搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However,our knowledge about the safe usage of anti-inflammatory drugs during pregnancy is still limited. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation,these models commonly lack microglia,which are susceptible to and promote inflammation. Furthermore,microglia are actively involved in neuronal development. Here,we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids. Once the outer plexiform layer forms,these hIPSC-derived microglia (iMG) fully integrate into the retinal organoids. Since the ganglion cell survival declines by this time in 3D-retinal organoids,we adapted the model into 2D and identify that the improved ganglion cell number significantly decreases only with iMG presence. In parallel,we applied the immunostimulant POLY(I:C) to mimic a fetal viral infection. While POLY(I:C) exposure alters the iMG phenotype,it does not hinder their interaction with ganglion cells. Furthermore,iMG significantly enhance the supernatant’s inflammatory secretome and increase retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably,while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG,ibuprofen rescues this effect only if iMG are present. Mechanistically,ibuprofen targets the enzymes cyclooxygenase 1 and 2 (COX1/PTGS1 and COX2/PTGS2) simultaneously,from which iMG mainly express COX1. Selective COX1 blockage fails to restore the calcium peak amplitude upon POLY(I:C) stimulation,suggesting ibuprofen’s beneficial effect depends on the presence and interplay of COX1 and COX2. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03366-x. View Publication

Cell fate progression of pluripotent progenitors is strictly regulated,resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study,we use human brain and retina organoid models and present single-cell profiling of H3K27ac,H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage,providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction,resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination. The mechanisms underlying human cell diversity are unclear. Here the authors provide a single-cell epigenome map of human neural organoid development and dissect how epigenetic changes control cell fate specification from pluripotency to distinct cerebral and retina neural types. View Publication

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3,SLMAP,and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states. View Publication

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis,osteogenesis,and spermatogenesis have been studied,the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here,using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls,we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover,loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically,CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes,which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex,through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity,which causes inhibition of AKT and ERK,leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development. View Publication

An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits,we investigated an in vitro neural tissue model for inter-regional connections,in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids,suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition,optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro. Connecting cerebral organoids with an axon bundle models inter-regional projections and enhances neural activity. Optogenetic stimulation induces short-term plasticity,offering insights into macroscopic circuit development and functionality. View Publication

ABSTRACTThe paradoxical exacerbation of cellular injury and death during reperfusion remains a problem in the treatment of myocardial infarction. Mitochondrial dysfunction plays a key role in the pathogenesis of myocardial ischemia and reperfusion injury. Dysfunctional mitochondria can be removed by mitophagy,culminating in their degradation within acidic lysosomes. Mitophagy is pivotal in maintaining cardiac homeostasis and emerges as a potential therapeutic target. Here,we employed beating human engineered heart tissue (EHT) to assess mitochondrial dysfunction and mitophagy during ischemia and reperfusion simulation. Our data indicate adverse ultrastructural changes in mitochondrial morphology and impairment of mitochondrial respiration. Furthermore,our pH-sensitive mitophagy reporter EHTs,generated by a CRISPR/Cas9 endogenous knock-in strategy,revealed induced mitophagy flux in EHTs after ischemia and reperfusion simulation. The induced flux required the activity of the protein kinase ULK1,a member of the core autophagy machinery. Our results demonstrate the applicability of the reporter EHTs for mitophagy assessment in a clinically relevant setting. Deciphering mitophagy in the human heart will facilitate development of novel therapeutic strategies. Summary: Mitochondrial dysfunction and lysosomal degradation of mitochondria (mitophagy) is induced after ischemia and reperfusion simulation in human engineered heart tissue,as shown with an endogenous pH-sensitive mitophagy reporter. View Publication

BackgroundRecent studies highlight the critical role of microglia in neurodegenerative disorders,and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches,yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia.MethodsWe developed the hCSF1Bdes mouse line,which is suitable as a new transplantation model and available to be crossed to any disease model of interest. The hCSF1Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additionally,we crossed this model with two humanized App KI mice,the AppHu and the AppSAA. Flow cytometry,immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease.ResultsOur results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1Bdes mice without triggering a NK-driven immune response. Furthermore,we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1Bdes and the crosses with the Alzheimer’s disease knock-in model AppSAA and the humanized App knock-in control mice,AppHu are deposited with EMMA and fully accessible to the research community.ConclusionThe hCSF1Bdes mouse is available for both non-profit and for-profit organisations,facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00823-2. View Publication

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however,its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here,we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids,revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation,expressed SOX2,and lacked CRX,consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas,particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities,demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1,Flavopiridol) and the cell-cycle inhibitor Volasertib,indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively,our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma,establishes a robust and clinically relevant human retinal organoid platform,and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype. View Publication

Homology Directed Repair (HDR) enables precise genome editing,but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes,such as Non-Homologous End Joining (NHEJ). In this study,we develop a functional,pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target,53BP1,identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing,synergize with a DNAPK inhibitor molecule,and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes. Subject terms: Targeted gene repair,Homologous recombination,High-throughput screening View Publication

Drugs work by binding to a specific 3D structure on a protein. Drug discovery has historically been driven by prior knowledge of function,either of a protein or chemical. This knowledge of function then drives investigations to probe chemical/protein interactions. We undertook a different approach. We first identified unique 3D structures,agnostic of function,and investigated whether they could lead us to innovative therapeutics. Using a synchrotron-based X-ray source,we first determined high-resolution structures of hundreds of proteins. With a supercomputer running analytical programs created by us,we identified novel 3D structures and screened for chemicals binding them. We then tested their ability to inhibit cancer growth without damaging normal cells. We identified a potent inhibitor of a deadly cancer,melanoma. It was not toxic to normal cells even at 2100-fold higher doses. It worked by inducing anoikis,a fundamental process of known importance for cancer. Therapeutics that selectively induce anoikis are needed. In summary,we demonstrate the power of using a 3D protein structure as the starting point to discover new biology and drugs. Drug discovery historically starts with an established function,either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function,we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds,we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures,we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast,prostate,colon,or lung cancer,and for toxicity on normal human bone marrow stem cells,both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound,Dxr2-017,exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays,Dxr2-017 had an IC50 of 12 nM against melanoma cells,while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis,a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable. View Publication

The increasing scarcity of organs and the significant morbidity linked to dialysis require the development of engineered kidney tissues from human-induced pluripotent stem cells. Integrative approaches that synergize scalable kidney organoid differentiation,tissue biomanufacturing,and comprehensive assessment of their immune response and host integration are essential to accomplish this. Here,we create engineered human kidney tissues composed of organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature,enhanced kidney cell apoptosis,and an inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent rapamycin,the induced immune response is greatly suppressed. Our model is a translational platform to study engineered kidney tissue immunogenicity and develop therapeutic targets for kidney rejection. Subject areas: Health sciences,Immunology,Bioengineering,Tissue engineering View Publication