Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate.

Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

iPSC-derived neural precursor cells engineering GBA1 recovers acid ß-glucosidase deficiency and diminishes α-synuclein and neuropathology.

Mutations in GBA1, encoding the lysosomal acid ß-glucosidase (GCase), cause neuronopathic Gaucher disease (nGD) and promote Parkinson disease (PD). The mutations on GBA1 include deletion and missense mutations that are pathological and lead to GCase deficiency in Gaucher disease. Both nGD and PD lack disease-modifying treatments and are critical unmet medical needs. In this study, we evaluated a cell therapy treatment using mouse iPSC-derived neural precursor cells (NPCs) engineered to overexpress GCase (termed hGBA1-NPCs). The hGBA1-NPCs secreted GCase that was taken up by adjacent mouse Gba -/- neurons and improved GCase activity, reduced GCase substrate accumulation, and improved mitochondrial function. Short-term in vivo effects were evaluated in 9H/PS-NA mice, an nGD mouse model exhibiting neuropathology and α-synuclein aggregation, the typical PD phenotypes. Intravenously administrated hGBA1-NPCs were engrafted throughout the brain and differentiated into neural lineages. GCase activity was increased in various brain regions of treated 9H/PS-NA mice. Compared with vehicle, hGBA1-NPC-transplanted mice showed ∼50% reduction of α-synuclein aggregates in the substantia nigra, significant reduction of neuroinflammation and neurodegeneration in the regions of NPC migration, and increased expression of neurotrophic factors that support neural cell function. Together, these results support the therapeutic benefit of intravenous delivery of iPSC-derived NPCs overexpressing GCase in mitigating nGD and PD phenotypes and establish the feasibility of combined cell and gene therapy for GBA1-associated PD.

A review of protocols for brain organoids and applications for disease modeling.

Recent breakthroughs in human stem cell technologies have enabled the generation of 3D brain organoid platforms for modeling human neurodevelopment and disease. Here, we review advances in brain organoid development, approaches for generating whole-brain or cerebral organoids and region-specific brain organoids, and their applications in disease modeling. We present a comprehensive overview of various brain organoid generation protocols, including culture steps, media, timelines, and technical considerations associated with each protocol, and highlight the advantages and disadvantages of each protocol. We also discuss the current limitations as well as increasing sophistication of brain organoid technology, and future directions for the field. These insights provide a valuable assessment of multiple commonly used brain organoid models and main considerations for investigators who are considering implementing brain organoid technologies in their laboratories.

Volatilomic Signatures of AGS and SNU-1 Gastric Cancer Cell Lines.

In vitro studies can help reveal the biochemical pathways underlying the origin of volatile indicators of numerous diseases. The key objective of this study is to identify the potential biomarkers of gastric cancer. For this purpose, the volatilomic signatures of two human gastric cancer cell lines, AGS (human gastric adenocarcinoma) and SNU-1 (human gastric carcinoma), and one normal gastric mucosa cell line (GES-1) were investigated. More specifically, gas chromatography mass spectrometry has been applied to pinpoint changes in cell metabolism triggered by cancer. In total, ten volatiles were found to be metabolized, and thirty-five were produced by cells under study. The volatiles consumed were mainly six aldehydes and two heterocyclics, whereas the volatiles released embraced twelve ketones, eight alcohols, six hydrocarbons, three esters, three ethers, and three aromatic compounds. The SNU-1 cell line was found to have significantly altered metabolism in comparison to normal GES-1 cells. This was manifested by the decreased production of alcohols and ketones and the upregulated emission of esters. The AGS cells exhibited the increased production of methyl ketones containing an odd number of carbons, namely 2-tridecanone, 2-pentadecanone, and 2-heptadecanone. This study provides evidence that the cancer state modifies the volatilome of human cells.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Cost-effectiveness analysis of a resource-intensive approach versus minimally invasive strategy for high-risk transcatheter aortic valve replacement patients.

Aortic stenosis has a high mortality rate in patients who do not receive aortic valve replacement. Previously, transcatheter aortic valve replacement (TAVR) was an intervention reserved for individuals deemed high-risk for surgery. Since that time, TAVR has increasingly been offered to lower risk patients, yet it is unclear whether TAVR will meet an acceptable cost-effectiveness threshold in this group. In this cost-effectiveness study, we employed a decision tree model with Monte Carlo probability sensitivity analysis to determine the incremental cost (in US$) per quality-adjusted life year (QALY) and life year (LY) of performing the TAVR procedure using the resource-intensive approach versus the minimally invasive strategy in high-risk surgical patients.

Implementing Research Shared (Core) Facility Billing Systems.

Research Shared (core) Facilities (RSF) operate as centers of expertise and help to accelerate basic and translational science. A centralized platform for unified ordering, equipment reservation, and the billing of services using an integrated software system is a valuable resource that many academic institutions should consider. This paper discusses considerations for best practices and identifies lessons learned from the implementation of two different software systems for RSF. After implementing two different centralized billing systems for RSF, this paper identifies considerations for best practices and discusses lessons learned.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

Monitoring the volatile language of fungi using gas chromatography-ion mobility spectrometry.

Fusarium oxysporum is a plant pathogenic fungus leading to severe crop losses in agriculture every year. A sustainable way of combating this pathogen is the application of mycoparasites-fungi parasitizing other fungi. The filamentous fungus Trichoderma atroviride is such a mycoparasite that is able to antagonize phytopathogenic fungi. It is therefore frequently applied as a biological pest control agent in agriculture. Given that volatile metabolites play a crucial role in organismic interactions, the major aim of this study was to establish a method for on-line analysis of headspace microbial volatile organic compounds (MVOCs) during cultivation of different fungi. An ion mobility spectrometer with gas chromatographic pre-separation (GC-IMS) enables almost real-time information of volatile emissions with good selectivity. Here we illustrate the successful use of GC-IMS for monitoring the time- and light-dependent release of MVOCs by F. oxysporum and T. atroviride during axenic and co-cultivation. More than 50 spectral peaks were detected, which could be assigned to 14 volatile compounds with the help of parallel gas chromatography-mass spectrometric (GC-MS) measurements. The majority of identified compounds are alcohols, such as ethanol, 1-propanol, 2-methyl propanol, 2-methyl butanol, 3-methyl-1-butanol and 1-octen-3-ol. In addition to four ketones, namely acetone, 2-pentanone, 2-heptanone, 3-octanone, and 2-octanone; two esters, ethyl acetate and 1-butanol-3-methylacetate; and one aldehyde, 3-methyl butanal, showed characteristic profiles during cultivation depending on axenic or co-cultivation, exposure to light, and fungal species. Interestingly, 2-octanone was produced only in co-cultures of F. oxysporum and T. atroviride, but it was not detected in the headspace of their axenic cultures. The concentrations of the measured volatiles were predominantly in the low ppbv range; however, values above 100 ppbv were detected for several alcohols, including ethanol, 2-methylpropanol, 2-methyl butanol, 1- and 3-methyl butanol, and for the ketone 2-heptanone, depending on the cultivation conditions. Our results highlight that GC-IMS analysis can be used as a valuable analytical tool for identifying specific metabolite patterns for chemotaxonomic and metabolomic applications in near-to-real time and hence easily monitor temporal changes in volatile concentrations that take place in minutes.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.

Comparative analysis of human microglial models for studies of HIV replication and pathogenesis.

BACKGROUND: HIV associated neurocognitive disorders cause significant morbidity and mortality despite the advent of highly active antiretroviral therapy. A deeper understanding of fundamental mechanisms underlying HIV infection and pathogenesis in the central nervous system is warranted. Microglia are resident myeloid cells of the brain that are readily infected by HIV and may constitute a CNS reservoir. We evaluated two microglial model cell lines (C20, HMC3) and two sources of primary cell-derived microglia (monocyte-derived microglia [MMG] and induced pluripotent stem cell-derived microglia [iPSC-MG]) as potential model systems for studying HIV-microglia interactions. RESULTS: All four microglial model cells expressed typical myeloid markers with the exception of low or absent CD45 and CD11b expression by C20 and HMC3, and all four expressed the microglia-specific markers P2RY12 and TMEM119. Marked differences were observed upon gene expression profiling, however, indicating that MMG and iPSC-MG cluster closely together with primary human microglial cells, while C20 and HMC3 were similar to each other but very different from primary microglia. Expression of HIV-relevant genes also revealed important differences, with iPSC-MG and MMG expressing relevant genes at levels more closely resembling primary microglia. iPSC-MG and MMG were readily infected with R5-tropic HIV, while C20 and HMC3 lack CD4 and require pseudotyping for infection. Despite many similarities, HIV replication dynamics and HIV-1 particle capture by Siglec-1 differed markedly between the MMG and iPSC-MG. CONCLUSIONS: MMG and iPSC-MG appear to be viable microglial models that are susceptible to HIV infection and bear more similarities to authentic microglia than two transformed microglia cell lines. The observed differences in HIV replication and particle capture between MMG and iPSC-MG warrant further study.

A benchmarking protocol for breath analysis: the peppermint experiment.

Sampling of volatile organic compounds (VOCs) has shown promise for detection of a range of diseases but results have proved hard to replicate due to a lack of standardization. In this work we introduce the 'Peppermint Initiative'. The initiative seeks to disseminate a standardized experiment that allows comparison of breath sampling and data analysis methods. Further, it seeks to share a set of benchmark values for the measurement of VOCs in breath. Pilot data are presented to illustrate the standardized approach to the interpretation of results obtained from the Peppermint experiment. This pilot study was conducted to determine the washout profile of peppermint compounds in breath, identify appropriate sampling time points, and formalise the data analysis. Five and ten participants were recruited to undertake a standardized intervention by ingesting a peppermint oil capsule that engenders a predictable and controlled change in the VOC profile in exhaled breath. After collecting a pre-ingestion breath sample, five further samples are taken at 2, 4, 6, 8, and 10 h after ingestion. Samples were analysed using ion mobility spectrometry coupled to multi-capillary column and thermal desorption gas chromatography mass spectrometry. A regression analysis of the washout data was used to determine sampling times for the final peppermint protocol, and the time for the compound measurement to return to baseline levels was selected as a benchmark value. A measure of the quality of the data generated from a given technique is proposed by comparing data fidelity. This study protocol has been used for all subsequent measurements by the Peppermint Consortium (16 partners from seven countries). So far 1200 breath samples from 200 participants using a range of sampling and analytical techniques have been collected. The data from the consortium will be disseminated in subsequent technical notes focussing on results from individual platforms.

Intravenous infusion of iPSC-derived neural precursor cells increases acid ß-glucosidase function in the brain and lessens the neuronopathic phenotype in a mouse model of Gaucher disease.

Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-ß-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4ß1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, -34%) and glucosylsphingosine (GS, -11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.

Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids.

Human organoid systems recapitulate in vivo organ architecture yet fail to capture complex pathologies such as inflammation and fibrosis. Here, using 11 different healthy and diseased pluripotent stem cell lines, we developed a reproducible method to derive multi-cellular human liver organoids composed of hepatocyte-, stellate-, and Kupffer-like cells that exhibit transcriptomic resemblance to in vivo-derived tissues. Under free fatty acid treatment, organoids, but not reaggregated cocultured spheroids, recapitulated key features of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes in a successive manner. Interestingly, an organoid-level biophysical readout with atomic force microscopy demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoids from patients with genetic dysfunction of lysosomal acid lipase phenocopied severe steatohepatitis, rescued by FXR agonism-mediated reactive oxygen species suppression. The presented key methodology and preliminary results offer a new approach for studying a personalized basis for inflammation and fibrosis in humans, thus facilitating the discovery of effective treatments.

Noncoding deletions reveal a gene that is critical for intestinal function.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis.

Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5-CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/ß-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.

Non-contact breath sampling for sensor-based breath analysis.

Breath analysis holds great promise for real-time and non-invasive medical diagnosis. Thus, there is a considerable need for simple-in-use and portable analyzers for rapid detection of breath indicators for different diseases in their early stages. Sensor technology meets all of these demands. However, miniaturized breath analyzers require adequate breath sampling methods. In this context, we propose non-contact sampling; namely the collection of breath samples by exhalation from a distance into a miniaturized collector without bringing the mouth into direct contact with the analyzing device. To evaluate this approach different breathing maneuvers have been tested in a real-time regime on a cohort of 23 volunteers using proton transfer reaction mass spectrometry. The breathing maneuvers embraced distinct depths of respiration, exhalation manners, size of the mouth opening and different sampling distances. Two inhalation modes (normal, relaxed breathing and deep breathing) and two exhalation manners (via smaller and wider lips opening) forming four sampling scenarios were selected. A sampling distance of approximately 2 cm was found to be a reasonable trade-off between sample dilution and requirement of no physical contact of the subject with the analyzer. All four scenarios exhibited comparable measurement reproducibility spread of around 10%. For normal, relaxed inspiration both dead-space and end-tidal phases of exhalation lasted approximately 1.5 s for both expiration protocols. Deep inhalation prolongs the end-tidal phase to about 3 s in the case of blowing via a small lips opening, and by 50% when the air is exhaled via a wide one. In conclusion, non-contact breath sampling can be considered as a promising alternative to the existing breath sampling methods, being relatively close to natural spontaneous breathing.