Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology

doi:10.21203/rs.3.rs-1846922/v1

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Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology

https://doi.org/10.21203/rs.3.rs-1846922/v1

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published 28 Jun, 2023

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Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca²⁺ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca²⁺ activated Cl⁻ channel Anoctamin 1 (ANO1) and the epithelial Na⁺ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl⁻ measurement have improved dynamic range, when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

Apical-out

3D culture

ductal cell

organoid

CFTR

Secretory epithelial cells act as a barrier between the lumen of different organs and the rest of the body, whereas they govern ion and fluid secretory processes to determine the amount and composition of the secreted juice. The pancreatic ductal epithelial cells secrete ~ 2 L HCO₃⁻ rich alkaline fluid daily that is essential to maintain physiological operation of the pancreas by flushing out bioactive molecules (such as digestive enzymes) from the ductal tree to the duodenum, preventing early proenzyme activation and thus tissue damage or failure ^1–3. On the other hand, impaired ductal secretion is a common feature in distinct pathophysiological conditions of the pancreas such as the genetic disorder cystic fibrosis (CF) and acute or chronic pancreatitis ^4–8. The vectorial ion and fluid secretion of the ductal cells are largely determined by the expression of ion channels and transporter proteins that display a strictly regulated apical-to-basal polarity ^1,9. According to the currently accepted model of ion secretion, the pancreatic ductal cells express the cystic fibrosis transmembrane regulator (CFTR) Cl⁻ channel and SLC26A6 Cl⁻/HCO₃⁻ exchanger on the apical membrane ¹. The functional interplay of these proteins and physical interaction of the R domain of CFTR and STAS domain of SLC26A6 enable the pancreatic ductal cells to generate a remarkably high intraluminal HCO₃⁻ concentration that is ~ 140 mM in humans ¹⁰. However currently the mechanism of the human HCO₃⁻ secretion is still debated and not completely understood.

One major bottleneck in the study of pancreatic secretory processes is the lack of human-relevant model systems that provide access to the apical membrane of ductal cells. Although, adherent cell lines and animal models are widely used and universal tools in both basic research and biomedical drug discovery they all have certain limitations ¹¹. Due to frequent species-specific differences, extrapolation of results from models or clinical translation of basic research findings remained a major unmet need. The recently developed patient-derived organoid cultures may provide a physiologically more relevant platform resolving previous difficulties ^12–14. Epithelial organoids are ex vivo three-dimensional (3D) cell cultures originated from Leucine-rich repeat-containing G-protein coupled receptor 5 positive (LGR5+) adult stem cells, which shows high self-renewal capacity in general ^15,16. Organoids can be established from human tissue samples and can be maintained for several passages making it easier to conduct studies where the availability of primary cells is limited, such as in case of the lung or pancreas ^12,17. Our group previously demonstrated that the expression, localization and function of key proteins involved in ductal fluid and ion secretion (CFTR, Na⁺/H⁺ exchanger 1. – NHE1 and electrogenic Na⁺/HCO₃⁻ cotransporter 1 – NBCe1) are equivalent in the pancreatic organoids and the primary isolated ductal fragments in mice ¹⁸. Moreover, the HCO₃⁻ secretion and CFTR activity is equivalent in the pancreatic organoids and the isolated ductal fragments. However, direct accessibility of the apical membrane which would be essential for basic physiological investigations and to develop drug screening assays is still difficult to implement. In addition, due to the vectorial ion and fluid transport into the lumen of the organoids, the increasing intraluminal pressure alone may be sufficient to negatively regulate epithelial secretory or barrier functions via Piezo1 mechanosensitive receptor ¹⁹. One possible strategy to overcome this could be the manipulation of the cell polarity in organoids, which was successfully applied in intestinal and airway organoids by the removal of the extracellular matrix (ECM) to investigate host-pathogen interactions and infectious diseases ^20–22. However, it is currently unknown how the polarity switch may alter the epithelial cell functions.

Therefore, we aimed to establish an advanced culturing method for human pancreas derived organoids based on ECM-removal-induced apical-to-basal polarity switching. Our results demonstrated that the cell and nuclei diameter in the apical-out organoids decreased indicating the elimination of the intraluminal pressure, whereas the resting intracellular Ca²⁺ concentration was more consistent in the apical-out organoids. Using this advanced model, we provided evidence about the expression and function of the Ca²⁺ activated Cl⁻ channel Anoctamin 1 (ANO1) and the epithelial Na⁺ channel (ENaC) in the ductal cells. Finally, we demonstrated that the available functional assays, such as forskolin-induced swelling, or intracellular Cl⁻ measurement have improved dynamic range, when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

L-WRN conditioned media

L-WRN cell line was grown in selection medium containing 10% FBS and 0.5–0.5 mg/ml G418 and Hygromycin B in ATCC-formulated DMEM ²³. The conditioned medium supplemented with 10% FBS and 1–1% kanamycin-sulfate and antibiotic-antimycotic solution has been collected three times from the date of plating every three days and pooled before further applications to minimalize batch-to-batch variation. Materials are listed in Supplementary Table 1.

Generation of human pancreas organoid cultures and manipulation of cell polarity

Human pancreatic tissue samples were collected from cadaver donors (Ethical approval No.: 37/2017-SZTE). Establishment and maintenance protocols were described previously ²³. Briefly, collected tissue samples were placed in splitting media (Supplementary Table 2.) and enzymatic digestion at 37^oC was performed in digestion media (Supplementary Table 3.). Efficiency of the tissue digestion was verified by stereomicroscopy every 10 minutes. The digested cell suspension was centrifuged (200 RCF, 10 min, 4ºC, Rotor radius: 180 mm) and the pellet was washed 3 times in total in wash media (Supplementary Table 4.) and resuspended in Matrigel. Next, 10 µl Matrigel was placed in each well of a 24-well cell culture plate and left for 10 minutes for solidification at 37ºC and 500 µl feeding media was applied in each well (Supplementary Table 5.). For culture splitting/subculturing, Matrigel removal and cell separation were performed simultaneously by using a 25V/V% TrypLE™ Express Enzyme in DPBS at 37ºC for 15-20 minutes in a vertical shaker followed by two times washing and plating steps as described above. Polarity change of fully grown organoids was induced by extracellular matrix removal with a 7-minute-long treatment of previously described digestion media (Supplementary Table 3.) at 37^oC followed by two gentle washing steps (parameters of centrifugation: 8 RCF, 10 min, 4ºC, Rotor radius: 180 mm). Organoids with retained cystic structure were replaced in 6-well plates pre-treated with Anti-Adherence Rinsing Solution and fed with feeding medium described above. All applied materials are listed in Supplementary Table 2-6.

Cryopreservation of primary epithelial cells

For cryopreservation organoids were digested to single-cell suspension by TrypLE™ Express Enzyme as described above. After 3 washing steps (50 RCF, 5 min, 4ºC, Rotor radius: 180 mm) the cell pellet was resuspended in Cryo medium based on feeding media and supplemented with 40% FBS and 5% DMSO. Cells were frozen in a propanol filled container (Nalgene Mr. Frosty, Sigma, C1562) according to the manufacturer’s protocol and stored in liquid nitrogen long-term. Materials are listed in Supplementary Table 7.

Gene expression analysis by qRT-PCR

Apical-in and apical-out hPOCs were used for RNA purification by NucleoZOL reagent. RNA concentration and purity of the samples were checked by NanoDrop spectrophotometer. In total, 1 μg purified mRNA was used for cDNA synthesis which was carried out by iScript cDNA Synthesis kit. RT-PCR reactions were performed by SsoAdvanced Universal SYBR Green Supermix and CFTR and ANO1 cDNA-specific primers. Relative gene expression analysis was performed by ΔΔCq technique. All the applied primers sets and reagents are listed in Supplementary Table 8-9.

Gene expression analysis of hPOCs by RNA-Seq

RNA was extracted from collected cell pellet by NucleoSpin RNA Plus kit according to the manufacturer’s protocol (Supplementary Table 10.). RNA-sequencing performed by Illumina NextSeq 500 instrument and data analyses process service were provided by DeltaBio2000 Ltd. Gene expression pattern was determined according to TPM (transcript/million) values (TPM data table 1).

siRNA transfection

Apical-out hPOCs were transfected with 50 nM siRNA or siGLOGreen transfection indicator and Lipofectamine 2000 in Feeding media (Supplementary Table 5) for 24h. Pre-designed siRNA sets used for gene specific knockdown are listed in Supplementary Table 11.

Immunofluorescent labelling and confocal microscopy

Immunofluorescent labelling on organoid cross sections was performed as previously described ²³. Briefly, organoids were frozen and sectioned in Shandon™ Cryomatrix™ at -20 °C. Sections (7 μm thick) were placed on microscope slides. Samples were fixed 4% PFA-PBS for 20 minutes followed by washing for 3x5 minutes in PBS. Antigen retrieval was performed in Sodium Citrate - Tween20 buffer (0.001 M Sodium Citrate Buffer, pH 6.0 and 0.05% Tween20) at 94 °C for 30 min followed by blocking step with 0.1% goat serum and 10% BSA in PBS for 2 hours at 37°C. Primary antibodies were applied overnight at 4°C while secondary antibodies were incubated for 2 hours at room temperature. Nuclear staining and mounting carried out simultaneously by ProLong™ Gold Antifade mounting medium with DAPI. Images were captured by a Zeiss LSM880 confocal microscope using a 40X oil immersion objective (Zeiss, NA: 1.4). Antibodies and materials applied are listed in Supplementary Table 12.

Scanning Electron Microscopy

Organoids were fixed with 2.5% (v/v) glutaraldehyde and 0.05 M cacodylate buffer (pH 7.2) in PBS overnight at 4 ◦C. 8 μL samples spotted onto a silicon disc coated with 0.01% (w/v) poly-l-lysine. Discs were washed twice with PBS and dehydrated with a graded ethanol series (30%, 50%, 70%, 80%, 100% ethanol (v/v), for 2 h each at 4 ◦C and 100% EtOH, for overnight). Following dehydration, samples were immersed for 5 mins in pure hexamethyldisilazane (HDMS) and air dried. All specimens were mounted on aluminium stubs using double-sided carbon tape and then were sputter coated with 15 nm gold in a sputter coater (Quorum Technologies, Laughton, East Sussex, UK) and observed under a Zeiss Sigma 300 Field-Emission scanning electron microscope (ZEISS). Materials are listed in Supplementary Table 13.

Fluorescent microscopy and reverse swelling assay

Apical-in and apical-out organoids were attached to poly-L-lysine coated cover glasses and were incubated in HEPES solution for 30 min at 37 °C with 5% CO₂. BCECF-AM, Fura2-AM (5-5 μmol/L) and MQAE (2 μmol/L) fluorescent indicators were applied for pH, Ca²⁺ and Cl^- measurements, respectively as previously described ¹⁸. After loading with the fluorescent indicator, the samples were mounted on an Olympus IX73 inverted microscope and were excited with a CoolLED PE-4000 (BCECF-AM) or a CoolLED pE-340fura (Fura2-AM and MQAE) illumination system. Filter combinations for each fluorescent dye were described previously ^18,24. Hamamatsu Orca-Flash 4.0 sCMOS camera and 20X water immersion objective (Olympus; NA: 0.8) with a temporal resolution of 1 sec were used for capturing fluorescent signals. Ratiometric image analysis for pH_i measurements was performed by Olympus Excellence software. Fluorescence signals of Fura2- and MQAE-loaded samples were normalized and represented as F1/F0 values. During reverse swelling assay transmission light illumination was applied. Images were taken in every minute and were analyzed and quantified by ImageJ software. Reagents and inhibitors applied in experiments are listed in Supplementary Table 14-15.

Statistical analysis

Data are expressed as means ± SEM. Significant difference between groups was determined by Mann-Whitney test. P < 0.05 was accepted as being significant. All statistical analysis were carried out by GraphPad Prism software (Version 8.3.1.).

Primary epithelial organoids established from human pancreatic tissue samples retain ductal characteristics and polarity

Pancreatic tissue samples were collected from 11 cadaver donors with no documented exocrine or endocrine pancreatic disease (6 male and 5 female patients; mean age 48 ± 9.798 years; mean BMI 25.72 kg/m²). After mechanical dissociation and enzymatic tissue digestion human pancreatic organoid cultures (hPOCs) were established in Matrigel and grown in feeding media containing Wnt3A/R-spondin/Noggin conditioned medium. Organoids were successfully generated from all 11 samples, which were subcultured and cryopreserved in large quantities after the first passaging step for further application (Fig. 1.A.). Cystic organoids appeared in the heterogeneous cell suspension on the second day of culture, while erythrocytes, acinar and stromal cells degraded. During passaging the organoids were enzymatically digested to single cells ensuring the clonal growth of the hPOCs (Fig. 1.B.). Cell fate of the cells was first investigated by RNA-sequencing, where the active expression of the adult stem cell marker LGR5 (Leucin-rich repeat-containing G-protein coupled receptor 5) and ductal markers such as cystic fibrosis transmembrane conductance regulator (CFTR), cytokeratin 19 (KRT19), occludin (OCLN), SRY-Box transcription factor 9 (SOX9), epithelial cell adhesion molecule (EPCAM), e-cadherin (CDH1), Hes family BHLH Transcription Factor 1 (HES1) was observed, while the absence of acinar- (amylase, AMY1A-C) endocrine- (Pancreatic polypeptide, PPY; Insulin, INS; Chromogranin A-B, (CHGA-B) and hematopoietic (vascular endothelial cadherin, CDH5) markers confirmed that the human pancreatic organoids containing ductal epithelial cells exclusively (Fig. 1.C). These ductal markers such as KRT19, SOX9, HNF1B and FOXA2 showed a typical localization pattern on the protein level. Moreover, the apical-to-basal polarity of the organoids was evidenced by the apical membrane localization of CFTR and OCLN and the basolateral expression of NBCe-1 (Fig. 1.D.).

Extracellular matrix removal induces a polarity switch that reduces the epithelial cell tension in the organoids

Previously, Co et al. successfully controlled the polarity of colon organoids by removal of the extracellular matrix scaffold ^20,21. These organoids switched polarity and maintained the barrier function and were particularly useful in studying host-pathogen interactions, however the epithelial cell functions, such as ion and fluid secretion were not assessed in those manuscripts. In case of human pancreatic organoids, we observed that the cystic forming structures kept in suspension culture undergo morphological changes after removal of the ECM and the typical cystic form was replaced by a denser structure formed by a columnar cell layer (Fig. 2.A.). Immunofluorescence staining of the apical CFTR, ACTIN and OCLN revealed that not only the morphology of the organoids changed but indeed a complete switch of the apical-to-basal polarity took place in the suspension culture (Fig. 2.A. and B.). The polarity switch was further confirmed by the detection of the brush border ¹⁸, a well-known apical membrane structure, on the outer surface of the organoids by scanning electron microscopy (Fig. 2.C.). Our group successfully used the apical-in pancreatic organoids to study the fluid and ion secretion of pancreatic ductal epithelial cells previously ^18,25. However, in this system the vectorial secretion of fluid maintains an elevated intraluminal pressure as the fluid is accumulated within the lumen, resulting in the cystic form, which may also affect the cell shape. Using immunofluorescent labelling of KRT19 and ACTIN and DAPI fluorescent stain, we compared the diameter of cells and nuclei in the apical-in and apical-out hPOCs. These images revealed that in the absence of a closed lumen (and thus the intraluminal pressure) the longitudinal diameter of the cells and nuclei significantly reduced leading to the formation of a columnal epithelial cell layer presumably due to the polarity-shift (Fig. 2.D.). Notably, hPOCs exhibit transcriptionally active PIEZO1 expression (Supplementary Fig. 1.), which is a mechanosensor mediating extracellular Ca²⁺ influx upon increased intraluminal tension ¹⁹. These results highlight that the removal of the ECM can induce a polarity shift in the hPOCs that eliminates the intraluminal pressure leading to a columnar epithelial morphology. The same morphology can be observed in primary ductal cells in their physiological environment ¹⁸.

The resting intracellular Ca²⁺ concentration is more consistent in apical-out hPOCs

Ca²⁺ signaling is one of the major signal transduction pathways regulating secretory processes in the exocrine pancreas whereas in pathophysiological conditions, inflammatory processes are always characterized by disturbed Ca²⁺ homeostasis ^6,26. Since the above-described intraluminal tension in the apical-in culture may influence the epithelial Ca²⁺ homeostasis we compared the Ca²⁺ signaling in apical-in and apical-out hPOCs. Evaluation of the resting intracellular Ca²⁺ levels revealed a significantly elevated basal Ca²⁺ level in apical-out hPOCs compared to apical-in organoids (Figure 3.A-B.). On one organoid multiple ROIs were selected during the measurement. To gain relevant data on the organoid level, all measured ROIs were translated into individual organoids where the difference was still significantly detectable (Figure 3.B.). However, if each organoid was plotted separately with the mean value of all ROIs, it is clearly shown that apical-in organoids have 2.8 times higher basal Ca²⁺ level deviation than their apical-in counterparts (Figure 3.C.). These observations suggest that the resting intracellular Ca²⁺ is more constant in the apical-out hPOCs. Notably, no significant difference was found in either the Ca²⁺ efflux or Ca²⁺influx rates in a similar evaluation process (Figure 3.D-E.), suggesting that the observed deviation in the basal intracellular Ca²⁺ in the apical-in organoids may be a result of a pre-stimulated state, which varies among different organoids. Since it was previously shown that ORAI1 CRAC channel is a promising therapeutic target to prevent epithelial cell Ca²⁺ overload we also investigated the channel localization in apical-out hPOCs which retained its apical membrane expression (Figure 3.F.) ²⁴. Taken together, our data suggest that resting intracellular Ca²⁺ level is more consistent in the apical-out hPOCs, which may be explained by the elimination of the intraluminal pressure and no pre-stimulation in this culture. Notably, this property of the apical-out organoids may eliminate the interfere with the detectable effect of pharmacons acting on the intracellular Ca²⁺ signaling making this culture a preferential choice for such experiments.

Functionally active Anoctamin 1 and ENaC are expressed in human pancreatic ductal cells

According to the currently accepted model of pancreatic ductal Cl^-/HCO₃^- secretion, CFTR plays a central role in this process, whereas other Cl^- channels, such as Ca²⁺ activated Cl^- channels (CaCC) are not considered ^27–30. To gain more insight, first we compared gene expression levels by RNA-sequencing - data were evaluated in TPM values - which revealed that Ano1 is the dominantly expressed CaCC channel in apical-in mouse organoids (Supplementary Figure 2.) (mPOCs) (45.73 times higher than Cftr) (Supplementary Figure 3), in the human hPOC model CFTR expression is higher than ANO1 (4.46 times), which is in line with the current literature. However, ANO1 expression is still considerable in hPOC suggesting that it may contribute to the secretory processes (Supplementary Figure 2.). To explore this possibility, we demonstrated the apical plasma membrane localization of ANO1 by immunohistochemistry (IHC) carried out on human pancreas tissue section (Figure 4.A.) and by immunofluorescent (IF) labelling performed on apical-out hPOCs (Figure 4.B.). The observed expression pattern suggests that ANO1 is dominantly present in the apical membrane, however it can be detected on the basolateral membrane as well. Next, we used an intracellular Cl^- ([Cl^-]_i) sensitive fluorescent indicator, MQAE to follow ANO1 driven Cl^- extrusion. Due to the chemical characteristics of MQAE, the emitted fluorescent signal inversely correlates with [Cl^-]_i. Apical-out hPOCs were challenged with 10 μM T16inhAO1, which is a pharmacological inhibitor of ANO1 during extracellular Cl^- removal from the HCO₃^-/CO₂^-buffered solution. These experiments revealed that 10 μM T16inhAO1 significantly reduced the intracellular Cl^- extrusion. Moreover in combination with 10 μM CFTR(inh)-172 the maximum response was further reduced (Figure 4.C.). To further assess the participation of ANO1 in apical Cl^- secretion, we capitalized the fact, that apical-out hPOCs are maintained in suspension culture that allows direct siRNA transfection of the organoids, which was limited by the presence of Matrigel in the previous culture technique. Therefore, we repeated this series of experiment with siRNA transfection where siCFTR and siANO1 alone and in combination significantly reduced Cl^- efflux compared to the control (siGloGreen). These experiments demonstrated that both siCFTR and siANO1 significantly decreased the apical Cl^- extrusion, which was further impaired by the combination treatment (Figure 4.D.). These results provide evidence to a significant contribution of ANO1 in Cl^- secretion of hPOCs, which may have a major and yet unrevealed physiological relevance.

Another ion channel that has a crucial role in the ion secretion in secretory epithelia is the epithelial sodium channel (ENaC). ENaC is usually expressed on the apical plasma membrane of the epithelial cells and participates in Na⁺ reabsorption ^31,32. However, previously its role was excluded in the pancreatic ductal cells ³³. ENaCs are heterotimers that are formed by α, β, and γ subunits (with a 1:1:1 stoichiometry) encoded by different genes (SCNN1A, SCNN1B, SCNN1D) ^34,35. Another subunit known as δ (SCNN1G) has been identified in humans ³⁶. It was previously shown that functional ENaC formed by single or only two subunits, exist in certain tissues but the exact structure and capacity of the channel in pancreatic ductal cells remained unexplored ³⁷. Interestingly, among 4 subunits, remarkable expression of SCNN1A and moderate expression of SCNN1D were detected suggesting heterotrimeric ENaC formation in hPOCs (Figure 4.E.). Moreover, the subunit α (SCNN1A) was detected in the apical membrane of polarity shifted hPOCs by immunofluorescent labelling (Figure 4.F.). Next, siRNA interference was applied to experimentally demonstrate the physiological function of the channel (Figure 4.G.). After 24h incubation with siSCNN1A and siSCNN1D, apical-out hPOCs were challenged by 100 μM amiloride from the apical membrane, which resulted in a rapid decrease in F495/F440 ratio, which suggest that the inhibition of ENaC activity indirectly influence the apical Cl^-/HCO₃^- exchange. In both treated group the maximum response was significantly decreased compared to the control (siGLO Green). These results suggest that SCNN1A and SCNN1D are constituted as functional heterotrimeric ENaC proteins in human pancreatic ductal cells.

Switching the polarity of organoids improve the performance of available functional assays

As mentioned above CFTR activity largely determines the ion and fluid transport in secretory epithelial cells, whereas restoration of the channel activity in cystic fibrosis is of great interest ⁶. To assess CFTR activity in epithelial cells a widely used indirect standard method is the forskolin-induced swelling (FIS) assay ³⁸. However, dynamic range of this technique is limited by the elevation of the intraluminal pressure within the organoids. Since the apical-out organoids are of switched polarity, reverse FIS assay was used to assess the activity of the wild type CFTR. 10 µM forskolin (FSK) decreased the relative volume of the apical-out organoids, which was abolished by the administration of 10 µM CFTR(inh)-172 (Figure 5.A.) suggesting that this technique is suitable to measure the activity of the wild type CFTR as well. Next, we wanted to compare further the performance of the apical-in and apical-out organoids in functional assays, therefore we compared CFTR activity by MQAE. The Cl^- extrusion was significantly decreased by 10 µM CFTR(inh)-172 suggesting the detected change is largely CFTR dependent in apical-in organoids (Figure 5.B-C.). However, when the same experiments were performed on apical-out organoids, the response to Cl^- removal was higher than in conventional organoids presumably caused by the direct apical perfusion and lack of intraluminal pressure which are clearly able to alter epithelial secretion (Figure 5.C, D.). Moreover, due to the enhanced resolution of the apical-out model, even the administration of a 20 µM CFTR(inh)-172 inhibitor could not completely abolish the Cl^- efflux process, confirming the involvement of additional channels, such as ANO1 (Figure 5.C.). Since the apical membrane and CFTR become directly available we also applied 10 μM VX-770 CFTR potentiator on apical-out hPOCs which significantly enhanced Cl^- efflux and kept the equilibrium elevated in [Cl^-]_i proving the applicability of apical-out hPOCs in in vitro drug testing assays (Figure 5.E.). Next Cl^- and HCO₃^- exchanger (CBE) CBE activity was measured by using BCECF pH-sensitive dye with CFTR inhibition in HCO₃^-/CO₂buffered solution. Cl^- withdrawal followed by direct apical perfusion of Cl^- containing HCO₃^-solution with simultaneous administration of 10 μM CFTR(inh)-172 decreased the slope of BCECF ratio (F495/F440) compared to the control group, which difference suggests indirect detection of CBE activity in hPOCs (Figure 5.F.). All dataset presented here suggest that hPOCs could provide significant benefits in the investigation of apical Cl^- transport mechanisms compared to conventional apical-in hPOCs.

In this manuscript we generated human pancreatic organoids and advanced the culture technique further to improve the accessibility of the apical membrane by manipulating the polarity of the cells. By switching apical-to-basal polarity the elongated epithelial cell morphology changed to a cuboidal shape in the apical-out organoids, whereas their basal intracellular Ca²⁺ level was more consistent. Using this novel model, we identified the expression and function of ANO1 and ENaC on the plasma membrane, that were never described in the pancreatic ductal cells. Finally, we demonstrated that functional assays (such as FIS, or CFTR activity measurements) display an improved dynamic range when performed using apical-out organoids.

In the recent years organoid cultures derived from tissue specific Lgr5 + adult stem cells emerged as novel models of organ development and disease ^10,11. By maintaining the activity of Wnt/β-Catenin signal transduction cascade – a key driver of most types of adult stem cells ¹² – organoid cultures (OCs) can be grown in vitro for long-term in 3D extracellular matrix based hydrogels; whereas, epithelial cells in the culture maintain the original cellular diversity and organization of the organ of origin ¹³. Originally small intestinal adult stem cells were used to generate crypt–villus like structures in vitro ¹⁴. Since then OCs have been established from a wide range of organs in the gastrointestinal tract, including colon, esophagus ¹⁰, stomach ¹⁵, liver and pancreas ¹⁶. Our group previously demonstrated that the apical-to-basal polarity of the epithelial cells, gene and protein expressions and ion transport activities in the mouse pancreatic organoids remarkably overlap with those observed in freshly isolated primary ductal fragments ¹⁸. These results highlighted that organoids are suitable to study epithelial cell functions as well. The original pancreatic organoid establishment protocol was based on manual picking of pancreatic ductal fragments after digesting the pancreatic tissue ³⁹. This step eventually requires manual work and experience; thus, it is not suitable for automatization. In the current study, we advanced this technique by using enzymatic digestion of the whole pancreatic tissue, without picking individual ductal fragments. With this technique we were able to generate pancreatic organoids that consisted solely of polarized epithelial cells with 100% efficiency. These epithelial cells expressed well-known ductal marker like CFTR, KRT19, SOX9, HNF1B or FOXA2, whereas non-ductal markers like amylase or insulin were completely absent. These results suggest that our advanced technique is suitable to generate pure pancreatic ductal cell cultures with high efficiency without the contamination of other cell types. Moreover, the elimination of the manual steps from the protocol may allow the automatization of the workflow for large scale manufacturing. Another well-known limitation of the use of organoids is the ECMs such as Matrigel, that is essential for the growth of organoids, however, significantly reduces the applicable molecular biology toolset. ECM can limit the diffusion of the test compounds and pharmacons due to the molecular size and charge specificity of the individual molecules, whereas it may also hinder the application of siRNAs and plasmids. In addition, in the conventional organoid cultures the apical membrane of the epithelial cells is not directly accessible for drugs. Moreover, due to the vectorial transport of ions and fluid the intraluminal pressure of the apical-in organoids is elevated without any pre-stimulation, which may affect the epithelial secretory processes. To overcome these limitations, we induced the switch of the apical-to-basal polarity of the organoids by replacing the established organoids into an ECM-free suspension culture. Immunostaining of CFTR, actin and occluding and visualization of the brush border on the outer surface of the apical-out organoids by scanning electron microscopy revealed a complete switch of the polarity after 48 h. This switch also led to a change of the epithelial cell morphology from an elongated to a cuboidal shape, suggesting that the elimination of the intraluminal pressure also affects the cell homeostasis. As an example to this, we demonstrated that the resting intracellular Ca²⁺ levels in unstimulated apical-out organoids were more consistent, compared to the apical-out organoids, whereas the intracellular Ca²⁺ release or extrusion was not changed. Ductal epithelial cells express the mechanosensitive receptor Piezo1, which is a Ca²⁺ channel and senses the intraluminal pressure and stretch. Activation of Piezo1 is able to trigger extrusion and apoptosis in crowded and cell division in sparse regions of the epithelial layer thus regulating epithelial turnover ⁴⁰. Moreover Piezo1 also plays a critical role in transducing mechanical signals into an intracellular inflammatory cascade leading to tissue inflammation ⁴¹. The activation of Piezo1 can regulate epithelial barrier functions negatively through Claudin-1 and induce epithelial dysfunction ¹⁹. Thus, the presence of intraluminal pressure in cystic organoids may therefore be a factor to be eliminated when comparing physiological and inflammatory conditions. Recently, Co et al. developed a technique to reverse enteroid polarity to study host-pathogen interactions ²⁰. They showed that apical-out enteroids maintain apical-to-basal polarity and barrier function in ECM-free media, differentiate into the major intestinal epithelial cell types, and exhibit polarized absorption of nutrients, whereas the bacterial entry mimics the in vivo entry process. Importantly, our technique overcomes several known limitations of the organoid cultures and improves the applicability of hPOCs in physiological studies and drug testing.

Next, we utilized the developed culture technique and the accessibility of apical membrane to investigate the ion transport of the polarized human pancreatic ductal cells. The current model of pancreatic ductal HCO₃⁻ secretion is reviewed in detail elsewhere ^1,42. Briefly, the secretion of the large amount of HCO₃⁻ depends on the interaction of the SLC26A6 Cl⁻/HCO₃⁻ exchanger and the CFTR Cl⁻ channel. SLC26A6 works with a 2HCO₃⁻:1Cl⁻ stoichiometry, moreover the STAS domain of the SLC26A6 interacts with the R domain of CFTR leading to increased activation ^10,43,44. In addition, stimulation of the cells with cAMP agonists leads to an increased HCO₃⁻ permeability of CFTR and a marked drop of the intracellular Cl⁻ concentration ⁴⁵. This activates the WNK1-OSR1/SPAK pathway, that ultimately led to phosphorylation of CFTR and a marked increase in the relative permeability of CFTR to HCO₃^{− 46}. In contrast to these, using our novel tool, we identified two ion channels that are expressed on the plasma membrane of the ductal cells and were never considered in pancreatic ion secretion. Our results revealed that ductal cells express functionally active ANO1, which is a Ca²⁺ activated Cl⁻ channel. ANO1 expression was only reported on the apical membrane of pancreatic acinar cells, in which HCO₃⁻ secretion via ANO1 attenuated the pH shift during acute pancreatitis ⁴⁷. In addition, we also demonstrated the expression and functional activity of ENaC on the apical membrane of the ductal cells. This was rather surprising, as researchers in the field agree, that ENaC does not participate in the ductal functions, whereas it is widely expressed in many types of secretory epithelial cells. Previous patch clamp studies on isolated rat ductal fragments showed no measurable effect of amiloride on ductal cells ³³. These results were not revised later in other species. Notably, our observations have limitations, that need to be considered. First, pancreatic ductal cells located at the different parts of the ductal tree exhibit different secretory activities. In the currently used model, we have no information exactly which part of the ductal tree is represented in the organoid culture. In addition, the participation of these channels in the physiological secretory processes needs further evaluation. On the other hand, the introduction of two novel ion channels will require a detailed revision of the secretory process, that will remarkably improve the current model and should lead to a better understanding of the human exocrine pancreatic ion secretion (Fig. 6.).

Finally, we provided examples, how this novel apical-out culture system could be used to improve the currently available functional assays. We demonstrated that the elimination of the intraluminal pressure remarkably improves the dynamic range of the FIS assay. FIS assay on human rectal organoids is used to predict drug response of CF patients ⁴⁸. In these screening assays researchers use rectal biopsy samples obtained from CF patients to generate organoid cultures to study the effect of CFTR correctors and predict drug response of the patients ⁴⁹, whereas it may not be suitable in patients with remaining CFTR function ⁵⁰. The advantage of our approach is twofold, first, the elimination of the ECM could make the assay suitable for automatization and large-scale screening, second the improved dynamic range may lead to a better resolution of the results and higher precision of the clinical predictions. In addition, we also demonstrated that intracellular Cl⁻ measurements on apical-out organoids provide a better dynamic range and an improved dose-response of the ductal epithelial cells.

Taken together the results presented here go far beyond our current conclusions. By eliminating the intraluminal pressure in conventional organoids, we have not only gained information about the real capabilities of apical channels, but also opened the door to multi-field translational research. Polarity-switched human pancreatic organoids offer new options for regenerative therapies for diabetes, acute or chronic pancreatitis or for cystic fibrosis of the pancreas.

Funding

The research was supported by the Momentum grant of the Hungarian Academy of Sciences (LP2017–18/2017 to JM), by funding from the Hungarian National Research, Development and Innovation Office (FK139269 to JM), by the National Excellence Program (20391-3/2018/FEKUSTRAT and TKP2021-EGA-28 to JM and UNKP-21-4-SZTE-116 to TM). The project has received funding from the EU’s Horizon 2020 research and innovation program under grant agreement No. 739593. The project was also supported by funding from the Hungarian Government (NTP-NFTÖ-B-0011 to ÁV, NTP-NFTÖ-21-B-0205 to AK and NTP-NFTÖ-21-B-0079 to VSZ)

Conflict of interest

The authors have no conflict of interest to declare.

Author contributions

ÁV and JM designed the research project; ÁV, TM, MG, AK, PS, VSz, KD, AF, FA and PP contributed to acquisition, analysis and interpretation of data for the work. GYF, ESZ, GYZ provided human pancreatic tissue samples. ÁV and JM drafted the work and all authors approved the final version of the manuscript, agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Data availability

The datasets generated during and/or analyzed during the current study are fully available upon contact with the corresponding author.

Ethics

Animals were used with adherence to the NIH guidelines and the EU directive 2010/63/EU. The study was approved by the National Scientific Ethical Committee on Animal Experimentation under license number XXI./2523/2018. The human sample collection and use including pancreas tissue samples from cadaver donors were executed in adherence with the EU standards and approved by the Regional Committee of Research Ethics of the Hungarian Medical Research Council under license number 37/2017-SZTE.

Acknowledgements

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Anion exchanger (AE), aquaporin (AQP), tight junction (TJ).

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Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology

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