Human Pluripotent Stem Cell-Derived Acinar/Ductal Organoids Generate

Downloaded from http://gut.bmj.com/ on June 1, 2017 - Published by group.bmj.com Gut Online First, published on October 7, 2016 as 10.1136/gutjnl-2016-312423 Pancreas ORIGINAL ARTICLE Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling Meike Hohwieler,1 Anett Illing,1 Patrick C Hermann,1 Tobias Mayer,1 Marianne Stockmann,1 Lukas Perkhofer,1 Tim Eiseler,1 Justin S Antony,2 Martin Müller,1 Susanne Renz,1 Chao-Chung Kuo,3 Qiong Lin,4 Matthias Sendler,5 Markus Breunig,1 Susanne M Kleiderman,1 André Lechel,1 Martin Zenker,6 Michael Leichsenring,7 Jonas Rosendahl,8 Martin Zenke,4 Bruno Sainz Jr,9 Julia Mayerle,5 Ivan G Costa,3 Thomas Seufferlein,1 Michael Kormann,2 Martin Wagner,1 Stefan Liebau,10 Alexander Kleger1 ▸ Additional material is ABSTRACT published online only. To view Objective The generation of acinar and ductal cells Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ from human pluripotent stem cells (PSCs) is a poorly gutjnl-2016-312423). studied process, although various diseases arise from this compartment. What is already known on this subject? Design We designed a straightforward approach to ▸ fi Human pluripotent stem cells (PSCs) present a For numbered af liations see direct human PSCs towards pancreatic organoids end of article. powerful tool for developmental studies and resembling acinar and ductal progeny. regenerative medicine. Correspondence to Results Extensive phenotyping of the organoids not ▸ Directed differentiation of PSCs towards Prof Dr Alexander Kleger, only shows the appropriate marker profile but also pancreatic cell fates requires the formation of Department of Internal ultrastructural, global gene expression and functional PDX1/NKX6.1-positive progenitor cells. Medicine I, University Medical ▸ fi Center Ulm, Albert-Einstein- hallmarks of the human pancreas in the dish. Upon Mutations in the cystic brosis transmembrane Allee 23, Ulm 89081, orthotopic transplantation into immunodeficient mice, conductance regulator (CFTR) perturb fluid Germany; these organoids form normal pancreatic ducts and acinar transport causing chronic airway infections or [email protected] tissue resembling fetal human pancreas without evidence biliary cirrhosis with variable phenotypes while of tumour formation or transformation. Finally, we fi SL and AK jointly supervised the pancreas is one of the rst organs affected. implemented this unique phenotyping tool as a model to this work and contributed fi equally. study the pancreatic facets of cystic fibrosis (CF). For the What are the new ndings? first time, we provide evidence that in vitro, but also in ▸ Efficient generation of high yields of pancreatic Received 13 June 2016 our xenograft transplantation assay, pancreatic progenitors from several human pluripotent Accepted 11 August 2016 commitment occurs generally unhindered in CF. stem cell lines. Importantly, cystic fibrosis transmembrane conductance ▸ PSC-derived pancreatic progenitors form regulator (CFTR) activation in mutated pancreatic pancreatic organoids that comprise acinar/ organoids not only mirrors the CF phenotype in ductal-like progeny and resemble human functional assays but also at a global expression level. pancreas upon orthotopic transplantation in We also conducted a scalable proof-of-concept screen in mice. CF pancreatic organoids using a set of CFTR correctors ▸ Induced PSCs from cystic fibrosis patients and activators, and established an mRNA-mediated gene display normal pancreatic commitment in vitro therapy approach in CF organoids. and in vivo at least until a fetal developmental Conclusions Taken together, our platform provides stage. novel opportunities to model pancreatic disease and ▸ Pancreatic organoids from patients with cystic development, screen for disease-rescuing agents and to fibrosis recapitulate defective CFTR function in test therapeutic procedures. vitro, allowing subsequent drug screening but also mRNA-mediated gene supplementation. How might it impact on clinical practice in the foreseeable future? INTRODUCTION ▸ Our system provides a novel approach to model To cite: Hohwieler M, Given their capacity to differentiate into every cell Illing A, Hermann PC, et al. human pancreatic development and disease. Gut Published Online First: type of the human body, human-induced pluripo- ▸ Humanised platform for (organ-specific and [please include Day Month tent stem cells (hiPSCs) provide a unique platform patient-specific) drug screening and testing of Year] doi:10.1136/gutjnl- for developmental studies and regenerative medi- therapeutic options in vitro and in vivo. – 2016-312423 cine.1 4 The generation of pancreatic progenitor Hohwieler M, et al. Gut 2016;0:1–14. doi:10.1136/gutjnl-2016-312423 1 Copyright Article author (or their employer) 2016. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Downloaded from http://gut.bmj.com/ on June 1, 2017 - Published by group.bmj.com Pancreas (PP) cells from PSCs follows the sequential induction of virtu- differentiation. The backbone medium for the first 6 days of dif- ally pure definitive endoderm (DE), foregut endoderm (GTE) ferentiation was BE1: MCDB131 (Invitrogen) with 0.8 g/L cell – and pancreatic endoderm (PE, figure 1A).5 7 Over the last culture tested glucose (Sigma), 1.174 g/L sodium bicarbonate decade, a series of studies have aimed at improving pancreatic (Sigma), 0.1% fatty acid free (FAF) BSA (A7030, Sigma), 2 mM 5–7 differentiation protocols. While most studies focused on the L-glutamine. Later differentiation was performed in BE3 as the generation of PDX1-positive PE,89true PP cells should coex- backbone medium: MCDB131 with 0.44 g/L glucose, 1.754 g/L 10 press both NKX6.1 and PDX1. While the exocrine and endo- sodium bicarbonate, 2% FAF-BSA, 2 mM L-glutamine, 44 mg/L crine lineages develop, NKX6.1 is still expressed in β cells, but L-ascorbic acid, 0.5× insulin-transferrin-selenium-ethanolamine becomes mutually exclusive with the expression of Ptf1a driving (ITS-X). Cells in differentiation were cultured at 37°C in a 5% 11 the exocrine lineage. Thus, the presence of NKX6.1 is one of CO2 incubator with daily media change. For the first day of dif- the key distinguishing features of these two lineages and hence ferentiation, cells were washed with phosphate buffered saline can be used to monitor the emergence of true progenitors. (PBS) (Sigma) and incubated with BE1 supplemented with Despite recent progress in differentiating PSCs towards endo- 3 mM GSK3β-inhibitor (CHIR99021) (Axon MedChem) and crine pancreatic progeny,12 13 the generation of ductal and 100 ng/mL Activin A. The next day the medium was replaced exocrine-like cells has not yet been adequately achieved, apart by BE1 with 100 ng/mL Activin A. After 3 days, media was from a recent study modelling human pancreatic cancer.14 changed to BE1 with 50 ng/mL KFG (Peprotech) for 2 days. Three-dimensional organoid models generated from PSCs can From day 6 until day 10, cells were cultured in BE3 medium faithfully model the in vivo situation,13 15 16 and disease-specific containing 0.25 mM SANT-1 (Sigma), 2 mM retinoic acid iPSCs allow the generation of distinct human disease (Sigma), 200 nM LDN-193189 (Sigma) and 500 nM models.12 13 15 Nevertheless, the generation of human pancreas PD0325901 (Calbiochem). At days 10–14, the cells received in mice upon xenotransplantation of non-transformed organoids BE3 supplemented with 50 ng/mL fibroblast growth factor has not been achieved to date. However, this would open up (FGF10) (Peprotech), 330 nM Indolactam V (Stem Cell entirely new research avenues. In addition, inherited pancreatic Technologies), 10 mM SB431542 (Axon MedChem) and add- diseases would benefit from in vivo gene supplementation strat- itional 16 mM glucose. An outline of the differentiation proto- egies as recently shown for a lung disease with specifically modi- col can also be found in figure 1A. fied mRNA, pre-evaluated in organoids.17 18 To tackle this unmet need in the pancreatic field, we describe herein a new PSC-based organoid system that was used to model pancreatic aspects of cystic fibrosis (CF). 3D pancreatic organoid culture CF is an inherited disease caused by either nonsense or mis- At the PP stage (day 12 of differentiation), cells were washed sense mutations in the cystic fibrosis transmembrane conduct- with PBS, incubated with TrypLE at 37°C for 5–6 min and care- ance regulator (CFTR) gene, resulting in complete absence of fully resuspended in DMEM/F12 resulting in clumps of 3–10 the protein, a misfolded polypeptide that is degraded by the cells. After centrifugation at 400 g for 5 min, the pellet was unfolded protein response, or a dysfunctional protein.19 CFTR washed in BE3 medium, centrifuged again and resuspended in encodes a chloride channel gated by cyclic AMP-dependent precooled day 12 medium (detailed above) supplemented with phosphorylation that is necessary for electrolyte and fluid 10 mM Rock inhibitor. The cell suspension was mixed on ice homeostasis of epithelia in various organs including the lung, with growth factor reduced matrigel at a 1:3 ratio and 25 mL liver, intestine and pancreas. Dysfunction of the CFTR leads to were transferred to a 48-well plate (NunclonΔSurface). the production of hyperviscous mucus causing chronic airway Following incubation for 10 min at 37°C, the solidified drop of infections or biliary cirrhosis with variable phenotypes.20 matrigel was overlayed with 200 mL of day 12 medium (see Although the pancreas is one of the first organs affected, knowl- above) containing 10 mM Rock inhibitor (always added for the edge about the pathophysiology of the pancreas during CF is first 4 days in three-dimensional (3D) culture), which was limited. Briefly, distinct CFTR genotypes have been shown to replaced the next day. On day 14, differentiation was continued not only increase the probability of developing either pancrea- with 10 ng/mL FGF2 and 10 mM Rock inhibitor in BE3 titis or perinatal exocrine insufficiency21 but also pancreatic medium.

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