技术资料

Cryopreservation in cryovials extends cell storage at low temperatures,and advances in organoid cryopreservation improve reproducibility and reduce generation time. However,cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles,we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs),early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs,including PSC cryopreservation kit,CryoStor CS10,3dGRO,and 10% DMSO,before being frozen overnight at ?80°C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type,organoid size,and CPA exposure duration on cell viability post-thaw. Additionally,the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably,smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 – 600 ?m. These cryopreserved early-stage HBOs successfully matured over 30 days,exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids,by integrating cryopreservation,thawing,culturing,staining,rinsing,and imaging processes within a single system,thereby preserving the 3D structure of the organoids. View Publication

AbstractBackgroundRodent models have been widely used to investigate skin development,but do not account for significant differences in composition compared to human skin. On the other hand,two-dimensional and three-dimensional engineered skin models still lack the complex features of human skin such as appendages and pigmentation. Recently,hair follicle containing skin organoids (SKOs) with a stratified epidermis,and dermis layer have been generated as floating spheres from human-induced pluripotent stem cells (hiPSCs).MethodsThe current study aims to investigate the generation of hiPSCs-derived SKOs using an air-liquid interface (ALI) model on transwell membranes (T-SKOs) and compares their development with conventional floating culture in low-attachment plates (F-SKOs).ResultsMature SKOs containing an epidermis,dermis,and appendages are created in both T-SKO and F-SKO conditions. It was found that the hair follicles are smaller and shorter in the F-SKO compared with T-SKOs. Additionally,the ALI conditions contribute to enhanced hair follicle numbers than conventional floating culture.ConclusionsTogether,this study demonstrates the significant influence of transwell culture on the morphogenesis of hair follicles within SKOs and highlights the potential for refinement of skin model engineering for advancing dermatology and skin research. View Publication

The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However,most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here,we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation,and the incorporation of newly synthesized histones at UV damage sites,that both prevent local mitotic phosphorylations on histone H3 serine residues. Functionally,this chromatin-marking pathway controls the segregation of UV damage in the cell progeny with consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions. The transmission of unrepaired DNA lesions through mitosis needs tight control. Here,the authors uncover a damaged chromatin marking mechanism driving the segregation of UV damage through mitosis with potential consequences on daughter cell fate. View Publication

A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this,the mechanism of how opioids impact the developing brain remains unknown. Here,we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model,we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1,two-tailed t-test,p =? 0.004; OPRK1,p =? 0.0003). Next,we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression,a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal,differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p =? 0.0496) and morphine exposure conditions (p,=? 0.0136,1-way ANOVA). Finally,we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA,F =? 16.05,p View Publication

AbstractNeurofilament light (NfL) concentration in CSF and blood serves as an important biomarker in neurology drug development. Changes in NfL are generally assumed to reflect changes in neuronal damage,while little is known about the clearance of NfL from biofluids. In a study of asymptomatic individuals at risk for prion disease,both blood and CSF NfL spiked in one participant following a 6-week course of minocycline,absent any other biomarker changes and without subsequent onset of symptoms. We subsequently observed high NfL after minocycline treatment in discarded clinical plasma samples from inpatients,in mouse plasma and in conditioned media from neuron–microglia co-cultures. The specificity and kinetics of NfL response lead us to hypothesize that minocycline does not cause or exacerbate neuronal damage,but instead affects NfL by inhibiting its clearance,posing a potential confounder for the interpretation of this important biomarker. Gentile et al. report that treatment with the drug minocycline may cause neurofilament light concentration to rise in CSF and blood. This effect appears mediated by changes in clearance of the protein,rather than release from tissue,confounding this biomarker normally held to report on neuronal health. View Publication

In the present study,we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely,in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons,while TH-positive catecholamine neurons were significantly underrepresented. Furthermore,high NMDAR1 expression was also detected in the KS spheroid,similarly to other patients-derived neuronal cultures,denoting high NMDAR1 expression as a general,KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat,rotenone,bardoxolone,and doxorubicin),and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions,drug development,and screening towards individual therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-72791-4. View Publication

Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date,the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation,motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity,MN were initially exposed once to 400 µM,600 µM,800 µM,or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS,XTT,IncuCyte,qRT-PCR,and Western Blot. For the first time,VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this,MN morphology and neurite network were altered time and concentration-dependently. Thus,MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future. View Publication

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However,we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis,particularly in the late stage,where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus,we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge,the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this,we generated inducible TRIB3 overexpression hESCs,named TRIB3tet-on OE H9,based on a Tet-On system. Then,we analyzed hemoglobin expression,condensed chromosomes,organelle clearance,and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry,enhanced hemoglobin and related protein expression in Western blot,decreased nuclear area size,promoted enucleation rate,decreased lysosome and mitochondria number,more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore,our study delineates the role of TRIB3 in terminal erythropoiesis,and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation. View Publication

Efficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However,administrated eosinophils might influx into inflammatory lungs,posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs,indicating a lower risk of causing airway disorders. Furthermore,these R-iEOs had enhanced anti-tumor functions,preferably accumulated at tumor sites,and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover,the chemo-trafficking signaling increased in R-iEOs,which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future. View Publication

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol,we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array,efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase,permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences,no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects. Key features • A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells • Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment • Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types. View Publication

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by sodium channel dysfunction. However,the clinical management of ARVC remains challenging. Identifying novel compounds for the treatment of ARVC is crucial for advancing drug development.PurposeIn this study,we aim to identify novel compounds for treating ARVC.MethodsMachine learning (ML) models were constructed using proteins analyzed from the scRNA-seq data of ARVC rats and their corresponding protein-protein interaction (PPI) network to predict binding affinity (BA). To validate these predictions,a series of experiments in cardiac organoids were conducted,including Western blotting,ELISA,MEA,and Masson staining to assess the effects of these compounds.ResultsWe first discovered and identified SCN5A as the most significantly affected sodium channel protein in ARVC. ML models predicted that Kaempferol binds to SCN5A with high affinity. In vitro experiments further confirmed that Kaempferol exerted therapeutic effects in ARVC.ConclusionThis study presents a novel approach for identifying potential compounds to treat ARVC. By integrating ML modeling with organoid validation,our platform provides valuable support in addressing the public health challenges posed by ARVC,with broad application prospects. Kaempferol shows promise as a lead compound for ARVC treatment. View Publication

Quantum dots,which won the Nobel Prize in Chemistry,have recently gained significant attention in precision medicine due to their unique properties,such as size-tunable emission,high photostability,efficient light absorption,and vibrant luminescence. Consequently,there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors,biomolecular imaging probes,and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs,which often require expensive and toxic precursors,as they offer several merits in eco-friendly synthesis,preparation from renewable sources,and cost-effective production. In this study,we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines,including human dermal fibroblasts,HeLa,cardiomyocytes,induced pluripotent stem cells,and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells,as they were taken up more by cells treated with tumor necrosis factor-? than untreated cells. In addition,our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents,which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases. View Publication