Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros

How well do brain organoids capture your brain?

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

Chapter 18 - Cerebral and noncerebral organoids

The advent of human induced pluripotent stem cell (hiPSC)-derived brain organoids represents a significant advance in environmental neurotoxicology, propelling the discipline toward human-relevant, mechanistic, and predictive in vitro paradigms. This review explores the utility of brain organoids in environmental neurotoxicology, which uniquely address critical limitations of traditional models by recapitulating key aspects of human brain development, including three-dimensional (3D) cytoarchitecture, multilineage cellular heterogeneity, and functional network activity. This review systematically elaborates on their construction principles, unique advantages in neurotoxicological research, and the significant progress made in elucidating mechanisms of toxicity. Notably, brain organoids exhibit enhanced sensitivity in identifying the subtle adverse outcomes of chronic, low-dose exposures to environmental contaminants, often eluding conventional approaches. Their key advantage lies in the greater capacity to deconstruct complex toxicological pathways, enabling precise tracing of adverse outcome pathways (AOPs). Future development requires enhancing model complexity through vascularization, promoting automation and standardization, and integrating artificial intelligence (AI) for data analysis. Concurrently, establishing sustained ethical oversight and standardized frameworks is essential to ultimately advance the field toward more precise and efficient hazard identification and risk characterization.Graphical abstractHighlights1. Human brain organoids bridge species gaps for more human-relevant neurotoxicity assessment.2. Brain organoids effectively recapitulate chronic, low-dose and mixture environmental risks.3. Integrating brain organoid data into the AOP frameworks enhances mechanistic understanding.4. Coupling brain organoids with organ-on-a-chip and AI advances next-generation risk assessment.

The Sumatran Rhinoceros Dicerorhinus sumatrensis is on the edge of extinction. The decline of this species was initially attributed to poaching and habitat loss, but evidence presented here indicates that reproductive failure has also been a significant cause of loss, and continues to affect wild populations. Indonesia’s remaining populations of Sumatran Rhino are small and scattered, with limited access to breeding opportunities with unrelated mates. This leaves them subject to inbreeding and isolation-induced infertility, linked to fertility problems analyzed here. Sumatran Rhino females in captivity showed high rates (>70%) of reproductive pathology and/or problems with conception, which has significantly hindered the breeding program. Technological advances enabling examination immediately after capture revealed similarly high rates and types of reproductive problems in individuals from wild populations. The last seven Sumatran Rhino females captured were from areas with small declining populations, and six had reproductive problems. Going forward, capturing similarly compromised animals will take up valuable space and resources needed for fertile animals. The high risk of infertility and difficulty of treating underlying conditions, coupled with the decreasing number of remaining animals, means that the success of efforts to build a viable captive population will depend upon utilizing fertile animals and applying assisted reproductive techniques. Decades of exhaustive in situ surveys have not provided information relevant to population management or to ascertaining the fertility status of individual animals. Thus the first priority should be the capture of individuals as new founders from areas with the highest likelihood of containing fertile rhinos, indicated by recent camera trap photos of mothers with offspring. In Sumatra these areas include Way Kambas and parts of the Leuser ecosystem.

Enhanced Radiation-Sparing Effects of Ultra-High Dose Rate Proton Radiation (FLASH-RT) in a Human Induced Pluripotent Stem Cell-Derived Cerebral Organoid Model

Human‐induced pluripotent stem cell (iPSC)‐derived brain organoids have attracted significant attention as promising models for drug screening owing to their remarkable resemblance to the human brain. The advent of disease model organoids, derived from patient's cells, has further elevated expectations for drug screening and personalized medicine. Nevertheless, the absence of a comprehensive platform for administering drugs and assessing their efficacy based on functional changes in brain organoids has remained a challenge. In this study, we introduce a one‐step drug screening system designed designed to investigate functional changes induced by diverse drug doses in multiple brain organoids, utilizing electrophysiological signal measurements. Our system comprises a specialized culture chamber with a microfluidic chip capable of accommodating 10 organoids and delivering varying doses of two drugs to each organoid. Additionally, we integrate a three dimentional microelectrode array (3D MEA) with ten shanks, enabling functional assessment of 10 brain organoids. This approach facilitates dose‐dependent drug screening across multiple organoids. We demonstrate the effectiveness of our system through real‐time analysis of neural activity changes triggered by different doses of pottassium chloride (KCl). Furthermore, sodium channel protein type 2 subunit alpha (SCN2A)‐epileptic organoids to demonstrate utility in disease‐model‐based drug screening. Our platform enables functional screening and personalized medicine using brain organoids.

A brain organoid is a self-organizing three-dimensional tissue derived from human embryonic stem cells or pluripotent stem cells and is able to simulate the architecture and functionality of the human brain. Brain organoid generation methods are abundant and continue to improve, and now, an in vivo vascularized brain organoid has been encouragingly reported. The combination of brain organoids with immune-staining and single-cell sequencing technology facilitates our understanding of brain organoids, including the structural organization and the diversity of cell types. Recent publications have reported that brain organoids can mimic the dynamic spatiotemporal process of early brain development, model various human brain disorders, and serve as an effective preclinical platform to test and guide personalized treatment. In this review, we introduce the current state of brain organoid differentiation strategies, summarize current progress and applications in the medical domain, and discuss the challenges and prospects of this promising technology.

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.

The future of cerebral organoids in drug discovery

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells.

Chapter 6 - Brain organoids: models of cell type diversity, connectivity, and disease phenotypes

Cellular complexity in brain organoids: Current progress and unsolved issues