Cerebral organoids transplantation improves neurological motor function in rat brain injury

Abstract Background and Purpose Cerebral organoids (COs) have been used for studying brain development, neural disorders, and species‐specific drug pharmacology and toxicology, but the potential of COs transplantation therapy for brain injury remains to be answered. Methods With preparation of traumatic brain injury (TBI) model of motor dysfunction, COs at 55 and 85 days (55 and 85 d‐CO) were transplanted into damaged motor cortex separately to identify better transplantation donor for brain injury. Further, the feasibility, effectiveness, and underlying mechanism of COs transplantation therapy for brain injury were explored. Results 55 d‐CO was demonstrated as better transplantation donor than 85 d‐CO, evidenced by more neurogenesis and higher cell survival rate without aggravating apoptosis and inflammation after transplantation into damaged motor cortex. Cells from transplanted COs had the potential of multilinage differentiation to mimic in‐vivo brain cortical development, support region‐specific reconstruction of damaged motor cortex, form neurotransmitter‐related neurons, and migrate into different brain regions along corpus callosum. Moreover, COs transplantation upregulated hippocampal neural connection proteins and neurotrophic factors. Notably, COs transplantation improved neurological motor function and reduced brain damage. Conclusions This study revealed 55 d‐CO as better transplantation donor and demonstrated the feasibility and efficacy of COs transplantation in TBI, hoping to provide first‐hand preclinical evidence of COs transplantation for brain injury.


| INTRODUC TI ON
With the evolution of stem cell technologies, increasing studies focus on the development of three-dimensional (3D) organ-like tissues, also known as organoids, from human-induced pluripotent stem cells (iPSCs) or isolated organ progenitor cells in vitro. 1 The generated organoids can mimic cytoarchitecture, cell-cell interactions and development of in-vivo organs, including organoids of gut, kidney, intestine, retina, and others. 1 Thus, the successful generation of cerebral organoids (COs) is a breakthrough in the field of brain research. 2 Unlike conventional cell culture with pure populations of particular stem cell-derived cell types, 1 the 3D COs generated from human PSCs contain diverse cell types, including neural progenitor cells (NPCs), neural stem cells (NSCs), mature and immature neurons, glial cells, etc They can form tissue morphology (up to 4 mm in diameter of single CO). 2,3 The COs also show robust neural connectivity and functionality to mimic in-vivo brain development and capture features of in-vivo brain regions. 2,[4][5][6] Cerebral organoids and brain-region-specific organoids have been used for studying brain development, neural disorders, and species-specific drug pharmacology and toxicology. For example, COs have been used for modeling human microcephaly induced by zika virus infection, 2 autism spectrum disorders induced by CRISPR/ Cas9-mediated gene mutation, 7 neurodegenerative microenvironment by using Alzheimer's patient-derived hiPSCs, 8 and developmental neurotoxicity with the exposure of rotenone, 9 alcohol, 10 vincristine, 11 and tranylcypromine. 12 There is no report of COs transplantation until the first study that establishes an in-vivo model of vascularized human brain organoids. 13 Transplantation of COs into retrosplenial cortex of immunodeficient mice shows progressive neural differentiation and maturation, and forms functional neuronal networks with host brain, but with no benefit on spatial learning ability. 13 A further study demonstrates that transplanted COs have higher cell survival rate, better multilineage neurodifferentiation and robust vascularization than NSCs transplantation in the mice brain. 14 Therefore, COs are an alternative source of NSCs as a tissue-based transplantation donor for neural repair. Traumatic brain injury (TBI) is a leading cause of death and disability in children and young adults worldwide. According to the World Health Organization, TBI will continue to be a major health problem and leading cause for disability by the year 2020. 15 Although extensive researches have been done, there is still no effective therapy for TBI. In the recent decades, cell transplantation has been proved to be an effective therapy for TBI, providing a promising regenerative medical strategy. [16][17][18] However, conventional cell-based transplantation faces the hurdles of poor cell survival and inadequate neural differentiation after transplantation. Transplantation of tissue has a higher cell survival rate as compared to transplantation of cell suspension grafts, 19 and COs transplantation has more advantages than NSCs transplantation. 14 We wonder whether transplanted COs could repair damaged brain tissue and improve dysfunction caused by brain injury. Meanwhile, as COs at different culture stage contain diverse mixture of NPCs/NSCs and neural cells, it is still unknown which culture stage of COs is the better donor for brain injury transplantation.
Our group previously performed several studies on brain injury and NSCs. [20][21][22][23] Here, with preparation of rat TBI model of motor dysfunction, we transplanted COs at different culture stage separately into damaged motor cortex to identify which is the better transplantation donor and explored the feasibility, effectiveness, and underlying mechanism of COs transplantation therapy for brain injury.

| Animals
All Sprague-Dawley rats (male, 250 ± 30 g) were purchased from Sino-British SIPPR/BK Lab Animal Ltd. All rats received humane care and were kept in a 12-hour light/dark cycle with free access to food and water throughout the study.

| Traumatic brain injury model
Traumatic brain injury model was prepared as previous report. 26 All rats were given Cyclosporin A (10 mg/kg, i.p.) on the day before surgery. A combination of ketamine (50 mg/kg), xylazine (2.6 mg/kg), and acepromazine (0.50 mg/kg) was used to anesthetize rats (i.p.).
As for craniotomy, a longitudinal incision (approximately 4 cm) was made along brain midline. A skull window (1.5 cm length, 0.6 cm breadth) in the right skull was made without damaging brain parenchyma. Under the stereotaxic apparatus, mechanical injury was made by biopsy punch to form a cavity of 3 mm diameter and 2 mm depth in the right motor cortex of rat brain. The center of a cavity in the TBI model for biological research located at 1.5 mm lateral to the midline and 0.5 mm posterior to bregma. The centers of two connected cavities in the TBI model for studying motor functional recovery located at 1.5 mm lateral to the midline, 1 mm anterior to bregma, and 2 mm posterior to bregma respectively.
All rats were randomly grouped. The rats in the Sham group underwent craniotomy without brain injury. The rats in the TBI group were performed as aforementioned without COs transplantation.
In the transplantation groups, COs at 55 and 85 days (namely 55 d-CO and 85 d-CO, respectively) were separately transplanted into the cavity of damaged motor cortex immediately after TBI surgery, namely 55 d-CO transplantation and 85 d-CO transplantation groups. The number of transplanted COs was as the same as the number of cavities that made in the motor cortex by biopsy punch.
After surgery, skull window was sealed with piece of skull and bone wax. The incision was sutured and covered with erythromycin ointment to prevent infection. All operations were performed under aseptic conditions. Cyclosporine A was intraperitoneally injected every other day until rats were sacrificed. There was no animal death in the rat TBI model with or without transplantation.

| Brain collection and immunofluorescence staining
Rat brains were harvested at the indicated day post implantation (dpi). Transcardial perfusion was performed with 4% paraformaldehyde (pH 7.4), and brains were collected carefully without disrupting lesioned site and transplanted COs and stored at -80°C. With 4% paraformaldehyde fixation at 4°C for 24 hours and tissue dehydration with 15%-30% sucrose/paraformaldehyde, brains were embedded in OCT compound (Tissue-plus, Fisher Healthcare) and sectioned into frozen coronal slices (8 μm thickness) in the cryostat (CM3050S; Leica Microsystems). As for immunofluorescence staining, nonspecific binding sites were blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 2 hours at room temperature.
Brain slices were then incubated with primary antibodies (Table S1) at 4°C overnight. After being washed by 1× PBS for three times, corresponding secondary antibodies (Alexa 488-conjugated and Cy3-conjugated, Table S1) were added and incubated for 2 hours at room temperature. Nuclei were stained with DAPI for 10 minutes.

| Histology examination and immunohistochemistry staining
Brains were collected and used for histology examination and immunohistochemistry staining as the above. 28 Briefly, with fixation with 4% paraformaldehyde at 4°C for 24 hours and tissue dehydration with 15%-30% sucrose/paraformaldehyde, brains were embedded and sectioned into paraffin-coated horizontal slices (8 μm thickness) containing brain injury sites. Then, brain slices were deparaffinized and stained with hematoxylin and eosin (HE). As for immunohistochemistry staining, brain slices were deparaffinized and retrieved antigen with citric acid buffer (PH 6.0) and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 2 hours at room temperature. Then, brain slices were incubated with specific primary antibodies (Table S1)

| Tissue lysate and immunoblotting
Tissue lysates and immunoblotting were performed as standard procedure. 29 Briefly, ipsilateral hippocampus was isolated from brain on the ice. The RIPA buffer (Beyotime Biotechnology) with protease inhibitor cocktail (Pierce) was used as lysis buffer. Protein extraction was collected after homogenate and centrifugation at 22 000 g, 4 ℃ for 20 minutes and stored at -80°C. The protein concentration was determined by enhanced BCA protein assay kit (Beyotime Biotechnology). After electrophoresis in 10% SDS-PAGE, proteins were transferred into nitrocellulose membranes and incubated with primary antibodies (Table S1) and corresponding secondary antibodies conjugated with Infrared-Dye (Li-Cor). The images of immunoblotting were obtained in the Odyssey Infrared Fluorescence Imaging System (Li-Cor). All immunoblotting experiments were repeated at least three times. The quantification of protein expression was analyzed in the Image J 1.5 software (Wayne Rasband, NIH) and analyzed by a blind observer.

| Behavior tests
In the behavior test of TBI model, modified neurological severity scores (mNSS) and beam walking test were used to evaluate the recovery of neurological motor function. 30,31 All animals were blinded to experimenter in all behavior tests. The baseline of neurological motor function of these rats before operation was similar.

| mNSS evaluation
Modified neurological severity scores (mNSS) were used to evaluate rat neuromuscular function. The mNSS evaluation indices were evaluated as follows: forelimb flexion (0 score, none; 0.5 score, slightly flexion; 1.0 score, the shoulder flexion can surround the entire the forelimb flexion); twist (0 score, none; 0.5 score, slightly twist; 1.0 score, forelimbs and heads can reach the hind limbs); side push (0 score, equal on both sides; 0.5 score, the ipsilateral weakened; 1.0 score, the ipsilateral has no resistance); circle (0 score, none; 0.5 score, large circle; 1.0 score, small circle); hind limb placement (0 score, rapid recovery; 0.5 score, recovery delay; 1.0 score, no recovery); and free activity (0 score, free activity; 0.5 score, reduced activity; 1.0 score, stimulating to be active; 2.0 score, stimulation is also inactive). mNSS scores were the sum of the above indexes.

| Beam walking test
All rats were trained for one week before TBI surgery to ensure all rats can walk through the balance beam smoothly. The balance beam used in this study was 2 cm width and 100 cm length. The beam walking test indices were evaluated as the follows: 0 score, smoothly through the balance beam without tumble; 1.0 score, smoothly cross the balance beam and less than 50% of the way with slip feet; 2.0 score, smoothly cross the balance beam and more than 50% of the way with slip feet; 3.0 sore, cross the balance beam but the ipsilateral limb does not help move forward; 4.0 score, cannot cross the balance beam but can balance on it; 5.0 score, falling from the balance beam.

| Randomization and blinding
All animals were randomly assigned into different groups. All researchers were blind to treatment or group throughout the behavioral testing, scoring, and statistical analysis.

| Statistical analysis
All data were shown as mean ± SEM. The line graphs were prepared in SigmaPlot 10.0 software (Systat Software Inc); the histograms were prepared in GraphPad Prism 7.0 statistical software (GraphPad Software, Inc). Statistical analyses were performed in the SPSS 11.0 software (SPSS Inc). Two-tailed Student's t-test was used for comparison between two groups. ANOVA comparison followed by Bonferroni post hoc tests was used for comparison of mean value among groups. P < .05 was considered statistically significant.  Subgranular zone (SGZ) of hippocampus and subventricular zone (SVZ) of lateral ventricles are known areas of neurogenesis. 32,33 Detection of neurogenesis in these two areas showed similar effects. BrdU + /Nestin + and BrdU + /DCX + cells were more in ipsilateral SGZ and SVZ in 55 d-CO and 85 d-CO transplantation groups than those in Sham and TBI groups, indicating enhanced neurogenesis by COs transplantation (Figures S1-S2).

| 55 d-CO is a better transplantation donor than 85 d-CO for neurogenesis and cell survival in rat brain injury
To determine which culture stage of COs has better proproliferation and prodifferentiation effects, we compared neurogenesis There was also no difference in rat serum ICAM-1 concentration among Sham, TBI, and 55 d-CO transplantation groups ( Figure S4C). Therefore, both 55 d-CO and 85 d-CO transplantation did not aggravate neural apoptosis and neuroinflammation after brain injury.
Taken together, 55 d-CO is a better transplantation donor for brain injury, which has more neurogenesis and more cell survival number than 85 d-CO after transplantation into damaged motor cortex. 55 d-CO was used for the following in-depth transplantation study.

| Vascularization between transplanted COs and host brain in rat brain injury
Previous study demonstrated the growth of vascular network between grafted COs and retrosplenial cortex of host brain. 13 As expected in the present study, there was vessel formation between transplanted COs and host brain by immunostaining STEM121 with endothelial markers CD31 or CD105 (also known as endoglin) ( Figure   S5A). The CD31 + endothelial cells showed overlap with STEM121 + F I G U R E 3 More cell survival from transplanted 55 d-CO than 85 d-CO in rat TBI model. A, Representative images of COs survival by immunostaining of human cytoplasmic marker (STEM121, green) at 7, 14 and 28 dpi in the transplantation periphery of ipsilateral cortex of 55 d-CO transplantation and 85 d-CO transplantation groups. STEM121 + cells distributed throughout the lesioned cavity. DAPI labels nuclei (blue). Scale bar: 50 μm. B, Quantitative analysis of STEM121 + cells per field in 55 d-CO transplantation and 85 d-CO transplantation groups. Immunostained positive cells were counted with six random microscope fields in the transplantation periphery of ipsilateral cortex and repeated with at least three independent animals per group. All data were shown as mean ± SEM and analyzed by ANOVA with Bonferroni posthoc tests. *P < .05 and **P < .01 vs 55 d-CO transplantation group. C, Representative images of COs survival by HE staining of horizontal sections with cavity in rat ipsilateral cortex. The cavity was filled with transplanted COs (dotted black lines). Scale bars: 500 μm cells in transplantation periphery of ipsilateral cortex, proving the vascularization between transplanted COs and host brain ( Figure   S5A). Vessel formation in transplanted COs was also confirmed by immunohistochemistry staining CD31 ( Figure S5B). The vascularization between transplanted COs and host brain plays a vital role in cells survival and further differentiation of transplanted COs.

| Cells from transplanted COs have the potential of multilineage differentiation to mimic brain cortical development and support motor cortex region-specific reconstruction via in situ differentiation and cell replacement in rat brain injury
Stem cell-based transplantation has been reported to support brain-region-specific reconstruction, functional replacement, and neural connection. 34,35 With immunostaining of human cell-de-

| Cells from transplanted COs show extensive migration into cortex, thalamus, and hippocampus along corpus callosum in rat brain injury
As there was decreased cell survival number of transplanted COs in the host brain over time ( Figure 3B), we wondered whether the decreased trend was caused by cell migration of transplanted COs into host brain. Immunostaining for human cytoplastic marker STEM121 demonstrated cells from transplanted COs distributed throughout the lesioned cavity at 7, 14, 28, and 56 dpi in ipsilateral cortex of host brain ( Figure 5). Cells from transplanted COs migrated into extensive regions of host brain along corpus callosum at 56 dpi and showed positive expression in ipsilateral cortical region ( Figure 5B1-B3,B1′-B3′), ipsilateral and contralateral thalamus ( Figure 5B4, 5B4′), and hippocampus ( Figure 5B3, B5-B7, 5B3′, 5B5′-B7′). Immunostaining for hNuclei, a human nuclear marker (another human cell marker different from human cytoplastic marker STEM121), proved the same cell migration of transplanted COs in the host brain ( Figure S6). Moreover, cells from transplanted COs existed in ipsilateral and contralateral SVZ of host brain ( Figure S7). Therefore, cells from transplanted COs have ability to migrate into extensive regions of host brain, suggesting potential integration and connection between transplanted COs and host brain.

| COs transplantation upregulates hippocampal neural connection proteins and neurotrophic factors in rat brain injury
We further explored possible mechanism by detecting hip-

| COs transplantation improves neurological motor function and reduces brain damage in rat brain injury
We next explored whether transplantation of 55 d-CO into damaged motor cortex could improve motor functional recovery and rescue brain damage. mNSS method was introduced to evaluate rat neurological function after TBI. Improved mNSS score was found from 5 dpi in transplantation group as compared to TBI group ( Figure 6H). Notably, COs transplantation recovered neurological function to normal level from 21 dpi, wherein no difference was found between Sham and transplantation groups ( Figure 6H).
Beam walking test confirmed the improvement of motor function in COs transplantation group as compared to TBI group ( Figure 6I).
At 42 dpi, we observed obvious brain atrophy in the damaged ipsilateral hemisphere of TBI group ( Figure 6G). Brain parenchyma in ipsilateral hemisphere of COs transplantation group showed relatively smooth and plump morphology with smaller cavity, proving the amelioration of brain damage ( Figure 6G). These results provide positive preclinical evidence for functional and morphological improvement of COs transplantation in brain injury therapy.

| D ISCUSS I ON
The present study demonstrates the feasibility, effectiveness, and mechanism of COs transplantation for brain injury therapy ( Figure 6J).

| The selection of research model for COs transplantation
Transplanted NSCs can reduce brain damage, enhance neural repair, provide trophic support, and improve functional recovery after brain injury. 16,17,31 As COs transplantation has higher cell survival rate, better multilineage neurodifferentiation, and robust vascularization than NSCs transplantation in the mouse brain, 14 we wondered the effect of COs transplantation on the repair of brain injury. So far, only two studies report COs transplantation and demonstrate the survival, differentiation, and vascularization of transplanted COs in retrosplenial cortex or frontoparietal cortex of mouse brain. 13,14 Considering the high disability and mortality of TBI and high incidence of TBI-induced motor dysfunction worldwide, here we made a direct mechanical cavity in the rat motor cortex to prepare TBI model of motor dysfunction and further explore the feasibility and potential mechanism of COs transplantation. In our study, the rat TBI model of motor dysfunction has advantages that: the cavity used for accommodating transplanted COs has abundant blood supply, contributing to the survival of transplanted COs; the model has no obvious change of neuroinflammation, providing a pure and simple model to mainly study the change of transplanted COs in the host brain; the simple model has performance of motor dysfunction, supporting the study of COs transplantation on neurological motor function recovery; the rats used for TBI model have no immunodeficiency to approach clinical practice.

| The identification of better COs transplantation donor for brain injury
Transplantation donor is an important parameter for preclinical transplantation study. 17 Current cell transplantation studies mainly use single type of NSCs or neural cells to repair brain injury. 17 However, the damaged tissues after brain injury contain diverse cell types rather than single cell types. COs consist of abundant neural cell types, representing an alternative transplantation donor for brain injury. As COs at different developmental stages are diverse in neural cell types, regional identities, and COs transplantation may be due to the use of immunosuppressive agent cyclosporin A throughout the study. Cyclosporin A has been F I G U R E 4 Cells from transplanted COs have the potential of multilineage differentiation to mimic brain cortical development and support motor cortex region-specific reconstruction in rat TBI model. A, Representative images of in-vivo differentiated COs by immunostaining of human cytoplasmic marker (STEM121, green) and neural stem cells (Nestin, red), neurons (Tuj1, red), or astrocytes (GFAP, red) at 7, 14, 28, and 56 dpi in 55 d-CO transplantation group. The cells of STEM121 + /Tuj1 + and STEM121 + /GFAP + gradually increased but STEM121 + /Nestin + cells gradually decreased until they disappeared. DAPI labels nuclei (blue). Scale bar: 50 μm. B, Quantification of the percentage of STEM121 + /Nestin + and STEM121 + /Tuj1 + cells in the in-vivo differentiated COs. Immunostained positive cells were counted with six random microscope fields in the transplantation periphery of ipsilateral cortex and repeated with at least three independent animals per group. All data were shown as mean ± SEM and analyzed by ANOVA with Bonferroni posthoc tests. *P < .05 and **P < .01 vs 55 d-CO transplantation group at 7 dpi; ## P < .01. N.S, not significant. C, Representative images of transplanted COs at 14, 28, and 56 dpi. Immunostaining for human cells by STEM121 (green) with motor neuronal progenitor cells (Olig2, red) and cholinergic neurons (Chat, red), and by hNuclei (red) with preplate/deep-layer neurons (TBR1,green), surface-layer neurons (SATB2, green), and glutamatergic neurons (vGlut1, green) showed in situ differentiation and cell replacement of transplanted COs in the damaged motor cortex. Scale bar: 50 μm A1 A2

A3 A4
widely used in clinical transplantation and reported to inhibit neuroinflammation after brain injury. 40

| The potential mechanism of COs transplantation for brain injury
In addition to above mentioned neurogenesis, cell apoptosis, and neuroinflammation mediated by COs transplantation, we further explored the potential mechanism of transplanted COs in brain injury. As expected, there is vascular connection between transplanted COs and host brain, which is important for promoting the survival of transplanted COs. According to recently reported article, the source of blood vessels was from the host brain. 13 The that are related to neurotransmitter release. It is worth mentioning that the differentiation trend of transplanted COs within host brain may need to be further explored. For example, whether the differentiation proportion of neuron/gliocyte and cholinergic neuron/noncholinergic neuron is close to brain physiology needs to be further clarified. Besides, cells from transplanted COs show extensive migration into cortex, thalamus, and hippocampus along corpus callosum after brain injury. The neuronal activity and functional connectivity between transplanted COs and host brain have been proved by electrophysiological recording and optogenetics. 13 Thus, the vascularization, differentiation, and migration of transplanted COs in the host brain support motor cortex regionspecific reconstruction after brain injury. to normal level, improves motor function, and reduces brain damage after brain injury, providing direct evidence for functional and morphological improvement of brain injury.
In the future study, many questions remain to be answered to explore the specific action mechanisms, optimal dose and delivery routes, therapeutic time window, and safety concerns of transplanted COs for brain injury therapy. Transplantation of modified cells with overexpression of growth or trophic factors is one way to improve the survival of grafts. 45,46 By introducing genetic manipulation or providing a scaffold environment for transplanted cells, 47,48 enhancing responsiveness and sensitivity of transplanted cells to endogenous signaling is another way to increase effectiveness. It is noteworthy that the culture of COs is based on the self-organization of hiPSCs, leading to the heterogeneity of each COs in cell composition, cell number, and so on. 3 The heterogeneity of COs may increase uncontrollability for transplantation therapy. As the recently cultured brain-region-specific COs has lower heterogeneity, 4 whether it is the better transplantation donor for brain-region-specific injury needs to be explored in the further study. In addition, the proliferation of NPCs/NSCs within transplanted COs needs to be controlled because of the possibility of tumorigenesis after transplantation, though we did not observe the phenomenon in our study.

| CON CLUS IONS
With identification of better transplantation donor of COs, the study gives the first demonstration of feasibility, effectiveness, and mechanism of COs transplantation in motor cortex brain injury, hoping to provide first-hand preclinical evidence of COs transplantation therapy for brain injury.
F I G U R E 5 Cells from transplanted COs migrate into cortex, thalamus, and hippocampus along corpus callosum in rat TBI model. A, Representative images of whole brain scan with COs transplantation at 7 and 14 dpi in 55 d-CO transplantation group. STEM121 (green), human cytoplasmic marker. Tissues inside the rectangle frame indicate transplanted COs in the host brain, wherein the right image is the high-magnification view of boxed area in the left image. DAPI labels nuclei (blue). Scale bars: 1000 μm in A1;A3; 200 μm in A2;A4. B, Representative images of migration of cell from transplanted COs into host brain at 56 dpi in 55 d-CO transplantation group. (B0 and B0′) Overall view of migration of cell from transplanted COs in rat brain. (B2 and B2′, B5 and B5′) Images showed corpus callosum as migration pathway of cells from transplanted COs into host brain. Cells from transplanted COs showed migration into cortical region (B1 and B1′), and migration into ipsilateral and contralateral hippocampus (B3 and B3′), thalamic nucleus (B4 and B4′), ipsilateral, and contralateral SGZ (B6 and B6′, B7 and B7′) in the host brain.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data that support the findings of this study are available from the corresponding author upon reasonable request.

O RCI D
Shu-Na Wang https://orcid.org/0000-0002-3091-1281 Jian-Sheng Lin https://orcid.org/0000-0002-2004-6079 Chao-Yu Miao https://orcid.org/0000-0002-8176-3434 F I G U R E 6 Cerebral organoids (COs) transplantation upregulates hippocampal neural connection proteins and neurotrophic factors, improves neurological motor function, and reduces brain damage in rat TBI model. A, Representative immunoblots of postsynaptic density protein 95 (PSD95, postsynaptic marker), synaptophysin (SYN, presynaptic marker), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), epidermal growth factor (EGF), and Tubulin (internal control protein as loading control) proteins at 14, 28, and 56 dpi in rat ipsilateral hippocampus of COs transplantation group. B-F, Quantitative analysis of protein expressions of PSD95, SYN, BDNF, NGF, and EGF normalized to Tubulin at 14, 28, and 56 dpi in rat ipsilateral hippocampus of COs transplantation group. All immunoblotting experiments in each group were repeated with four times. All data were mean value that normalized to protein expression in Sham group. G, Representative images of whole brain at 42 dpi in the rat TBI and TBI transplanted with COs groups. The cavity in transplantation group was smaller than that in TBI group. Scale bars: 1 cm. H-I, Rat mNSS score and beam walking test performance were recorded at 2, 5, 7, 11, 14, 21, 28, 35, and 42 dpi in rat TBI model. All data were shown as mean ± SEM and analyzed by ANOVA with Bonferroni posthoc tests, n = 8. *P < .05 and **P < .01 vs Sham group; # P < .05 and ## P < .01 vs TBI group. N.S, not significant. J, The proposed mechanism for the efficacy of COs transplantation in brain injury therapy. Cell number and composition are different in 55 d-CO and 85 d-CO. 55 d-CO is a better transplantation donor than 85 d-CO for cell survival and neurogenesis. Intracerebral transplantation of 55 d-CO into damaged motor cortex can improve neurological motor function and rescue brain damage via activation of exogenous neural repair and upregulation of neural connection proteins and neurotrophic factors