技术资料

The search for an effective cell replacement therapy for diabetes has driven the development of “perfect” pancreatic islets from human pluripotent stem cells (hPSCs). These hPSC-derived pancreatic islet-like β cells can overcome the limitations for disease modelling,drug development and transplantation therapies in diabetes. Nevertheless,challenges remain in generating fully functional and mature β cells from hPSCs. This review underscores the significant efforts made by researchers to optimize various differentiation protocols aimed at enhancing the efficiency and quality of hPSC-derived pancreatic islets and proposes methods for their improvement. By emulating the natural developmental processes of pancreatic embryogenesis,specific growth factors,signaling molecules and culture conditions are employed to guide hPSCs towards the formation of mature β cells capable of secreting insulin in response to glucose. However,the efficiency of these protocols varies greatly among different human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) lines. This variability poses a particular challenge for generating patient-specific β cells. Despite recent advancements,the ultimate goal remains to develop a highly efficient directed differentiation protocol that is applicable across all genetic backgrounds of hPSCs. Although progress has been made,further research is required to optimize the protocols and characterization methods that could ensure the safety and efficacy of hPSC-derived pancreatic islets before they can be utilized in clinical settings. View Publication

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents,including histamine,serine proteases,and various eicosanoids and cytokines. As such,a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology,assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells,cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive,requiring only 500 cells per data point,which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation,to enable innovative drug discovery efforts to be prosecuted. View Publication

Platelets are key mediators of atherothrombosis,yet,limited tools exist to identify individuals with a hyperreactive platelet phenotype. In this study,we investigate the association of platelet hyperreactivity and cardiovascular events,and introduce a tool,the Platelet Reactivity ExpreSsion Score (PRESS),which integrates platelet aggregation responses and RNA sequencing. Among patients with peripheral artery disease (PAD),those with a hyperreactive platelet response (>60% aggregation) to 0.4 µM epinephrine had a higher incidence of the 30 day primary cardiovascular endpoint (37.2% vs. 15.3% in those without hyperreactivity,adjusted HR 2.76,95% CI 1.5–5.1,p = 0.002). PRESS performs well in identifying a hyperreactive phenotype in patients with PAD (AUC [cross-validation] 0.81,95% CI 0.68 –0.94,n = 84) and in an independent cohort of healthy participants (AUC [validation] 0.77,95% CI 0.75 –0.79,n = 35). Following multivariable adjustment,PAD individuals with a PRESS score above the median are at higher risk for a future cardiovascular event (adjusted HR 1.90,CI 1.07–3.36; p = 0.027,n = 129,NCT02106429). This study derives and validates the ability of PRESS to discriminate platelet hyperreactivity and identify those at increased cardiovascular risk. Future studies in a larger independent cohort are warranted for further validation. The development of a platelet reactivity expression score opens the possibility for a personalized approach to antithrombotic therapy for cardiovascular risk reduction. Platelet hyperreactivity is associated with cardiovascular events in patients with PAD. Here the authors derive and validate a circulating platelet genetic signature to discriminate platelet hyperreactivity and cardiovascular risk. View Publication

Human pluripotent stem cells offer a scalable platform to study genetic and signalling mechanisms governing cell lineage decisions during differentiation. Genome-wide and single-cell transcriptomics technologies likewise offer high-throughput analysis of heterogeneous cell differentiation states. While in vivo development has been extensively characterised using these technologies,there remains a need for comprehensive single-cell transcriptomic profiling of stem cell differentiation from pluripotency. Understanding gene expression changes governing differentiation in vitro is key to developing high fidelity differentiation protocols and understanding fundamental mechanisms of development. We generated a single-cell RNA sequencing time course to study the role of developmental signalling pathways on multilineage diversification from pluripotency in vitro. The combined dataset of over 60,000 cells spans cell types from a time course of differentiation across all germ layers,ranging from gastrulation cell states to progenitor and committed cell types. These data provide a diverse benchmarking reference point to compare against in vivo development and advance understanding of signalling regulation of differentiation,providing insights into protocol development,drug screening,and regenerative medicine applications. View Publication

Various systems for differentiating hematopoietic cells from human pluripotent stem cells (PSCs) have been developed,although none have been fully optimized. In this report,we describe the development of a novel three-dimensional system for differentiating hematopoietic cells from PSCs using collagen sponges (CSs) reinforced with poly(ethylene terephthalate) fibers as a scaffold. PSCs seeded onto CSs were differentiated in a stepwise manner with appropriate cytokines under serum-free and feeder-free conditions. This process yielded several lineages of floating hematopoietic cells repeatedly for more than 1 month. On immunohistochemical staining,we detected CD34+ cells and CD45+ cells in the surface and cavities of the CS. Taking advantage of the portability of this system,we were able to culture multiple CSs together floating in medium,making it possible to harvest large numbers of hematopoietic cells repeatedly. Given these findings,we suggest that this novel three-dimensional culture system may be useful in the large-scale culture of PSC-derived hematopoietic cells. View Publication

Polycomb group protein BMI1 plays an important role in cellular homeostasis by maintaining a balance between proliferation and senescence. It is often overexpressed in cancer cells and is required for self-renewal of stem cells. At present,very little is known about the signaling pathways that regulate the expression of BMI1. Here,we report that BMI1 autoactivates its own promoter via an E-box present in its promoter. We show that BMI1 acts as an activator of the WNT pathway by repressing Dickkopf (DKK) family of WNT inhibitors. BMI1 mediated repression of DKK proteins; in particular,DKK1 led to up-regulation of WNT target c-Myc,which in turn further led to transcriptional autoactivation of BMI1. Thus,a positive feedback loop connected by the WNT signaling pathway regulates BMI1 expression. This positive feedback loop regulating BMI1 expression may be relevant to the role of BMI1 in promoting cancer and maintaining stem cell phenotype. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods,which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence,focusing on measurable parameters of hindgut spheroids,the intermediate step between definitive endoderm and mature organoids. We found that 13{\%} of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number,diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation,we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 $\mu$m and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids,respectively,thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication