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.

Keywords: brain injury; cerebral organoids; functional recovery; neural repair; transplantation.

Figure 1

Comparison of cell number and composition in COs at 55 and 85 d. A, Schematic diagram of COs culture. The day of embryonic bodies (EBs) initially made from hESCs was defined as day 0. EBs gradually showed bright surface with relative dark center from 1 to 8 d after induction (DAI). After Matrigel embedding for expanding neuroepithelial buds, well‐defined polarized neuroepithelium‐like structures resembled neural tubes at 15 DAI. Scale bars: 100 μm. B, Immunostaining for neural progenitor cells (SOX2, green) and neurons (Tuj1, red) in COs at 30 and 60 DAI. DAPI labels nuclei (blue). Scale bar: 100 μm. C, Immunostaining for COs at 75 DAI with forebrain (Foxg1, red) and choroid plexus (TTR, red). Scale bars: 50 μm. D, Cell number in COs at 55 and 85 d after induction (namely 55 d‐CO and 85 d‐CO). ^** P < .01. All data were shown as mean ± SEM and analyzed by Student's t‐test, n = 8. E, Cell composition in 55 d‐CO and 85 d‐CO. Nestin (red), neural stem cells; NeuN (red), mature neurons; Tuj1 (red), neurons; GFAP (red), astrocytes; SOX2 (green), neural progenitor cells; TBR1 (green), preplate/deep‐layer neurons; SATB2 (green), surface‐layer neurons; vGlut1 (green), excitatory glutamatergic neurons. Scale bar: 50 μm

Figure 2

More neurogenesis in 55 d‐CO than 85 d‐CO transplantation periphery of ipsilateral cortex in rat TBI model. A, Illustration of COs transplantation into damaged motor cortex in the rat TBI model. The lesioned cavity was made at the motor cortex (1.5 mm lateral to midline, 0.5 mm posterior to bregma) by biopsy punch. B, Representative images of neurogenesis in the transplantation periphery of ipsilateral cortex by immunostaining of proliferated neural stem cells (BrdU⁺/Nestin⁺, red and green, respectively), migrated newborn neurons (BrdU⁺/DCX⁺, red and green, respectively), and differentiated mature neurons (BrdU⁺/NeuN⁺, red and green, respectively) at 7, 14, 28, and 56 dpi in Sham, TBI, 55 d‐CO transplantation, and 85 d‐CO transplantation groups. BrdU, proliferation marker. DAPI labels nuclei (blue). Scale bars: 50 μm. C‐E, Quantitative analysis of neurogenesis by counting BrdU⁺/Nestin⁺, BrdU⁺/DCX⁺, and BrdU⁺/NeuN⁺ cells in the transplantation periphery of ipsilateral cortex at 7, 14, 28, and 56 dpi. Immuno‐stained 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, **P < .01 vs TBI group; #P < .05, ##P < .01. N.S, not significant

Figure 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

Figure 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

Figure 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. Scale bars: 1000 μm in B0,B0′; 200 μm in B3,B3′,B4,B4′,B5,B5′; 100 μm in B1,B1′,B2,B2′,B6,B6′,B7,B7′

Figure 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