e5 
  117. Leite NC, Sintov E, Meissner TB, et al. Modeling type 1 diabetes in vitro using human pluripotent stem cells. Cell Rep 2020;32:107894