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Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs—productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated levels of phosphorylated ERK1/2. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05493-9.

SummaryMolecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons. Graphical abstract Highlights•Single cell analyses reveal putative ciliary margin (pCM) presence in retinal organoids•PCM harbors early RPCs which differentiate to late RPCs and retinal neurons•Single cell ATAC-seq data reveal novel TF binding motifs in RPCs and retinal neurons•RO development largely recapitulates retinogenesis Genetics; Molecular biology; Neuroscience; Cell biology; Omics

Natural killer (NK) cells are cytotoxic immune cells that can eliminate target cells without prior stimulation. Human induced pluripotent stem cells (iPSCs) provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers. In this in vitro study, a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells, and distinct maturational stages of iPSC-NK were characterized. Mature cells of CD56bright CD16bright phenotype showed upregulation of CD56, CD16, and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure, while exposure to aggressive atypical teratoid/rhabdoid tumor (ATRT) cell lines enhanced NKG2D and NKp46 expression. Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-? secretion in activated NK cells. CD56bright CD16bright iPSC-NK cells showed a ratio-dependent killing of ATRT cells, and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT. The iPSC-NK cells were also cytotoxic against other brain, kidney, and lung cancer cell lines. Further NK maturation yielded CD56?ve CD16bright cells, which lacked activation markers even after exposure to interleukins or ATRT cells - indicating diminished cytotoxicity. Generation and characterization of different NK phenotypes from iPSCs, coupled with their promising anti-tumor activity against ATRT in vitro, offer valuable insights into potential immunotherapeutic strategies for brain tumors. Graphical abstractImage 1 Highlights•Natural killer (NK) cells were derived from human induced pluripotent stem cells (iPSCs) in the absence of feeder cells.•Various maturational subtypes of iPSC-NK cells were characterized, and the phenotypic and functional properties were studied.•iPSC-NK cells of CD56bright CD16bright phenotype expressed activation markers in response to interleukin stimuli.•iPSC-NK cells were cytotoxic toward human atypical teratoid and rhabdoid tumor (ATRT) cells and other human cancer cells.•The cytotoxicity of iPSC-NK cells against various cancer cells in vitro might be translated into an in vivo immunotherapy.

Ribosomal RNA (rRNA) genes exist in multiple copies arranged in tandem arrays known as ribosomal DNA (rDNA). The total number of gene copies is variable, and the mechanisms buffering this copy number variation remain unresolved. We surveyed the number, distribution, and activity of rDNA arrays at the level of individual chromosomes across multiple human and primate genomes. Each individual possessed a unique fingerprint of copy number distribution and activity of rDNA arrays. In some cases, entire rDNA arrays were transcriptionally silent. Silent rDNA arrays showed reduced association with the nucleolus and decreased interchromosomal interactions, indicating that the nucleolar organizer function of rDNA depends on transcriptional activity. Methyl-sequencing of flow-sorted chromosomes, combined with long read sequencing, showed epigenetic modification of rDNA promoter and coding region by DNA methylation. Silent arrays were in a closed chromatin state, as indicated by the accessibility profiles derived from Fiber-seq. Removing DNA methylation restored the transcriptional activity of silent arrays. Array activity status remained stable through the iPS cell re-programming. Family trio analysis demonstrated that the inactive rDNA haplotype can be traced to one of the parental genomes, suggesting that the epigenetic state of rDNA arrays may be heritable. We propose that the dosage of rRNA genes is epigenetically regulated by DNA methylation, and these methylation patterns specify nucleolar organizer function and can propagate transgenerationally.

Using live-cell microscopy, we find that loss of VPS13C in human neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal tethers, leading to impaired lysosomal motility and defective lysosomal function as well as a decreased phospho-Rab10-mediated lysosomal stress response. Loss-of-function mutations in VPS13C are linked to early-onset Parkinson’s disease (PD). While VPS13C has been previously studied in non-neuronal cells, the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy, we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts, leading to impaired lysosomal motility and cellular distribution, as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD.

This study investigates the impact of sodium channel protein type 1 subunit alpha (SCN1A) gene knockout (SCN1A KO) on brain development and function using cerebral organoids coupled with a multiomics approach. From comprehensive omics analyses, we found that SCN1A KO organoids exhibit decreased growth, dysregulated neurotransmitter levels, and altered lipidomic, proteomic, and transcriptomic profiles compared to controls under matrix-free differentiation conditions. Neurochemical analysis reveals reduced levels of key neurotransmitters, and lipidomic analysis highlights changes in ether phospholipids and sphingomyelin. Furthermore, quantitative profiling of the SCN1A KO organoid proteome shows perturbations in cholesterol metabolism and sodium ion transportation, potentially affecting synaptic transmission. These findings suggest dysregulation of cholesterol metabolism and sodium ion transport, with implications for synaptic transmission. Overall, these insights shed light on the molecular mechanisms underlying SCN1A-associated disorders, such as Dravet syndrome, and offer potential avenues for therapeutic intervention.

This study presents a comprehensive transcriptomic analysis of feeder-free extended pluripotent stem cells (ffEPSCs) and their parental human embryonic stem cells (ESCs), providing new insights into understanding human early development and cellular heterogeneity of pluripotency. Leveraging Smart-seq2-based single-cell RNA sequencing (scRNA-seq), we have compared gene expression profiles between ESCs and ffEPSCs and uncovered distinct subpopulations within both groups. Through pseudotime analysis, we have mapped the transition process from ESCs to ffEPSCs, revealing critical molecular pathways involved in the shift from a primed pluripotency to an extended pluripotent state. Additionally, we have employed repeat sequence analysis based on the latest T2T database and identified the stage-specific repeat elements contributing to regulating pluripotency and developmental transitions. This dataset deepens our understanding on early pluripotency and highlights the role of repeat sequences in early embryonic development. Our findings thus offer valuable resources for researchers in stem cell biology, pluripotency, early embryonic development, and potential cell therapy and regenerative medical applications.

This study describes novel interactors of the retinal Crumbs complex and reveals homo- and heterotypic interactions of CRB1 and CRB2 that are not significantly affected by patient-associated mutations. Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1–CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1–CRB2 interactions. We observed no or a mild loss of CRB1–CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

Under specific conditions, cultured human embryonic stem cells (hESCs) corresponding to primed post-implantation epiblasts can be converted back to a ‘naïve pluripotency’ state that resembles the pre-implantation epiblasts. The core pluripotency factor OCT4 is known to be crucial in regulating different states of pluripotency, but its potential regulatory role in human naïve pluripotency remains unexplored. In this study, we systematically mapped out phosphorylation sites in OCT4 protein that are differentially phosphorylated between two states of pluripotency, and further identified ATR as a key kinase that phosphorylated OCT4 in naïve but not primed hESCs. The kinase activity levels of ATR in naïve hESCs were higher than those in primed hESCs. Ablating cellular ATR activity significantly halted the induction of naïve hESCs from their primed counterparts, and increased early apoptotic death of naïve hESCs upon UV and CPT treatment. Thus, our work reveals the importance of ATR activity in safeguarding human naïve pluripotency, and implicates a potential association of OCT4 phosphorylation, DNA damage sensing and repairing system in regulating different states of pluripotency during early development.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus strains that cause severe infections. Bicomponent PVL kills phagocytes depending on cell surface receptors, such as complement 5a receptor 1 (C5aR1). How the PVL-receptor interaction enables assembly of the leukocidin complex, targeting of membranes, and insertion of a pore channel remains incompletely understood. Here, we demonstrate that PVL binds the anionic phospholipids, phosphatidic acid, and cardiolipin, under acidic conditions and targets lipid bilayers that mimic lysosomal and mitochondrial membranes, but not the plasma membrane. The PVL–lipid interaction was sufficient to enable leukocidin complex formation as determined by neutron reflectometry and the rupture of model membranes, independent of protein receptors. In phagocytes, PVL and its C5aR1 receptor were internalized depending on sphingomyelin and cholesterol, which were dispensable for the interaction of the toxin with the plasma membrane. Internalized PVL compromised the integrity of lysosomes and mitochondria before plasma membrane rupture. Preventing the acidification of organelles or the genetic loss of PVL impaired the escape of intracellular S. aureus from macrophages. Together, the findings advance our understanding of how an S. aureus toxin kills host cells and provide key insights into how leukocidins target membranes. Staphylococcus aureus secretes toxins, such as Panton-Valentine leukocidin (PVL), to kill immune cells, including macrophages. This study shows that PVL binds phosphatidic acid and cardiolipin in acidic conditions, targeting lysosomal and mitochondrial membranes (but not the plasma membrane) to promote bacterial escape.

Human induced pluripotent stem cells (iPSCs) have great potential in research, but pluripotency testing faces challenges due to non-standardized methods and ambiguous markers. Here, we use long-read nanopore transcriptome sequencing to discover 172 genes linked to cell states not covered by current guidelines. We validate 12 genes by qPCR as unique markers for specific cell fates: pluripotency (CNMD, NANOG, SPP1), endoderm (CER1, EOMES, GATA6), mesoderm (APLNR, HAND1, HOXB7), and ectoderm (HES5, PAMR1, PAX6). Using these genes, we develop a machine learning-based scoring system, “hiPSCore”, trained on 15 iPSC lines and validated on 10 more. hiPSCore accurately classifies pluripotent and differentiated cells and predicts their potential to become specialized 2D cells and 3D organoids. Our re-evaluation of cell fate marker genes identifies key targets for future studies on cell fate assessment. hiPSCore improves iPSC testing by reducing time, subjectivity, and resource use, thus enhancing iPSC quality for scientific and medical applications. Quality control, including pluripotency testing of human iPSCs lacks standardization. Here, authors identify and validate gene markers to develop the machine learning-based hiPSCore to streamline pluripotency testing and elevate iPSC quality.

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity. Calcium is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. Here we show a robust pathway for new calcium signaling-based cardiac regenerative strategies. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC) induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD or nifedipine induced CM cell cycle by modulating calcineurin activity. Moreover, ectopic expression of RRAD/CDK4/CCND in combination induced CM proliferation in vitro and in vivo, improved cardiac function and reduced scar size post-myocardial infarction.