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BackgroundPhotoreceptor death leads to inherited blinding retinal diseases,such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances,developing effective treatments is urgent. This study evaluates the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF),which are known to restrict common cell death pathways associated with retinal diseases.MethodsWe tested chemically synthesized peptides (17-mer and H105A) with affinity for the PEDF receptor,PEDF-R,delivered as eye drops to two RP mouse models: rd10 (phosphodiesterase 6b mutation) and RhoP23H/+ (rhodopsin P23H mutation). Additionally,we engineered AAV-H105A vectors for intravitreal delivery in RhoP23H/+ mice. To assess peptide effects in human tissue,we used retinal organoids exposed to cigarette smoke extract,a model of oxidative stress. Photoreceptor survival,morphology and function were evaluated.ResultsHere we show that peptides 17-mer and H105A delivered via eye drops successfully reach the retina,promote photoreceptor survival,and improve retinal function in both RP mouse models. Intravitreal delivery of a AAV-H105A vector delays photoreceptor degeneration in RhoP23H/+ mice up to six months. In human retinal organoids,peptide H105A specifically prevents photoreceptor death induced by oxidative stress,a contributing factor to RP progression.ConclusionsPEDF peptide-based eye drops offer a promising,minimally invasive therapy to prevent photoreceptor degeneration in retinal disorders,with a favorable safety profile. Plain language summaryRetinitis pigmentosa (RP) is a rare inherited condition that causes the gradual death of photoreceptors (light-sensing cells) in the eye,leading to vision loss. There is currently no cure. This study tested a potential treatment using small protein fragments (peptides) from PEDF,a protective protein naturally found in the eye. Researchers delivered these peptides through eye drops or gene therapy in mouse models of RP and to human retinal organoids (lab-grown retina tissue). Mice treated early maintained healthy vision cells,while untreated mice experienced rapid cell loss and vision decline. These results suggest that peptide-based eye drops could be a simple,safe,and effective way to slow vision loss in patients with RP. Bernardo-Colón et al. evaluate small peptides derived from the neurotrophic region of pigment epithelium-derived factor (PEDF) as potential therapeutics for retinitis pigmentosa using mouse models and human retinal organoids. A significant delay in photoreceptor death with eye drop or gene therapy delivery is seen. View Publication

miRNA,short non-coding RNA,are rapidly emerging as important regulators in cell homeostasis,as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC),whose axons connect the eye to the brain,are central nervous system cells of great interest,yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line,human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC,with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA’s role in RGC function,as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration. View Publication

Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern,severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript,the small nuclear RNA U4atac,is involved in the excision of ~850 minor introns. Here,we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC,instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts,we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities,leading to aneuploidy,cell cycle arrest and cell death. In cortical organoids,we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes,by observing delayed apico-basal polarization of NSC. Altogether,these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids,thus impeding their overall growth and shedding light on mechanisms leading to microcephaly. Author summaryPrimary microcephaly is defined as a severe reduction of the brain size that occurs prenatally. Variants in about 50 genes have been associated to primary microcephaly,and most of them encode proteins that regulate cell cycle,notably by participating to centrosome biogenesis. Intriguingly,some other genes involved in the process of minor splicing,such as RNU4ATAC,are also related to primary microcephaly without clear understanding of the underlying pathophysiological mechanisms. In our previous work,we discovered that alterations of minor splicing result into dysfunction of the centrosome/cilium complex. Here,we further feed this link between minor splicing and centrosome/primary cilium by reporting the particular case of a patient who presents with all features of the rare RNU4ATAC-associated syndrome,called the Taybi-Linder syndrome,and yet,is homozygous for the only recurrent pathogenic variant in the centrosomal RTTN gene. Hence,to decipher the underlying cellular mechanisms,we generated unique human neuronal cellular models–iPSC-derived neural stem cells (NSC) and cortical organoids–and unveiled the combination of events that contribute to the depletion of the NSC pool and explain RTTN-associated microcephaly. Our work gives thus precious hints for the understanding of the Taybi-Linder syndrome physiopathology. View Publication

SummaryHuman pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment.For complete details on the use and execution of this protocol,please refer to Wells et al.1 Graphical abstract Highlights•Brief pulse of Ngn2 induces neural progenitor cells from human stem cells•Guidance on expanding,freezing,and thawing SNaP cells for future use•Immunostaining-based assays assess cell identity and differentiation potential Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment. View Publication

The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications,as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines,robustly identifies inefficient gRNAs across different published datasets,and is easily accessible via online genome browser tracks. In addition,we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a,which may be relevant for safety in gene therapy applications. Reliable prediction of guide RNA (gRNA) activity is key for efficient CRISPR gene editing. Here,the authors show that efficiency of gRNAs is often underestimated when only indels are scored and introduce tools for predicting activity of chemically synthesized gRNAs and HDR efficiency. View Publication

Background: Duchenne muscular dystrophy (DMD),which affects 1 in 3500 to 5000 newborn boys worldwide,is characterized by progressive skeletal muscle weakness and degeneration. The reduced muscle regeneration capacity presented by patients is associated with increased fibrosis. Satellite cells (SCs) are skeletal muscle stem cells that play an important role in adult muscle maintenance and regeneration. The absence or mutation of dystrophin in DMD is hypothesized to impair SC asymmetric division,leading to cell cycle arrest. Methods: To overcome the limited availability of biopsies from DMD patients,we used our 3D skeletal muscle organoid (SMO) system,which delivers a stable population of myogenic progenitors (MPs) in dormant,activated,and committed stages,to perform SMO cultures using three DMD patient-derived iPSC lines. Results: The results of scRNA-seq analysis of three DMD SMO cultures versus two healthy,non-isogenic,SMO cultures indicate reduced MP populations with constant activation and differentiation,trending toward embryonic and immature myotubes. Mapping our data onto the human myogenic reference atlas,together with primary SC scRNA-seq data,indicated a more immature developmental stage of DMD organoid-derived MPs. DMD fibro-adipogenic progenitors (FAPs) appear to be activated in SMOs. Conclusions: Our organoid system provides a promising model for studying muscular dystrophies in vitro,especially in the case of early developmental onset,and a methodology for overcoming the bottleneck of limited patient material for skeletal muscle disease modeling. View Publication

To accurately study human organ function and disease ‘in the dish’,it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver,which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however,severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore,the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study,we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore,we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue,with the rationale that by taking such an approach,we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue. View Publication

During gastrulation,the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT),which is marked by cadherin switching,where E-Cadherin is downregulated,and N-Cadherin is upregulated. To understand the relationships between differentiation,EMT,and cadherin switching,we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols,as well as variants in which signaling through key pathways including Activin,BMP,and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus,cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently,E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall,cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation. View Publication

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by profound neuronal and cognitive decline,with increasing evidence implicating astrocyte dysfunction in disease pathology. While traditional therapeutic approaches have primarily targeted neurons,the crucial role of astrocytes in metabolism,neurotransmission,amyloid-beta clearance,and neuroinflammation underscores their potential as therapeutic targets. In this study,we employed a multiomic integrative analysis combining transcriptomic and kinomic profiling of human induced pluripotent stem cell (hiPSC)-derived astrocytes from patients with familial AD (fAD) compared to healthy controls (HCs). Our transcriptomic analysis identified 1249 significantly differentially expressed genes,highlighting a pronounced upregulation of inflammatory genes (SERPINA3,IL6R,IL1RAP,TNFRSF11A) and a concomitant downregulation of genes essential for synaptic support and ion channel function (STMN2,NMNAT2,SCN2A,GRIN1). Kinomic profiling revealed dysregulated kinase activities within DYRK,GSK,and MAPK families,further implicating altered kinase signaling pathways in astrocyte dysfunction. Integration of these datasets pinpointed critical molecular hubs,notably within the PI3K signaling and inflammatory pathways,highlighting targets such as JAK2,STAT3,and AKT1 as potential modulators of disease progression. Furthermore,leveraging the Library of Integrated Network-Based Cellular Signatures (LINCS) platform,we identified chemical perturbagens,including fluticasone propionate and Akt inhibitors,capable of reversing the transcriptomic signatures associated with fAD astrocytes. This integrative multiomic approach not only enhances our understanding of astrocyte-specific molecular mechanisms in AD but also provides novel targets for therapeutic intervention aimed at mitigating astrocyte-driven neurodegeneration. Highlights•Familial AD astrocytes display significant pro-inflammatory transcriptomic and kinomic dysregulation.•PI3K and inflammatory signaling pathways are highly dysregulated in familial AD astrocytes.•Expression of inflammatory markers such as SERPINA3,IL6R,and TNFRSF11A is increased in familial AD astrocytes.•Kinase activity analysis identifies DYRK,GSK,and MAPK pathways as key dysregulated axes in familial AD astrocytes.•Potential astrocyte-specific therapeutic approaches to AD include targeting PI3K,JAK,and STAT3. View Publication

SummaryLineage-specific differentiation of human induced pluripotent stem cells (hiPSCs) relies on complex interactions between biochemical and physical cues. Here we investigated the ability of hiPSCs to undergo lineage commitment in response to inductive signals and assessed how this competence is modulated by substrate stiffness. We showed that Activin A-induced hiPSC differentiation into mesendoderm and its derivative,definitive endoderm,is enhanced on gel-based substrates softer than glass. This correlated with changes in tight junction formation and extensive cytoskeletal remodeling. Further,live imaging and biophysical studies suggested changes in cell motility and interfacial contacts underlie hiPSC layer reshaping on soft substrates. Finally,we repurposed an ultra-soft silicone gel,which may provide a suitable substrate for culturing hiPSCs at physiological stiffnesses. Our results provide mechanistic insight into how epithelial mechanics dictate the hiPSC response to chemical signals and provide a tool for their efficient differentiation in emerging stem cell therapies. Graphical abstract Highlights•Tuning of substrate stiffness can enhance mesendoderm/endoderm hiPSC differentiation•Altered tight junction formation drives increased differentiation on soft substrates•Changes in cell motility and interfacial contacts underlie hiPSC layer remodeling Mechanobiology; Stem cells research; Biophysics View Publication

BackgroundGenome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified a plethora of risk loci. However,the disease variants/genes and the underlying mechanisms have not been extensively studied.MethodsBulk ATAC-seq was performed in induced pluripotent stem cells (iPSCs) differentiated various brain cell types to identify allele-specific open chromatin (ASoC) SNPs. CRISPR-Cas9 editing generated isogenic pairs,which were then differentiated into glutamatergic neurons (iGlut). Transcriptomic analysis and functional studies of iGlut co-cultured with mouse astrocytes assessed neuronal excitability and lipid droplet formation.ResultsWe identified a putative causal SNP of CLU that impacted neuronal chromatin accessibility to transcription-factor(s),with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. And,neuronal CLU facilitated neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes caused astrocytes to uptake less glutamate thereby altering neuron excitability.ConclusionsFor a strong AD-associated locus near Clusterin (CLU),we connected an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00840-1. View Publication

BackgroundStem cell-derived secreted factors could protect neurons in neurodegenerative disease or after injury. The exact neuroprotective components in the secretome remain challenging to discover. Here we developed a cell-to-cell interaction model to identify a retinal ganglion cell (RGC)-protective factor derived from induced pluripotent stem cells (iPSCs).MethodsPrimary RGCs were co-cultured with iPSCs or treated with iPSC-conditioned media in vitro. Cell viability were assayed using live-cell staining,and culture supernatant were analyzed via multiplexed antibody-based assays and ELISA. In vivo tests were carried out under mouse optic nerve crush model and RGC transplantation study in rats. Paired t-tests were used for data analysis between two groups.ResultsRGC viability was significantly enhanced when iPSCs were first stimulated with RGC-derived supernatant before iPSC-conditioned medium was collected and added into RGC culture. A significant increase of stem cell growth factor-beta (SCGF-?) concentration was detected in the latter conditioned medium. SCGF-? enhanced RGC survival in vitro and in vivo,and RGC-derived interleukin-12(p70) (IL-12[p70]) promotes secretion of iPSC-derived SCGF-?. Downstream of this IL-12(p70)-to-SCGF-? axis,ngn2 was significantly upregulated,and was found both necessary and sufficient for RGC survival.ConclusionThis study addresses a longstanding question of how neurons and stem cells interact to promote neuroprotection,and define a novel molecular interaction pathway whereby RGC’s secretion of IL-12(p70) enhances iPSCs’ secretion of SCGF-?,and SCGF-? protects RGCs via upregulating ngn2,suggesting that neurons may call on stem cells for their own protection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04198-5. View Publication