Engineering a microwell duct-on-chip technology to translate exocrine pancreatic organoids to a cancer model

Das duktale Adenokarzinom der Bauchspeicheldrüse (PDAC) ist eine der tödlichsten Erkrankungen der exokrinen Bauchspeicheldrüse, für die uns relevante Frühdiagnosemarker fehlen. Um PDAC-Marker zu identifizieren, werden in vitro kultivierte exokrine Pankreasmodelle aus dem frühestmöglichen, präkanzerösen Stadium benötigt. Die Übertragung der Pankreasgang-Differenzierung von humanen pluripotenten Stammzellen (hiPSCs) in in vitro-Krankheitsmodelle erfordert ein umfassendes Verständnis der Entwicklungsbahnen von pankreasspezifischen Zelltypen. In dieser Arbeit wurde eine Microwell-Chip-Technologie mit definierten mikrostrukturierten Strukturen entwickelt, um aus hiPSC differenzierte Vorläuferzellen des Pankreas (PP) in einer 3-dimensionalen Zellkultur zu assemblieren. Die Vorteile der Chip-Plattform sind i) die parallele Bildung von Hunderten gleichgroßer 3D-Zellaggregate, ii) eine Matrigel-freie Mikroumgebung, iii) die Kompatibilität mit hochauflösender Bildgebung, iv) die einfache Anwendbarkeit für verschiedene nachfolgende Analysen mit minimaler Störung und v) die Möglichkeit, Ko-Kulturen zu etablieren. Der Chip wurde verwendet, um in weniger als 6 Stunden tausende von 3D-Zellaggregaten aus etwa 600 PPs zu bilden. In den folgenden 14 Tagen wurden die 3D-PP-Kulturen mit einem definierten Wachstumsfaktorprotokoll in pankreatische dukt-ähnliche Organoide differenziert. Zeitaufgelöste Einzelzell-Transkriptionsprofile und Immunfluoreszenz von gereinigten dukt-ähnlichen Organoiden der Bauchspeicheldrüse zeigten die Entstehung von zwei Arten von duktalen Vorläufern, Zwischenstufen, und reifen duktalen Zellen und wenigen nicht-duktalen Zelltypen. Entsprechende dynamische Transkriptionsstadien wiesen auf definierte Differenzierungsrouten der duktalen Zellen hin, die in zwei entweder CFTR+ oder Mucin+ Subpopulationen resultieren. Diese Subpopulationen wurden bereits in primären Einzelzelltranskriptomen des Pankreas gefunden[4]. Die Integration unseres Einzelzelldatensatzes mit drei primären Pankreasdatensätzen[4-6] zeigte, dass unsere dukt-ähnlichen Zellen zusammen mit primären duktalen Zellen zu den beiden Subpopulationen clustern. Außerdem konnten die Marker der Subpopulationen in einem reanalysierten Primärdatensatz[5] erneut identifiziert und in menschlichem Primärgewebe angefärbt werden. Darüber hinaus wurde die Duct-on-Chip-Plattform genutzt, um Organoid-Ko-Kulturen mit humanen Stellat-Zellen zu etablieren. Als zusätzliche Anwendung ermöglichte die Matrigel-freie Chip-Technologie die Entnahme des Sekretoms und Proteoms der Organoide. In Verbindung mit dem Einzelzell-Transkriptom und der klinischen Validierung ermöglichten uns diese Sekretomstudien die Entdeckung eines beispielhaften frühen PDAC-Marker namens FLNB, welcher sowohl in Biopsien als auch im peripheren Blut von Patienten im Frühstadium nachweisbar ist. Zusammenfassend zeigt diese Arbeit die erfolgreiche Herstellung von Pankreas dukt-ähnlichen Organoiden aus hiPSCs, die ein Reifestadium aufweisen, welches mit dem des fötalen Pankreas vergleichbar ist. Durch die Kombination von zeitaufgelöster Einzelzelltranskriptomik mit verschiedenen Analysemethoden, Sekretomstudien, Proteomstudien und klinischer Validierung auf unserem Microwell-Chip wurde ein patientenspezifisches Duktmodell und ein potenzielles Krebsdiagnoseinstrument entwickelt.Pancreatic ductal adenocarcinoma (PDAC) is one of the most severe diseases of the exocrine pancreas, for which relevant early diagnostic markers are still missing. To identify PDAC biomarkers, experimental models employing in vitro cultivation of exocrine pancreas models require as early as possible precancerous stages. The translation of pancreatic ductal differentiation of human pluripotent stem cells (hiPSCs) into in vitro disease models requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. In this study, a microwell chip technology exhibiting defined microstructured patterns to assemble hiPSC-derived pancreatic progenitor cells (PP) into a 3-dimensional cell culture was developed. The advantages of the chip platform are i) the parallel formation of hundreds of equally sized 3D cell aggregates, ii) a Matrigel-free microenvironment, iii) the compatibility with high-resolution imaging, iv) simple applicability for several downstream analyses with minimal perturbation, and v) the possibility to establish co-cultures. The chip was used to generate thousands of 3D cell aggregates from approximately 600 PPs, in less than six hours. For the following 14 days, the 3D PP cultures were differentiated towards pancreatic ductal-like organoids by employing defined growth factor protocols. Time-resolved single-cell transcriptional profiling and immunofluorescence of cleared pancreatic duct-like organoids revealed the emergence of two types of ductal progenitors, intermediates, mature duct-like cells, and a few non-ductal cell types. Corresponding dynamic transcriptional stages indicated defined differentiation routes of duct-like cells, cumulating in two either CFTR+ or mucin+ subpopulations, which have been found before in primary single-cell transcriptomes of the pancreas[4]. The integration of the PDLO single-cell dataset into three primary pancreas datasets[4-6] showed that the duct-like cells clustered together with primary ductal cells into the two subpopulations. Furthermore, the markers of the subpopulations could be reidentified in a reanalyzed primary dataset[5] and subjected to confirmation by immunofluorescence in primary human tissue. Additionally, the duct-on-chip platform was exploited to establish organoid co-cultures with stellate cells. As an additional application, the Matrigel-free chip technology allowed the characterization of secretome and proteome. Together with the single-cell transcriptome and clinical validation, these secretome studies revealed an exemplary early PDAC marker, called FLNB, which is detectable in biopsies and early-stage patients' peripheral blood. In conclusion, this study reports the successful engineering of pancreatic duct-like organoids from hiPSCs, which show a maturation stage comparable to the fetal pancreas. By combining time-resolved single-cell transcriptomics with different analysis methods, secretome, proteome and clinical validation on our microwell chip, a patient-specific duct model and a potential cancer diagnostic tool was developed

Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn13-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn13-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Determination of the strong coupling constant αs from transverse energy–energy correlations in multijet events at s√=8 TeV using the ATLAS detector

Measurements of transverse energy–energy correlations and their associated asymmetries in multi-jet events using the ATLAS detector at the LHC are presented. The data used correspond to s√=8 TeV proton–proton collisions with an integrated luminosity of 20.2 fb−1 . The results are presented in bins of the scalar sum of the transverse momenta of the two leading jets, unfolded to the particle level and compared to the predictions from Monte Carlo simulations. A comparison with next-to-leading-order perturbative QCD is also performed, showing excellent agreement within the uncertainties. From this comparison, the value of the strong coupling constant is extracted for different energy regimes, thus testing the running of αs(μ) predicted in QCD up to scales over 1 TeV . A global fit to the transverse energy–energy correlation distributions yields αs(mZ)=0.1162±0.0011(exp.) +0.0084−0.0070(theo.) , while a global fit to the asymmetry distributions yields a value of αs(mZ)=0.1196±0.0013(exp.) +0.0075−0.0045(theo.)