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Modeling Synaptic Maturation From Growth Cone to Synapse in Human Organoids.
Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However, methodologies for isolating functional synaptic structures from these models remain limited. Here, we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air-liquid interface cerebral organoids (ALI-COs) at distinct developmental stages (Day 90 and 150). Notably, the method avoids density gradients, requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g., GAP43) and classical synaptosomal proteins (e.g., PCLO, BSN, SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane-enclosed GCPs with fibrous content and mitochondria in Day 90 isolates, and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl-induced depolarization, which triggered phosphorylation changes in growth cone proteins (GAP43, MARCKS, MARCKSL1), cytoskeletal regulators (DCLK1, SHTN1, MARK4, MAP1B) and protein kinases (CAMK2G, PRKCE) in Day 90 GCPs, as well as classical synaptic vesicle cycle proteins (SYN1, DNM1, RPH3A) at Day 150. Overall, this study establishes a centrifugation-based protocol for isolating growth cones and immature synapses from human organoids, capturing key stages of synaptic development and enabling scalable, patient-compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders.
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Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.
Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.
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Human neural organoid modeling of diffuse midline glioma captures the complexity of patient tumors.
BACKGROUND: Diffuse Midline Glioma H3K27-altered (DMG) is an extremely aggressive and lethal childhood brain cancer that grows within the midline structure of the brain. Current treatment options are only palliative, making DMG in desperate need for therapeutic breakthroughs. One of the major challenges limiting the study of DMG is the lack of reliable preclinical models. In-vivo mouse models are expensive and technically challenging and in-vitro cell culture models lack the essential components of tumor microenvironment (TME) needed to recapitulate the complex biology of these tumors. Scalable human planar neural organoids (PNOs) with multi-cellular make-up can serve as a cost effective and reliable model system to capture DMG biology and allow effective species matched drug testing in-vitro. METHODS: Using 3 separate DMG patient derived xenograft (PDX) cell lines, we spatially profiled a novel scalable human iPSC-derived PNO system containing neurons, functional astrocytes and microglia using the NanoString GeoMx spatial transcriptomics system. RESULTS: We found that all three cell lines interact with and integrate into the human PNOs, demonstrating favorable growth conditions in a complex co-culture. Across spatially resolved regions of interest (ROI’s) tumor cells individually interact with microglia and astrocytes and transcriptomic profiling of these mixed cell ROI’s shows differences in the genetic signatures of both the normal cells (microglia/astrocytes) and the tumor cells. When compared to biopsies obtained directly from DMG patients, DMG cells within PNOs correlate strongly at both transcriptomic and proteomic levels. The multi-cellular PNOs also enabled drug target bystander toxicity screening not possible in a traditional tumor cell only monoculture. CONCLUSION: This study provides a proof-of-concept for scalable PNO modeling for DMG and underscores the translational relevance of this model system.
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Mechanisms of Activation and Serotonin Release From Human Enterochromaffin Cells.
BACKGROUND & AIMS: Gastrointestinal (GI) enterochromaffin (EC) cells are specialised sensors of luminal stimuli. They secrete most of the body's serotonin (5-HT), and are critical for modulating GI motility, secretion, and sensation, while also signaling satiety and intestinal discomfort. The aim of this study was to investigate mechanisms underlying the regulation of human EC cells, and the relative importance of direct nutrient stimulation compared with neuronal and paracrine regulation. METHODS: Intestinal organoids from human duodenal biopsies were modified using CRISPR-Cas9 to specifically label EC cells with either the fluorescent protein Venus or the cyclic adenosine monophosphate (cAMP) sensor Epac1-S-H187. EC cells were purified by fluorescence-activated cell sorting for analysis by bulk RNA sequencing and liquid chromatography mass spectrometry peptidomics. The function of human EC cells was studied using single-cell patch clamp, calcium and cAMP imaging, and 5-hydroxytryptamine (5-HT) enzyme-linked immunosorbent assays (ELISAs). RESULTS: Human EC cells showed expression of receptors for nutrients (including GPR142, GPBAR1, GPR119, FFAR2, OR51E1, OR51E2), gut hormones (including SSTR1,2&5, NPY1R, GIPR) and neurotransmitters (ADRA2A, ADRB1). Functional assays revealed EC responses (calcium, cAMP, and/or secretion) to a range of stimuli, including bacterial metabolites, aromatic amino acids, and adrenergic agonists. Electrophysiological recordings showed that isovalerate increased action potential firing. CONCLUSIONS: 5-HT release from EC cells controls many physiological functions and is currently being targeted to treat disorders of the gut-brain axis. Studying ECs from human organoids enables improved understanding of the molecular mechanisms underlying EC cell activation, which is fundamental for the development of new strategies to target 5-HT-related gut and metabolic disorders.
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Integration and Differentiation of Transplanted Human iPSC-Derived Retinal Ganglion Cell Precursors in Murine Retinas.
Optic neuropathy such as glaucoma, stemming from retinal ganglion cell (RGC) degeneration, is a leading cause of visual impairment. Given the substantial loss of RGCs preceding clinical detection of visual impairment, cell replacement therapy emerges as a compelling treatment strategy. Human-induced pluripotent stem cells (hiPSCs) serve as invaluable tools for exploring the developmental processes and pathological mechanisms associated with human RGCs. Utilizing a 3D stepwise differentiation protocol for retinal organoids, we successfully differentiated RGC precursors from hiPSCs harboring a BRN3B-GFP RGC reporter, verified by GFP expression. Intravitreal transplantation of enriched RGC precursors into healthy or N-methyl-D-aspartate (NMDA)-injured mice demonstrated their survival, migration, and integration into the proper retinal layer, the ganglion cell layer, after 3 weeks. Notably, these transplanted cells differentiated into marker-positive RGCs and extended neurites. Moreover, enhanced cell survival was observed with immunosuppressive and anti-inflammatory treatments of the host prior to transplantation. These data underscore the potential of transplanted RGC precursors as a promising therapeutic avenue for treating degenerative retinal diseases resulting from RGC dysfunction.
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Ceritinib inhibits growth and ACTH production of PitNETs: Insights from patient-derived organoids.
ACTH-secreting pituitary neuroendocrine tumors (PitNETs), also known as Cushing's disease (CD), are a rare condition lacking effective pharmacological treatments. Drug development for CD has been limited by the absence of suitable preclinical research models. Here, we established a patient-derived organoid (PDO) model and used it for screening tyrosine kinase inhibitors for CD. Ceritinib emerged as a promising therapeutic candidate due to its potent and consistent efficacy across PDOs from different patients in vitro, as well as in vivo in a xenograft mouse model. Multi-omics analyses and molecular validation revealed that Ceritinib exerts its effects by inhibiting phosphorylation within the PI3K-Akt signaling pathway. Specifically, Akt1 was identified as a key mediator, as its knockdown attenuated the suppressive potency of Ceritinib on both tumor growth and ACTH synthesis. Furthermore, Akt1-mediated regulation of ACTH was found to occur via Nur77, the transcriptional activator of POMC. In conclusion, Ceritinib effectively suppresses tumor growth and ACTH production in PDOs of both functioning and silent CD, positioning it as a promising therapeutic agent for CD.
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Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins.
The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However, it remains unknown whether analogous developmental processes are manifested in the human brain. Here we report oligodendrogenesis in forebrain organoids, generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) versus dorsal forebrain organoids (DFOs). Interestingly, oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore, dorsally derived oligodendroglial cells outcompete ventrally derived oligodendroglia and become dominant in FFOs after long-term culture. Thus, our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally and dorsally derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects.
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RNF213 deficiency in human iPSC-derived vascular organoids captures key feature of moyamoya disease vasculopathy.
OBJECTIVES: This study aimed to establish a human iPSC-derived model of Moyamoya disease (MMD) by generating RNF213-knockdown vascular organoids to capture key disease-relevant vascular features. METHODS: RNF213-knockdown iPSC lines were established using lentiviral shRNA and selected with puromycin. Knockdown efficiency was validated by qRT-PCR and Western blot. Vascular organoids were differentiated from the engineered iPSCs using the STEMdiff™ Blood Vessel Organoid Kit according to the manufacturer's protocol. Organoids were analyzed by immunofluorescence for vascular markers (CD31, COL4A1, PDGFR-β, α-SMA) and transcriptomic profiling was performed using DRUG-seq. RESULTS: Three distinct RNF213-knockdown iPSC clones were successfully generated, exhibiting varying knockdown efficiencies. qPCR analysis demonstrated RNF213 mRNA reductions of approximately 22% (V1), 70% (V2), and 88% (V3), with corresponding decreases in protein levels confirmed by Western blot. Although V2 and V3 clones displayed more substantial molecular knockdown, organoids derived from the V1 clone manifested the most severe phenotypic abnormalities, characterized by disrupted CD31+ endothelial networks and Collagen IV+ basement membranes. Transcriptomic profiling identified 1543 differentially expressed genes, with enrichment analysis revealing significant dysregulation of angiogenesis-related pathways. This included marked upregulation of pro-angiogenic factors (CCL5, EREG) and downregulation of inhibitory factors (TNMD), effectively recapitulating key molecular features of Moyamoya disease. CONCLUSION: We developed a novel human vascular organoid model with RNF-213 deficiency that captures key features of MMD vasculopathy, providing a valuable platform for mechanistic studies and drug screening.
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Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures.
Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.
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Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.
Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.