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Page 264 of 548-
From Amyloid Exposure to Genetic Causality: Converging hiPSC-Based Models of Alzheimer's Disease.
Alzheimer's disease (AD) is a neurodegenerative disorder in which genetic susceptibility, aging, protein aggregation, and neuroglial dysfunction converge to drive progressive neuronal loss. Although animal models and conventional cell systems have provided fundamental insights into pathomechanisms, they only partially reproduce the human-specific and multifactorial nature of AD. Human induced pluripotent stem cell (hiPSC)-based models have emerged as a powerful platform to investigate disease-relevant processes within a patient-specific genetic background while enabling the generation of multiple neural cell types and multicellular systems. In this review, we present the evolution of hiPSC-based AD modeling, from reductionist paradigms based on exogenous amyloid-β exposure to CRISPR-engineered isogenic models addressing causal mutations and genetic risk variants associated with either familial or sporadic forms. We examine the contribution of three-dimensional brain organoids and neuroglial co-culture systems in recapitulating cell-cell interactions, neuroinflammatory responses, and tissue-level pathological processes. We focus on current challenges, including the limited representation of neuronal aging, variability among organoids, and the need for standardized protocols. Finally, we discuss the translational potential of hiPSC-derived systems for target identification, drug discovery, and regenerative medicine. Collectively, these advances position hiPSC-based platforms as essential complementary models for elucidating AD pathogenesis and accelerating the development of precision therapeutic strategies.
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Neurospheres Induced from Human Adipose-Derived Stem Cells as a New Source of Neural Progenitor Cells.
Human adipose-derived stem cells are used in regenerative medicine for treating various diseases including osteoarthritis, degenerative arthritis, cartilage or tendon injury, etc. However, their use in neurological disorders is limited, probably due to the lack of a quick and efficient induction method of transforming these cells into neural stem or progenitor cells. In this study, we reported a highly efficient and simple method to induce adipose-derived stem cells into neural progenitor cells within 12 hours, using serum-free culture combined with a well-defined induction medium (epidermal growth factor 20 ng/ml and basic fibroblast growth factor, both at 20 ng/ml, with N2 and B27 supplements). These adipose-derived stem cell-derived neural progenitor cells grow as neurospheres, can self-renew to form secondary neurospheres, and can be induced to become neurons and glial cells. Real-time polymerase chain reaction showed significantly upregulated expression of neurogenic genes Sox2 and Nestin with a moderate increase in stemness gene expression. Raybio human growth factor analysis showed a significantly upregulated expression of multiple neurogenic and angiogenic cytokines such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic growth factor, nerve growth factor, basic fibroblast growth factor and vascular endothelial growth factor etc. Therefore, adipose-derived stem cell-derived neurospheres can be a new source of neural progenitor cells and hold great potential for future cell replacement therapy for treatment of various refractory neurological diseases.
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Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSC-derived retinal organoids and retina-on-chip.
Mutations in the tripeptidyl peptidase 1 (TPP1) gene lead to neuronal ceroid lipofuscinosis type 2 (CLN2), characterized by lysosomal accumulation of lipofuscins predominantly in the brain and retina. The ocular phenotype is characterized by outer retinal degeneration that leads to vision loss. Leveraging human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs), retinal pigmented epithelial cells, and the retina-on-chip system, we establish an in vitro CLN2 model that recreates the principal histological hallmarks, namely the accumulation of subunit C of mitochondrial ATP synthase (SCMAS) and lipids mainly in the outer retina. Furthermore, single-cell RNA sequencing reveals a dysregulation of translational and mitochondrial function in CLN2 cones. Finally, adeno-associated virus (AAV)-mediated TPP1 gene therapy can restore TPP1 expression and decrease and even prevent SCMAS accumulations. Our study uses an innovative human-relevant microphysiological retinal disease models, uncovers previously uncharacterized mechanisms of CLN2 pathophysiology, and demonstrates the potential of AAV9.hCLN2 gene therapy for CLN2 disease, potentially treating patient blindness.
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[Generating pancreatic islets organoids: Langerhanoids].
The extension of islet transplantation to a wider number of Type 1 diabetic patients is compromised by the scarcity of donors, the reduced ex vivo survival of pancreatic islets and the use of immunosuppressive treatments. Islets of Langerhans isolated from brain-dead donors are currently the only cell source for transplantation. Thus, it is crucial to find an alternative and an abundant source of functional insulin secreting cells not only for clinical use but also for the development of research dedicated to the screening of drugs and to the development of new therapeutic targets. Several groups around the world, including ours, develop 3D culture models as Langerhanoids that closely mimick human pancreatic islets physiology. In this review, we describe recent advances to mimic the pancreatic niche (extracellular matrix, vascularization, microfluidics) allowing better functionality of Langerhanoids.
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Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin.
Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.
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Evidence for the expression of Thy-l-analogous structures on human thymic epithelial cells.
Cultured human thymic tissue was examined electron microscopically and analysed for the expression of the Thy-l-analogous antigen. It was found that almost all cultured cells were of epithelial appearance, exhibiting both tonofibrils and well-developed demosomes. Absorption of an anti-human brain serum highly purified for antibodies directed against the non-species-specific determinant of the Thy-l-analogous antigen with these cultured cells revealed its presence on thymic epithelial cells. Using the indirect peroxidase-labelled antibody technique, this molecule could be localized on the non-attached cell surface of human thymic epithelial cells with the preference of their cell processes. The biological significance of the expression of this antigen on the thymic epithelial cells of various species for cellular interaction within the thymus is discussed.
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Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes.
Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.
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Bulk RNA sequencing analysis of developing human induced pluripotent cell-derived retinal organoids.
Retinogenesis involves the transformation of the anterior developing brain into organized retinal lamellae coordinated by intricate gene signalling networks. This complex process has been investigated in several model organisms such as birds, fish, mammals and amphibians, yet many facets of retinal development are different in humans and remain unexplored. In this regard, human pluripotent stem cell (hPSC)-derived 3D retinal organoids and Next Generation Sequencing (NGS) have emerged as key technologies that have facilitated the discovery of previously unknown details about cell fate specification and gene regulation in the retina. Here we utilized hPSCs integrated with fluorescent reporter genes (SIX6-p2A-eGFP/CRX-p2A-h2b-mRuby3) to generate retinal organoids and carry out bulk RNA sequencing of samples encompassing the majority of retinogenesis (D0-D280). This data set will serve as a valuable reference for the vision research community to characterize differentially expressed genes in the developing human eye.
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Transcriptome and metabolome analyses reveal the effects of formula and breast milk on the growth and development of human small intestinal organoids.
Breast milk is widely acknowledged as the ideal nutritional resource for infants and can well meet the nutritional requirements for baby's growth and development. Infant formula is a substitute for breast milk, designed to closely mimic its composition and function for breast milk. Most of the previous studies used tumor colorectal cancer cell lines to study the nutritional potency of formula and its components, so realistic data closer to the baby could not be obtained. Small intestinal organoids, derived from differentiated human embryonic stem cells, can be used to simulate nutrient absorption and metabolism in vitro. In this experiment, we used small intestinal organoids to compare the nutrient absorption and metabolism of three infant formulae for 0-6 months with breast milk samples. Transcriptome and metabolome sequencing methods were used to analyze the differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs). The pathways related to DEGs, DEMs were enriched using GO, KEGG, GSEA and other methods to investigate their biological characteristics. We have found that both formula and breast milk promote the development of the infant's immune system, nutrient absorption and intestinal development. In PMH1 we found that the addition of oligofructose to milk powder promoted lipid metabolism and absorption. In PMH2 we found that whey protein powder favours the development of the immune system in infants. In PMH3 we found that oligogalactans may act on the brain-gut axis by regulating the intestinal flora, thereby promoting axon formation and neural development. By linking these biological properties of the milk powder with its composition, we confirmed the effects of added ingredients on the growth and development of infants. Also, we demonstrated the validity of small intestine organoids as a model for absorption and digestion in vitro. Through the above analyses, the advantages and disadvantages of the roles of formula and breast milk in the growth and metabolism of infants were also compared.
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Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids.
Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120 days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly.