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

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

Blood transfusion plays a vital role in modern medicine,but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution,yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development,and EPO is among the most expensive media components. To address this challenge,we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells,we achieve equivalent erythroid differentiation,transcriptomic changes,and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors,potentially improving scalable manufacturing of a wide range of clinically relevant cell types. Shortages of donor blood for transfusions can have severe medical consequences,and ex vivo production of red blood cells offers a potential solution. Here authors developed synthetic EPO receptors,which allow erythropoiesis (red blood cell production) without the need for expensive EPO. View Publication

BackgroundThe epoxyeicosatrienoic acids (EETs) are derivatives of the arachidonic acid metabolism with anti-inflammatory activities. However,their efficacy is limited due to the rapid hydrolysis by soluble epoxide hydrolase (sEH). Inhibition of sEH has been shown to stabilize the EETs and reduce neuroinflammation in A? mouse models of Alzheimer’s disease (AD). However,the role of the sEH-EET signaling pathway in other CNS cell types and neurodegenerative conditions are less understood.MethodsHere we investigated the mechanisms and functional role of the sEH-EET axis in tauopathy by treating PS19 mice with a small molecule sEH inhibitor TPPU and by crossing the PS19 mice with Ephx2 (gene encoding sEH) knockout mice. This was followed by single-nucleus RNA-sequencing (snRNA-seq),biochemical and immunohistochemical analysis,and behavioral assessments. Additionally,we examined the effects of the sEH-EET pathway in primary microglia cultures and human induced pluripotent stem cell (iPSC)-derived neurons exhibiting seeding-induced Tau inclusions.ResultssEH inhibition improved cognitive function,rescued neuronal cell loss,and reduced Tau pathology and microglial reactivity. snRNA-seq revealed that TPPU treatment upregulated genes involved in actin cytoskeleton and excitatory synaptic pathways. Treatment of human iPSC-derived neurons with TPPU enhanced synaptic density without affecting Tau accumulation,suggesting a cell-autonomous neuroprotective effect of sEH blockade. Furthermore,sEH inhibition reversed disease-associated and interferon-responsive microglial states in PS19 mice,while EET supplementation promoted Tau phagocytosis and clearance in primary microglia cultures.ConclusionThese findings demonstrate that sEH blockade or EET augmentation confers therapeutic benefit in neurodegenerative tauopathies by simultaneously targeting neuronal and microglial pathways.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00844-x. View Publication

BackgroundThe simultaneous differentiation of human pluripotent stem cells (hPSCs) into both endodermal and mesodermal lineages is crucial for developing complex,vascularized tissues,yet poses significant challenges. This study explores a method for co-differentiation of mesoderm and endoderm,and their subsequent differentiation into pancreatic progenitors (PP) with endothelial cells (EC).MethodsTwo hPSC lines were utilized. By manipulating WNT signaling,we optimized co-differentiation protocols of mesoderm and endoderm through adjusting the concentrations of CHIR99021 and mTeSR1. Subsequently,mesoderm and endoderm were differentiated into vascularized pancreatic progenitors (vPP) by adding VEGFA. The differentiation characteristics and potential of vPPs were analyzed via transcriptome sequencing and functional assays.ResultsA low-dose CHIR99021 in combination with mTeSR1 yielded approximately 30% mesodermal and 70% endodermal cells. Introduction of VEGFA significantly enhanced EC differentiation without compromising PP formation,increasing the EC proportion to 13.9%. Transcriptomic analyses confirmed the effectiveness of our protocol,showing up-regulation of mesodermal and endothelial markers,alongside enhanced metabolic pathways. Functional assays demonstrated that vPPs could efficiently differentiate into insulin-producing ?-cells,as evidenced by increased expression of ?-cell markers and insulin secretion.ConclusionOur findings provide a robust method for generating vPPs,which holds significant promise for regenerative medicine applications,particularly in diabetes treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04120-5. View Publication

Amyotrophic lateral sclerosis (ALS) involves motor neuron death due to mislocalized TDP-43. Pathologic TDP-43 associates with stress granules (SGs),and lowering the SG-associated protein ataxin-2 (ATXN2) using Atxn2-targeting antisense oligonucleotides prolongs survival in TAR4/4 sporadic ALS mice but failed in clinical trials likely due to poor target engagement. Here we show that an AAV with potent motor neuron transduction delivering Atxn2-targeting miRNAs reduces Atxn2 throughout the central nervous system at doses 40x lower than published work. In TAR4/4 mice,miAtxn2 increased survival (50%) and strength,and reduced motor neuron death,inflammation,and phosphorylated TDP-43. TAR4/4 transcriptomic dysregulation recapitulated ALS gene signatures that were rescued by miAtxn2,identifying potential therapeutic mechanisms and biomarkers. In slow progressing hemizygous mice,miAtxn2 slowed disease progression,and in ALS patient-derived lower motor neurons,our AAV vector transduced >95% of cells and potently reduced ATXN2 at MOI 4 logs lower than previously reported. These data support AAV-RNAi targeting ATXN2 as a translatable therapy for sporadic ALS. Amado et al. develop a gene therapy for sporadic ALS using motor neuron-targeting AAVs to deliver RNAi targeting ataxin-2. In a mouse model,survival,strength,and disease-related pathology are improved; and human motor neurons are strongly transduced. View Publication

SummaryBackgroundPresence of nerves in tumours,by axonogenesis and neurogenesis,is gaining increased attention for its impact on cancer initiation and development,and the new field of cancer neuroscience is emerging. A recent study in prostate cancer suggested that the tumour microenvironment may influence cancer progression by recruitment of Doublecortin (DCX)-expressing neural progenitor cells (NPCs). However,the presence of such cells in human breast tumours has not been comprehensively explored.MethodsHere,we investigate the presence of DCX-expressing cells in breast cancer stromal tissue from patients using Imaging Mass Cytometry. Single-cell analysis of 372,468 cells across histopathological images of 107 breast cancers enabled spatial resolution of neural elements in the stromal compartment in correlation with clinicopathological features of these tumours. In parallel,we established a 3D in vitro model mimicking breast cancer neural progenitor-innervation and examined the two cell types as they co-evolved in co-culture by using mass spectrometry-based global proteomics.FindingsStromal presence of DCX + cells is associated with tumours of higher histological grade,a basal-like phenotype,and shorter patient survival in tumour tissue from patients with breast cancer. Global proteomics analysis revealed significant changes in the proteomic landscape of both breast cancer cells and neural progenitors in co-culture.InterpretationThese results support that neural involvement plays an active role in breast cancer and warrants further studies on the relevance of nerve elements for tumour progression.FundingThis work was supported by the 10.13039/501100005416Research Council of Norway through its Centre of Excellence funding scheme,project number 223250 (to L.A.A),the 10.13039/100008730Norwegian Cancer Society (to L.A.A. and H.V.),the Regional Health Trust Western Norway (Helse Vest) (to L.A.A.),the 10.13039/501100008728Meltzer Research Fund (to H.V.) and the 10.13039/100000002National Institutes of Health (NIH)/10.13039/100000057NIGMS grant R01 GM132129 (to J.A.P.). View Publication

Adherent two-dimensional human gastruloids have provided insights into early human embryogenesis. Even though the model system is highly reproducible,no available automated technology can screen and sort large numbers of these near-millimeter-sized complex structures for large-scale assays. Here,we developed a microraft array-based technology to perform image-based assays of large numbers of fixed or living gastruloids and sort individual gastruloids for downstream assays,such as gene expression analysis. Arrays of 529 indexed magnetic microrafts each (789?µm side length) possessing flat surfaces were photopatterned with a central circular region (500?µm diameter) of extracellular matrix with an accuracy of 93?±?1% to form a single gastruloid on each raft. An image analysis pipeline extracted features from transmitted light and fluorescence images of the gastruloids. The large microrafts were released and collected by an automated sorting system with efficiencies of 98?±?4% and 99?±?2%,respectively. The microraft array platform was used to assay individual euploid and aneuploid (possessing abnormal numbers of chromosomes) gastruloids with clear phenotypic differences. Aneuploid gastruloids displayed significantly less DNA/area than euploid gastruloids. However,even gastruloids with the same condition displayed significant heterogeneity. Both noggin (NOG) and keratin 7 (KRT7),two genes involved in spatial patterning within gastruloids,were upregulated in aneuploid relative to that in the euploid gastruloids. Moreover,relative NOG and KRT7 expressions were negatively correlated with DNA/area. The microraft arrays will empower novel screens of single gastruloids for a better understanding of key mechanisms underlying phenotypic differences between gastruloids. View Publication

BackgroundChronic lung disease of prematurity,called bronchopulmonary dysplasia (BPD),lacks effective therapies,stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD,but current protocols do not accurately replicate the distal niche environment of the native lung. Herein,we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.MethodsHuman PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry,immunofluorescence,and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6),E-cadherin (CDH1),NK2 Homeobox 1 (NKX2-1),HT2-280,surfactant proteins B (SFTPB) and C (SFTPC).ResultsWe observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally,we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids,mimicking in situ fetal respiratory movements,increased AEC2 differentiation without affecting proximal epithelial differentiation.ConclusionOur data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03890-2. View Publication

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However,the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis,potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing,which will ultimately increase editing efficiency. In our study,we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines. View Publication

Coronary microvascular disease (CMD) and its progression towards major adverse coronary events pose a significant health challenge. Accurate in vitro investigation of CMD requires a robust cell model that faithfully represents the cells within the cardiac microvasculature. Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) offer great potential; however,they are traditionally derived via differentiation protocols that are not readily scalable and are not specified towards the microvasculature. Here,we report the development and comprehensive characterisation of a scalable 3D protocol enabling the generation of phenotypically stable cardiac hPSC-microvascular-like ECs (hPSC-CMVECs) and cardiac pericyte-like cells. These were derived by growing vascular organoids within 3D stirred tank bioreactors and subjecting the emerging 3D hPSC-ECs to high-concentration VEGF-A treatment (3DV). Not only did this promote phenotypic stability of the 3DV hPSC-ECs; single cell-RNA sequencing (scRNA-seq) revealed the pronounced expression of cardiac endothelial- and microvascular-associated genes. Further,the generated mural cells attained from the vascular organoid exhibited markers characteristic of cardiac pericytes. Thus,we present a suitable cell model for investigating the cardiac microvasculature as well as the endothelial-dependent and -independent mechanisms of CMD. Moreover,owing to their phenotypic stability,cardiac specificity,and high angiogenic potential,the cells described within would also be well suited for cardiac tissue engineering applications.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09929-5. View Publication