A spinal cord organoid culture medium and culture method with an enriched population of excitatory interneurons with thoracic spinal cord characteristics

Abstract

This invention relates to the field of organoid culture technology, specifically providing a culture medium and method for spinal cord organoids enriched with excitatory interneurons exhibiting characteristics of the thoracic segment of the spinal cord. The culture medium comprises: a composition for pluripotent stem cells differentiating into neural mesodermal progenitor cells; a composition determining the dorsal-ventral and rostral-caudate fate of the spinal cord organoids; and a composition promoting the neuralization and neuronal maintenance of the spinal cord organoids. This induction method enables the organoids to exhibit thoracic spinal cord characteristics in the early 20 days of differentiation, with prominent developmental features of spinal cord interneurons. The spinal cord organoids of this invention show mature spinal cord excitatory interneurons and glial cells detected on day 40 of induction, possessing mature neurophysiological activity, and can stably maintain activity and function during long-term in vitro culture. The organoid culture method provided by this invention is highly efficient and simple, and has potential application value in the field of organoid construction.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a spinal cord organoid culture medium and culture method that is enriched with excitatory interneurons characteristic of the thoracic segment of the spinal cord. Background Technology

[0002] Spinal cord injury (SCI) can lead to transient or permanent disability and a range of serious complications, resulting in a significant global disease burden. Severe SCI results in loss of sensory and motor function below the level of innervation. Current clinical treatment strategies seek to neuroprotect early injury, but primary and secondary injury factors often lead to irreversible local neuronal necrosis, followed by failure of neural circuit reconstruction due to local axonal regeneration inhibitory substances, immune environment imbalance, and fibrous scarring. Therefore, given the limited effectiveness of conventional treatments, finding effective spinal cord regeneration or replacement therapies is crucial. Cell therapy is considered highly promising.

[0003] In recent decades, numerous clinical cell infusion therapies utilizing neural stem / progenitor cells and mesenchymal stem cells have been reported to help improve patients' neuromotor status. However, the results are highly variable, and there has been no evidence that stem / progenitor cells regulate neural circuit plasticity or help restore higher pathway function, possibly due to limited cell survival, differentiation failure, and immune rejection of xenogeneic cells. Although neural stem cells have demonstrated good graft and host integration capabilities and neuronal differentiation potential, their efficacy has not been proven in large-scale clinical trials.

[0004] Spinal cord organoids (SCOs) based on human pluripotent stem cells have recently emerged as an emerging approach to cell therapy for spinal cord injury. Due to their unique hybrid cell therapy comprising well-defined populations of neurons, glial cell subsets, and neural progenitor cells, they hold promise for advanced functional reconstruction. Furthermore, the cell source of SCOs—human induced pluripotent stem cells (hiPSCs)—avoids ethical concerns and may become a preferred therapy in regenerative medicine. Existing results demonstrate that SCOs exhibit neuronal survival and migration in animal models of complete spinal cord injury, promoting partial motor recovery. However, this research is still in its early exploratory stages, and large-scale clinical / preclinical trials have not yet confirmed the definitive efficacy of this therapy.

[0005] Currently, there is no unified standard for induction protocols for spinal cord organoids, and issues such as excessively long induction cycles and the need to improve neuronal differentiation efficiency remain. Furthermore, the functions of specific neuronal subtypes in spinal cord injury repair are not yet fully understood. More importantly, homologous graft generation protocols for different spinal cord segments (cervical / thoracic / lumbar) have not yet been established. Some studies have indicated that functional regeneration of the corticospinal tract (CST) requires cell grafts with posterior neural tube characteristics, rather than cephalic features, highlighting the importance of homologous graft therapy strategies for spinal cord injuries.

[0006] Spinal cord injury treatment focuses on the reconstruction of motor function, and the transplantation of active motor neurons has been explored, but its efficacy appears to be limited. Excitatory interneurons are components of the central pattern generator that controls respiration and movement, playing a crucial role in the recovery of spinal cord function. Furthermore, V2a interneurons generated from human pluripotent stem cells have been shown to survive in the cervical spinal cord of mice and form synapses with host spinal cord neurons, helping to restore the rhythmic movement of respiratory muscles.

[0007] Therefore, the purpose of this invention is to develop a functional spinal cord organoid culture system that matches the identity of the thoracic segment of the spinal cord and is enriched with interneurons. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a spinal cord organoid culture medium and method, which constructs a spinal cord organoid with the characteristics of thoracic and spinal cord excitatory interneurons. This can simulate the development process and cellular composition of spinal cord organoids enriched with thoracic excitatory interneurons, providing an in vitro research platform for the pathogenesis of human spinal cord-related diseases and a research tool for the prevention and treatment of these diseases.

[0009] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a spinal cord organoid culture medium enriched with excitatory interneurons characteristic of the thoracic segment of the spinal cord, the culture medium comprising: Compositions for differentiating pluripotent stem cells into neural mesodermal progenitor cells, comprising a TGF-β pathway inhibitor and a WNT pathway activator; compositions for determining the dorsoventral and rostral fate of spinal cord organoids, comprising an SHH activator; Notch signaling inhibitors for promoting specific differentiation of interneurons; and compositions for promoting neuralization and neuronal maintenance of spinal cord organoids, comprising epidermal growth factor (EGF), glial cell-derived neurotrophic factor (GDNF), and L... Ascorbic acid (AA), brain-derived neurotrophic factor (BDNF), and nerve growth factor-3.

[0010] Preferably, the culture medium further includes: Culture medium 1 containing TGF-β pathway inhibitor SB431542 and WNT pathway activator CHIR99021; 3D embryoid culture medium containing Blebbistatin 2; Contains epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), and L... Culture medium containing ascorbic acid AA, SHH activator SAG / PUR and Notch signaling inhibitor DAPT; Contains brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor-3, and L. Culture medium for ascorbic acid AA 4.

[0011] Preferably, the culture medium 1 further includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol.

[0012] Preferably, the culture medium 2 further includes Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol medium.

[0013] Preferably, the culture medium 3 further includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol.

[0014] Preferably, the culture medium 4 further includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol.

[0015] Preferably, the concentration of GlutaMAX in the culture media 1, 3, and 4 is 0.8%-1.2%; for example, it can be 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or 1.2%, etc., preferably 1%.

[0016] Preferably, the concentration of non-essential amino acid NEAA in culture media 1, 3, and 4 is 0.8%-1.2%; for example, it can be 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or 1.2%, preferably 1%.

[0017] Preferably, the concentration of dimercaptoethanol in culture media 1, 3, and 4 is 0.05-0.15 mM; for example, it can be 0.05 mM, 0.08 mM, 0.1 mM, 0.12 mM, 0.13 mM, or 0.15 mM, etc., preferably 0.1 mM.

[0018] In the culture media 1, 3, and 4, the concentration of N2 cell culture additive is 1.5%-2.5%; for example, it can be 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%, preferably 2%.

[0019] Preferably, the concentration of B27 cell culture additive in culture media 1, 3, and 4 is 0.8%-1.2%; for example, it can be 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or 1.2%, etc., preferably 1%.

[0020] Preferably, the concentration of SB431542 in the culture medium 1 is 5-15 μM, for example, it can be 5 μM, 6 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM or 15 μM, etc.; preferably 10 μM.

[0021] Preferably, the concentration of CHIR99021 in the culture medium 1 is 2-5 μM, for example, it can be 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM or 5 μM, etc.; preferably 3 μM.

[0022] Preferably, the concentration of Blebbistatin in the culture medium 2 is 15-25 ng / ml, for example, it can be 15 ng / ml, 18 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml, etc.; preferably 10 ng / ml.

[0023] Preferably, the concentration of epidermal growth factor (EGF) in the culture medium 3 is 15-25 ng / ml, for example, it can be 15 ng / ml, 18 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml, etc.; preferably 20 ng / ml.

[0024] Preferably, the concentration of brain-derived neurotrophic factor (BDNF) in the culture medium 3 is 15-25 ng / ml, for example, it can be 15 ng / ml, 18 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml, etc.; preferably 20 ng / ml.

[0025] Preferably, in the culture medium 3, L The concentration of ascorbic acid AA is 150-250 nM, for example, it can be 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM or 250 nM, etc.; preferably 200 nM.

[0026] Preferably, the concentration of SHH activator SAG in the culture medium 3 is 40-60 μM, for example, it can be 40 nM, 42 nM, 45 nM, 48 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM or 60 nM, etc.; preferably 50 μM.

[0027] Preferably, the concentration of SHH activator PUR in the culture medium 3 is 0.08-0.12 μM, for example, it can be 0.08 μM, 0.085 μM, 0.09 μM, 0.1 μM, 0.105 μM, 0.11 μM, 0.115 μM or 0.12 μM, etc.; preferably 0.1 μM.

[0028] Preferably, the concentration of the Notch signaling inhibitor DAPT in the culture medium 3 is 0.8-1.2 μM, for example, it can be 0.8 μM, 0.85 μM, 0.9 μM, 1 μM, 1.05 μM, 1.1 μM, 1.15 μM or 1.2 μM, etc.; preferably 1 μM.

[0029] Preferably, the concentration of brain-derived neurotrophic factor (BDNF) in the culture medium 4 is 10-30 ng / ml, for example, it can be 10 ng / ml, 12 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 18 ng / ml, 20 ng / ml, 22 ng / ml, 25 ng / ml, 26 ng / ml, 28 ng / ml or 30 ng / ml, etc.; preferably 20 ng / ml.

[0030] Preferably, the concentration of glial cell-derived neurotrophic factor (GDNF) in the culture medium 4 is 10-30 ng / ml, for example, it can be 10 ng / ml, 12 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 18 ng / ml, 20 ng / ml, 22 ng / ml, 25 ng / ml, 26 ng / ml, 28 ng / ml or 30 ng / ml, etc.; preferably 20 ng / ml.

[0031] Preferably, the concentration of nerve growth factor-3 in the culture medium 4 is 10-30 ng / ml, for example, it can be 10 ng / ml, 12 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 18 ng / ml, 20 ng / ml, 22 ng / ml, 25 ng / ml, 26 ng / ml, 28 ng / ml or 30 ng / ml, etc.; preferably 20 ng / ml.

[0032] Preferably, in the culture medium 4, L The concentration of ascorbic acid AA is 150-250 nM, for example, it can be 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM or 250 nM, etc.; preferably 200 nM.

[0033] In a second aspect, the present invention provides a method for culturing spinal cord organoids using the spinal cord organoid culture medium as described in the first aspect, the method comprising the following steps: (1) Culture human pluripotent stem cells to 80%-90% confluence; (2) The human pluripotent stem cells obtained in step (1) are cultured and induced to differentiate in culture medium 1 of the spinal cord organoid culture medium to form early embryoids; (3) The early embryoids obtained in step (2) are cultured in culture medium 2, culture medium 3 and culture medium 4 in sequence to induce differentiation and complete the spinal cord organoid culture.

[0034] Preferably, the sequential culturing and induction of differentiation specifically involves: The culture time in the culture medium 1 is 3-5 days, preferably 4 days; The culture time in the culture medium 2 is 0.5-1.5 days, preferably 1 day; The culture time in the culture medium 3 is 10-20 days, for example, it can be 10 days, 12 days, 14 days, 15 days, 16 days, 18 days or 20 days, preferably 15 days; The culture time in the culture medium 4 is 20-300 days, for example, it can be 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 120 days, 150 days, 180 days, 200 days, 220 days, 250 days, 280 days or 300 days, preferably 40 days; the organoids obtained by culture in the culture medium of the present invention can reach functional maturity on the 40th day, and can be maintained in vitro for up to 300 days to maintain function.

[0035] As a preferred technical solution of the present invention, the method for culturing spinal cord organoids with characteristics of thoracic spinal cord segments and excitatory interneurons specifically includes the following steps: 1. First, perform 2D induction culture, the specific steps of which are as follows: Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) were cultured in ncTarget hPSC medium in Matrigel-coated 6-well plates to 80% confluence, then separated with EDTA digestion solution, and then subcultured in ncTarget hPSC medium containing the non-muscle myosin IIA inhibitor Blebbistatin in Matrigel-coated 6-well plates. When the cell confluence reaches 80%, the 2D cells are cultured in medium 1 to induce differentiation.

[0036] 2. Next, the early embryoid body is formed, and the specific steps are as follows: After 2D-induced embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) were separated into single cells using TrypLE digestion solution, 9000 cells per well were resuspended in a low-adhesion 96-well U-shaped culture plate, and cultured in medium 2 for 1 day to form early embryoids.

[0037] 3. Next, the embryoids were cultured and induced to differentiate sequentially in culture medium 3-4, as follows: The obtained embryos were suspended in culture medium 3 and cultured for 15 days; then transferred to culture medium 4 and cultured for 20-300 days.

[0038] Thirdly, the present invention also provides the application of the spinal cord organoid culture medium as described in the first aspect in the preparation of spinal cord organoids.

[0039] The above technical solution has the following advantages or beneficial effects: (1) The present invention provides a method and culture medium for culturing spinal cord organoids, which can effectively promote the efficient differentiation of pluripotent stem cells under three-dimensional culture conditions.

[0040] (2) Compared with the prior art, this induction scheme enables the organoids to exhibit obvious spinal cord thoracic segment characteristics and spinal cord interneuron development characteristics (IN) in the early differentiation stage (20 days). The spinal cord organoids of this invention showed mature spinal cord glutamatergic V2a interneurons and glial cells on the 40th day of induction, with mature neurophysiological activity, and can stably maintain activity and function in long-term in vitro culture.

[0041] (3) The organoid culture method provided by the present invention is simple and efficient, and has potential application value in the field of organoid construction. Attached Figure Description

[0042] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0043] Figure 1 This is a flowchart illustrating the construction of spinal cord organoids in an embodiment of the present invention.

[0044] Figure 2 This is a schematic diagram of the RT-PCR verification results of characteristic markers of the rostral end of the spinal cord in the early stage of spinal cord organoid differentiation 20 days in an embodiment of the present invention; wherein, scheme #1: SB431542 (10 μM) + CHIR99021 (1.5 μM), scheme #2: SB431542 (10 μM) + CHIR99021 (3 μM).

[0045] Figure 3 This is a schematic diagram of the RT-PCR verification results of markers for the p2 ventral domain (NKX6-1), V2a interneuron (CHX10), V2b interneuron (GATA3), and motor neuron (ISL-1) on day 20 of spinal cord organoid differentiation in this embodiment of the invention; wherein, scheme #1: SB431542 (10 μM) + CHIR99021 (1.5 μM), scheme #2: SB431542 (10 μM) + CHIR99021 (3 μM).

[0046] Figure 4 VSX2 on day 20 of the spinal cord organoid in this embodiment of the invention. + ISL-1 + Statistics on the proportion of positive cells stained by immunofluorescence.

[0047] Figure 5 This is day 12 of spinal cord organoid differentiation in this embodiment of the invention, targeting neural mesodermal progenitor cells (SOX2). + / T + ) and neural tube epithelial morphology (ZO-1) + / SOX2 +Immunofluorescence staining images.

[0048] Figure 6 These are immunofluorescence staining images of V2a interneurons (VSX2) and motor neurons (ISL-1) on day 20 of spinal cord organoid differentiation in this embodiment of the invention. The bottom images represent magnified portions of the dashed boxes in the top images.

[0049] Figure 7 Immunofluorescence staining images of glutamatergic V2a interneurons (VSX2 / vGLUT2) and glial cells (S100β) on day 40 of spinal cord organoid differentiation in this embodiment of the invention.

[0050] Figure 8 This is a screenshot of a calcium ion imaging video taken on day 40 of spinal cord organoid differentiation in an embodiment of the present invention.

[0051] Figure 9 This diagram shows the neural electrical activity patterns on days 30, 40, and 50 of spinal cord organoid differentiation in this embodiment of the invention. The diagram illustrates a representative thermogram (top) of electrical activity at each time point and the electrical oscillation trajectory of a single electrode (middle), where red dots indicate more than 5 spike events within 100 ms, and a screenshot of a calcium ion imaging video of the electrical signal spike (bottom).

[0052] Figure 10 The figures illustrate the changes in neural activity patterns and average discharge frequency before and after drug treatment on day 40 of spinal cord organoid differentiation in this embodiment of the invention. Figure a shows the neural discharge frequency; figure b shows the number of spikes in each electrical activity burst; figure c shows the average burst rate of organoid electrical activity; and figure d shows the synchronization rate between electrodes. Changes in SCO discharge frequency were observed after treatment with the following drugs: 6 μM TTX (Figure e), 100 μM CNQX (Figure f), 100 μM AP-5 (Figure g), 10 μM baclofen (Figure h), and 100 μM baclofen (Figure i). Detailed Implementation

[0053] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product manual.

[0054] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. Specifically, the human pluripotent stem cell line involved in this invention, "Pluripotent Stem Cell-9 (ESC9; DC-02)" and its accompanying culture medium, were purchased from Zhong Sheng Su Yuan Biotechnology Co., Ltd. (Anhui, China).

[0055] Example 1: Construction of spinal cord organoids This embodiment provides a method for culturing spinal cord organoids with characteristics of the thoracic spinal cord and excitatory interneurons. The specific process for constructing spinal cord organoids is as follows: Figure 1 As shown, it includes the following steps: 1. Perform 2D induction culture 1.1 Pluripotent stem cells-9 (ESC9; DC-02) were cultured in ncTarget hPSC medium in Matrigel-coated 6-well plates to 80% confluence, then separated with EDTA digestion solution, and subcultured in ncTarget hPSC medium containing 2.5 μM Blebbistatin in Matrigel-coated 6-well plates.

[0056] 1.2 When the cell confluence reaches 80%, 2D cells are cultured in medium 1 to induce differentiation, initially counted as day 0. The medium consists of: Neurobasal medium, 2% N2 cell culture additive, 1% B27 cell culture additive, 1% non-essential amino acid NEAA, 1% GlutaMAX, 0.1 mM dimercaptoethanol, 10 μM SB431542, and 3 μM CHIR99021.

[0057] 2. Formation of early embryoids On day 4, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) induced by 2D induction were separated into single cells using TrypLE digestion solution and resuspended at 9000 cells per well in a low-adhesion 96-well U-shaped culture plate. The cells were cultured in medium 2 for 1 day, which consisted of Neurobasal medium, 2% N2 cell culture additive, 1% B27 cell culture additive, 1% non-essential amino acid NEAA, 1% GlutaMAX, 0.1 mM dimercaptoethanol, and 10 μM Blebbistatin to form early embryoids.

[0058] 3. Embryoids are cultured sequentially in medium for 3-4 hours to induce differentiation. 3.1 On day 5, the culture medium was changed to medium 3 and cultured for 15 days. The medium was changed every other day. The composition of the medium was: Neurobasal medium, 2% N2 cell culture additive, 1% B27 cell culture additive, 1% non-essential amino acid NEAA, 1% GlutaMAX, 0.1 mM dimercaptoethanol, 20 ng / ml EGF, 20 ng / ml BDNF, 200 nM AA, 50 μM MSAG, 0.1 μM PUR, and 1 μM DAPT.

[0059] 3.2 On day 20, the culture medium was changed to medium 4, specifically: Neurobasal medium, 2% N2 cell culture additive, 1% B27 cell culture additive, 1% non-essential amino acid NEAA, 1% GlutaMAX, 0.1 mM dimercaptoethanol, 20 ng / ml BDNF, 20 ng / ml GDNF, 20 ng / ml NT-3, and 200 nM AA, to maintain organoid function. The culture time was 20-300 days.

[0060] The specific components used in the culture are shown in Table 1 below: Table 1

[0061] Example 2: RT-PCR Validation of Characteristic Markers of the Coracocaudal End of the Spinal Cord in Early Differentiation In this embodiment, step 1.2 of Example 1 is initially counted as day 0. Organoid RNA samples were collected on day 20 from the start of construction and subjected to reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from the samples according to the instructions of the RNeasy® Plus Micro Kit RNA extraction kit. The specific procedures are as follows: After sampling at different time points, samples were transferred to EP tubes, 350 μl of RLT Plus buffer was added for lysis, and the samples were thoroughly mixed using a shaker.

[0062] Transfer the lysate to a gDNA Eliminator column placed in a 2 ml collection tube, centrifuge at 12000 rpm / min for 30 s, discard the column, and retain the filtrate.

[0063] Add 350 μl of 70% ethanol to the filtrate and mix thoroughly.

[0064] Transfer the lysis buffer to an RNeasy MinElute column placed in a 2 ml collection tube. Centrifuge at 12000 rpm / min for 15 s and discard the filtrate.

[0065] Add 700 μl of buffer RW1 to the RNeasy MinElute column, centrifuge at 12000 rpm / min for 15 s, and discard the filtrate.

[0066] Add 500 μl of RPE buffer to the RNeasy MinElute column, centrifuge at 12000 rpm / min for 15 s, and discard the filtrate.

[0067] Add 500 μl of 80% ethanol to the RNeasy MinElute column, centrifuge at 12000 rpm / min for 2 min, and discard the 2 ml collection tube and filtrate.

[0068] Transfer the membrane to a new 2 ml collection tube on the RNeasy MinElute column, centrifuge at 12000 rpm / min for 5 min to dry the membrane, and discard the 2 ml collection tube and filtrate.

[0069] Transfer the RNeasy MinElute column to a new 1.5 ml collection tube, add 14 μl of RNase-free water directly to the membrane, and centrifuge at 12000 rpm / min for 1 min to elute RNA; determine the RNA concentration using a micro-ultraviolet spectrophotometer.

[0070] Reverse transcription was performed using the HiScript® III All-in-one RT SuperMix Reverse Transcription Kit, specifically as follows: Prepare a reaction mixture of 800 ng of RNA and 20 μl of total volume according to the RT-PCR reverse transcription kit instructions. Denature the RNA in a 50°C metal bath for 15 min. Then extend the reaction at 85°C for 5 s to obtain the cDNA product.

[0071] The cDNA obtained by reverse transcription was amplified according to the GenStar qPCR Mix kit instructions. The amplification reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, and repeated for 40 cycles.

[0072] The expression level of the target gene was normalized using the same GAPDH internal reference gene in the same sample, and 2 The relative expression levels of each gene were calculated using the ΔCt method.

[0073] Specifically, the study detected rostral developmental markers HOXA2, HOXB4, HOXC5, HOXC6, HOXA7, HOXB7, and HOXC9; spinal cord functional domain precursor marker NKX6-1; and spinal cord functional domain maturation markers CHX10, GATA3, and ISL1.

[0074] The primers used (SEQ ID NO.1-24) are listed in Table 2: Table 2

[0075] The above polymerase chain reaction (RT-PCR) was used to obtain Figure 2 , Figure 3 The results in.

[0076] in, Figure 2 The results showed that early thoracic spinal cord features induced by regimen #2 SB431542 (10 μM) + CHIR99021 (3 μM) were obvious. Figure 3 The results showed that the CHX10 expression levels in both groups were significantly higher than those in ISL-1 and GATA3 (by approximately 900 and 140 times, respectively).

[0077] Example 3: Results of immunofluorescence staining for characteristic biomarkers Step 1.2 of Example 1 is initially counted as day 0. Organoid samples from day 12, day 20, and day 40 of the #2 induction protocol are collected for immunofluorescence staining. The specific operation is as follows: Organoid samples were fixed overnight in 4% PFA solution, dehydrated in 30% sucrose solution, and then embedded in OCT frozen sections; the samples were sectioned to a thickness of 16 μm using a cryostat. Use 10% goat serum and 0.1% Triton Block with X-PBS solution at room temperature for 2 hours, then dilute the primary antibody in blocking buffer and incubate overnight at 4°C; dilute the secondary antibody with blocking buffer, incubate at room temperature for 2 hours, wash, and stain the nucleus with DAPI. Specifically, detect the neural progenitor marker SOX2; the neurotransmitter marker VGLUT2; and the glial marker S100β.

[0078] The above immunofluorescence staining was used to obtain... Figures 4-7 The results in.

[0079] in, Figure 4 Displaying spinal cord organoids on day 20, VSX2. + The proportion of positive cells was significantly higher; Figure 5 SOX2 and ZO1 staining revealed neural progenitor cells and neuroepithelial markers on day 12 of organoid induction.

[0080] Figure 6 Staining of ISL1 and VSX2 showed extensive distribution of CHX10+ interneurons (~53.73%) in both groups on day 20 of organoid induction, while the number of motor neurons (approximately 4.93%) was relatively small, indicating specific differentiation of V2a interneurons.

[0081] Figure 7 The interneurons expressing vGLUT2 and VSX2 confirmed the glutamatergic interneuron subtype, and S100β demonstrated the presence of glial cell populations in the periphery and core region of the sphere.

[0082] Example 4: Calcium Imaging Experiment of Organoid Samples Step 1.2 of Example 1 is initially counted as day 0. Organoid samples collected 40 days after construction are used for calcium imaging. The specific operation is as follows: Fluo-4 was diluted to a 10 μM working solution in medium 7 and incubated at 37°C and 5% CO2 for 30 min, followed by washing with DPBS for 15 min. Spontaneous calcium activity in organoids was recorded using the Harmony high-content imaging system (one frame every 1–2 s).

[0083] The calcium imaging obtained above Figure 8 The results showed that spinal organoid neurons exhibited active calcium transient activity as early as day 40.

[0084] Example 5: Experiment on Neural Electrical Activity of Organoid Samples Step 1.2 of Example 1 is initially counted as day 0. Organoid samples are collected on days 30, 40, and 50 after construction for multi-channel electrophysiological recording. The specific operation is as follows: Each well of the six-well MEA plate (Axion Biosystems) contains 64 low-impedance electrodes. Organoids are placed into the MEA wells to cover as many of the 64 microelectrodes as possible.

[0085] Eight minutes of spontaneous electrical activity recording of organoid neurons was performed using the Maestro pro MEA system and AxIS software (Axion Integrated Studio Navigator 1.5, Axion Biosystems). The above multi-channel electrophysiological recordings yielded the following results. Figure 9 The results showed that spinal organoid neurons exhibited active electrophysiological activity as early as day 40, demonstrating physiological maturity.

[0086] like Figure 10 As shown in Figures a to d, the application of the voltage-sensitive sodium channel blocker tetrodotoxin (TTX) significantly inhibited the electrical activity of SCO, confirming that the electrical signal is a result of neuronal action potentials. Figure 10 Treatment with AMPA receptor antagonists TTX (Figure e), CNQX (Figure f), and NMDA receptor antagonist AP-5 (Figure g) significantly reduced the neural firing rate, confirming the presence of excitatory synaptic transmission in SCO.

[0087] Treatment with the GABA receptor antagonist bicuculline (Figure h) or the GABA receptor agonist baclofen (Figure i) had no significant effect on the discharge frequency of SCO, confirming that inhibitory synaptic signaling is absent or present in SCO.

[0088] It should be noted that this invention induces stem cells into spinal cord organoids with excitatory interneurons enriched, exhibiting characteristics of the thoracic spinal cord, by using different culture media at different stages and different combinations of cytokines and small molecule compounds at each stage of differentiation induction. This invention employs specific component formulations, which produce synergistic effects, inducing stem cells into spinal cord organoids with motor activity.

[0089] The present invention also conducted a component comparison experiment. In Comparative Example 1, SB431542 (TGF-β pathway inhibitor) in culture medium 1 was replaced with LDN193189 (BMP signaling inhibitor).

[0090] In Comparative Example 2, culture medium 4 did not contain glial cell-derived neurotrophic factor GDNF, and the content of BDNF was increased to 40 ng / ml.

[0091] In Comparative Example 3, culture medium 4 did not contain nerve growth factor-3, and the GDNF content was increased to 40 ng / ml.

[0092] In Comparative Example 4, culture medium 3 did not contain the SHH activator PUR, and the SAG content was increased to 40 ng / ml.

[0093] The specific formulations of the culture media used in each comparative example will not be described in detail here.

[0094] In the comparative culture medium, the combination used in this invention was not followed, and as a result, the stem cells were not successfully induced into spinal cord organoids with an abundance of excitatory interneurons characteristic of the thoracic spinal cord.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for spinal cord organoids enriched with excitatory interneurons characteristic of the thoracic segment of the spinal cord, characterized in that, The spinal cord organoid culture medium comprises: A composition for the differentiation of pluripotent stem cells into neural mesodermal progenitor cells, comprising a TGF-β pathway inhibitor and a WNT pathway activator; A composition that determines the fate of the dorsal and ventral sides and the rostellate tail of spinal cord organoids, comprising SHH activator SAG and SHH activator PUR; And, compositions that promote spinal cord organoid neuronization and neuronal maintenance, comprising Notch signaling inhibitors, epidermal growth factor (EGF), glial cell-derived neurotrophic factor (GDNF), and L... Ascorbic acid (AA), brain-derived neurotrophic factor (BDNF), and nerve growth factor-3.

2. The culture medium as described in claim 1, characterized in that, The spinal cord organoid culture medium includes culture medium 1, culture medium 2, culture medium 3 and culture medium 4; The culture medium 1 includes: TGF-β pathway inhibitor SB431542 and WNT pathway activator CHIR99021; The culture medium 2 includes: Blebbistatin; The culture medium 3 includes: epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), and L... Ascorbic acid AA, SHH activator SAG, SHH activator PUR, and Notch signaling inhibitor DAPT; The culture medium 4 includes: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor-3, and L... Ascorbic acid AA.

3. The culture medium as described in claim 2, characterized in that, The culture medium 1 also includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol; The culture medium 2 also includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acids NEAA, GlutaMAX, and dimercaptoethanol; The culture medium 3 also includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol; The culture medium 4 also includes: Neurobasal basal medium, N2 cell culture additive, B27 cell culture additive, non-essential amino acid NEAA, GlutaMAX, and dimercaptoethanol.

4. The culture medium as described in claim 3, characterized in that, The concentration of GlutaMAX in culture media 1, 3 and 4 is 0.8%-1.2%.

5. The culture medium as described in claim 3, characterized in that, The concentration of non-essential amino acids (NEAA) in culture media 1, 3, and 4 is 0.8%-1.2%. In culture media 1, 3 and 4, the concentration of dimercaptoethanol is 0.05-0.15 mM; In culture media 1, 3, and 4, the concentration of N2 cell culture additive is 1.5%-2.5%. The concentration of B27 cell culture additive in culture media 1, 3 and 4 is 0.8%-1.2%.

6. The culture medium as described in claim 3, characterized in that, In culture medium 1, the concentration of SB431542 is 5-15 μM; In the culture medium 1, the concentration of CHIR99021 was 2-5 μM.

7. The culture medium as described in claim 3, characterized in that, In the culture medium 3, the concentration of epidermal growth factor (EGF) is 15-25 ng / ml; In the culture medium 3, the concentration of brain-derived neurotrophic factor BDNF is 15-25 ng / ml; In the culture medium 3, L The concentration of ascorbic acid AA is 150-250 nM; In the culture medium 3, the concentration of SHH activator SAG is 40-60 μM; In culture medium 3, the concentration of SHH activator PUR is 0.08-0.12 μM; In culture medium 3, the concentration of the Notch signaling inhibitor DAPT was 0.8-1.2 μM; In the culture medium 4, the concentration of brain-derived neurotrophic factor BDNF is 10-30 ng / ml; In the culture medium 4, the concentration of glial cell-derived neurotrophic factor GDNF is 10-30 ng / ml; In the culture medium 4, the concentration of nerve growth factor-3 is 10-30 ng / ml; In the culture medium 4, L The concentration of ascorbic acid AA is 150-250 nM.

8. A method for culturing spinal cord organoids using the spinal cord organoid culture medium as described in any one of claims 1-7, characterized in that, The method includes the following steps: (1) Culture human pluripotent stem cells to 80%-90% confluence; (2) The human pluripotent stem cells obtained in step (1) are cultured and induced to differentiate in culture medium 1 of the spinal cord organoid culture medium to form early embryoids; (3) The early embryoids obtained in step (2) are cultured in culture medium 2, culture medium 3 and culture medium 4 in sequence to induce differentiation and complete the spinal cord organoid culture.

9. The method according to claim 8, characterized in that, The specific timeframe for the culture-induced differentiation is as follows: The culture time in culture medium 1 is 3-5 days; The culture time in culture medium 2 is 0.5-1.5 days; The culture time in culture medium 3 is 10-20 days; The culture time in the culture medium 4 is 20-300 days.

10. The use of the spinal cord organoid culture medium as described in any one of claims 1-7 in the preparation of spinal cord organoids.

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