Protocol for human brain organoid transplantation into a rat visual cortex to model neural repair.

Human stem-cell-derived organoids represent a promising substrate for transplantation-based neural repair. Here, we describe a protocol for transplanting forebrain organoids into an injured adult rat visual cortex. This protocol includes surgical details for craniectomy, aspiration injury, organoid transplantation, and cranioplasty. This platform represents a valuable tool for investigating the efficacy of organoids as structured grafts for neural repair. For complete details on the use and execution of this protocol, please refer to Jgamadze et al.1.

Cell Replacement Therapy for Brain Repair: Recent Progress and Remaining Challenges for Treating Parkinson's Disease and Cortical Injury.

Neural transplantation represents a promising approach to repairing damaged brain circuitry. Cellular grafts have been shown to promote functional recovery through "bystander effects" and other indirect mechanisms. However, extensive brain lesions may require direct neuronal replacement to achieve meaningful restoration of function. While fetal cortical grafts have been shown to integrate with the host brain and appear to develop appropriate functional attributes, the significant ethical concerns and limited availability of this tissue severely hamper clinical translation. Induced pluripotent stem cell-derived cells and tissues represent a more readily scalable alternative. Significant progress has recently been made in developing protocols for generating a wide range of neural cell types in vitro. Here, we discuss recent progress in neural transplantation approaches for two conditions with distinct design needs: Parkinson's disease and cortical injury. We discuss the current status and future application of injections of dopaminergic cells for the treatment of Parkinson's disease as well as the use of structured grafts such as brain organoids for cortical repair.

Structural and functional integration of human forebrain organoids with the injured adult rat visual system.

Brain organoids created from human pluripotent stem cells represent a promising approach for brain repair. They acquire many structural features of the brain and raise the possibility of patient-matched repair. Whether these entities can integrate with host brain networks in the context of the injured adult mammalian brain is not well established. Here, we provide structural and functional evidence that human brain organoids successfully integrate with the adult rat visual system after transplantation into large injury cavities in the visual cortex. Virus-based trans-synaptic tracing reveals a polysynaptic pathway between organoid neurons and the host retina and reciprocal connectivity between the graft and other regions of the visual system. Visual stimulation of host animals elicits responses in organoid neurons, including orientation selectivity. These results demonstrate the ability of human brain organoids to adopt sophisticated function after insertion into large injury cavities, suggesting a translational strategy to restore function after cortical damage.

Modeling traumatic brain injury with human brain organoids.

Traumatic brain injury (TBI) remains a prominent public health concern despite several decades of attempts to develop therapies for the associated neurological and cognitive deficits. Effective models of this condition are imperative for better defining its pathophysiology and testing therapeutics. Human brain organoids are stem cell-derived neural tissues that recapitulate many of the steps of normal neurodevelopment, resulting in the reproduction of a substantial degree of brain architecture. Organoids are highly relevant to clinical conditions because of their human nature and three-dimensional tissue structure, yet they are easier to manipulate and interrogate experimentally than animals. Thus, they have the potential to serve as a novel platform for studying TBI. In this article, we discuss available in vitro models of TBI, active areas of inquiry on brain organoids, and how these two concepts could be merged.

Functional Cortical Axon Tracts Generated from Human Stem Cell-Derived Neurons.

IMPACT STATEMENT: Axon regeneration is negligible in the adult mammalian brain, and thus, white matter damage often leads to permanent neurological deficits. A novel approach for axon repair is the generation of axon tracts in the laboratory setting followed by transplantation of these constructs. This article details a human substrate for this repair strategy. Using the technique of axon stretch growth, functional cortical axon tracts are generated from human pluripotent stem cells at rates of up to 1 mm/day. These results form the basis of a potential patient-specific protocol for cerebral axon transplantation after injury.

Bundled Three-Dimensional Human Axon Tracts Derived from Brain Organoids.

Reestablishing cerebral connectivity is a critical part of restoring neuronal network integrity and brain function after trauma, stroke, and neurodegenerative diseases. Creating transplantable axon tracts in the laboratory is an unexplored strategy for overcoming the common barriers limiting axon regeneration in vivo, including growth-inhibiting factors and the limited outgrowth capacity of mature neurons in the brain. We describe the generation, phenotype, and connectivity of constrained three-dimensional human axon tracts derived from brain organoids. These centimeter-long constructs are encased in an agarose shell that permits physical manipulation and are composed of discrete cellular regions spanned by axon tracts, mirroring the separation of cerebral gray and white matter. Features of cerebral cortex also are emulated, as evidenced by the presence of neurons with different cortical layer phenotypes. This engineered neural tissue represents a first step toward potentially reconstructing brain circuits by physically replacing neuronal populations and long-range axon tracts in the brain.

Engineering advanced neural tissue constructs to mitigate acute cerebral inflammation after brain transplantation in rats.

Stroke, traumatic brain injuries, and other similar conditions often lead to significant loss of functional brain tissue and associated disruption of neuronal signaling. A common strategy for replacing lost neurons is the injection of dissociated neural stem cells or differentiated neurons. However, this method is unlikely to be suitable for replacing large brain cavities, and the resulting distribution of neurons may lack the necessary architecture to support appropriate brain function. Engineered neural tissues may be a viable alternative. Cell death is a prominent concern in neuronal grafting studies, a problem that could be magnified with the transplantation of engineered neural tissues. Here, we examined the effect of one contributor to cell death, acute cerebral inflammation, on neuronal survival after the transplantation of bioengineered constructs based on silk scaffolds. We found evidence of a high degree of inflammation and poor neuronal survival after introducing engineered constructs into the motor cortex of rats. Integrating a corticosteroid (methylprednisolone) into the constructs resulted in significantly improved neuron survival during the acute phase of inflammation. The improved construct survival was associated with decreased markers of inflammation and an anti-inflammatory state of the immune system due to the steroid treatment.

Neural Substrate Expansion for the Restoration of Brain Function.

Restoring neurological and cognitive function in individuals who have suffered brain damage is one of the principal objectives of modern translational neuroscience. Electrical stimulation approaches, such as deep-brain stimulation, have achieved the most clinical success, but they ultimately may be limited by the computational capacity of the residual cerebral circuitry. An alternative strategy is brain substrate expansion, in which the computational capacity of the brain is augmented through the addition of new processing units and the reconstitution of network connectivity. This latter approach has been explored to some degree using both biological and electronic means but thus far has not demonstrated the ability to reestablish the function of large-scale neuronal networks. In this review, we contend that fulfilling the potential of brain substrate expansion will require a significant shift from current methods that emphasize direct manipulations of the brain (e.g., injections of cellular suspensions and the implantation of multi-electrode arrays) to the generation of more sophisticated neural tissues and neural-electric hybrids in vitro that are subsequently transplanted into the brain. Drawing from neural tissue engineering, stem cell biology, and neural interface technologies, this strategy makes greater use of the manifold techniques available in the laboratory to create biocompatible constructs that recapitulate brain architecture and thus are more easily recognized and utilized by brain networks.

Thermoswitching microgel carriers improve neuronal cell growth and cell release for cell transplantation.

Successful cell replacement therapy in the central nervous system (CNS) depends on both the transplanted cell type and the cell delivery method. It was established that differentiated neurons are the most desirable cell source; however, they are highly sensitive to dissociation shear; removing them from the growth surface inflicts serious damage, rendering them less viable for transplantation. Pilot experiments using glass colloids as injectable cell carriers for cell transplantation in the adult rat hippocampus have greatly improved neuron survival and long-term neuron integration. However, these early studies have highlighted glass particle shortcomings. They are uncompressible, and, thus, only a small number of beads can be injected, limiting the transplanted cell number. Moreover, they remain permanently in the brain. To improve colloidal carriers properties for cell transplantation and establish a basis for the next generation of cell delivery supports, we have designed a broadly applicable engineering strategy to enable neuronal cell growth on and release from hydrogel particles before transplantation. Here, we describe poly(N-isopropylacrylamide) (pNIPAM) particle preparation, and we demonstrate that these hydrogel particles both facilitate manipulation of neurons and enable the increase in the number of viable transplanted cells in the young adult rat hippocampus. The absence of long-term cell association to beads suggested that pNIPAM thermoswitching properties enable the separation of cells from the beads during injection, which minimizes the number of injected carriers. Contrary to observations with glass carriers, no particle clumping was observed at the injection site, which indicates minimal risk of long-term inflammatory responses. Taken together, the properties of hydrogel particles make them a promising micro-carrier to improve neuronal cell transplantation.

Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain.

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.