Application of human retina organoid in preparation of medicine for treating traumatic optic neuropathy

Abstract

The invention provides an application of a human retina organ in preparation of a medicament for treating traumatic optic neuropathy. According to the invention, the induced and differentiated human retina-like organ is applied to the optic nerve injury area, and the eye and brain nerve connection can be functionally reconstructed, so that the damaged visual function is recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of cell therapy, and in particular to use of human retinal organoids in the preparation of cells for treating traumatic optic neuropathy. Background Art

[0002] Traumatic optic neuropathy (TON) is a serious disease in which the optic nerve is damaged due to trauma to the head or eye. It is estimated that the annual incidence of TON is about 5 to 15 cases per 100,000 people, and it is particularly common in traffic accidents, falls, and sports activities. The main symptoms of TON include vision loss, visual field defects, decreased contrast sensitivity, and loss of light reflex. Clinical signs include weakened or absent pupillary light reflex, optic disc pallor, and optic nerve atrophy. TON causes the patient's eye-brain nerve connection to be interrupted and visual function is irreversibly damaged. There is currently no effective treatment.

[0003] The optic nerve is a pure white matter structure of the central nervous system composed of retinal ganglion cell axons and glial cells, and its regenerative capacity is extremely limited. At present, the clinical treatments for TON mainly include glucocorticoid therapy, surgical decompression, neurotrophic factors, and hyperbaric oxygen therapy. However, these clinical treatments cannot effectively promote optic nerve regeneration and restore visual function. In the research field, emerging interventions such as conventional cell therapy, gene therapy, and activation of endogenous stem cells not only fail to overcome obstacles such as low survival rate, tumor risk, the need for pre-activation, and endogenous stem cell depletion, but also fail to achieve stable and effective functional reconstruction of eye-brain nerve connections.

[0004] Cell therapy is a therapeutic strategy with great potential for clinical transformation. Since the 1980s, scientists have studied various seed cells for repairing optic nerve damage, including umbilical cord blood stem cells, mesenchymal stem cells, fetal retinal ganglion cells, and retinal ganglion cells derived from human pluripotent stem cells. However, due to the lack of suitable regenerative factors in the injury environment, the number, distance, and orientation of axon growth of donor retinal ganglion cells are still very limited. When embedded in a 3D printed fibrin matrix modified with growth factors, the axon growth and synaptic connection ability of donor neurons in the host tissue were significantly improved. However, the microenvironment of optic nerve development during embryonic development is very complex, including cell interactions between retinal ganglion cells, retinal ganglion cells and retinal progenitor cells, retinal ganglion cells and glial cells, and interactions with growth factors in the microenvironment such as FGF, Netrin-1, and the Ephrin family. At present, there is no ideal solution that can simultaneously provide retinal ganglion cells at a suitable differentiation stage and their complete developmental microenvironment. Therefore, developing a new therapeutic approach that can simultaneously provide retinal ganglion cells at an appropriate differentiation stage and a complete developmental microenvironment has become an urgent problem to be solved in the current field of optic nerve injury treatment.

[0005] Human retinal organoids (HROs) are tiny embryonic retinal tissues derived from human pluripotent stem cells, containing a large number of retinal stem cells, retinal ganglion cells and their appropriate developmental microenvironment in the human embryonic period. With the help of the in vivo vascular system and immune cells, HROs can further improve their developmental microenvironment, promote their neuronal differentiation and maturation, and become a tissue model closer to the in vivo development of the human central nervous system. Therefore, HROs have great clinical application and translational potential. Previous animal experiments have shown that when HROs are transplanted into the degenerated retina or damaged visual cortex of model animals, they can complete structural and functional integration with the host and improve the visual function of the host. At the same time, a recent clinical trial study has shown that after HROs were transplanted into the retina of patients with retinitis pigmentosa, they survived for more than 2 years without immune rejection and tumor formation, proving their safety in clinical application. HROs transplantation also has great application prospects in the treatment of optic nerve damage diseases. Previous studies have transplanted human retinal organoids into the vitreous cavity (in front of the retina) of glaucoma macaques, and observed that the axons of retinal ganglion cells in human retinal organoids projected long distances along the retinal nerve fiber bundles to the optic disc, indicating that retinal ganglion cells in human retinal organoids can use host microenvironment signals to guide their axons to grow in a directional manner toward the target area in the brain. Therefore, human retinal organoid transplantation is a treatment with great clinical translation prospects. In theory, it can be used to repair optic nerve damage caused by trauma and functionally reconstruct interrupted eye-brain nerve connections, but no related research has been reported so far. Summary of the invention

[0006] The present invention relates to a novel treatment method based on human retinal organoids (hROs) for repairing optic neuropathy caused by trauma. The present invention restores impaired visual function by applying induced differentiated human retinal organoids to the damaged area of ​​optic nerve to reconstruct functional eye-brain nerve connections.

[0007] The present invention discloses the use of human retinal organoids in the preparation of a method for treating traumatic optic neuropathy, wherein the human retinal organoids contain a neural retina having a typical epithelial structure, and in one embodiment according to the present invention, the neural retina is rich in Chx10 + Retinal progenitor cells and ATOH7 + Brn3b + Retinal ganglion cells.

[0008] According to one embodiment of the present invention, the human retinal organoid is prepared by the following method:

[0009] 1) Human pluripotent stem cells were cultured in 5% CO 2 , incubated in a first differentiation medium at 37° C.; preferably, the human pluripotent stem cells are WAe009-A cell line;

[0010] 2) After 6 days of induction in the first differentiation medium, recombinant human BMP4 was added to the incubated first culture medium to form a second differentiation medium, wherein the final concentration of recombinant human BMP4 in the second differentiation medium was 1.5 nM;

[0011] 3) On the 18th day of differentiation induction, the cells undergoing differentiation were transferred to a long-term culture medium and cultured until the 30th to 40th day.

[0012] The first differentiation medium comprises 45% V / V Iscove's modified Dulbecco's medium (IMDM, Gibco), 45% V / V Hams F12 (Gibco), Glutamax (Gibco), 1% chemically defined lipid concentrate (Gibco), 10% Knockout serum replacement (KSR, Gibco), 450 μM monothioglycerol (Sigma-Aldrich), 100 U / ml penicillin (Gibco), 20 mM Y-27632 (Sigma-Aldrich) and 100 μg / ml streptomycin (Gibco);

[0013] The second differentiation medium is supplemented with recombinant human BMP4 on the basis of the first differentiation medium, and the final concentration of the recombinant human BMP4 is 1.5 nM;

[0014] The long-term culture medium contained DMEM / F12-Glutamax medium, 1% V / V N2 supplement (Gibco), 10% V / V fetal bovine serum (FBS, Gibco), 0.5 μM retinoic acid (Sigma-Aldrich), 0.1 mM taurine (Sigma-Aldrich), 0.25 μg / ml Fungizone (Gibco), 100 U / ml penicillin and 100 μg / ml streptomycin.

[0015] In one embodiment according to the present invention, a half-medium liquid change operation is performed every 3 days during the culture process.

[0016] In one embodiment of the present invention, the human retinal organoids are cut into tissue blocks of 0.5 mm*0.5 mm*0.5 mm before use, or cut into tissue blocks according to the application object.

[0017] In one embodiment according to the present invention, the human retinal organoid is applied inside the optic nerve sheath or outside the optic nerve sheath.

[0018] In one embodiment according to the present invention, the invention further comprises the combined administration of an immunosuppressant.

[0019] In one embodiment according to the present invention, the immunosuppressant is cyclosporine.

[0020] In one embodiment of the present invention, the traumatic optic neuropathy includes lesions caused by complete optic nerve severance, partial optic nerve severance or blunt trauma.

[0021] The beneficial effects of the above technical solution of the present invention are as follows:

[0022] The human retinal organoid culture medium combination provided by the present invention can effectively induce differentiation to obtain human retinal organoids. The obtained human retinal organoids can rebuild the interrupted eye-brain nerve connection and restore the visual function and vision-related behavior of the damaged optic nerve. According to different types and degrees of traumatic optic neuropathy, therapeutic effects can be produced by applying human retinal organoids inside or outside the optic nerve sheath. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The design of the human retinal organoid application and detection method, the experimental process and the gross anatomical results of optic nerve repair described in the present invention;

[0024] Figure 2 This is an image of a human retinal organoid transplant applied to the inside of the optic nerve sheath;

[0025] Figure 3This is the result of OCT detection of the thickness of the rat optic disc and retinal ganglion cell complex;

[0026] Figure 4 This is a graph showing the results of the visual sensitivity and contrast sensitivity test of a small animal visual motion instrument;

[0027] Figure 5 This is a diagram showing the results of the pupillary direct light reflex test using a small animal visual pupillometer.

[0028] Figure 6 This is the result diagram of the visual electrophysiological test of flash visual evoked potential (FVEP) and flash optic nerve evoked potential (FONP);

[0029] Figure 7 This is a graph showing the results of small animal vision-related animal behavior tests (light-dark preference test and visual cliff test);

[0030] Figure 8 This is a diagram showing the effectiveness of CTB anterograde tracing in detecting eye-brain nerve connections;

[0031] Fig. 9 Figure 2 shows the test results of restoring partial visual function in the optic nerve crush injury model using human retinal organoid transplantation. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0033] Experimental design:

[0034] The most severe rat model of traumatic optic neuropathy was constructed; human retinal organoids were transplanted into the site of optic nerve damage in traumatic optic neuropathy; 30 days after surgery, the visual function of rats in the normal group (only optic nerve exposure without injury), disease group (optic nerve exposure and injury), and treatment group (optic nerve exposure, injury, and human retinal organoid tissue transplantation) was systematically and objectively evaluated through small animal optical coherence tomography, optokinetography, small animal pupillometer, visual electrophysiology, in vivo electrophysiology of the superior colliculus, vision-related animal behavior detection, and CTB anterograde tracing of the nerve tract, to verify the effectiveness of human retinal organoid transplantation in repairing traumatic optic neuropathy ( Figure 1 )

[0035] Statistical analysis

[0036] The present invention uses SPSS 30.0.0 to perform statistical analysis on the result data, and all data are expressed as mean ± standard deviation. The comparison between the groups uses one-way ANOVA single factor analysis of variance. When the ANOVA result shows significance (P<0.05), the significant difference between the groups is further determined by Tukey's multiple comparison test. The following marks are used in the charts and result descriptions: P<0.05: *, P<0.01: **, P<0.001: ***, if the P value is ≥0.05, it is marked as ns, indicating that the difference is not statistically significant.

[0037] Example 1. Human pluripotent stem cells induced differentiation of retinal organoids

[0038] The present invention uses the human pluripotent stem cell WAe009-A cell line (RRID: CVCL_9773, purchased from WiCell Research Institute) as an example of human pluripotent stem cells.

[0039] The WAe009-A cell line was maintained in Essential 8 medium (purchased from Gibco) and Vitronectin (purchased from Gibco) culture system without feeder layer. During maintenance culture, WAe009-A cell colonies were passaged every 4-5 days and treated with Versene (purchased from Gibco) at 37°C for 5 minutes. The detached cell clumps were broken into small pieces by gentle pipetting. At each passage, morphologically identifiable differentiated cells were removed mechanically.

[0040] The present invention refers to the previously reported SFEBq induction method as a typical induction method for human retinal organoids (Kuwahara, A., Ozone, C., Nakano, T., Saito, K., Eiraku, M., and Sasai, Y. 2015. Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue. Nat Commun 6, 6286.).

[0041] First, WAe009-A cell colonies were dissociated into single cells using TrypLE Express (purchased from Gibco) and quickly reaggregated in a low cell adhesion 96-well plate with V-bottom conical wells (purchased from Sumitomo Bakelite). 10,000 cells per well were cultured in retinal organoid differentiation medium supplemented with 20 mM Y-27632 (purchased from Sigma-Aldrich) at 5% CO.2 , incubated at 37°C. The differentiation medium components included 45% Iscove's modified Dulbecco's medium (IMDM, purchased from Gibco), 45% Hams F12 (F12, Gibco), Glutamax, 1% chemically defined lipid concentrate (purchased from Gibco), 10% Knockout serum replacement (KSR, purchased from Gibco), monothioglycerol (450 μM, purchased from Sigma-Aldrich), 100 U / ml penicillin and 100 μg / ml streptomycin (purchased from Gibco). On the 6th day of induction (D6), recombinant human BMP4 (purchased from PeproTech) was added to the culture medium at a final concentration of 1.5 nM (55 ng / ml), and the culture medium was half-changed every 3 days. Human retinal organoids were transferred to ultra-low adhesion 9 cm suspension culture dishes (purchased from Corning) on ​​the 18th day of induction (D18) and continued to be cultured using long-term culture medium. The long-term culture medium components include DMEM / F12-Glutamax culture medium (purchased from Gibco), 1% N 2 Supplement (purchased from Gibco), 10% fetal bovine serum (FBS, purchased from Gibco), 0.5 μM retinoic acid (purchased from Sigma-Aldrich), 0.1 mM taurine (purchased from Sigma-Aldrich), 0.25 μg / ml Fungizone (purchased from Gibco), 100 U / ml penicillin and 100 μg / ml streptomycin (purchased from Gibco). Human retinal organoids that meet the experimental standards should contain neural retina with typical epithelial structure at day 30 to 40, and the neural retina is rich in Chx10 + Retinal progenitor cells and ATOH7 + Brn3b + Retinal ganglion cells. Before surgery, the neural retina of human retinal organoids is cut into tissue blocks larger than about 0.5mm*0.5mm*0.5mm, or cut into tissue blocks according to the application object, for the next transplantation application experiment.

[0042] Example 2. Construction of the most severe traumatic optic neuropathy rat model

[0043] In order to strictly verify the effectiveness of human retinal organoid transplantation in repairing traumatic optic neuropathy, the present invention uses two methods to construct rat models of optic nerve injury with different degrees of severity to simulate the most challenging traumatic optic neuropathy in clinical practice.

[0044] The present invention constructs the most severe traumatic optic neuropathy rat model, that is, completely severing the optic nerve fibers by total optic nerve transection combined with partial optic nerve parenchyma resection, the steps are as follows:

[0045] 1) Long-Evans rats (purchased from Jicui Yaokang) 5-6 weeks after birth and weighing 200-250 g were selected, half male and half female. The rats were anesthetized by inhalation of 3% isoflurane (purchased from Baxter), and after disinfection with iodine, 0.4% oxybuprocaine hydrochloride eye drops (purchased from Santen) were instilled into the conjunctival sac for local anesthesia.

[0046] 2) The left optic nerve was exposed through the conjunctival approach, the optic nerve sheath was cut 1.5 mm behind the bulb, the length was about 0.5 mm, the optic nerve substance was cut by intraocular shears about 0.5-0.8 mm, and micro curved forceps were used to polish the inner wall of the optic nerve sheath three times to ensure that the optic nerve fibers were completely severed, thereby simulating complete optic nerve rupture injury. This operation is designed to reproduce the most thorough and irreversible traumatic optic neuropathy to verify the ultimate effect of treatment.

[0047] The operation method of the optic nerve crush injury model of the present invention (simulating common clinical optic nerve blunt contusion) is as follows:

[0048] The optic nerve is clamped with microscopic toothed forceps for 10 seconds to induce partial damage to the optic nerve fibers. This method removes less optic nerve parenchyma, preserves some nerve fiber structures, simulates a relatively mild injury scenario, and is used to evaluate the repair effect of treatment under different degrees of injury.

[0049] Example 3. Application of human retinal organoid transplantation

[0050] Using the traumatic optic neuropathy rat model constructed in Example 2 above, the human retinal organoid neural retinal tissue blocks prepared before surgery were transplanted into the optic nerve injury site and implanted into the cavity of the optic nerve sheath, i.e., human retinal organoid optic nerve sheath transplantation ( Figure 2 ); If it is an optic nerve crush injury model, the human retinal organoid neural retinal tissue block is directly transplanted and applied to the optic nerve sheath outside the optic nerve injury site. Tobramycin dexamethasone eye ointment is given after the operation, and the immunosuppressant cyclosporine (10 mg / kg / day) is given regularly for at least 1 month after the operation.

[0051] Example 4. Small Animal Optical Coherence Tomography

[0052] The fifth-generation Phoenix small animal retinal imaging system (purchased from Phoenix Research Labs, USA) was used to detect the thickness of the retinal ganglion cell complex (RGCC) in the rat retina.

[0053] Before imaging, compound tropicamide eye drops (purchased from Santen) were used to dilate the pupil, and 0.4% oxybuprocaine hydrochloride eye drops (purchased from Santen) were used for local anesthesia, and lubricants (such as methylcellulose) were applied to the rat cornea to prevent drying. Subsequently, LE rats anesthetized by intraperitoneal injection of Avertin (purchased from Laite) (200 mg / kg) were placed on the bracket of the imaging device, ensuring that the eyes were facing the OCT optical system. High-resolution OCT imaging was performed using the Phoenix system, the focus was adjusted to obtain retinal images, and the B-scan mode was used for scanning. The software tool provided by the device automatically identified and manually corrected the boundaries of the nerve fiber layer, ganglion cell layer, and inner plexiform layer to measure the thickness of the ganglion cell complex. Multiple repeated scans were performed for each rat, and the average thickness around the optic disc was calculated. During the experiment, the rats were ensured to be properly anesthetized to reduce image distortion caused by eye movement, and the equipment was calibrated before imaging to ensure measurement accuracy. After the experiment, the rats' eyes were rinsed with saline to remove residual drugs.

[0054] The results are as follows Figure 3 As shown, human retinal organoid transplantation can preserve retinal ganglion cell complex thickness after traumatic optic neuropathy.

[0055] The structural integrity of the retinal ganglion cell complex (GCC) at different time points after optic nerve injury was evaluated by small animal optical coherence tomography (OCT). Representative OCT images showed that the thickness of the retinal ganglion cell complex was significantly reduced at postoperative day 30 (PSD30) in the disease group compared with the normal group, indicating a large loss of retinal ganglion cells after optic nerve injury. In contrast, the treatment group that received human retinal organoid transplantation showed a significant protective effect of GCC thickness at both PSD30 and PSD90. In particular, at PSD30, the thickness of the GCC layer in the treatment group was significantly higher than that in the disease group, although still lower than that in the normal group, indicating that the retinal structure was partially restored. At PSD90, the GCC thickness in the treatment group was only slightly but not statistically significant lower than that at PSD30, showing the persistence of the transplantation effect over a longer period of time. Quantitative analysis further verified the above results. The average GCC thickness in the normal group was approximately 78 μm, which was significantly higher than that in the disease group at PSD30 (approximately 30 μm, P<0.01). In the treatment group, the GCC thickness at PSD30 was close to 60 μm, which was significantly improved compared with the disease group (P<0.01). This effect was basically maintained at PSD90 (about 55 μm). There was no significant difference between PSD30 and PSD90 in the treatment group (ns), indicating the stability of structural protection. Dissection on the 30th day after surgery revealed that the optic nerve tissue near the retina in the disease group had completely degenerated, while the optic nerve in the treatment group showed an anatomically repaired appearance, and the gross structure of the optic nerve tissue near the retina remained intact ( Figure 3 ).

[0056] Example 5. Small Animal Vision Instrument

[0057] The visual acuity of LE rats was tested using the Optodrum Plus system (purchased from Striatech, Germany). Before the test, the healthy eye of the rat (uniformly the right eye) was sutured and covered with a black patch to avoid the contribution of the healthy eye to the visual acuity test. During the test, the rat was placed on the central platform of the Optodrum Plus system to ensure that its head was centered and could rotate freely. In a quiet environment, radial sinusoidal stripes rotating at 12° / s were presented through the system software. When the rat can perceive the movement of the stripes, a spontaneous optokinetic response (OKR) will appear with the direction of rotation of the stripes. Visual acuity measurement is performed by gradually increasing the spatial frequency of the stripes to determine the highest spatial frequency that the rat can perceive (unit: cycles / degree, cpd). The visual threshold is defined as the highest frequency at which the rat can still produce a stable optokinetic response at a given spatial frequency.

[0058] The contrast sensitivity test method is similar to the vision test, using the same equipment and experimental conditions. Before the test, the healthy eye of the rat (uniformly the right eye) was sutured and covered with a black patch to avoid the contribution of the healthy eye to the contrast sensitivity. The spatial frequency of the stripes was fixed in the Optodrum Plus system, and the contrast of the stripes was gradually reduced by adjusting the contrast level, from 100% to detect the contrast sensitivity threshold of the rat. At each spatial frequency, the rat was recorded for conditions in which it could still produce a visual motion response under the lowest contrast conditions. The contrast sensitivity threshold is defined as the lowest contrast level that a rat can perceive at a specific spatial frequency, and the reciprocal of the contrast sensitivity threshold is the contrast sensitivity. The equipment was calibrated before each experiment to ensure the accuracy of the data.

[0059] The results are as follows Figure 4 As shown, human retinal organoid transplantation significantly improved visual function in traumatic optic neuropathy, including visual acuity and contrast sensitivity.

[0060] The recovery of visual function was evaluated by visual actigraphy. Visual acuity and contrast sensitivity were measured by changing the frequency and contrast of the stripe pattern, respectively. The results showed that compared with the normal group, the visual acuity and contrast sensitivity of rats in the disease group (PSD30) were significantly decreased, while the treatment group showed significant improvement in visual function at PSD30. In the normal group, the visual acuity reached about 0.58 cycle / degree, while the visual acuity in the disease group was significantly reduced to 0 cycle / degree (P<0.001). In contrast, the visual acuity of the treatment group at PSD30 was significantly restored to about 0.14 cycle / degree, which was significantly higher than that of the disease group (P<0.001), but still lower than that of the normal group. The average contrast sensitivity of the normal group was about 7, while that of the disease group was only about 0 (P<0.001). The contrast sensitivity of the treatment group at PSD30 was restored to about 1.5, which was significantly higher than that of the disease group (P<0.001), but still lower than that of the normal group ( Figure 4 ).

[0061] Example 6. Small Animal Pupillometer

[0062] use The A-2000 small animal pupillometer system (purchased from Neurotics, USA) was used to detect the direct light reflex (Pupillary Light Reflex, PLR) of rats. Before the test, the healthy eye of the rat (uniformly the right eye) was sutured and covered with a black patch to avoid the indirect light reflex produced by the healthy eye. The experiment was carried out in a low-illuminance red light environment (<5lux). No mydriasis was performed before the test to ensure a natural pupil reflex. Rats received an intraperitoneal injection of Avertin (purchased from Laite) for anesthesia (200mg / kg) to stabilize the head position without inhibiting pupillary response. The rats were placed in On the A-2000 system platform, software-controlled light stimulation (wavelength 622nm, light flux 40lumin) was used to illuminate the left eye of LE rats, and the original data of pupil diameter changes were dynamically recorded. The final data were compared and analyzed with relative pupil diameter values. Each rat was tested three times. During the experiment, the intensity and time of light stimulation were ensured to be consistent. After the experiment, the rat eyes were rinsed with saline to remove residual irritants.

[0063] The results are as follows Figure 5 As shown, human retinal organoid transplantation partially restores the pupillary direct light reflex in traumatic optic neuropathy.

[0064] The direct pupillary light reflex (PLR) function of rats in different groups was detected by a small animal pupillometer. Continuous red light stimulation of 622nm was used and the pupil reflex changes were recorded by an infrared camera to evaluate the recovery of optic nerve function. The experiment showed that in the normal group, the pupil contracted rapidly after light stimulation, and no obvious pupil contraction reaction was observed in the rats in the disease group at PSD30, while obvious pupil contraction reaction could be observed in the rats in the treatment group at PSD30.

[0065] The direct pupillary light reflex of the normal group rats showed 100% contraction response, while all rats in the disease group could not produce pupillary contraction response at PSD30. The contraction response ratio of the treatment group increased significantly to more than 52% ( Figure 5 ). The pupils of the normal group contracted rapidly and with a large amplitude, while the disease group showed no significant contraction response at PSD30. In contrast, the treatment group showed a certain degree of pupil contraction response at PSD30, although the contraction amplitude and speed were lower than those of the normal group ( Figure 5 ). The average contraction latency of the normal group was less than 1 second, while the rats in the disease group failed to trigger any pupil constriction response at PSD30 (N / A). The contraction latency of the treatment group was about 7 seconds, which was significantly longer than that of the normal group (P<0.001). The pupil constriction amplitude of the normal group was about 0.54, while it was significantly reduced to 0.1 in the disease group (P<0.001). The contraction amplitude of the treatment group at PSD30 recovered to about 0.5, which was significantly higher than that of the disease group (P<0.001), and there was no statistical difference compared with the normal group (ns)( Figure 5 ).

[0066] Example 7. Visual electrophysiology

[0067] The flash visual evoked potential (FVEP) of rats was detected using a visual electrophysiological system.

[0068] Before the test, the healthy eye of the rat (uniformly the right eye) was sutured and covered with a black patch to avoid FVEP produced by the healthy eye. Before the test, 1% compound tropicamide eye drops (purchased from Santen) were used to dilate the pupil, and 0.4% oxybuprocaine hydrochloride eye drops (purchased from Santen) were used for corneal surface anesthesia. To ensure the stability of the test, the rats received intraperitoneal injection of avertin (purchased from Laite) anesthesia (200 mg / kg) to keep quiet. Subsequently, the rats were fixed on the electrophysiological testing platform, the recording electrode was placed above the visual cortex (occipital area), the reference electrode was placed on the nasal bone or forehead, and the ground electrode was placed on the tail. The flash stimulation was controlled by the system software, and the light intensity was set to 10-1000cd·s / m 2The flash frequency was 1 Hz and the duration was 10 milliseconds. The potential waveform within 50-150 milliseconds after stimulation was recorded, and the latency and amplitude of the P1 wave were analyzed to evaluate the optic nerve function. Each rat was tested three times. During the test, the consistency of light stimulation intensity and electrode position was ensured, and the system was calibrated before each experiment to ensure signal accuracy. After the experiment, the rat's eyes were rinsed with saline and properly placed after it woke up.

[0069] The flash optic nerve potential (FONP) of rats was detected using a visual electrophysiological system.

[0070] Before the test, the healthy eye of the rat was covered with a black patch to avoid possible influence of the healthy eye. Before the test, 1% compound tropicamide eye drops (purchased from Santen) were used to dilate the pupil, and 0.4% oxybuprocaine hydrochloride eye drops (purchased from Santen) were used for corneal surface anesthesia. To ensure the stability of the test, the rats received intraperitoneal injection of avertin (purchased from Laite) anesthesia (200 mg / kg) to keep quiet. First, the optic nerve of the left eye of the LE rat was exposed through the conjunctival approach, and the recording electrode was placed inside the optic nerve orbit (if it was a treatment group, it was placed on the optic nerve behind the transplant area), the reference electrode was placed at the equator of the eyeball (close to the sclera), and the ground electrode was placed at the tail. During the experiment, sufficient hemostasis was performed to keep the orbit dry, and there was no accumulation of blood or tissue fluid to cause short circuit. Subsequently, the rat was fixed on the electrophysiological detection platform, and the flash stimulation was controlled by the system software, and the light intensity was set to 10-1000cd·s / m 2 The flash frequency was 1 Hz and the duration was 10 milliseconds. The potential waveform within 50-150 milliseconds after stimulation was recorded, and the amplitude of the FONP wave was analyzed to evaluate the optic nerve function. Each rat was tested three times. During the test, the consistency of light stimulation intensity and electrode position was ensured, and the system was calibrated before each experiment to ensure signal accuracy. After the experiment, the rat's eyes were rinsed with saline, tobramycin dexamethasone eye ointment was applied, and the rat was properly placed after waking up.

[0071] The results are as follows Figure 6 As shown, human retinal organoid transplantation can restore some visual electrophysiological functions of traumatic optic neuropathy.

[0072] In order to evaluate the recovery of optic nerve conduction function, the present invention detects each group of rats by flash visual evoked potential (FVEP) and flash optic nerve potential (FONP).

[0073] The FVEP results showed that in the normal group, the N1 and P1 waves of the FVEP waveform could be clearly observed, while in the rats in the disease group (PSD30), the waveform almost disappeared, indicating that the optic nerve conduction function was severely impaired. The treatment group showed a certain degree of waveform recovery at PSD30, but its amplitude and latency were still different from those of the normal group. The N1 latency of the normal group was about 35ms, while the N1 latency of the disease group was significantly prolonged to more than 43ms (P<0.01). The N1 latency of the treatment group recovered to about 39ms at PSD30, which was significantly longer than that of the normal group (P<0.05), but there was no statistically significant difference compared with the disease group (ns). The average amplitude of the normal group reached about 13μV, while that of the disease group was only about 2μV (P<0.001). The amplitude of the treatment group recovered significantly to about 8μV at PSD30, which was significantly higher than that of the disease group (P<0.001), but still lower than that of the normal group (P<0.01) ( Figure 6 ).

[0074] FONP has a higher signal-to-noise ratio and lower variability than FVEP, and is more objective and reliable than FVEP in evaluating the conduction function of the optic nerve. The FONP results showed that in the normal group, the FONP waveform could be clearly observed; in the rats in the disease group (PSD30), the waveform was completely extinguished, indicating that the conduction function of the optic nerve was completely interrupted; and the treatment group showed a certain degree of waveform recovery at PSD30. The FONP waveform amplitude of the normal group was about 14μV, the FONP waveform amplitude of the disease group could not be detected, and the FONP waveform amplitude of the treatment group at PSD30 was about 2.8μV, which was significantly lower than that of the normal group (P<0.005), indicating that the optic nerve in the treatment group partially recovered the nerve conduction function ( Figure 6 ).

[0075] In addition, if Fig. 9 As shown, human retinal organoid transplantation can also improve visual function in the optic nerve crush injury model.

[0076] Flash visual evoked potential (FVEP) was used to detect functional recovery, thereby evaluating the therapeutic effect of human retinal organoid transplantation in the optic nerve crush injury model. FVEP functional testing found that the normal group showed a clear N1-P1 waveform 30 days after surgery, while there was almost no obvious FVEP waveform response in the disease group. The treatment group showed a partially recovered N1-P1 waveform, and the amplitude was significantly improved compared with the disease group. Quantitative analysis of the N1-P1 amplitude found that the average amplitude of the normal group reached about 13μV, while that of the disease group was only about 2μV (P<0.001). The amplitude of the treatment group recovered to about 13μV, which was significantly higher than that of the disease group (P<0.001), but there was no significant difference compared with the normal group (ns)( Fig. 9 )

[0077] Example 8. Vision-related animal behavior detection

[0078] Dark-Light Preference Test: In order to evaluate the light sensitivity and dark tendency of rats, a light-light preference test was performed. A square behavioral experimental box contains a completely dark area (darkroom) in the middle, and the rest are bright areas (white light, 300 lux). Before the test, the healthy eye of the rat (the right eye) was sutured and covered with a black patch to avoid the visual-related behavioral effects of the healthy eye. Each rat was gently placed in the center of the experimental box to adapt to the dark environment for 5 minutes, and then the lighting was suddenly turned on (white light, 300 lux). The camera system was used to record whether the rat entered or passed through the dark room within 30 seconds to evaluate its ability to move toward light or darkness. Each rat repeated the task independently 4 times. The success rate of successfully completing the light-dark preference test was recorded.

[0079] Visual Cliff Test: In order to evaluate the depth perception ability and visual behavior of rats, a visual cliff test was used. The experimental apparatus consists of a transparent elevated platform with a visual pattern on one side of the platform to simulate a safe area and a transparent cliff on the other side to simulate a visual drop. After the experimental rats were adapted to dark adaptation conditions for 10 minutes, they were gently placed in the center of the platform. The movement trajectory and selection behavior of each rat on the platform were recorded by a camera system, and the number of times it chose the visual pattern area or the cliff area within 5 minutes was observed. The proportion of rats choosing the safe area and the hesitation time were used as indicators to evaluate their visual perception ability.

[0080] The results are as follows Figure 7 As shown, human retinal organoid transplantation improves vision-related behavioral responses in traumatic optic neuropathy.

[0081] In order to evaluate the effect of human retinal organoid transplantation on the recovery of visual function in rats with optic nerve injury, the light-dark preference test and the visual cliff test were used to detect the recovery of visual behavior. In the light-dark preference test, the rats in the normal group showed a strong photosensitive response and quickly entered the shielding box to avoid light stimulation in almost all experiments (100%). However, the rats in the disease group (PSD30) almost lost this response, and the response rate dropped significantly to 0%. In contrast, the response rate of the treatment group at PSD30 was significantly restored, reaching an average of more than 50%, which was not statistically significant compared with the normal group (ns), but was significantly higher than the disease group (P<0.01). The visual cliff test is used to evaluate the ability of rats to perceive depth. In the visual cliff test, rats in the normal group tended to stay in the safe light-colored area (staying time ratio of about 80%), while the proportion of staying time in the disease group at PSD30 was significantly reduced to about 40%, and the treatment group significantly increased the time spent in the light-colored area at PSD30 (about 58%, P<0.01), which was significantly higher than the disease group (P<0.001), but still lower than the normal group (P<0.05) ( Figure 7 )

[0082] Example 9. Anterograde tracing of CTB neural tracts

[0083] To evaluate the regeneration of nerve fibers and the neural connectivity of target areas in the brain after optic nerve injury in rats, cholera toxin B subunit conjugate (Alexa Fluor TM 555, CTB555, purchased from Invitrogen) for anterograde nerve tract tracing.

[0084] After the rats were anesthetized by intraperitoneal injection of Avertin (purchased from Laite, 250 mg / kg), the left sclera was exposed by micromanipulation, and 1% CTB was injected into the vitreous cavity through the limbus of the cornea and sclera using a 10 μL microsyringe. The total injection volume was 3 μL and the injection rate was 0.2 μL / min. After the injection was completed, the needle was kept at the injection site for 1 minute to prevent reflux, and antibiotic eye ointment was used to cover to prevent infection. Seven days after the injection of CTB, anesthesia was performed by overdose of isoflurane and cardiac perfusion fixation was performed, first with 0.9% saline, and then with 4% paraformaldehyde (purchased from Solebao) for perfusion fixation. Subsequently, the rat brain and optic nerve tissue were removed, fixed in 4% paraformaldehyde for 4 hours, and then transferred to 30% sucrose solution for dehydration overnight. The tissue sections were 30 μm thick and mounted on slides. After the slices were washed with PBS, CTB-positive labeled nerve fibers were observed under a Zeiss LSM 880 fluorescence microscope, and high-resolution images were taken, followed by image analysis using ImageJ software. CTB injections were performed under sterile conditions to ensure that the microinjection needle was stable to avoid damaging the retina. At the same time, the rats' eyes were closely monitored to prevent infection. All experimental operations followed the ethical requirements for animal experiments.

[0085] The results are as follows Figure 8 As shown, human retinal organoid transplantation is used to reconstruct eye-brain neural connections in traumatic optic neuropathy.

[0086] The CTB anterograde tracing technique was used to track the eye-brain nerve connection after human retinal organoid transplantation to evaluate the repair effect of human retinal organoid transplantation on the eye-brain nerve connection. The experiment marked and imaged the optic nerve and superior colliculus 30 days after surgery (PSD30). At PSD30 in the treatment group, a large number of linear CTB signals were observed to successfully cross the optic nerve injury site and extend distally along the optic nerve trajectory; in the disease group, no linear CTB signals were observed to cross the optic nerve injury site at PSD30. Distribution of CTB-labeled superior colliculus axon terminals: Rats in the treatment group showed significant CTB-positive fiber terminals or neuronal cell bodies in the superior colliculus region, indicating that the eye-brain nerve connection after CTB repair successfully reached its original target area (superior colliculus of the midbrain), while there were no significant CTB-positive fiber terminals or neuronal cell bodies in the superior colliculus region of rats in the disease group ( Figure 8 ).

[0087] The above examples demonstrate that the human retinal organoids provided by the present invention have the following beneficial effects:

[0088] 1. The application of human retinal organoid transplantation effectively protected the retinal structure of rats after traumatic optic neuropathy, especially the retinal ganglion cell complex representing the optic nerve fibers.

[0089] 2. The application of human retinal organoid transplantation significantly improved the visual function of rats after traumatic optic neuropathy, including visual acuity and contrast sensitivity, indicating the therapeutic effect of human retinal organoid transplantation in the repair of traumatic optic neuropathy.

[0090] 3. The application of human retinal organoid transplantation significantly improved the direct pupillary light reflex function of rats with traumatic optic neuropathy, showing its therapeutic effect in restoring the optic nerve conduction function.

[0091] 4. The application of human retinal organoid transplantation significantly improved the visual electrophysiological functions of rats with traumatic optic neuropathy, including flash visual evoked potential and flash optic nerve potential, which indicates that the application of human retinal organoid transplantation has a therapeutic effect in promoting the recovery of optic nerve function.

[0092] 5. The application of human retinal organoid transplantation significantly improved the vision-related behavioral responses of rats after traumatic optic neuropathy, indicating that it has a therapeutic effect in promoting the recovery of visual function.

[0093] 6. The application of human retinal organoid transplantation can repair the interrupted eye-brain nerve connection pathway after traumatic optic neuropathy.

[0094] 7. According to the different types and degrees of traumatic optic neuropathy, the therapeutic effect can be achieved by optimizing the transplantation application site (inside or outside the optic nerve sheath) and transplanting human retinal organoid tissue into the optic nerve sheath or outside the optic nerve sheath.

[0095] 8. In summary, transplanting human retinal organoids into the site of optic nerve damage in traumatic optic neuropathy can reconstruct the interrupted eye-brain nerve connection and restore the visual function and vision-related behavior of the damaged optic nerve.

[0096] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of human retinal organoids in the preparation of a method for treating traumatic optic neuropathy, wherein: The human retinal organoids contain a neural retina with a typical epithelial structure, preferably, the neural retina is rich in Chx10 + Retinal progenitor cells and ATOH7 + Brn3b + Retinal ganglion cells.

2. The use according to claim 1, wherein The human retinal organoids are prepared by the following method: 1) incubating human pluripotent stem cells in a first differentiation medium under the conditions of 5% CO2 and 37°C; preferably, the human pluripotent stem cells are WAe009-A cell line; 2) After 6 days of induction in the first differentiation medium, recombinant human BMP4 was added to the incubated first culture medium to form a second differentiation medium, wherein the final concentration of recombinant human BMP4 in the second differentiation medium was 1.5 nM; 3) On the 18th day of differentiation induction, the cells undergoing differentiation were transferred to a long-term culture medium and cultured until the 30th to 40th day. The first differentiation medium comprises 45% V / V Iscove's modified Dulbecco's medium (IMDM), 45% V / V Hams F12, Glutamax, 1% chemically defined lipid concentrate, 10% Knockout serum replacement (KSR), 450 μM monothioglycerol, 100 U / ml penicillin, 20 mM Y-27632 and 100 μg / ml streptomycin; The second differentiation medium is supplemented with recombinant human BMP4 on the basis of the first differentiation medium, and the final concentration of the recombinant human BMP4 is 1.5 nM; The long-term culture medium contained DMEM / F12-Glutamax medium, 1% V / V N2 supplement, 10% V / V fetal bovine serum (FBS), 0.5 μM retinoic acid, 0.1 mM taurine (Sigma-Aldrich), 0.25 μg / ml Fungizone, 100 U / ml penicillin and 100 μg / ml streptomycin.

3. The use according to claim 2, wherein During the culture process, half of the culture medium was replaced every 3 days.

4. The use according to claim 2 or 3, wherein The human retinal organoids are cut into tissue blocks of 0.5 mm*0.5 mm*0.5 mm before use, or cut into tissue blocks according to the application object.

5. The use according to any one of claims 2 to 4, wherein The human retinal organoids are applied inside the optic nerve sheath or outside the optic nerve sheath.

6. The use according to claim 5, wherein Co-administration of immunosuppressants is also contemplated.

7. The use according to claim 6, wherein The immunosuppressant is cyclosporine.

8. The use according to any one of claims 1 to 7, wherein The traumatic optic neuropathy includes complete optic nerve severance, partial optic nerve severance and blunt trauma lesions.

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