A preparation of spinal cord organoids based on trichostatin a pretreatment and applications thereof

Abstract

The present application relates to the technical field of medicine, discloses a kind of spinal cord organoid preparation based on trichostatin A pretreatment, including human induced pluripotent stem cell differentiation obtained human spinal cord organoid, trichostatin A and GelMA hydrogel;Wherein, the trichostatin A is used to carry out pretreatment to the human spinal cord organoid before transplantation, to activate NRF2 / HO-1 signal pathway;And its application in preparing drug for treating spinal cord injury.The application combines TSA pretreatment and hSCOs transplantation for the first time, realizes the double advantages of "drug pretreatment enhances cell therapy", clearly that TSA plays the role of antioxidation and anti-apoptosis through NRF2 / HO-1 signal pathway, significantly enhances the survival ability of hSCOs in injury microenvironment, and has good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a spinal cord organoid preparation based on trogostatin A pretreatment and its application. Background Technology

[0002] Spinal cord injury (SCI) is a devastating trauma to the central nervous system, often resulting in permanent motor and sensory dysfunction due to the disruption of neural pathways. Current clinical treatment primarily relies on surgical decompression. However, the efficacy of administering high-dose methylprednisolone shortly after injury has limitations, and its use has some side effects. Therefore, there is an urgent need to develop novel and effective treatments targeting the core pathological mechanisms of SCI.

[0003] Cell-based regenerative therapy is a promising direction in the treatment of spinal cord injury, encompassing exogenous cell transplantation and enhancement of endogenous stem cell function. Current technologies achieve better regenerative therapeutic effects by selecting different cell types and states, as well as different treatment methods and intervention timing.

[0004] Transplanted cells can replace damaged and lost neural tissue, but cell transplantation therapy also faces several challenges, such as local tumor formation, poor cell differentiation, enhanced immune response of transplanted cells, and low cell survival rates, all of which require further solutions. After transplantation, organoid core cells sometimes die in large numbers due to ischemia, hypoxia, and inflammatory environments; maintaining their survival is a primary challenge. Furthermore, the cascade of secondary damage following primary SCI injury is a major cause of further deterioration in neurological function.

[0005] Therefore, there is a need for a cell preparation that reduces side effects after cell transplantation, and a treatment method that can reduce the cascade of secondary damage by regulating the microenvironment at the injury site, thereby improving treatment efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a spinal cord organoid preparation based on trogostatin A pretreatment and its application, in order to solve the technical problems in the prior art where secondary SCI injury is difficult to solve by cell transplantation alone, and where there are strong side effects after cell transplantation.

[0007] To solve the above-mentioned technical problems, the present invention specifically provides the following technical solution: This invention provides a spinal cord organoid preparation based on quabustatin A pretreatment, comprising human spinal cord organoids obtained from differentiation of human induced pluripotent stem cells, quabustatin A, and GelMA hydrogel; The trogostatin A is used to pretreat the human spinal cord organoids before transplantation to activate the NRF2 / HO-1 signaling pathway.

[0008] As a preferred embodiment of the present invention, the concentration of the quabustatin A is 3-7 μM.

[0009] The present invention provides a pharmaceutical composition for treating spinal cord injury, comprising the above-mentioned spinal cord organoid preparation and a pharmaceutically acceptable carrier or scaffold.

[0010] This invention provides the application of the above-mentioned spinal cord organoid preparation in the preparation of a drug for treating spinal cord injury.

[0011] As a preferred embodiment of the present invention, the treatment includes promoting the recovery of nerve function, reducing oxidative stress and apoptosis, inhibiting glial scar formation and neuroinflammation, and promoting axonal regeneration.

[0012] This invention provides a method for preparing spinal cord organoids for treating spinal cord injuries, comprising the following steps: S1. Differentiation of human spinal cord organoids from human induced pluripotent stem cells; S2. On day 24 of differentiation, human spinal cord organoids were pretreated with trichostatin A to activate the NRF2 / HO-1 signaling pathway. S3. Load the pretreated human spinal cord organoid into a hydrogel to form an implantable complex.

[0013] As a preferred embodiment of the present invention, the pretreatment includes the following conditions: the concentration of the quabustatin A is 3-7 μM, and the treatment time is 6-24 hours.

[0014] As a preferred embodiment of the present invention, the hydrogel is a GelMA hydrogel; The GelMA hydrogel contains the photoinitiator LAP; The crosslinking conditions for the GelMA hydrogel are irradiation with 405nm ultraviolet light for 20-40 seconds.

[0015] Compared with the prior art, the present invention has the following advantages: This invention is the first to propose a method for treating spinal cord injury by pretreating human spinal cord organoids with trichostatin A and transplanting them. It is the first to combine TSA pretreatment with hSCOs transplantation, achieving the dual advantages of "drug pretreatment enhancing cell therapy". It clarifies that TSA exerts its antioxidant and anti-apoptotic effects through the NRF2 / HO-1 signaling pathway, and the mechanism was verified by the inhibitor ML385. The mechanism is clear, showing that TSA pretreatment significantly enhances the survival ability of hSCOs in the injury microenvironment, while improving the local microenvironment, promoting nerve regeneration and functional recovery. It has good prospects for clinical translation and provides a new strategy and experimental basis for the treatment of spinal cord injury. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0017] Figure 1 This invention provides a schematic diagram of a multi-level induction method for generating human spinal cord organoids from human pluripotent stem cells; Figure 2 This invention provides a schematic diagram of the experimental design and treatment strategy for generating human spinal cord organoids using iPSCs; Figure 3 This invention provides optical micrographs of hSCOs at different stages; Figure 4 Immunofluorescence staining images of dorsal and ventral pattern markers OLIG2, NKX6.1, PAX6 and SOX2 in spinal cord organoids on day 14 are provided for this invention. Scale bar = 100 μM. Figure 5 Immunofluorescence staining images of Tuj1, Syn and NF, markers of neuronal maturation in spinal cord organoids, on day 24 are provided for this invention. Scale bar = 100 μM. Figure 6 This invention provides a statistical chart of the average fluorescence intensity analysis of various immune antibodies; Figure 7 This invention provides an appropriate concentration statistical plot (AC) for screening and modeling oxidative stress-induced grouping by using CCK-8 to detect the toxic effects of hydrogen peroxide and TSA on hSCOs. Figure 8 ROS immunofluorescence pattern is provided for this invention; Figure 9 This invention provides a statistical chart of the average fluorescence intensity analysis of ROS; Figure 10 This invention provides a statistical graph of ROS expression intensity analysis using flow cytometry. Figure 11 This invention provides statistical charts analyzing hSCOsSOD activity and MDA content in different subgroups. The data are expressed as mean ± SD (n=3 for each group). Figure 12 This invention provides a graph showing the effect of different concentrations of TSA on the expression of oxidative stress-related proteins; Figure 13 Statistical analysis and graphs of different protein expression results are provided for this invention; Figure 14To provide a basis for this invention, the Nrf2 channel inhibitor ML385 was added simultaneously, four subgroups were selected as the optimal TSA concentration, and the expression maps of oxidative stress-related proteins were measured. Figure 15 This invention provides statistical analysis charts of protein expression results from four subgroups. Figure 16 An expression map of apoptosis-related proteins in grouped organoids is provided for the present invention. Figure 17 Statistical analysis graphs of apoptosis-related protein expression are provided for this invention; Figure 18 The present invention provides flow cytometry apoptosis results of organoids in different groups, and the data are expressed as mean ± SD (n=3 per group). Figure 19 This invention provides statistical charts of BBB scores for each group of rats; Figure 20 This invention provides statistical charts of the inclined plane test scores for each group of rats; Figure 21 This invention provides gait analysis and statistical results graphs for each group; Figure 22 This invention provides H&E and Nissl staining results and statistical analysis charts of neurons in each group of rats; Figure 23 This invention provides representative immunofluorescence images of Tuj1+ neurons (red) and GFAP+ astrocytes (green) at the lesion site; Figure 24 This invention provides a statistical chart for quantitative analysis of the fluorescence intensity of Tuj1 and GFAP; Figure 25 This invention provides TUNEL staining images of spinal cord injury sites; Figure 26 This invention provides a statistical chart for quantitative analysis of TUNEL fluorescence intensity; Figure 27 This invention provides an immunofluorescence staining (red) image of NF200+ neurofilaments; Figure 28 This invention provides a statistical chart for quantitative analysis of NF fluorescence intensity; Figure 29 This invention provides a representative immunofluorescence image of CD68+ macrophages / microglia (green) in the center of the lesion; Figure 30 This invention provides a statistical chart of quantitative analysis of CD68+ cells in each region. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Following primary injury to spinal cord injury (SCI), the ensuing cascade of secondary injuries is the primary cause of further deterioration in neurological function. In this process, oxidative stress triggered by microenvironmental dysregulation plays a central role. The explosive accumulation of reactive oxygen species (ROS) exceeds endogenous antioxidant defenses, leading to lipid peroxidation, protein / DNA damage, and mitochondrial dysfunction, ultimately driving neuronal and glial cell death processes such as apoptosis and pyroptosis.

[0020] Traditional drug screening models (such as single-cell lines or animal models) have limitations, including significant interspecies differences and the inability to simulate the complex three-dimensional microenvironment of the human body. The emergence of human induced pluripotent stem cell-derived spinal cord organoids (hSCOs) has revolutionized SCI research. These organoids highly mimic human spinal cord tissue in terms of three-dimensional structure, cellular diversity, and function, making them ideal humanized models for studying pathological mechanisms and conducting preclinical drug evaluation.

[0021] Based on human spinal cord organoid technology, this invention creatively proposes a pretreatment technology for the panhistone deacetylase inhibitor trachostatin A (TSA), and provides a spinal cord organoid preparation based on trachostatin A pretreatment.

[0022] In this formulation, the present invention combines organoid formulations based on organoid transplantation technology with a therapeutic approach targeting oxidative stress pathways, thereby enabling its application as a highly promising therapeutic strategy in the treatment of spinal cord injury.

[0023] Specifically, the spinal cord organoid preparation based on quabustatin A pretreatment proposed in this invention includes human spinal cord organoids obtained from the differentiation of human induced pluripotent stem cells, quabustatin A, and GelMA hydrogel; wherein, quabustatin A is used to pretreat the human spinal cord organoids before transplantation.

[0024] HDACs (such as HDAC2) are abnormally expressed in neurons after SCI, participating in epigenetic regulation and exacerbating damage. TSA exerts a neuroprotective effect by inhibiting HDACs, reducing oxidative stress, suppressing inflammation, and reducing apoptosis. Pretreatment with trichostatin A improves the local microenvironment for cell growth and increases organoid survival.

[0025] Furthermore, TSA can also activate the NRF2 / HO-1 signaling pathway, enhance cellular antioxidant defense capabilities, reduce ROS accumulation, inhibit apoptosis, alleviate inflammatory responses, and promote axonal regeneration, thereby exerting antioxidant and anti-apoptotic effects and weakening oxidative stress-mediated secondary spinal cord injury.

[0026] It is evident that pretreatment with trogostatin A has a significant synergistic effect, not only significantly enhancing the survival ability of spinal cord organoids in the injury microenvironment, but also improving the local microenvironment and promoting nerve regeneration and functional recovery.

[0027] Spinal cord organoid technology and trichostatin A pretreatment technology can be used as a pharmaceutical composition for the treatment of spinal cord injury, including human spinal cord organoids derived from human induced pluripotent stem cell differentiation, trichostatin A, GelMA hydrogel, and pharmaceutically acceptable carriers or scaffolds.

[0028] This drug combination is used to prepare a treatment for spinal cord injury, which includes promoting the recovery of nerve function, reducing oxidative stress and apoptosis, inhibiting glial scar formation and neuroinflammation, and promoting axonal regeneration.

[0029] like Figure 1 and Figure 2 As shown, the present invention further provides a method for preparing this spinal cord organoid for treating spinal cord injury, comprising the following steps: S1. Differentiation of human spinal cord organoids from human induced pluripotent stem cells; S2. On day 24 of differentiation, human spinal cord organoids were pretreated with trichostatin A to activate the NRF2 / HO-1 signaling pathway. The concentration of trichostatin A was 3-7 μM and the treatment time was 6-24 hours. S3. Load the pretreated human spinal cord organoids into a hydrogel to form an implantable complex, which can be used for subsequent experiments or implantation.

[0030] Methods for inducing human induced pluripotent stem cells to differentiate into human spinal cord organoids, such as... Figure 1 As shown.

[0031] Specifically, in step S2, the hSCOs from day 24 are pretreated with 5 μMTSA for 12 h and then uniformly mixed with a 5% GelMA hydrogel precursor containing 0.25% LAP photoinitiator.

[0032] In step S3, the cell-loaded hydrogel suspension was injected into a polydimethylsiloxane (PDMS) mold and crosslinked under 405 nm UV light for 30 seconds to form an hSCOs / GelMA complex. This complex was then cultured in neurobasal™-A / B27 medium for 24 hours for subsequent experiments or implantation.

[0033] TSA pretreatment significantly enhanced the survival of hSCOs in the injury microenvironment, while improving the local microenvironment and promoting nerve regeneration and functional recovery.

[0034] The following are specific examples. These examples utilize hiPSCs-derived hSCOs to establish an H2O2 oxidative damage model and, in combination with the NRF2-specific inhibitor ML385, systematically evaluate the neuroprotective effect of TSA and clarify whether it works by regulating the NRF2 pathway. In vivo validation and efficacy evaluation: TSA-pretreated hematopoietic stem cells were transplanted into a spinal cord injury animal model, and their role in promoting neurological function recovery was evaluated in vivo.

[0035] 1. Stem cell culture Human induced pluripotent stem cells (hiPSCs) are derived from foreskin fibroblasts of healthy male newborns from ATCC (Xiamen IMOCELL Biotechnology Co., Ltd.), and this cell line was used in all experiments.

[0036] Cells were cultured in pre-coated Matrigel® T25 flasks under standard conditions of 37°C and 5% CO2 in hESC / iPSC complete medium (IMOCELL, #IM-014), with the medium changed daily. When cell confluence reached approximately 80%, iPSCs were digested and passaged using iPSC passage digestion solution (IMOCELL, #IMC-014-E) and reseeded at appropriate densities into Matrigel-coated culture dishes.

[0037] To ensure the stability of the cell genome, the number of passages of hiPSCs used in all experiments was limited to no more than 6.

[0038] 2. Spinal cord organoid culture and induced differentiation Spinal cord organoids were constructed using a three-dimensional suspension culture system based on the aforementioned hiPSCs.

[0039] The steps are as follows: First, hiPSCs were digested into a single-cell suspension using a dissociation reagent (IMOCELL, #IMC-014-E) and seeded into 96-well plates with ultra-low adsorption substrate at a density of 9000 cells per well.

[0040] Subsequently, staged differentiation was carried out in N2B27 basal medium (a 1:1 mixture of DMEM / F-12 [Gibco, #11320] and Neurobasal™ medium (Gibco, #21103]), with 0.5% N2 (ThermoFisher, #17502048), 1% vitamin A-free B27 (Invitrogen, #12587010), 1M 1-glutamine (BDBiosciences, #35050061), 0.1M β-mercaptoethanol (Macklin, #35050061, #M81300) and 0.5μM ascorbic acid (MCE, #HY-B0216).

[0041] The differentiation process was divided into three stages: in the first stage (Days 0-2), 10 μM MY-27632 (MCE, #HY-10583), 20 ng / mL bFGF (Novoprotein, #C024) and 3 μM CHIR99021 (MCE, #HY-10182) were added to promote neuroectodermal specialization; in the second stage (Days-15), 10 μM MSB431542 (MCE, #HY-10431) and 0.2 μM DN193189 (MCE, #HY-12021) were added to inhibit the TGF-β / BMP signaling pathway. 100 nM retinoic acid (MCE#h-14649) and 500 nM MSAG (MCE#h-12848) were used in combination to co-induce spinal motor neuron differentiation. At the same time, 10 ng / mL BDNF (Novoprotein, #C340) and 20 ng / mL LGDNF (Novoprotein, #C338) were added in phase III (Days 15 and later) to support neuronal maturation and long-term survival.

[0042] Throughout the differentiation process, 50% of the culture medium was replaced by gravity sedimentation every 48 hours.

[0043] 3. Pretreatment On day 24 of differentiation, human spinal cord organoids were pretreated with 5 μM trichostatin A for 12 hours.

[0044] 4. Preparation of hSCOs / GelMA composite structure The hiPSCs-derived hSCOs differentiated to day 24 were uniformly mixed with a 5% GelMA hydrogel precursor (GelMA, EFL, #GM60) containing 0.25% LAP photoinitiator.

[0045] The cell-loaded hydrogel suspension was injected into a polydimethylsiloxane (PDMS) mold and crosslinked under 405 nm UV light for 30 seconds to form an hSCOs / GelMA complex. This complex was then cultured in neurobasal™-A / B27 medium for 24 hours for subsequent experiments or implantation.

[0046] The experimental results are further disclosed below to verify the protective effect of TSA against oxidative stress and its mechanism.

[0047] 1. Detection methods for oxidative stress and apoptosis-related indicators Establish experimental groups: To investigate the protective effect of TSA against oxidative stress and its mechanism, the differentiated and mature hSCOs (approximately day 30) from Example 1 were randomly divided into four groups: NC group (routine culture), model group (80 μM H2O2 treatment for 6 hours), TSA (MCE, #HY-15144) pretreatment group (5 μM H2O2 pretreatment for 12 hours, H2O2 treatment for 6 hours), and TSA+ML385 (MCE, #HY-100523) group (5 μM H2O2 + 10 μM RF2 inhibitor ML385 pretreatment for 12 hours, H2O2 treatment for 6 hours).

[0048] All treatment conditions were optimized through preliminary experiments; and cell viability was determined using the CCK-8 assay: 10 μL of CCK-8 reagent (BestBio, #BB4281) was added to each well after treatment. After incubation at 37°C for 2 h, the absorbance of 100 μL of supernatant was measured at 450 nm to assess relative cell viability.

[0049] (1) Detection of reactive oxygen species (ROS): The DCFH-DA fluorescent probe method (BestBio, #BB-46051) was used. After treatment, hSCOs were frozen sections or prepared into single-cell suspensions and incubated with 10 μM DCFH-DA probe at 37℃ in the dark for 20 min. After washing with PBS, the average fluorescence intensity was observed and photographed by fluorescence microscopy or quantitatively analyzed by flow cytometry.

[0050] (2) Determination of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content: Processed hSCOs were collected and total protein was extracted by ultrasonic disruption. After determining protein concentration using the BCA method, enzyme markers were read at 450 nm and 532 nm using the WST-1 SOD assay kit (Beyotime, #S0101) and the TBA MDA assay kit (Beyotime, #S0131), respectively, according to the instructions. SOD activity units (U / mgprot) and MDA content (nmol / mgprot) were measured.

[0051] (3) Apoptosis assay: The treated hSCOs were collected into a single-cell suspension and double-stained using the Annexin V-FITC / PI apoptosis detection kit (BestBio, #BB-4101). After staining, the cells were incubated in the dark and immediately analyzed by flow cytometry to distinguish between live cells (Annexin V- / PI-), early apoptotic cells (Annexin V+ / PI-), and late apoptotic / necrotic cells (Annexin V+ / PI+).

[0052] 2. Western Imprint Method hSCOs from each group were collected, and total protein was extracted using RIPA lysis buffer (ServiceBio, #G5002). After quantification by BCA method (ServiceBio, #G2026), an equal volume of protein (20 μg) was subjected to SDS-PAGE (Beyotime, #P0015L) electrophoresis and transferred to a PVDF membrane (Millipore, #IPVH00010). The membrane was blocked with 5% skim milk (BSA; Beyotime, #P0231) at room temperature for 1 hour, and then incubated overnight at 4°C with the corresponding primary antibody (e.g., NRF2, HO-1, β-actin, etc.). After washing with TBST, the secondary antibody was labeled with HRP (ServiceBio, #GB23303; 1:20,000 dilution) and incubated at room temperature for 1 hour. The band gray values ​​were quantitatively analyzed using ECL chemiluminescent substrate (Millipore, #WBULS0500) with β-actin as an internal control.

[0053] 3. Immunofluorescence analysis hSCOs or animal spinal cord tissue were fixed in 4% paraformaldehyde (Servicebio, #G1101), dehydrated with sucrose, and the tissue samples were embedded in a compound for optimal cutting temperature (Biosharp, #BL557A) to prepare 10 μM thick frozen sections.

[0054] Each fraction was blocked using antigen retrieval, 0.3% Triton X-100 permeabilizer, and QuickBlock™ blocking buffer (Beyotime, #P0231), and incubated overnight at 4°C with primary antibodies (such as Tuj-1, GFAP, NRF2, etc.). Subsequently, the cells were incubated for 2 hours at room temperature in the dark with the corresponding fluorescent secondary antibodies (Huabio, #HA1121 and #HA1126), and the nuclei were restained with DAPI (Beyotime, #C1005). After sealing, images were acquired using a fluorescence microscope under the same exposure parameters, and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0055] 4. Animal experiments Twenty-four female SD rats (200–250 g) were randomly divided into four groups (n=6): sham operation group, spinal cord injury model group (SCI), spinal cord organoid therapy group (hSCOs), and TSA-pretreated spinal cord organoid therapy group (TSA+hSCOs). After anesthesia with intraperitoneal injection of sodium pentobarbital (30 mg / kg), the spinal cord was exposed via T10 laminectomy, and moderate contusion was induced using an Allen's percussion instrument (5 g weight dropped from a height of 10 cm).

[0056] The sham surgery group underwent only laminectomy without spinal cord injury. Immediately postoperatively, the hCSOs and TSA+hCSOs groups received their respective implants via GelMA hydrogel (Plan 1). Postoperative care included manual bladder emptying and prophylactic penicillin G (50,000 IU / kg, IM) three times daily for 7 days. Wound integrity and systemic infection status were monitored daily. Standard food and water were provided under controlled conditions (22±1°C, 12h light-dark cycle).

[0057] The experimental animal protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Wannan Medical College, Wuhu City (No.: 15815861), and all procedures were approved by the Institutional Animal Care and Use Committee of WNMC (Approva#WNMC-AWE-2023455).

[0058] 5. Functional recovery and histological assessment Four weeks post-surgery, two observers unaware of the rat group assignments assessed the rats weekly using the following methods: BBB motor score for hindlimb motor function openness assessment (0-21 points); inclined plane test: recording the maximum tilt angle at which the animal could maintain a stable posture; and gait analysis: quantifying toe dragging rate using a plantar ink walking test. Four weeks later, after cardiac perfusion fixation, the spinal cord of the injured segment was harvested, paraffin-embedded, sectioned, and stained with H&E and Nissl to assess the lesion cavity volume and neuronal viability. Simultaneously, immunofluorescence staining analysis (e.g., Tuj-1, GFAP, CD68) was performed to observe neuronal and glial cell activation and inflammatory infiltration.

[0059] II. Data Statistics and Results Analysis: All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 10.3.1 software. Normality of the data was tested (Shapiro-Wilk test) and chi-square test (Brown-Forsythe test). Unpaired t-tests (Welch corrected) were then performed for comparisons between two groups. One-way ANOVA was performed for comparisons among multiple groups, followed by Tukey's post-hoc test. A statistically significant difference was considered to be *p < 0.05.

[0060] 1. Differentiation of human spinal cord organoids (hSCOs) This embodiment employs a staged induction protocol to differentiate human induced pluripotent stem cells (hiPSCs) into spinal cord organoids (… Figure 1-3 (This simulates the complex three-dimensional environment of the human spinal cord.)

[0061] On day 14 of differentiation, immunofluorescence analysis confirmed that the organoids had successfully established a dorsal-ventral pattern and expressed key progenitor cell markers: OLIG2 (ventral motor neuron progenitor marker), NKX6.1 (ventral neural tube pattern marker), PAX6 (dorsoventral boundary marker), and SOX2 (neural stem cell marker). Figure 4 ).

[0062] On day 24 of differentiation, the organoids further matured, with high expression of neuronal marker Tuj-1 (β-III tubulin), synaptic marker Syn, and neurofilament protein NF. Figure 5 ).

[0063] Quantitative analysis of mean fluorescence intensity confirmed the stable expression of these neuronal markers, indicating that the spinal motor neuron lineage is maturing. Figure 6 ).

[0064] Human spinal cord organoids were successfully created on the surface.

[0065] 2. The neuroprotective effect of TSA against oxidative stress injury is concentration-dependent. In order to determine the modeling conditions, Figure 7 A showed that 80 μM H₂O₂ significantly reduced cell viability without affecting the structural integrity of organoids; therefore, 80 μM H₂O₂ was chosen as the induction concentration for the oxidative stress model. Figure 7 A).

[0066] Toxicity tests showed that when the TSA concentration was greater than 10 μM, TSA exhibited significant cytotoxicity. Figure 7 B).

[0067] Further screening showed that 5 μMTSA pretreatment most effectively reversed H2O2-induced cell viability decline, while 10 μMTSA pretreatment produced the opposite effect. Figure 7 (C) This indicates that the effect of TSA has a concentration-dependent biphasic characteristic, and the concentration of TSA needs to be controlled at 5 μM-10 μM, and further, not exceeding 7 μM.

[0068] Regarding oxidative stress indicators, the H2O2 model group showed significantly increased ROS and MDA levels and decreased SOD activity. Figure 8-11Pretreatment with 5 μM TSA effectively reversed the above changes, but 10 μM TSA exacerbated oxidative stress damage, further confirming that appropriate concentrations of TSA have a protective effect on hSCOs.

[0069] In summary, TSA pretreatment at an appropriate concentration (5 μM) can effectively alleviate H2O2-induced oxidative stress damage in hSCOs, while TSA pretreatment at a higher concentration (10 μM) may aggravate H2O2-induced oxidative stress damage, indicating that TSA has a biphasic concentration effect.

[0070] 3. TSA exerts its antioxidant effect by activating the NRF2 / HO-1 pathway. Western blot analysis revealed that, compared with the NC group, the expression of NRF2 and its downstream protein HO-1 in the cell nucleus was downregulated, while the expression of the cytoplasmic repressor KEAP1 was upregulated in the H2O2 model group. Figure 12-13 ).

[0071] Further investigation revealed that 5 μMTSA pretreatment significantly promoted NRF2 nuclear translocation, upregulated HO-1, and inhibited KEAP1, while 10 μMTSA pretreatment did not show these effects, suggesting that the antioxidant effect of TSA is closely related to the activation of the NRF2 / HO-1 pathway.

[0072] It is worth noting that the NRF2 inhibitor ML385 can completely reverse the TSA-induced upregulation of the NRF2 / HO-1 axis and downregulation of the KEAP1 axis. Figure 14-15 Ultimately, it was proven that this signal axis is a key mechanism for TSA-mediated antioxidant protection.

[0073] 4. TSA inhibits oxidative stress-induced apoptosis through the NRF2 / HO-1 axis. The optimal concentration (5 μM) of TSA significantly inhibited H2O2-induced apoptosis in hSCOs cells by activating the NRF2 / HO-1 signaling pathway.

[0074] Western blotting analysis showed that H2O2-induced model group showed upregulation of pro-apoptotic proteins Bax and Cleaved Caspase-3, and downregulation of anti-apoptotic protein Bcl-2. Treatment with 5 μM TSA effectively reversed these protein expression changes, while the NRF2 inhibitor ML385 completely blocked this protective effect. Figure 16-18 ).

[0075] Annexin V / PI flow cytometry analysis further confirmed that TSA significantly reduced the H2O2-induced increase in apoptosis rate, and ML385 also eliminated this effect. Figure 18 ).

[0076] 5. TSA pretreatment with hSCOs promotes motor function recovery and tissue repair in rats with spinal cord injury. This invention establishes a rat spinal cord contusion model and divides the animals into four groups: sham operation group, spinal cord injury model group (SCI), hSCOs treatment group (hSCOs), and TSA pretreatment hSCOs group (TSA+hSCOs).

[0077] Behavioral assessment 4 weeks post-surgery showed severe impairment of hindlimb motor function in the SCI group rats. Compared with the SCI group, the BBB scores and maximum dwell angle in the inclined plane test of the hSCOs group and the TSA+hSCOs group were significantly improved from the 2nd week post-surgery. Figures 19-20 ).

[0078] Gait analysis: The paw ink application experiment showed that rats in the SCI group dragged their hind limbs significantly. Compared with the model group, the toe dragging was significantly reduced in the hSCOs group and the TSA+hSCOs group, with the TSA+hSCOs group showing slightly better improvement than the hSCOs group. Figure 21 ).

[0079] Histopathology: H&E and Nissl staining showed large cavities in the lesion center of the SCI group, residual neuronal edema, and cerebral degeneration. The lesion volume was significantly smaller in the hSCOs group and the TSA+hSCOs group, and the number of surviving neurons around the cavity was significantly greater than that in the model group. Figure 22 ).

[0080] The above results confirm that the TSA+hSCOs group is more effective in promoting the recovery of motor function and the preservation of tissue structure after spinal cord injury.

[0081] 6. TSA pretreatment of hSCOs can reduce scar formation and apoptosis of astrocytes after spinal cord injury. To further evaluate its long-term repair effect, immunofluorescence analysis was performed 4 weeks after the injury.

[0082] Immunofluorescence staining analysis of cellular responses, astrocyte activation, and neurofibrillary growth at the injury site: Compared with the Sham group, the SCI group showed significant proliferation of GFAP-positive reactive astrocytes (glial scar) in the injury area, while Tuj-1-positive neurofibrils were sparse. Treatment with hSCOs and TSA+hSCOs significantly inhibited GFAP overexpression and promoted the growth of Tuj-1-positive fibers. Figure 23 , Figure 24 However, the TSA+hSCOs group is more effective.

[0083] This indicates that, compared with hSCOs alone, TSA-pretreated hSCOs are more effective in improving the injury microenvironment, inhibiting the formation of harmful glial scars, and supporting the survival / regeneration of nerve fibers.

[0084] TUNEL staining showed a large number of apoptotic cells in the damage center of the SCI group. The TSA+hSCOs group significantly reduced the number of TUNEL-positive cells. Figure 25 , 26 This result is consistent with the in vitro experimental results, confirming that TSA-pretreated hSCOs also have significant anti-apoptotic neuroprotective effects in vivo.

[0085] 7. TSA pretreatment of spinal cord organoids promotes axonal regeneration and reduces neuroinflammation. NF200 (neurofilament protein) staining showed that, compared with the Sham group, almost no intact neurofilaments were detected in the lesion area of ​​the SCI group. In contrast, sparse but clear NF200-positive neofilaments were observed in the hSCOs group around the lesion area, and the NF200-positive neofilaments were more prominent in the lesion area of ​​the TSA+hSCOs group. Figure 27 , 28 ).

[0086] CD68 staining (labeling macrophages / microglia) showed that the SCI group had a large number of activated CD68-positive cells in the damage center, and the TSA+hSCOs treatment group significantly reduced the infiltration of CD68-positive cells. Figure 29 , Figure 30 These results indicate that TSA-pretreated spinal organoid therapy not only provides neuroprotection in the acute phase, but also creates a favorable microenvironment for nerve repair in the subacute phase by reducing neuroinflammation and promoting axonal regeneration.

[0087] III. Analysis of Experimental Results This invention systematically studies the enhancing effect of TSA pretreatment on the antioxidant stress capacity of hSCOs and its therapeutic potential in spinal cord injury repair by combining a three-dimensional human spinal cord organoid model with a rat spinal cord injury model.

[0088] The results showed that the protective effect of TSA against oxidative stress damage in hSCOs exhibited a typical concentration-dependent biphasic characteristic, with 5 μM being the optimal protective concentration. Secondly, its protective mechanism depended on the activation of the NRF2 / HO-1 antioxidant signaling pathway, thereby inhibiting apoptosis. Finally, compared with untreated hSCOs, transplantation of hSCOs pretreated with 5 μM TSA was more effective in promoting the recovery of motor function in SCI rats, reducing histopathological damage, inhibiting glial scar formation and neuroinflammation, and promoting axonal regeneration.

[0089] 1. The neuroprotective effect of TSA exhibits a biphasic concentration-dependent effect. This invention reveals for the first time the "double-edged sword" nature of TSA in neuroprotection. Pretreatment with low concentration (5 μM) TSA effectively antagonizes H2O2-induced oxidative damage and apoptosis, while pretreatment with high concentration (10 μM) exhibits toxicity and exacerbates oxidative damage.

[0090] The underlying mechanism is that appropriate concentrations of TSA promote the transcription of protective genes (such as NRF2 target genes) by moderately regulating histone acetylation levels; however, excessively high concentrations may lead to non-specific acetylation modifications, interfering with core cellular processes (such as DNA repair and mitochondrial function) or inducing autophagy / apoptosis-related pathways, which may cause cytotoxicity. This underscores the importance of determining a precise dose window when applying HDACi to the treatment of neural prostheses.

[0091] 2. The NRF2 / HO-1 pathway is the core hub for TSA to exert its antioxidant and anti-apoptotic effects. This invention powerfully demonstrates, through molecular biology experiments and intervention with a specific inhibitor (ML385), the central role of the NRF2 / HO-1 pathway in TSA-mediated neuroprotection. Under oxidative stress, KEAP1-mediated NRF2 degradation is exacerbated, leading to impaired nuclear translocation and reduced expression of antioxidant response elements (AREs)-driven protective genes (such as HO-1).

[0092] This invention proposes that 5 μMTSA can effectively reverse this process, promoting NRF2 nuclear accumulation and HO-1 upregulation while downregulating KEAP1. Activation of NRF2 can not only directly scavenge ROS and upregulate antioxidant enzymes such as SOD, but its downstream product HO-1 can also decompose toxic heme into bilirubin and carbon monoxide, which have anti-inflammatory and antioxidant effects, forming a multi-layered protective network.

[0093] Furthermore, ML385 completely blocked the anti-apoptotic effect of TSA, clarifying the causal chain of "TSA→NRF2 activation→oxidative stress relief→mitochondrial pathway apoptosis inhibition (restoring the Bcl-2 / Bax ratio and reducing Caspase-3 activation)," providing a solid molecular mechanism explanation for the neuroprotective effect of TSA.

[0094] 3. TSA pretreatment can enhance the in vivo repair effect of hSCOs. This invention successfully translates in vitro mechanism research results into animal models and demonstrates that TSA pretreatment is an effective "enhancement" strategy. Sole transplantation of hSCOs has shown some repair effects, indicating their potential to replace neurons, secrete neurotrophic factors, or serve as biological scaffolds. However, TSA-pretreated hSCOs demonstrate better overall efficacy.

[0095] This advantage stems from the two key capabilities that pretreatment endows transplanted cells with: (1) Enhanced inherent resilience: In the early stages of transplantation, cells are exposed to severe stresses such as ischemia and inflammation. TSA-pretreated hSCOs activate the NRF2 / HO-1 pathway, acting like "cellular armor" to better survive in the harsh injury microenvironment, thus enabling more cells to perform their functions. This explains the advantages of the TSA+hSCOs group in reducing apoptosis (TUNEL staining) and preserving neurons (Nissl staining).

[0096] (2) Positive microenvironmental regulation: TSA pretreatment enhanced the ability of hSCOs to secrete beneficial factors or inhibit harmful signals, possibly through epigenetic regulation. This was reflected in the more significant inhibition of reactive astrocyte proliferation (reduced GFAP) and macrophage / microglia infiltration (reduced CD68) in the TSA+hSCOs group. Inhibition of glial scar formation and neuroinflammation is crucial for creating a microenvironment that allows axonal regeneration.

[0097] This invention proposes that the regeneration of NF200-positive neurofilaments is the result of the combined action of this improved microenvironment and surviving neuronal graft cells.

[0098] In summary, this study demonstrates that appropriate TSA pretreatment can significantly enhance the resistance of human spinal cord organoids to oxidative stress and apoptosis by activating the NRF2 / HO-1 signaling pathway. Transplanting these "enhanced" organoids into spinal cord-injured rats more effectively promotes the recovery of motor function, with the mechanism involving synergistic effects such as improved transplant cell survival, inhibition of glial scar formation and neuroinflammation, and promotion of axonal regeneration. This strategy provides a new approach for optimizing stem cell or organoid transplantation for spinal cord injury treatment—namely, pre-enhancing the therapeutic resilience of transplanted cells through pharmacological pretreatment—and possesses significant translational medical potential.

[0099] The embodiments disclosed in this invention utilize human spinal cord organoids (hSCOs) derived from human pluripotent stem cells (iPSCs) to simulate the oxidative stress environment after spinal cord injury (SCI) to investigate whether TSA pretreatment can exert a protective effect by regulating the NRF2 / HO-1 pathway. Human spinal cord organoids (hSCOs) are generated from iPSCs using a stage-specific model to evaluate the effects of TSA pretreatment on in vitro anti-oxidative stress and anti-apoptosis. Specifically, in a rat T10 contusion model, sham-operated (Sham), injury (SCI), hSCOs transplantation, and TSA-pretreated hSCOs transplantation (TSA+hSCOs) groups were established, and cells were implanted using GelMA hydrogel. The Basso-Beattie-Bresnahan (BBB) ​​motor score and slant plane test were used to assess motor function recovery, and tissue repair, neuronal regeneration, and inflammation were analyzed.

[0100] The results showed that TSA treatment significantly improved organoid cell viability and reduced apoptosis and ROS accumulation. The mechanism was to promote NRF2 nuclear translocation and upregulate HO-1 expression, while the inhibitor ML385 reversed this protective effect.

[0101] This embodiment describes a spinal cord organoid preparation based on trichostatin A pretreatment and its application. TSA can enhance the antioxidant stress and anti-apoptotic capacity of spinal cord organoids by activating the NRF2 / HO-1 pathway. TSA-pretreated organoid transplantation combines the advantages of drug protection and cell regeneration, providing a new strategy and experimental basis for the treatment of spinal cord injury. By loading and solidifying the organoids with GelMA hydrogel and transplanting them to the spinal cord injury site, the survival rate of spinal cord organoids can be improved and the microenvironment of the injury site can be improved, thereby treating spinal cord injury.

[0102] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. A spinal cord organoid preparation based on a pretreatment with Trichostatin A, characterized in that, These include human spinal cord organoids derived from human induced pluripotent stem cell differentiation, trogostatin A, and GelMA hydrogel; The trogostatin A is used to pretreat the human spinal cord organoids before transplantation to activate the NRF2 / HO-1 signaling pathway.

2. The spinal cord organoid preparation based on trogostatin A pretreatment according to claim 1, characterized in that, The concentration of the quabustatin A was 3-7 μM.

3. A pharmaceutical composition for treating spinal cord injury, characterized in that, This includes the spinal cord organoid formulations as described in claim 1 or 2, as well as pharmaceutically acceptable carriers or scaffolds.

4. The use of a spinal cord organoid formulation as described in claim 1 or 2 in the preparation of a medicament for treating spinal cord injury.

5. The application according to claim 4, characterized in that, The treatment includes promoting the recovery of nerve function, reducing oxidative stress and apoptosis, inhibiting glial scar formation and neuroinflammation, and promoting axonal regeneration.

6. A method for preparing spinal cord organoids for treating spinal cord injury, characterized in that, Includes the following steps: S1. Differentiation of human spinal cord organoids from human induced pluripotent stem cells; S2. On day 24 of differentiation, human spinal cord organoids were pretreated with trichostatin A to activate the NRF2 / HO-1 signaling pathway. S3. Load the pretreated human spinal cord organoid into a hydrogel to form an implantable complex.

7. A method for preparing spinal cord organoids for treating spinal cord injury according to claim 6, characterized in that, The pretreatment includes the following conditions: the concentration of the quabustatin A is 3-7 μM, and the treatment time is 6-24 hours.

8. A method for preparing spinal cord organoids for treating spinal cord injury according to claim 6, characterized in that, The hydrogel is a GelMA hydrogel. The GelMA hydrogel contains the photoinitiator LAP; The crosslinking conditions for the GelMA hydrogel are irradiation with 405nm ultraviolet light for 20-40 seconds.

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