Info
Background:
Stroke is a leading cause of death and disability worldwide, with over 12 million annual incidents, of which ischemic stroke accounts for more than 60%. Current treatments are limited by a narrow therapeutic window and lack long-term efficacy. Cerebral organoids are three-dimensional stem cell-derived constructs that recapitulate key structural and cellular features of the brain. Compared with neural stem cells, organoids offer greater cellular diversity and organized architecture, enabling systematic functions. Previous studies have shown that transplanted cerebral organoids can reconstruct neural circuits and repair brain damage, with superior efficacy over neural stem cells alone. Nevertheless, the in vivo mechanisms remain poorly understood. Positron emission tomography (PET) molecular imaging provides a non-invasive tool to longitudinally assess neural function, synaptic density, and neuroinflammation, thereby enabling visualization of the transplantation process in vivo.
Hypothesis/Objective:
This study aims to establish stable human induced pluripotent stem cell (iPSC)-derived cerebral organoids, transplant them into rat models of ischemic stroke, and employ PET molecular imaging combined with ex vivo techniques to monitor therapeutic processes and elucidate host-organoid interactions.
Methods:
Human iPSCs carrying a GFP reporter gene were passaged and induced to form embryoid bodies, which were then directed toward neuroectodermal differentiation. After 8 weeks of suspension culture with agitation, mature brain organoids were generated. In vivo, a focal cortical infarction model was established in rats using photothrombotic occlusion, followed by transplantation of organoids into the infarcted area. Longitudinal PET imaging with [¹⁸F]FDG, [¹⁸F]SynVesT-1, and [¹⁸F]DPA-714 was performed before infarction, before transplantation, and at 2, 4, and 8 weeks post-transplantation to monitor glucose metabolism, synaptic density, and neuroinflammation. Concurrent neurobehavioral tests (cylinder and forelimb placement) were conducted at the same time points. At endpoint, immunofluorescence staining assessed organoid survival, differentiation, and maturation, as well as synaptic connectivity with host neurons. Single-nucleus RNA sequencing further characterized the cellular composition of transplanted organoids and host microenvironment alterations at single-cell resolution.
Results:
Immunofluorescence and single-nucleus RNA sequencing confirmed that the generated organoids exhibited mature, heterogeneous cellular compositions with complex intercellular networks prior to transplantation. In the rat model, longitudinal PET imaging revealed dynamic changes in glucose metabolism, synaptic density, and neuroinflammation over 8 weeks, which significantly correlated with improved neurobehavioral outcomes. Endpoint ex vivo analyses demonstrated that transplanted organoids survived, differentiated, and matured, with evidence of synaptic connections established between organoid-derived neurons and host circuits. Single-nucleus RNA sequencing further revealed transplantation-induced remodeling of host cell-type composition and intercellular communication, indicating a favorable shift in the local brain microenvironment. Collectively, these findings provide convergent evidence that the transplanted organoids achieved structural integration and microenvironmental modulation.
Conclusion:
This study demonstrates that human iPSC-derived cerebral organoids can be reliably generated and, upon transplantation into ischemic stroke rats, survive, differentiate, and functionally integrate into host neural circuitry. The combination of longitudinal PET imaging and ex vivo single-cell analyses provides comprehensive evidence that organoid transplantation restores synaptic density and glucose metabolism, attenuates neuroinflammation, and remodels the host microenvironment. These multifaceted effects are closely associated with improved functional recovery, elucidating the reparative mechanisms of organoid-based therapy. Our findings offer critical experimental support for the future translational application of cerebral organoid transplantation in cerebrovascular diseases.
Image/Figure:
Click to view full size
Click to view full size
Image/Figure Caption:
a. Immunofluorescence staining of neurons in brain organoids; b. Dynamic monitoring of synaptic density changes via [¹⁸F]SynVesT-1 PET imaging following brain organoid transplantation for the treatment of ischemic stroke in rats; c. Predicted number (left) and strength (right) of intercellular interactions among various cell populations in brain organoids; d. UMAP plot of cellular populations in the microenvironment after brain organoid transplantation.
Author
Jing Wang
Zhejiang Unviersity