**3. Neural organoids in regenerative medicine**

Neural organoids, which recapitulate the process of native neurogenesis in the development of CNS, have been applied in a large variety of areas including simulating brain development and retinogenesis. Moreover, emerging organoid-based cell transplantation has made considerable progress in reconstruction of lost neural circuits, damaged neural cortex and visual function, which facilitates the application of 3D organoid systems in disease modeling and regenerative medicine. Representative examples are involved in two aspects: (a) isolating neural progenitor cells (NPCs) from neural organoids; (b) transplanting neural organoids in immunodeficient animals. The stem cells in the organoids derived from hPSCs present a higher survival rate and closer connection with the surrounding tissues in the host. Distinct from conventional stem cell therapy usually focusing on specific populations of stem cells or NPCs, neural organoids offer an entire set of cell types of the human organs.

#### **3.1 Brain organoid system in regenerative medicine**

Brain disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI), and stroke, along with several other chronic neurodegenerative disorders, are debilitating diseases that have few treatment options. Stem cell

therapy is likely to provide beneficial effects for the indications of these diseases. The current understanding of brain diseases is mainly based on traditional 2D monocultural cells, animal models, and postmortem examination. Because of the inherent species differences between animals and humans and the individual differences among genetic and environmental backgrounds, it remains a challenge to investigate brain development and associated disorders. To establish better models of human brain development, stem-cell-based 3D brain organoids systematically decipher the developmental rules, presenting the 3D architectures and physiology of the brain. These generated brain organoids show robust neuronal subtypes and glial subtypes and functionality to mimic *in vivo* neural connectivity [24, 25]. In comparison with 2D monocultural stem cell cultures, physiologic conditions closer to the human organism are provided by organoids to support cell-cell and cell-matrix interactions. Therefore, as an ideal cell source, brain organoids have great potential for the reconstruction of lost neural circuits. Brain organoids transplantation strategy is expected to become an effective treatment for neurological defects after brain injury (**Figure 1b**).

Recently, in two studies, scientists transplanted hPSC-derived cerebral organoids into mouse cerebral cortex and successfully generated vascularized organoids, which promoted the progressive neuronal differentiation and maturation and increased cell survival [26, 27]. They observed the widespread axonal extension and precise synaptic connectivity outside the graft area; however, the region-specific long projections and synaptogenesis mapping were not reported in the two studies. Previously, reported approaches produced brain organoids with large lumens and tubes, which results in insufficient oxygen and nutrients support in increasing metabolic needs, making them difficult to apply in transplantation therapy [10, 28]. Recently, an optimized culture protocol was developed to efficiently generate small human cerebral organoids, which presents the benefit of alleviating the risk of cell overgrowth and safety concerns after injecting into the mouse medial prefrontal cortex [29]. The transplanted cerebral organoids extended projections to basal brain regions and generated human glutamatergic neurons with mature electrophysiology [29]. Moreover, mice transplanted with cerebral organoids show potentiated auditory startle fear response, indicating that the organoids can be functionally integrated into preexisting host mouse neural circuits via building up bidirectional synaptic connections, which provides crucial therapeutic strategy for neurological diseases [29].

However, owing to the cellular composition of brain, organoids dramatically changes along the time course of the development, it is necessary to demonstrate which stage of the organoids is best suitable for transplantation. To address this limitation, Kitahara et al. transplanted hESC-derived cerebral organoids at 6w or 10w into mouse cerebral cortex and found that 6w-organoids extend more axons along corticospinal tracts but caused graft overgrowth with higher populations of proliferative cells while axonal extensions from 10w-organoids were smaller in number but enhanced after brain injury 1 week [30]. A similar study reported that 55d and 85d-cerebral organoids were transplanted into damaged motor cortex, indicating that 55d-cerebral organoids can be used as a better transplantation donor for traumatic brain injury (TBI) [31]. Cells from the transplanted cerebral organoids have the capability to support region-specific reconstruction of damaged brain cortex, upregulate hippocampal neural connection protein and neurotrophic factor, and improve of damaged motor cortex. It is also reported that cerebral organoids were transplanted at 55 days to explore the feasibility of organoid transplantation in stroke [32]. Cerebral organoids were transplanted at 6 h or 24 h after middle cerebral artery occlusion (MCAO) surgery, resulting in reducing brain infarct volume and improving *Applications of Neural Organoids in Neurodevelopment and Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.104044*

neurological motor function. Furthermore, they also observed that the transplanted cerebral organoids were also related with increased neurogenesis, synaptic reconstruction, axonal regeneration and angiogenesis, decreased neural apoptosis, and rescued more survival neurons after stroke [32]. Although a few works with respect to transplanting brain organoid system were reported, it still has promising technologies in the future treatment of central nervous system diseases. Hence, the effects of the developmental organoid stage and host brain environment should be accurately evaluated when developing an organoid-replacement therapy for brain injury.

#### **3.2 Retinal organoid in regenerative medicine**

Retinal degenerative diseases, such as glaucoma, RP, and Age-Related Macular Degeneration (AMD), usually lead to irreversible blindness. So does the importance of finding a viable treatment. Regardless of the underlying etiology of retinal degeneration, the common endpoint is the loss of photoreceptors and underlying RPE. Cell replacement strategy provides a good solution for the treatment of retinal degeneration. Although plenty of studies have been made to understand the cellular and molecular mechanisms of retinal disorders, our knowledge is still in its infancy, and the immortalized retinal cell lines have not recapitulated the developmental stages of the human native retina. The new methodological advances in inducing hPSCs into human retina tissues have opened new possibilities for basic research on investigating the therapies or treatments in regenerative medicine [18, 33]. The generated retinal organoids closely resemble many aspects of the real human retina, including retinaspecific ribbon synapse [34] and physiological-relevant response to light stimuli [35], which empower researchers to explore the pathogenesis of retinal diseases and pursue cell/tissue transplantation for developing novel treatment options. Because retinal organoids contain all the cell types of human retina, it plays a primary role in the field of transplantation therapy. In this section, we focus on single-cell suspensions isolated from retinal organoids and application of retinal organoids sheets transplantation used for cell therapies in regenerative medicine (**Figure 1b**).

#### *3.2.1 Retinal organoids as a cell source for therapeutic transplantation*

During the previous decade, the aborted human fetal tissues and the hPSCderived retinal progenitors were two cell sources for transplantation. Representative retinal cell replacement clinical trials are transplantation of hPSC-derived RPE for the treatment of retinal diseases, including AMD and Stargardt disease [36–38]. It has been proved that the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. In 2006, stemcell-derived precursor photoreceptors were first integrated into the outer retinal layer of degenerating retina and had success in improving vision [39, 40]. However, the strong ethical restrictions and limited cell sources remain a challenge in current transplantation therapies. The retinal organoids that contain abundant retinal progenitor cells (RPCs) can act as an ideal cell source transplantation in retinal degenerative diseases. Zou et al. transplanted effectively purifying RPCs with the surface markers (C-Kit+/SSEA4−) into the retinal degeneration models of rats and mice, showing benefits to the improvement of vision and preservation of the retinal structure [41]. The RGCs are the earliest differentiated cells closely associated with glaucoma. But the population of RGCs in retinal organoids is not substantial as they gradually degenerate following long-term culture time. Thus, prolonging the

survival time of RGCs may provide the possibility for RGC replacement therapy. Several approaches have been taken to improve the short life of implanted RGCs and the length of axons, such as adding extrinsic growth factors [20], combining 2D and 3D protocols [42], and co-culturing with Müller glia [43]. In another animal study, by transplanting purified rod photoreceptors isolated from retinal organoids in defective S- and M- cone opsins, Nrl-/- mouse retinas can restore rod-mediated visual function and be incorporated into the host retina with forming synaptic-like structures in close apposition to mouse interneurons [44]. Interestingly, recent studies contradicted the common view that transplanted photoreceptors integrate into the photoreceptor layer of recipients. They demonstrated that the material transfer between donor rod photoreceptors and host photoreceptors leads to the acquisition of proteins originally expressed only by donor cells [45, 46]. Thus, the mechanism of the photoreceptor transplantation demands reinterpretation.

#### *3.2.2 Retinal organoid sheet transplantation for improving visual function*

A retinal sheet derived from cultured retinal organoids or fetal retina is another approach to preserving the neural circuitries and improving visual function. Cell suspension strategies consist of transplanting purified photoreceptor precursor cells, whereas retinal sheet transplantations engraft retinal organoids containing both photoreceptor cells and inner retinal neurons. The inner neurons located in the transplanted retinal sheet, which serves as a scaffold and nurturing microenvironment, are conductive to outer layer retinal cells in differentiation and maturation, preserving normal lamination structures. It is reported that the retinal sheet graft can produce less immune activation that enhances life span and the survival rate of transplanted cells, providing suitability approach for therapies of late-stage retinal diseases.

Furthermore, several studies have demonstrated that the transplantation of hPSCderived RPE cells in AMD patients shows promising outcomes in clinical trials, such as improvement in retinal integrity, maintainability in visual acuity, and increase in vision-related quality of life [47]. Currently, the transplantation of early-stage retinal organoid sheets is verified to establish connections more effectively with host retinal degeneration, and these connections show higher survival rate over time. A series of studies have been performed to investigate whether the transplantation of retinal organoid sheets can differentiate, integrate, and improve visual function in animal models with severe retinal degeneration [48–50]. In 2016, Shirai et al. dissected "retinas" from organoids to get transparent and continuous neural retina sheets and transplanted them into two primate models with retinal degeneration. In both monkey and rat, grafted hESC-retina differentiated into a range of retinal cell types, such as photoreceptors. The photoreceptors were proved to have migrated to the outer nuclear layer and the host-graft synaptic connections were established in those animal models [51]. Similar results were achieved in another study of transplanting the sheets dissected from hESC-derived retinal organoids into retinal degenerate rats [48]. In addition, to enhance functional integration of transplanted retinal sheet, a method in which a genetic modification was used to reduce ON-bipolar cells resulted in efficiently restoring RGC light responsiveness in degenerated retina [52]. However, in those studies, the absence of a well-defined RPE monolayer presents a main limiting factor for retinal sheet transplantation. To overcome this limitation, hESC-derived retinal organoids and RPE monolayer were combined using different bio-adhesives to transplant into immunodeficient Royal College of Surgeons (RCS) rats. The co-grafts were observed to reconstruct the severely damaged retina structure

#### *Applications of Neural Organoids in Neurodevelopment and Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.104044*

and improve visual function [53]. Those studies demonstrate the clinical feasibility of hPSC-derived retinal organoids sheet transplantation and provide practical tools to optimize transplantation strategies for future clinical applications.

In retinal tissue engineering, biomaterials are utilized to optimize the models of the human retina. A growing number of biomaterials, especially synthetic polymer scaffolds, such as biodegradable polycaprolactone (PCL) and polylactic-coglycolic acid (PLGA), have been widely used. The remarkable properties of defined synthetic polymer substrate are thin and biodegradable, which can be placed into the retinal subretinal space with minimal physical distortion [54]. In terms of the report, transplanting mouse RPCs cultured on biodegradable thin-film PCL scaffolds with varying surface topographies into the retinal subretinal space help newly integrated mRPCs exhibit potential to guide stem cell differentiation toward photoreceptor fate and to help cells localized to the outer nuclear layer [55]. Another study implanted the human retinal organoids, which are seeded on PLGA sheets into both normal and Chronic Ocular Hypertension (OHT) rhesus monkey retinas. They found that despite the need of immunosuppression for dexamethasone after transplantation, survival and differentiation into retinal tissue were successfully improved [56]. Subsequently, the same group proved again that with the support of PLGA sheets, retinal organoids showed active proliferation, migration and projection of axons into the host optic nerve after transplanting into OHT rhesus monkey eyes [57].
