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These datasets underpin the analysis presented in the agency's work. Most data may be viewed interactively on screen and downloaded in Excel format. All countries. Topics A-Z. The content in this section is aimed at anyone involved in planning, implementing or making decisions about health and social responses. Best practice. We have developed a systemic approach that brings together the human networks, processes and scientific tools necessary for collecting, analysing and reporting on the many aspects of the European drugs phenomenon. Explore our wide range of publications, videos and infographics on the drugs problem and how Europe is responding to it. All publications. More events. More news. We are your source of drug-related expertise in Europe. We prepare and share independent, scientifically validated knowledge, alerts and recommendations. About the EUDA. The analysis of municipal wastewaters for drugs and their metabolic products to estimate community consumption is a developing field, involving scientists working in different research areas, including analytical chemistry, physiology, biochemistry, sewage engineering, spatial epidemiology and statistics, and conventional drug epidemiology. This page presents the findings from studies conducted since Data from all studies can be explored through an interactive tool, and a detailed analysis of the findings of the most recent study, in , is presented. See our wastewater analysis hub page for more resources on this topic. Please note that due to the large amount of data involved processed on this page, it may take some moments before all content appears. In this section you can explore the data from the most recent study in , as well as from previous studies. Each study reveals a picture of distinct geographical and temporal patterns of drug use across European cities. Clicking on a symbol in the graph or the map will show more detailed information for a given wastewater treatment plant. You can also select a site from the drop-down menu. Preparing the data The data explorer is designed to work with most modern browsers but if this message is still visible after 1 minute, we recommend trying again with another browser. The findings of the largest European project to date in the emerging science of wastewater analysis are presented in this section. The results provide a valuable snapshot of the drug flow through the cities involved, revealing marked geographical variations. Wastewater analysis is a rapidly developing scientific discipline with the potential for monitoring real-time data on geographical and temporal trends in illicit drug use. Originally used in the s to monitor the environmental impact of liquid household waste, the method has since been used to estimate illicit drug consumption in different cities Daughton, ; van Nuijs et al. It involves sampling a source of wastewater, such as a sewage influent to a wastewater treatment plant. This allows scientists to estimate the quantity of drugs consumed by a community by measuring the levels of illicit drugs and their metabolites excreted in urine Zuccato et al. In , a Europe-wide network Sewage analysis CORe group — Europe SCORE was established with the aim of standardising the approaches used for wastewater analysis and coordinating international studies through the establishment of a common protocol of action. The first activity of the SCORE group was a Europe-wide investigation, performed in in 19 European cities, which allowed the first ever wastewater study of regional differences in illicit drug use in Europe Thomas et al. That study included the first intercalibration exercise for the evaluation of the quality of the analytical data and allowed a comprehensive characterisation of the major uncertainties of the approach Castiglioni et al. A standard protocol and a common quality control exercise were used in all locations, which made it possible to directly compare illicit drug loads in Europe over a one-week period during 10 consecutive years van Nuijs et al. Raw hour composite samples were collected during a single week between March and May in the majority of the cities. These samples were analysed for the urinary biomarkers i. In addition, the samples were analysed for the main urinary metabolites i. The specific metabolite of heroin, 6-monoacetylmorphine, has been found to be unstable in wastewater. Consequently, the only alternative is to use morphine, although it is not a specific biomarker and can also be excreted as a result of therapeutic use. The project findings revealed distinct geographical and temporal patterns of drug use across European cities see the data explorer. The annual SCORE wastewater sampling presented here, from 88 cities, showed that, overall, the loads of the different stimulant drugs detected in wastewater in varied considerably across study locations, although all illicit drugs investigated were found in almost every city that participated. For the first time, data from outside Europe is also shown and compared against European cities. The BE loads observed in wastewater indicate that cocaine use remains highest in western and southern European cities, in particular in cities in Belgium, the Netherlands and Spain. Low levels were found in the majority of the eastern European cities, although the most recent data continues to show signs of increase. The loads of amphetamine detected in wastewater varied considerably across study locations, with the highest levels being reported in cities in the north and east of Europe, as in previous years. Amphetamine was found at much lower levels in cities in the south of Europe, although with the most recent data showing some signs of increase. The observed methamphetamine loads in the other locations were very low, although most recent data show signals of increases in central European cities. For the second time, ketamine loads are being published. The highest mass loads were found in the wastewater in cities in Belgium, France, the Netherlands, and Spain. The study highlighted differences between these cities within the same country, which may be explained in part by the different social and demographic characteristics of the cities universities, nightlife areas and age distribution of the population. Interestingly, in the majority of countries with multiple study locations, no marked differences were found when comparing large cities to smaller locations for all substances. In addition to geographical patterns, wastewater analysis can detect fluctuations in weekly patterns of illicit drug use. More than three quarters of cities show higher loads of amphetamine, BE, ketamine and MDMA in wastewater during the weekend Friday to Monday than during weekdays. Seventy-three cities have participated in at least five of the annual wastewater monitoring campaigns since This allows for time trend analysis of drug consumption based on wastewater testing. Cannabis is Europe's most commonly used illicit drug, with an estimated However, both the level of use and trends in use reported in recent national data appear heterogeneous. In wastewater, cannabis use is estimated by measuring its main metabolite, THC-COOH, which is the only suitable biomarker found so far. Although it is excreted in a low percentage and more research is still needed Causanilles et al. In , there were diverging trends with 20 cities out of 51 reporting an increase in THC-COOH loads in wastewater samples, and 15 a decrease. Low levels were found in the majority of the eastern European cities, but the most recent data continues to show signs of increases. When comparing to study locations outside the European Union, cities in Brazil, Switzerland and in the United States show similar levels of use as the cities in Europe with the highest loads. A relatively stable picture of cocaine use was observed between and in most cities. The data revealed further increases in cocaine residues in most cities when compared to data, with 49 out of 72 cities reporting an increase, while 13 cities reported no change and 10 cities reported a decrease. An overall increase is seen for all 10 cities with data for both and These 7 cities were selected owing to the availability of annual data from to Contrary to previous years, in most countries with multiple study locations, no marked differences were found when comparing large cities to smaller locations. More than three quarters of cities show higher loads of BE in wastewater during the weekend Friday to Monday than during weekdays, which may reflect a pattern of more recreational use. A recent European project on wastewater found crack cocaine residues in all 13 participating cities and for all sampling days, with the highest loads reported in Amsterdam and Antwerp. Where data is available, when comparing to study locations outside the European Union, only cities in Switzerland show similar levels of use as the cities in Europe with the highest loads, while all the other location show low levels of MDMA use. General population surveys in many countries showed that MDMA prevalence was declining from peak levels attained in the early to mids. In recent years, however, the picture has remained mixed with no clear trends. Where prevalence is high, this may reflect MDMA no longer being a niche or subcultural drug limited to dance clubs and parties, but now being used by a broader range of young people in mainstream nightlife settings, including bars and house parties. Looking at longer-term trends in wastewater analysis, in most cases the loads increased between , and have fluctuated after this. In , possibly due to the fact that in the majority of countries nightlife was largely closed for long periods, almost half of the cities 24 of 49 reported a decrease with 18 reporting an increase. In , 38 out of 58 cities, reported a decrease. In , 28 out of 62 cities reported an increase and 27 a decrease. Of the 69 cities that have data on MDMA residues in municipal wastewater for and , 42 reported an increase mostly in northern Europe , 11 a stable situation and 16 a decrease mostly in cities in southern and central Europe. Of the 9 cities with data for both and , 9 had higher MDMA loads in than in As for cocaine, and contrary to previous years, in most countries with multiple study locations, no marked differences were found when comparing large cities to smaller locations. More than three quarters of cities showed higher loads of MDMA in wastewater during the weekend Friday to Monday than during weekdays, reflecting the predominant use of ecstasy in recreational settings. Amphetamine and methamphetamine, two closely related stimulants, are both consumed in Europe, although amphetamine is much more commonly used. Methamphetamine consumption has historically been restricted to Czechia and, more recently, Slovakia, although recent years have seen increases in use in other countries. The loads of amphetamine detected in wastewater varied considerably across study locations, with the highest levels reported in cities in the north and east of Europe. Amphetamine was found at much lower levels in cities in the south of Europe, although the most recent data shows some signs of increase. To examine the data, use the data explorer , also available on this page. Underlying data is available in source data. The observed methamphetamine loads in the other locations were very low to negligible, although most recent data show signals of increases in central European cities. Overall, the data related to amphetamine and methamphetamine from the 11 monitoring campaigns showed no major changes in the general patterns of use observed, although since increases were observed in several cities for both substances in regions where use has traditionally been low to negligible. Of the 65 cities with data on amphetamine residues in municipal wastewater for and , 26 reported an increase, 13 a stable situation and 26 a decrease. Of the 67 cities that have data on methamphetamine residues in municipal wastewater for and , 15 reported an increase, 13 a stable situation and 39 a decrease. In , methamphetamine use was found to be distributed more evenly over the whole week than in previous years, possibly reflecting the use of these drugs being associated with more regular consumption by a cohort of high-risk users. For amphetamine, more than three quarters of cities show higher loads during the weekend Friday to Monday than during weekdays. In , low levels of ketamine residues in municipal wastewater were reported by 49 cities, although with signals of increases. Of the 22 cities that have data on ketamine residues for and , 12 reported an increase, 8 a stable situation and 2 a decrease. The highest mass loads were detected in cities in Belgium, France, the Netherlands, and Spain. More than three quarters of cities showed higher loads of ketamine in wastewater during the weekend Friday to Monday than during weekdays, reflecting the predominant use of ketamine in recreational settings. Wastewater analysis offers an interesting complementary data source for monitoring the quantities of illicit drugs used at the population level, but it cannot provide information on prevalence and frequency of use, main classes of users and purity of the drugs. Additional challenges arise from uncertainties associated with the behaviour of the selected biomarkers in the sewer, different back-calculation methods and different approaches to estimate the size of the population being tested Castiglioni et al. The caveats in selecting the analytical targets for heroin, for example, make monitoring this drug in wastewater more complicated compared to other substances Been et al. Also, the purity of street products fluctuates unpredictably over time and in different locations. Furthermore, translating the total consumed amounts into the corresponding number of average doses is complicated, as drugs can be taken by different routes and in amounts that vary widely, and purity levels fluctuate Zuccato et al. Efforts are being made to enhance wastewater monitoring approaches. For example, work has been undertaken on overcoming a major source of uncertainty related to estimating the number of people present in a sewer catchment at the time of sample collection. This involved using data from mobile devices to better estimate the dynamic population size for wastewater-based epidemiology Thomas et al. Wastewater-based epidemiology has established itself as an important tool for monitoring illicit drug use and future directions for wastewater research have been explored EMCDDA, First, wastewater analysis has been proposed as a tool to address some of the challenges related to the dynamic new psychoactive substances NPS market. This includes the large number of individual NPS, the relatively low prevalence of use and the fact that many of the users are actually unaware of exactly which substances they are using. A technique has been established to identify NPS that involves the collection and analysis of pooled urine from stand-alone portable urinals from nightclubs, city centres and music festivals, thereby providing timely data on exactly which NPS are currently in use at a particular location Archer et al. The project applied innovative analytical chemical and epidemiological methods and a robust risk-assessment procedure to improve the identification of NPS, to assess risks, and to estimate the extent and patterns of use in specific groups e. Second, in addition to estimating illicit drug use, wastewater-based epidemiology has been successfully applied in recent years to providing detailed information on the use and misuse of alcohol Boogaerts et al. Furthermore, wastewater analysis can potentially provide information on health and illness indicators within a community Kasprzyk-Hordern et al. Third, the potential for wastewater-based epidemiology to be used as an outcome measurement tool, in particular in the evaluation of the effectiveness of interventions that target drug supply e. Close collaboration between the different stakeholders involved, including epidemiologists, wastewater experts and legal authorities, is highly recommended in order to start examining these potential wastewater-based epidemiology applications EMCDDA, High levels of MDMA were recorded during the whole monitoring period in one city in the Netherlands, suggesting continuous discharges of unconsumed MDMA from sources within the wastewater catchment area, indicating drug production was taking place in this region. Fourth, by back-calculating the daily sewer loads of target residues, wastewater analysis can provide total consumption estimates, and specific efforts are now being directed towards finding the best procedures for estimating annual averages. It is envisaged that findings from wastewater analysis can help to further develop work in this area. Finally, new methods such as enantiomeric profiling have been developed to determine if mass loads of drugs in wastewater originated from consumption or from the disposal of unused drugs or production waste. It is now important to assess the possible utility of wastewater analysis to report on drug supply dynamics, including synthetic drug production Emke et al. For example, recent malfunctioning of a small wastewater treatment plant in the Netherlands was caused by direct discharges in the sewage system of chemical waste from a drug production site. Further analysis revealed the actual synthesis process used to manufacture the corresponding drugs. The study confirmed that the chemical waste from the illegal manufacturing of stimulants will result in a specific chemical fingerprint that can be tracked in wastewater and used for forensic purposes. Such profiles can be used to identify drug production or synthesis waste disposal in the wastewater catchment area Emke et al. Wastewater analysis has demonstrated its potential as a useful complement to established monitoring tools in the drugs area. It has some clear advantages over other approaches as it is not subject to response and non-response bias and can better identify the true spectrum of drugs being consumed, as users are often unaware of the actual mix of substances they take. This tool also has the potential to provide timely information in short timeframes on geographical and temporal trends. In order to check the quality and accuracy of data, further comparisons between wastewater analysis and data obtained through other indicators are needed. As a method, wastewater analysis has moved from being an experimental technique to being a new method in the epidemiological toolkit. Its rapid ability to detect new trends can help target public health programmes and policy initiatives at specific groups of people and the different drugs they are using. In addition to the glossary below, see also Frequently-asked questions on wastewater-based epidemiology and drugs. Traces of drugs consumed will end up in the sewer network either unchanged or as a mixture of metabolites. Metabolites, the end products of metabolism, are the substances produced when the body breaks drugs down. Wastewater analysis is based on the fact that we excrete traces in our urine of almost everything we consume, including illicit drugs. The target drug residue is what remains in the wastewater after excretion and is used to quantify the consumption of illicit drugs in the population. Analytical chemists look for urinary biomarkers measurable characteristics to calculate population drug use in wastewater samples, which can be the parent drug i. Enantiomeric profiling is an analytical chemistry technique used to determine if studied drugs in wastewater originate from consumption or direct disposal eq. It is based on the fact that chiral molecules if only one chiral centre is present exist as two enantiomers opposite forms which are non-superimposable mirror images of each other. As the enantiomeric ratio will change after human metabolism, the enantiomeric fraction can be used to determine whether the studied drugs in wastewater originate from consumption. In order to estimate levels of drug use from wastewater, researchers attempt first to identify and quantify drug residues, and then to back-calculate the amount of the illicit drugs used by the population served by the sewage treatment plants Castiglioni et al. This approach involves several steps see figure. Initially, composite samples of untreated wastewater are collected from the sewers in a defined geographical area. The samples are then analysed to determine the concentrations of the target drug residues. A correction factor for each drug is taken into account as part of the calculation. In a last step, the result is divided by the population served by the wastewater treatment plant, which shows the amount of a substance consumed per day per 1 inhabitants. Population estimates can be calculated using different biological parameters, census data, number of house connections, or the design capacity, but the overall variability of different estimates is generally very high. Although primarily used to study trends in illicit drug consumption in the general population, wastewater analysis has also been applied to small communities, including workplaces, schools Zuccato et al. Using this method in small communities can involve ethical risks Prichard et al. In the SCORE group published ethical guidelines for wastewater-based epidemiology and related fields Prichard et al. The objective of these guidelines is to outline the main potential ethical risks for wastewater research and to propose strategies to mitigate those risks. Archer, J. Bade, R. Baz-Lomba, J. Been, F. Bijlsma, L. Boogaerts, T. Castiglioni, S. Causanilles, A. Daughton, C. Emke, E. Hall, W. Kasprzyk-Hordern, B. Kinyua, J. Krizman-Matasic, I. Lai, F. Mardal, M. Mastroianni, N. Prichard, J. Reid, M. Senta, I. Thomaidis, N. Thomas, K. Yang, Z. Zuccato, E. Show source tables. You can download the source data for drugs in wastewater in cities from our our data catalogue or use the links below to directly download the CSV files. Homepage Quick links Quick links. GO Results hosted on duckduckgo. Main navigation Data Open related submenu Data. Latest data Prevalence of drug use Drug-induced deaths Infectious diseases Problem drug use Treatment demand Seizures of drugs Price, purity and potency. Drug use and prison Drug law offences Health and social responses Drug checking Hospital emergencies data Syringe residues data Wastewater analysis Data catalogue. Selected topics Alternatives to coercive sanctions Cannabis Cannabis policy Cocaine Darknet markets Drug checking Drug consumption facilities Drug markets Drug-related deaths Drug-related infectious diseases. Recently published Findings from a scoping literature…. Penalties at a glance. Frequently asked questions FAQ : drug…. FAQ: therapeutic use of psychedelic…. Viral hepatitis elimination barometer…. EU Drug Market: New psychoactive…. EU Drug Market: Drivers and facilitators. Statistical Bulletin home. Quick links Search news Subscribe newsletter for recent news Subscribe to news releases. Breadcrumb Home Publications Wastewater analysis and drugs — a European multi-city study. On this page. Wastewater analysis and drugs — a European multi-city study. PDF is being prepared. This make take up to a minute. Once the PDF is ready it will appear in this tab. Sorry, the download of the PDF failed. Introduction The analysis of municipal wastewaters for drugs and their metabolic products to estimate community consumption is a developing field, involving scientists working in different research areas, including analytical chemistry, physiology, biochemistry, sewage engineering, spatial epidemiology and statistics, and conventional drug epidemiology. Page last updated: 20 March World view Europe South America Oceania. Complete source data for all wastewater measurments, all cities, all years CSV format Wastewater treatment centres information table CSV format Changes in the mean weekly measurements by targeted substance, from wastewater analyses in selected European cities between and CSV format Aggregated trends in cocaine residues in 7 EU cities, to CSV format.

Pluripotent Stem-Cell Platforms for Drug Discovery

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Official websites use. Share sensitive information only on official, secure websites. Correspondence: Kevin G. Chen, M. Use of human pluripotent stem cells hPSCs and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery hPDD. Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR , the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis. Human pluripotent stem cells hPSCs are cells that possess the capacity to self-renew and to differentiate into all cell types of the adult body, which collectively include human embryonic stem cells hESCs and induced pluripotent stem cells hiPSCs \[ 1 — 3 \]. Encouragingly, this hPDD strategy has been further emphasized and actively implemented since the establishment of iPSCs by genetically reprogramming adult mouse fibroblasts in and human somatic cells in to an embryonic stem cell-like state \[ 1 , 2 , 4 \] Figure 1. Schema of drug developmental timeline, projected phases, and their corresponding or expected achievements through hPSC-based drug discovery hPDD : A Stage of infancy — : characterizing hPSC properties; improving hPSC growth, differentiation, cell maturation conditions; applying stem-cell niche concept for coculture, iPSC-disease modeling; and premature drug screening; B Stage of adolescence — : Optimizing stem-cell niche signaling in vitro to guide multidimensional e. Although hPDD was widely used for drug discovery between the years and , substantial experimental variability and reproducibility were encountered, hindering its application. Notable hPDD challenges have been: i choice of appropriate hPSC cultures and differentiation platforms due to a lack of understanding of the advantages and limitations of these platforms in hPDD Figures 2 and 3 \[ 5 , 6 \], 2 heterogeneity and genomic instability arising from hPSC culture and differentiation \[ 7 — 10 \], 3 that disease models established from hPSCs do not fully recapitulate diseases \[ 11 — 13 \], and 4 problems with drug-screening designs e. Since , understanding of hPSCs has increased, allowing efficient hPSC expansion, maintenance, and differentiation \[ 5 \]. We predict that hPSC-derived systems will be routinely used to accurately model diseases and for effective evaluation of drug efficacy Figure 1. Schema of 2D- and 3D-dimenisonal culture platforms. B Flow chart of 2D-monolayer and 3D-organoid culture: top panel Heterogeneous hPSC colonies top view of one representative colony in a culture dish , showing different cellular states in the periphery and center of the colony, can be homogenized as a 2D-monolayer for high-content imaging and high-throughput drug screening HTS. The graphs depict a sectional view of cells and 2D- and 3D-culture environments supplemented with cell culture medium, growth factors, extracellular matrices, and drugs. In this review, we do not intend to provide a comprehensive review of hPDD, as other similar reviews have already been published Supplemental Table 1. Instead, we will discuss the heterogeneity and reproducibility of hPSCs and their differentiation platforms related to hPDD; roles of morphogenesis , organogenesis , and tissue integration ; maturity and functionality of differentiated cells in multi-dimensional e. Experimental results obtained from in vitro hPSC culture and differentiation platforms are highly variable due to the existence of several pluripotent states e. Thus, heterogeneity both in pluripotency and differentiation is one of the leading causes of experimental variability, reproducibility, and reliability. In fact, many uncontrollable variables in hPSC culture systems stem from an insufficient understanding of optimal growth conditions to support physiologically relevant hPSCs and differentiated cells in vitro. In general, hPSC-based models can be classified into four major categories: colonies, 2D-monolayers, suspension, and 3D-culture-based organoids. The locations of CFTR mutations of interest, which are associated with cystic fibrosis, are indicated by asterisks. NCM culture was developed for homogeneous hPSC expansion, maintenance, and differentiation, aiming to minimize cellular heterogeneity and experimental variability \[ 18 \]. It is based on dissociated single cells grown on suitable extracellular matrices e. The addition of these inhibitors enhances the initial hour single-cell plating efficiency, thereby greatly reducing the heterogeneity of the culture, increasing hPSC expansion, and improving recovery rates after cryopreservation Figure 3 \[ 18 , 21 \]. Hence, NCM culture significantly increases the reproducibility and efficiency of hPDD studies compared to colony-type culture and embryoid body EB suspension culture Figures 2 and 3. Thus far, 2D-monolayer-based methods, including NCM, have been widely used for hPDD in various cellular and genetic models, including neuroectodermal disease \[ 22 — 26 \], mesoderm-derived cardiomyocytes and cardiomyopathy \[ 27 — 30 \], and several hepatic pathological conditions \[ 31 — 33 \]. To better understand the role of 2D-monolayer culture in hPDD, here, we highlight a few representative cell types derived from three germ-layer lineages. In their study, Studer, Lee, and colleagues screened compounds and achieved a target hit rate of 0. Thus, this study highlights the utility of an hiPSC-based 2D-monolayer model in identifying potential drug candidates and revealing molecular mechanisms for the therapy of neuronal diseases. It is believed that conventional 3D-EB derived cardiomyocytes or cardiac tissues are better choices for hPDD than those of 2D-monolayers in terms of their cell maturity and electrophysiological and functional suitability \[ 34 — 36 \]. Nevertheless, Lacone and coworkers implemented 2D-monolayer-based high-content phenotypic drug screening for diabetic cardiomyopathy using patient-specific hiPSC-derived cardiomyocytes. They screened compounds in a chemical library, examined their effects on subcellular structure and sarcomeric integrity of cardiomyocytes, and attained a target hit rate of 5. This work empowers conventional phenotypic assays with a 2D-monolayer platform for therapeutic tactics to treat complicated metabolic diseases. Moreover, 2D-monolayer culture, especially NCM, is particularly suitable for conducting low-throughput screening and up to HTS in endoderm-based epithelial cell models, since an epithelial monolayer is the essential element of many endodermal tissues. In addition, current hepatic differentiation protocols based on hPSC colony-type culture and 3D-EB differentiation do not easily produce homogeneous and fully mature hepatocytes in a small-well e. Hence, this improved 2D-NCM protocol might facilitate compound screening to identify small molecules that regulate liver differentiation, metabolism, and response to hepatitis C virus infection \[ 37 \]. Furthermore, 2D-NCM-based HTS was implemented to identify drugs that rescue alpha-1 antitrypsin deficiency, a liver disease without effective drugs or alternative therapies. Jang, Ye, and colleagues screened clinical compounds in an established chemical library and obtained a target hit rate of 8. Taken together, the above studies indicate the usefulness of 2D-monolayers in hPDD by offering proof-of-concept screens, confirming the activity of approved drugs in desired cells, demonstrating the predictive power of cytotoxicity of drugs, and identifying candidate drugs for therapeutics. Thus far, a limited number of clinical drugs obtained by 2D-monolayer systems are undergoing clinical trials \[ 38 \]. One such drug, termed RG \[ 25 \] and intended to treat spinal muscular atrophy, has failed in a Phase I clinical trial ClinicalTrials. Compared with conventional 3D-culture systems e. These limitations include the absence of a 3D in vivo environment that mimics developmental and disease niches, less mature differentiated adult tissues e. Nonetheless, the above mentioned major hurdles could be minimized or even circumvented. This study suggests that 2D-monolayer culture and hPDD should be carried out in physiological conditions e. Regarding increasing cellular maturity, Herron et al. Thus, these modifications are improving the development of functionally compatible adult tissues for hPDD. Guided by this consideration, we have developed xeno-free and chemically defined conditions to avoid complicating interactions between ROCKi and drugs used for screening \[ 45 \]. Hence, colony-type cultures are not suitable for genetic engineering by transfection with DNAs or RNAs via lipofectamine reagents or for transduction by viral particles \[ 18 , 45 — 47 \]. Conventionally, genetic analyses of hPSCs largely rely on isolating and characterizing individual stable clones, which takes several months. In contrast, NCM culture platforms considerably improve the efficiency of genetic engineering, permitting genetic manipulation of hPSCs and subsequent functional assays within 1 week \[ 18 , 48 \]. These simple platforms have been widely used for high-content imaging and HTS despite their limitations. However, 2D-monolayer culture and differentiation also constitute the initial important steps necessary to generate 3D-organoids, based on epithelial layer formation Figure 2B \[ 49 , 50 \]. Precise integration of these 2D-monolayer units into a complex 3D-culture system Figure 4 , which can capitulate physiological morphogenesis and organogenesis in a culture dish and a screen plate , would greatly expand avenues for hPDD. Schema of multi-dimensional culture, differentiation, and drug discoveries based on pluripotent and multipotent stem cell models: A—E Human somatic cells and adult stem cells aSCs as cell resources for reprogramming to generate iPSCs, followed by differentiation toward progenitors and functional mature cells; A—G Human adult cells or tissues as cell resources for isolation of progenitor and aSCs, followed by expansion and differentiation toward functional mature cells; H 3D coordinates based on x, y, and z axis. Of note, the dimension of time in 4D-organoids includes both the experimental i. Moreover, organoid-based assays could also be implemented via venous and luminal output modules as indicated. Abbreviations: a, small arteries; EL, epithelial lumen; ESF, extrinsic signaling factors that are absent in 3D- and 4D-organoid culture; v, small veins. Applying the principles of developmental biology to hPSC culture may facilitate recapitulation of hPSC-based morphogenesis and organogenesis \[ 7 \]. In an early study, differentiation of hESCs to cardiomyocytes was achieved by coculture with visceral endoderm-like cells \[ 51 \]. To recapitulate physiologically relevant micro-environmental cues for hPDD, we need to optimize extracellular matrices, the concentration gradients of growth factors from cells or tissues, nutrients in growth medium, oxygen and carbon dioxide concentrations, and temperatures that cells would experience in vivo \[ 7 , 52 \]. Growth conditions that integrate autocrine, paracrine, and telecrine signals provide exogenous growth factors, ligands, and cell-permeable small molecules that can mimic in vivo microenvironments. These conditions may help facilitate growth of 3D-cocultures and the establishment of hPSC-derived organoids. Supplementation of desired growth factors in the presence of pathway-specific small molecules significantly increases or decreases the maturation process of differentiated cells. Highly controllable 3D-organoids, using micro-bioreactor platforms including microfluidic bioreactors, are making hPDD possible at different scales. Organoids are now defined as stem-cell-derived 3D tissues with organ-specific cell types in vitro \[reviewed in \[ 6 , 56 — 59 \]. In organoids, only stem cells are capable of self-renewal. In general, organoid cultures depend on the support form extracellular matrices and niche factors provide in the medium. Moreover, stem cells in organoids can differentiate toward adult tissues, which exhibit similar functionality as the tissue of origin, under proper differentiation conditions. There are diverse types of organoids based on their cell or tissue origins, which include hESC-, hiPSC-, adult stem cell aSC -, and primary tissue-derived organoids. Self-organized functional organoids have been generated using robust protocols for directed differentiation of hPSCs Figure 2B. These organoids include: hepatic endoderm and functional liver buds with vasculature i. Organoids retain the genetic and epigenetic signatures of original tissues or tumors, and as such, can be used to model human development and diverse types of diseases, including cystic fibrosis \[reviewed in \[ 6 , 56 — 59 \]. Organoid systems have also been used to study infectious diseases to understand host—microbe interactions \[ 57 \], such as replication of Norovirus in human intestinal stem cell-derived enteroid culture \[ 66 \]. Brain organoids generated from hPSC-derived forebrain-specific regions and hPSC-derived neural stem cells can mimic the phenotypes of in vivo and in vitro Zika virus infection and damage \[ 67 , 68 \]. Moreover, forebrain organoids have been used to identify promising small-molecule inhibitors of Zika virus infection and induced neural cell death \[ 69 \]. Thus, multi-dimensional organoids can be used to improve potential drug efficacy at the organ level. Furthermore, organoid conditions could be adapted to micro-bioreactor platforms, with various controllable parameters e. In brief, 3D-organoids are generated by self-organizing propagated and differentiated cells, including cocultured cells, in a three-dimensional geometric space Figure 4J, 4H. Moreover, to enable the generation of physiologically-relevant organoids, we further propose the concept of 5D-organoids Figure 4L. Theoretically, 5D-organoids could be established, based on 4D-organoid culture, by adding another dimensional complexity, which embraces many intercellular, immunological, and inflammatory signals that are absent from current 4D-organoid cultures \[ 6 , 7 \]. Thus, accurately providing additional extrinsic signaling factors such as diverse types of cytokines in 4D-organoid culture would facilitate the derivation of highly mature and physiologically-compatible 5D-organoids for regenerative medicine and drug screens. Clearly, the fidelity, maturity, and functionality of organoids are important for successful hPPD, and will be discussed next. Although multi-dimensional hPSC platforms and 3D-cocultures are relatively easy to construct, such systems may not resemble live organisms in terms of physiological barriers, epigenetic factors, and immunomodulatory effects stimulated by in vivo environmental cues. Thus, they may not accurately represent human tissues in vivo. Immature cellular structures, similar to embryonic or neonatal tissues, are frequently encountered, which are largely associated with inadequate development of stem-cell niches. Therefore, these hPSC-derived models should be carefully evaluated and further optimized based on imitating stem-cell niche signals. Stem-cell niches are a specific microenvironment that allows stem cells to be maintained in an undifferentiated state \[ 73 — 75 \]. Stem-cell niches function in a highly dynamic manner at different developmental stages of embryonic and postnatal tissues. We have recently proposed a paralogous stem-cell niche PSN concept that clarifies the coupling and uncoupling mechanisms between a specific stem-cell niche and its dynamic regeneration sites at different developmental stages \[ 76 \]. Developmentally, PSNs are gradually transformed analogous niches within an individual species during development. After the first epithelial-mesenchymal transition EMT in the primitive streak, progressive variations of cell identity give rise to a cluster of adjacent stem-cell niches i. Notably, PSNs are distinct from each other in terms of their niche signaling pathways that are required for the commitment of a specific cell lineage. For example, long-term intestinal epithelial culture in vitro is made possible under a Wnt-dependent stem-cell niche \[ 77 \]. Clearly, the above examples provide important insights into mechanisms regarding the dependency or independency of stem-cell niches in cellular differentiation and 3D-tissue organization. Therefore, it is important to provide basic niche components e. Identification and implementation of the mechanisms behind faithful niche models may encourage maturity and functionality in hPSC-derived organoids for use in in vitro drug discovery. To assess the usefulness of a hPDD model, it is essential to examine the protein expression of multiple panels of tissue-specific regulators, enzymatic chemical metabolizers, and drug transporters. These regulatory proteins and drug transporters are key determinants of intracellular drug concentrations and activities. Critical proteins must also be expressed at the right time and in precise locations at physiologically relevant concentrations to faithfully implement their default functions. An unsatisfactory combination of any of the above parameters could undermine the success of any type of drug-discovery study. Moreover, compared with in vivo molecular standards, any altered expression of the predominant enzymes \[e. Similarly, altered ATP-binding cassette ABC transporters, which determine intracellular drug and ion concentrations \[ 83 — 85 \], would dramatically affect the clearance of various drugs in cultured cells and hPSC-derived organoids. Therefore, prior to any major drug-discovery projects, it is essential to determine the optimal maturity and functionality of differentiated cells and organoids in vitro and to characterize the hPSC-derived organoid tissues using native tissues as standards for comparison. Careful attention must be given to drug regulators and metabolizers e. It is well established that ABC multidrug transporters play essential roles in both the distribution and efflux of drugs in a broad range of cell types and tissues under both physiological and pathological conditions \[reviewed in \[ 84 — 89 \]. Some ABC transporters are also critical components of the blood-brain, blood-testicular, and blood-placental barriers \[ 85 \]. It is known that ABC transporters are major determinants of intrinsic and acquired cancer drug resistance \[ 84 , 88 \]. Altered or suboptimal expression of ABC transporters could hinder hPDD studies from achieving enhanced or decreased drug efflux in those hPSC-derived organoids of interest. As an example of the role of ABC transporters with regards to the functional maturity of differentiated cells e. These ABC transporters have been shown to have a major effect on the drug-discovery pipeline using other cellular models at different stages. Thus, experimental data obtained from organoids with altered ABC transporter expression could potentially provide inaccurate information concerning preclinical drug testing, since these transporters affect accumulation of drugs in target tissues. Many pre-clinical drugs such as a substantial percentage of effective kinase inhibitors and drugs in the clinic are substrates of ABC transporters. These chemical substrates can be differentially exported from diverse types of cells and tissues by ABC transporters. This differential efflux of intracellular drugs may confuse the interpretation of drug-screening data and increase the risk of drug resistance in clinical trials. Furthermore, mutant cells sometimes bear critical ABC transporter mutations at drug-binding sites and pockets, or polymorphisms that affect substrate recognition \[ 92 — 96 \]. When using hPSC-derived 3D-organoids for drug screening and discovery, researchers should perform routine examinations to determine if preclinical drug candidates are bound by ABC transporters on cell surface membranes, are accumulated intracellularly, and are effluxed from various types of cells including hepatocytes that are enriched with ABC transporter expression. We have chosen to highlight CF-organoids in this review because drug-discovery studies using these organoids have confronted all the problems discussed in the previous sections. Significantly, CF-organoid studies have provided proof-of-concept examples from experimental investigation to clinical applications, and have thus accelerated the development of translational medicine. Therefore, CF-organoids serve as a good model for discussion. CF is a life-threatening autosomal recessive disorder that affects approximately 70,—, individuals worldwide www. It is likely that patient groups with mixed drug responses may have considerable genetic heterogeneity, with rare mutations and complex alleles, all of which are difficult to recapitulate and assess pre-clinically in animal models. With regard to these complications, CF-organoid-based disease modeling offers an opportunity to identify responders for personalized medicine. Simple CF intestinal organoids have a structure similar to that of a cellular cyst, in which the apical plasma membrane is located on the luminal side of the organoids Figure 5A, 5B. Using these intestinal organoids, several powerful functional CFTR assays have been established \[ 97 \]. Through the FIS assay, CFTR activity can be directly determined by quantifying the amount of fluid secreted from the cells into the lumen and by measuring the size of the organoid lumen after adding the adenylyl cyclase activator, forskolin Figure 5B. Hence, a rapid swelling phenotype is expected in healthy organoids after forskolin induction, whereas decreased or diminished swelling may be observed in CF organoids \[ 97 \]. As a result, defective processing of CFTR to the cell membranes of these airway epithelial cells was reversed to the wild-type level. CFTR chloride current was observed in response to increased cAMP stimulation mediated by a cocktail including forskolin, similar to that observed in wild-type lung epithelial cells derived from hiPSCs. With a similar CRISPR approach, Clevers and colleagues were also able to revert the defect of the rapid chloride-channel swelling response in intestinal organoids, derived from intestinal stem cells of CF patients, to normal conditions \[ 99 \]. The rapid swelling phenotype seen in healthy organoids was restored in CF patient organoids in the presence of both VX and VX Thus, the FIS assay is an accurate functional assay of CF drugs in organoids, which could be used for drug screens and drug repurposing efforts using patient-specific organoids in personalized medicine. In these assays, homozygous mutations with a specific splicing defect in the CFTR gene \[ \] showed no response to the CF drugs tested. This was apparently due to a defect in CFTR protein synthesis from immature transcripts. Thus, this genotype could be used as a negative control when performing this assay. Interestingly, the rectal organoids derived from these patients showed a strong response to VX alone, but not to VX or Orkambi in the FIS assay \[ \]. This example highlights the clinical potential of an organoid-based assay for evaluation of drug efficacy and drug repurposing screen. We suggest that CFTR mutational organoids can be classified, patterned, assayed, and used for predicting the outcomes of CF drugs or drug combinations. Despite the growing enthusiasm of applying organoids for hPDD, organoid-based technologies appear to have several inevitable limitations. The absence of immunological and inflammatory responses that occur in a human body may be a major limiting factor. There are also limitations in using organoids for HTS assays in their current platforms due to spherical and often complicated cellular structures Figure 3 and see Outstanding Questions. Can human pluripotent stem cell hPSC -based drug discovery hPDD provide convincing platforms for in vitro drug discovery and drug repurposing with greatly reduced use of animal models? Could hPDD become a routine practice for any new drug discovery or drug repurposing at a reasonable cost? Can experimental variability be minimized and avoided with better designed hPSC models? Can physiologically relevant organoids be generated in vitro for modeling of diverse diseases and for drug screens? How can hPDD be optimized through existing coherent animal and human datasets, drug bank databases, and high-resolution protein structures to accelerate drug discovery for personalized medicine at a molecular level? How can hPDD be used to complement or even replace preclinical drug development? Would it be possible to establish complicated organoid models to simulate human body physiology and to predict or monitor diverse clinical trials e. Many unique and unknown structural features of proteins e. Moreover, this is multi-disciplinary work that requires cooperation between chemists, structural biologists, and stem-cell biologists, and physicians. In this collaboration team, chemists and structural biologists work on designing or modifying drugs based on high-resolution of desired protein structures. Stem-cell biologists focus on optimizing hPSC-derived organoid models for identifying lead compounds and testing their cytotoxicity and efficacy in animal models. Finally, clinicians are responsible for implementing clinical trials to determine drug safety and efficacy in patients and then to feedback this information to the collaboration team for further drug assessments. Human pluripotent stem-cell derived organoids faithfully retain genetic mutations of original tissues. These organoids can be used to model human development and diverse types of diseases caused by mutations in one or multiple genes. Multi-dimensional organoids can be used for in vitro drug screens and for evaluating drug efficacy at the organ level. There are many organs and tissues that express abundant ABC multidrug transporters. Physiologically or pathologically relevant organoids with optimal membrane expression of ABC transporters might provide accurate information about intracellular drug concentrations. Thus, organoid-based drug-transport assays may be particularly useful for determining whether pre-clinically developed drugs are substrates of one or more ABC transporters. This information may help clinicians overcome potential drug-transporter-mediated drug resistance in patients. In clinics, patient groups often have considerable genetic heterogeneity, with various rare mutations and complex alleles that are difficult to assess pre-clinically in animal models. CFTR-organoid-based disease modeling and drug screens provide a convincing proof-of-concept evidence that mutational organoids from these patient groups might be classified, assayed, and used for predicting the outcomes of drugs or drug combinations and for identifying responders. Different types of drug-response data from patients can be integrated with molecular and atomic structures of specific drug targets e. This is a multi-disciplinary effort that requires the cooperation between physicians, stem-cell biologists, and structural biologists, which would facilitate the implementation of precision medicine at a molecular level. Experimental variability could be minimized and avoided with better designed hPSC models and physiologically relevant organoids. The availability of multi-dimensional coculture systems, hPSC-based disease modeling, hPSC- and aSC-derived organoids, and informative functional assays in vitro , offers the possibility to optimize hPDD through existing coherent animal and human datasets, the drug bank database, and high-resolution protein structures. We believe that the use of patient-relevant organoids as a disease model system will revolutionize the methods for drug discovery and development as evidenced in studies using mutation-specific CF-organoids for evaluation of drug efficacy. Human pluripotent stem cells hPSCs and their differentiated derivatives represent valuable cell resources for organogenesis, disease modeling, and hPSC-based drug discovery hPDD in vitro. Abundant contributing variables, including the dimensionality of cell-culture methods, cell maturity and functionality, and experimental variability, epitomize the major challenges of hPDD research. Targeting various CFTR mutations in organoids derived from patients with cystic fibrosis provides a proof-of-principle drug discovery model, defining a potential pathway to the implementation of hPDD for personalized medicine. Ronald D. We thank Dr. Richard Leapman and Dr. Pingyu Zhong for critical discussion and comments on the manuscript, and George Leiman for editorial assistance. Alan Koretsky for his strong support of this work. Morphogenesis is induced and controlled by many cellular and developmental programs. This distinct pluripotency also extends to pluripotent stem cells that have identical pluripotent features under cell culture conditions. Compared to 4D-organoids, 5D-organoids have one-dimensional higher complexity, which is regulated by providing additional extrinsic signaling factors in 4D-organoids to generate highly physiologically-relevant organoids. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Trends Mol Med. Published in final edited form as: Trends Mol Med. Find articles by Kevin G Chen. Find articles by Barbara S Mallon. Find articles by Kyeyoon Park. Find articles by Pamela G Robey. Find articles by Michael M Gottesman. Find articles by Wei Zheng. Issue date Sep. PMC Copyright notice. The publisher's version of this article is available at Trends Mol Med. Open in a new tab. Similar articles. 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