Human iPSC-derived Organoids Research Articles

Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models.

The human photoreceptor function is dependent on a highly specialised cilium. Perturbation of cilial function can often lead to death of the photoreceptor and loss of vision. Retinal ciliopathies are a genetically diverse range of inherited retinal disorders affecting aspects of the photoreceptor cilium. Despite advances in the understanding of retinal ciliopathies utilising animal disease models, they can often lack the ability to accurately mimic the observed patient phenotype, possibly due to structural and functional deviations from the human retina. Human-induced pluripotent stem cells (hiPSCs) can be utilised to generate an alternative disease model, the 3D retinal organoid, which contains all major retinal cell types including photoreceptors complete with cilial structures. These retinal organoids facilitate the study of disease mechanisms and potential therapies in a human-derived system. Three-dimensional retinal organoids are still a developing technology, and despite impressive progress, several limitations remain. This review will discuss the state of hiPSC-derived retinal organoid technology for accurately modelling prominent retinal ciliopathies related to genes, including RPGR, CEP290, MYO7A, and USH2A. Additionally, we will discuss the development of novel gene therapy approaches targeting retinal ciliopathies, including the delivery of large genes and gene-editing techniques.

Loss of non-motor kinesin KIF26A causes congenital brain malformations via dysregulated neuronal migration and axonal growth as well as apoptosis.

Kinesins are canonical molecular motors but can also function as modulators of intracellular signaling. KIF26A, an unconventional kinesin that lacks motor activity, inhibits growth-factor-receptor-bound protein 2 (GRB2)- and focal adhesion kinase (FAK)-dependent signal transduction, but its functions in the brain have not been characterized. We report a patient cohort with biallelic loss-of-function variants in KIF26A, exhibiting a spectrum of congenital brain malformations. In the developing brain, KIF26A is preferentially expressed during early- and mid-gestation in excitatory neurons. Combining mice and human iPSC-derived organoid models, we discovered that loss of KIF26A causes excitatory neuron-specific defects in radial migration, localization, dendritic and axonal growth, and apoptosis, offering a convincing explanation of the disease etiology in patients. Single-cell RNA sequencing in KIF26A knockout organoids revealed transcriptional changes in MAPK, MYC, and E2F pathways. Our findings illustrate the pathogenesis of KIF26A loss-of-function variants and identify the surprising versatility of this non-motor kinesin.

Revisiting the role of Notch in nephron segmentation confirms a role for proximal fate selection during mouse and human nephrogenesis.

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human induced pluripotent stem cells (iPSCs) to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the demonstration that nicastrin (NCSTN)-deficient human iPSC-derived organoids differentiate into TFA2B+ distal tubule and CDH1+ connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.

During embryogenesis, paracrine signaling between tissues in close proximity contributes to the determination of their respective cell fate(s) and development into functional organs. Organoids are invitro models that mimic organ formation and cellular heterogeneity, but lack the paracrine input of surrounding tissues. Here, we describe a human multilineage iPSC-derived organoid that recapitulates cooperative cardiac and gut development and maturation, with extensive cellular and structural complexity in both tissues. We demonstrate that the presence of endoderm tissue (gut/intestine) in the organoids contributed to the development of cardiac tissue features characteristic of stages after heart tube formation, including cardiomyocyte expansion, compartmentalization, enrichment of atrial/nodal cells, myocardial compaction, and fetal-like functional maturation. Overall, this study demonstrates the ability to generate and mature cooperative tissues originating from different germ lineages within a single organoid model, an advance that will further the examination of multi-tissue interactions during development, physiological maturation, and disease.

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

SO038A TRANSLATIONAL KIDNEY ORGANOID SYSTEM BOLSTERS HUMAN RELEVANCE OF CLINICAL DEVELOPMENT CANDIDATE

Abstract Background and Aims A major challenge in drug discovery is gaining confidence in the human relevance of pre-clinical animal studies. While human iPSC-derived organoids offer exciting opportunities to address this, concerns about applicability and scalability remain. Here, we report a high-throughput human organoid platform for assessment of kidney disease targeting compounds. Method In vitro & in vivo transplanted under athymic rat kidney capsule, differentiated organoids were characterized using single cell RNA sequencing (scRNA-Seq), NanoString, & immunofluorescence techniques. Immunofluorescence quantitative analysis of aggregated actin per in vitro organoid in a protamine sulfate (PS) podocyte injury model was used to evaluate efficacy of GFB-887, a sub-type selective, small molecule transient receptor potential canonical 5 (TRPC5) inhibitor. In pharmacokinetic studies, GFB-887 was orally administered into rats, then the presentation of GFB-887 was measured in rat plasma, rat kidney, & in vivo transplanted human organoids. In pharmacodynamic studies of transplanted human organoids, rats were co-perfused with GFB-887 & PS or orally dosed with GFB-887 prior to PS, then immunofluorescence quantitative analysis of mean synaptopodin intensity in podocytes of the in vivo transplanted organoids was used to evaluate efficacy of GFB-887. Results We confirmed platform reproducibility by scRNA-Seq and derived a NanoString panel for efficient quality control. Organoid transplantation in rats for 2 to 4 weeks promoted organoid maturation and vascularization. In functional studies, cyclosporine A (CsA), a calcineurin inhibitor clinically utilized for the treatment of nephrotic syndrome, and GFB-887, a novel sub-type selective TRPC5 inhibitor currently in clinical development, protected in vitro kidney organoids from injury. Pharmacodynamic studies with GFB-887 delivered orally to rats were also successfully performed in human transplanted organoids. Conclusion These data show how human organoids can deliver confidence in taking development candidate compounds to the clinic, fulfilling their promise to revolutionize drug discovery.

3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.