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The dose response curve is the gold standard for measuring the effect of a drug treatment,but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns,but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose,calculate classical pharmacological parameters such as the EC50,and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib,nilotinib,dasatinib and PD0325901) across a six-logarithm dose range,using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets,dose-dependent effects on cellular processes were identified using automated pathway analysis,and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover,these methods are automated,robust to non-optimized assays,and could be applied to other sources of quantitative data. View Publication

Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs),including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),large numbers of PSC-derived cell products are in demand for applications in drug screening,disease modeling,and especially cell therapy. In stem cell-based therapy,tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO,0.86 $$m) for MRI analysis. The protocol described PSC expansion and differentiation into NPs,and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders. View Publication

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders,with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs’ anti-inflammatory properties compared with two-dimensional (2D) cultures,the differentially expressed miRNAs were examined. Thus,we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1),a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably,iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly,iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD),improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized,off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD. View Publication

IntroductionEmerging biotechnologies are increasingly being explored for food production,including the development of cell-cultivated meat. Conventional approaches typically rely on satellite cell (SC) biopsies,which present challenges in scalability. Bovine induced pluripotent stem cells (biPSCs) represent a promising alternative due to their capacity for self-renewal and developmental plasticity.MethodsThis study utilized both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs suitable for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors pMaster K and pCXB-EBNA1,leading to the emergence of putative iPSC colonies 13 days post-nucleofection. A clonal line,bFF-iPSCs pMK,was selected for further analysis.ResultsThe bFF-iPSCs pMK line expressed key pluripotency markers including alkaline phosphatase (AP),OCT4,SOX2,and NANOG,and was stably maintained for over 33 passages,although episomal plasmids remained detectable. in vitro myogenic differentiation was assessed by comparing this line to a previously established lentiviral reprogrammed line (bFF-iPSCs mOSKM). Both lines exhibited downregulation of pluripotency markers and upregulation of the early myogenic marker PAX3. By day 30,the bFF-iPSCs pMK line formed elongated,multinucleated cells characteristic of myotubes and displayed a corresponding gene expression profile.DiscussionThese results provide new insights into bovine in vitro myogenesis and its application in cultured meat production. While promising,the study also highlights the difficulty in achieving complete myogenic differentiation,indicating a need for further optimization of differentiation protocols. Graphical abstract View Publication

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach,we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However,both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore,signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo,we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically,BMP signaling is integrated by SOX2. The interpretation of the key developmental signal BMP remains poorly understood. Here,the authors show that the total time-integrated signaling controls differentiation in a stem cell embryo model and provide a possible mechanism. View Publication

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation,which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing,in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent,species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members,implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead,mis-splicing was detected in human and mouse neural tissue,revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together,these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology. THOC6 is required for TREX tetramer formation. Analysis of pathogenic THOC6 variants differentiate the conserved mRNA export functions of TREX dimers and RNA processing functions of TREX tetramers underlying THOC6 Intellectual Disability Syndrome. View Publication

To comprehend the volumetric neural connectivity of a brain organoid,it is crucial to monitor the spatiotemporal electrophysiological signals within the organoid,known as intra-organoid signals. However,previous methods risked damaging the three-dimensional (3D) cytoarchitecture of organoids,either through sectioning or inserting rigid needle-like electrodes. Also,the limited numbers of electrodes in fixed positions with non-adjustable electrode shapes were insufficient for examining the complex neural activity throughout the organoid. Herein,we present a magnetically reshapable 3D multi-electrode array (MEA) using direct printing of liquid metals for electrophysiological analysis of brain organoids. The adaptable distribution and the softness of these printed electrodes facilitate the spatiotemporal recording of intra-organoid signals. Furthermore,the unique capability to reshape these soft electrodes within the organoid using magnetic fields allows a single electrode in the MEA to record from multiple points,effectively increasing the recording site density without the need for additional electrodes. Conventional platforms for electrophysiological recording of organoids have limited recording site density. Here,the authors present the magnetically reshapable 3D liquid metal-based electrode array for high-resolution analysis on neural activities of brain organoids. View Publication

Centrioles define centrosome structure and function. Deregulation of centriole numbers can cause developmental defects and cancer. The p53 tumor suppressor limits the growth of cells lacking or harboring additional centrosomes and can be engaged by the “mitotic surveillance” or the “PIDDosome pathway”,respectively. Here,we show that early B cell progenitors frequently present extra centrioles,ensuing their high proliferative activity and related DNA damage. Extra centrioles are efficiently cleared during B cell maturation. In contrast,centriole loss upon Polo-like kinase 4 (Plk4) deletion causes apoptosis and arrests B cell development. This defect can be rescued by co-deletion of Usp28,a critical component of the mitotic surveillance pathway,that restores cell survival and maturation. Centriole-deficient mature B cells are proliferation competent and mount a humoral immune response. Our findings imply that progenitor B cells are intolerant to centriole loss but permissive to centriole amplification,a feature potentially facilitating their malignant transformation. Centrioles organize chromosome segregation,migration,and the immune synapse. Here,Schapfl et al. show that B cell progenitors tolerate centriole overamplification,but not loss,while mature B cells can mount a humoral immune response in their absence. View Publication

This study aimed to identify unique characteristics in the peripheral blood mononuclear cells (PBMCs) of endometriosis patients and develop a non-invasive early diagnostic tool. Using single-cell RNA sequencing (scRNA-seq),we constructed the first single-cell atlas of PBMCs from endometriosis patients based on 107,964 cells and 25,847 genes. Within CD16+ monocytes,we discovered JUP as a dysregulated gene. To assess its diagnostic potential,we measured peritoneal fluid (PF) and serum JUP levels in a large cohort of 199 patients including 20 women with ovarian cancer (OC). JUP was barely detectable in PF but was significantly elevated in the serum of patients with endometriosis and OC,with levels 1.33 and 2.34 times higher than controls,respectively. Additionally,JUP was found in conditioned culture media of CD14+/CD16+ monocytes aligning with our scRNA-seq data. Serum JUP levels correlated with endometriosis severity and endometrioma presence but were unaffected by dysmenorrhea,menstrual cycle,or adenomyosis. When combined with CA125 (cancer antigen 125) JUP enhanced the specificity of endometriosis diagnosis from 89.13% (CA125 measured alone) to 100%. While sensitivity remains a challenge at 19%,our results suggest that JUP’s potential to enhance diagnostic accuracy warrants additional investigation. Furthermore,employing serum JUP as a stratification marker unlocked the potential to identify additional endometriosis-related genes,offering novel insights into disease pathogenesis. View Publication

SummarymRNA-based in vivo chimeric antigen receptor (CAR)-T cell engineering offers advantages over ex vivo therapies,including streamlined manufacturing and transient expression. However,current delivery methods require antibody-modified vehicles with manufacturing challenges. In this study,inspired by cardiolipin,we identify cardiolipin-like di-phosphoramide lipids that improve T cell transfection without targeting ligands,both in vitro and in vivo. The T cell-favored tropism is likely due to the lipid’s packing,shape,and rigidity. Encapsulating circular RNA further prolongs mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles,we deliver mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR),alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single-cell sequencing in humans confirms uPAR’s relevance to senescence and inflammation in RA. To facilitate clinical translation,we screen and humanize single-chain variable fragments (scFvs) against uPAR,establishing a PL40 mRNA-encoded humanized uPAR CAR with potential for treating aging-inflamed disorders. Graphical abstract Highlights•Cardiolipin-mimic phosphoramide (CAMP) LNPs transfect T cells without antibody modification•Circular mRNA prolongs mRNA expression•Senolytic in vivo CAR-T treats inflamm-aging disease (liver fibrosis and rheumatoid arthritis)•Develop humanized anti-human uPAR scFv Zhang et al. develop Cardiolipin-mimic phosphoramide (CAMP) lipids,which enable T cell transfection without antibody modification. Using CAMP-based LNPs,they generate senolytic CAR-T cells in vivo to target inflamm-aging diseases. Additionally,they employ circular mRNA to prolong transgene expression. The authors also engineer a humanized anti-human uPAR scFv for clinically relevant applications. View Publication