技术资料

Developmental and epileptic encephalopathies (DEEs) feature altered brain development,developmental delay and seizures,with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A,encoding a membrane trafficking protein,and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1,components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles,biasing daughter cells towards a more fate-committed state,ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families. Developmental and epileptic encephalopathies are devastating neurological disorders. Here,the authors establish a cohort of patients with variants in the gene DENND5A and use human stem cells to discover a disease mechanism involving altered cell division. 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

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However,generation of human AID cell lines is challenging,especially in human embryonic stem cells (hESCs). Here,we develop HiHo-AID2,a streamlined procedure for rapid,one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines,including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03187-w. 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

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

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

SUMMARYHuman neurogenesis is disproportionately protracted,lasting >10 times longer than in mouse,allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord,expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called vpMN (ventral motor neuron progenitor) that uniquely extends and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2,which in vpMNs is co-expressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study,we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that NKX2-2 represses proneural gene NEUROG2 by two distinct,Notch-independent mechanisms that are respectively apparent in rodent and human spinal progenitors: in rodents (and chick),NKX2-2 represses Olig2 and the motor neuron lineage through its tinman domain,leading to loss of Neurog2 expression. In human vpMNs,however,NKX2-2 represses NEUROG2 but not OLIG2,thereby allowing motor neurogenesis to proceed,albeit in a delayed and protracted manner. Interestingly,we found that ectopic expression of tinman-mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs,repressing Neurog2 but not Olig2,and leading to delayed and protracted motor neurogenesis. Our studies identify a Notch- and tinman-independent mode of Nkx2-2-mediated Neurog2 repression that is observed in human spinal progenitors,but is normally masked in rodents and chicks due to Nkx2-2’s tinman-dependent repression of Olig2. 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

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

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

Enhancing RuvBL1,but particularly RuvBL2 expression,reduces toxic dipeptide repeat proteins in vitro and in vivo models of C9orf72-linked ALS/FTD,suggesting that modulating RuvBL1/2 levels could be a promising therapeutic approach for C9ALS/FTD. A G4C2 hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore,decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study,we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2,which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1,but to a greater extent RuvBL2,reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines,primary neurons from the C9-500 transgenic mouse model,and patient-derived iPSC motor neurons. In vivo,we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in our Drosophila model was sufficient to rescue a number of DPR-related motor phenotypes. Thus,modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD. View Publication

This study describes an OTULIN-related autoinflammatory syndrome (ORAS) patient with two rare heterozygous variants of OTULIN (p.P152L and p.R306Q); the latter is a de novo variant that acts in a dominant-negative manner to cause ORAS. OTULIN-related autoinflammatory syndrome (ORAS),a severe autoinflammatory disease,is caused by biallelic pathogenic variants of OTULIN,a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here,we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells,which confirms that the patient has ORAS. However,although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN,the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis. Graphical Abstract View Publication