Neural stem cell induced differentiation and clinical application method for refractory epilepsy
By differentiating patient-derived pluripotent stem cells into GABAergic inhibitory interneurons and transplanting them to the epileptic lesion area, combined with closed-loop intervention, the effectiveness and safety issues of refractory epilepsy treatment were resolved, significantly reducing seizure frequency and improving quality of life.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-15
- Publication Date
- 2026-04-07
AI Technical Summary
In the current technology, drug treatment for patients with refractory epilepsy is not effective, and surgical treatment is risky and difficult to implement, which makes it difficult to control epileptic seizures, seriously affects the quality of life and may cause complications.
The patient's own induced pluripotent stem cells were differentiated into GABAergic inhibitory interneurons, which were then transplanted to the epileptic lesion area under the guidance of stereotactic electroencephalography. This was combined with antiepileptic drugs and neurorehabilitation training for closed-loop intervention to achieve the reconstruction and inhibitory balance of the neural network.
It significantly reduces the frequency of seizures in refractory epilepsy, improves the safety and effectiveness of treatment, reduces drug side effects, improves the recovery of neurological function, and dynamically adjusts the treatment plan to ensure stable results.
Smart Images
Figure CN121801835A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy. Background Technology
[0002] Epilepsy is a common chronic neurological disorder affecting hundreds of millions of people worldwide. Among these, refractory epilepsy accounts for approximately 20%-30% of all epilepsy cases. Despite receiving regular treatment with at least two appropriate antiepileptic drugs, patients with refractory epilepsy often struggle to effectively control their seizures, severely impacting their quality of life and placing a heavy burden on themselves and their families. Long-term, frequent seizures can also lead to complications such as cognitive impairment and behavioral abnormalities, further reducing a patient's ability to live independently and their social adaptability.
[0003] Currently, drug therapy remains the primary treatment for epilepsy. However, for patients with refractory epilepsy, due to the complex pathogenesis, drugs often fail to precisely target the pathogenic sites, leading to poor efficacy. Long-term use of antiepileptic drugs can also induce drug resistance, causing previously effective drugs to gradually lose their ability to control seizures. Furthermore, the side effects of these drugs can cause additional harm to the patient's health. For some patients with refractory epilepsy who do not respond to drug therapy, surgical treatment is necessary. The purpose of surgery is primarily to remove or destroy the epileptic focus to reduce or stop seizures. However, not all patients are suitable for surgery. On the one hand, in some patients, the epileptic focus is located in important functional areas of the brain, and surgical removal may lead to severe neurological dysfunction, such as impairment of language or motor function. On the other hand, in some patients, the epileptic focus is difficult to locate precisely, making surgical treatment difficult to implement. In addition, some patients may still experience epilepsy recurrence after surgery. Summary of the Invention
[0004] The purpose of this invention is to provide a method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy, thereby solving the technical problems existing in the prior art.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:
[0006] A method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy, comprising the following steps: S1: Cell source and preparation, using induced pluripotent stem cells (iPSCs) derived from the patient, obtained by reprogramming skin fibroblasts and other somatic cells; placing the iPSCs in a matrix gel-coated culture dish and preparing them in a culture medium containing basic fibroblast growth factor; S2: Constructing an induction differentiation system, differentiating the iPSCs into GABAergic inhibitory interneurons, and further differentiating the GABAergic inhibitory interneurons; S3: Cell purification and expansion, using immunomagnetic beads to purify and enrich cells expressing GABAergic neuron markers; S4: Cell quality control, performing quality control on the purified cells, including cell shape and purity, cell viability and quantity, functional testing, tumorigenicity testing, and sterility and microbial testing.
[0007] Furthermore, in step S1, the reprogramming process involves collecting a small number of skin fibroblasts from patients with refractory epilepsy and inducing them into induced pluripotent stem cells in the laboratory using reprogramming factors Oct4, Sox2, Klf4, and c-Myc. During the reprogramming process, viral vector transduction or non-viral vector transduction methods can be used to introduce the above transcription factors into the fibroblasts. The transduced cells are then cultured in a stem cell culture medium containing basic fibroblast growth factor to form induced pluripotent stem cell clones.
[0008] Furthermore, the culture medium in the culture dish in step S1 is: DMEM / F12 basal medium, supplemented with N2 and B27 nutrient supplements, basic fibroblast growth factor, and penicillin / streptomycin dual antibiotics.
[0009] Further, step S2 includes the following steps: S2.1: Neural progenitor cell induction, induced pluripotent stem cells are digested from the culture dish and seeded at a preset density on a culture plate pre-coated with matrix gel, and cultured in an induction medium containing recombinant human activin A and recombinant human basic fibroblast growth factor; S2.2: Neuron induction and differentiation, neurospheres are collected and further expanded in a neural stem cell culture medium containing epidermal growth factor and basic fibroblast growth factor. After culturing for 7-9 days, retinoic acid is added to induce neural stem cells to differentiate into neurons; S2.3: Directed differentiation of GABAergic interneurons, based on S2.2, growth factors and small molecule compounds related to inhibitory neuron differentiation are added to promote the directed differentiation of cells into GABAergic interneurons.
[0010] Furthermore, in step S4, cell viability and quantity are detected using trypan blue staining to determine cell survival rate; the functional assays include electrophysiological recording and neurotransmitter release analysis.
[0011] Further, the clinical application method includes the following steps: T1: Clinical assessment, including a detailed history of epilepsy and assessment of seizure type, clarifying the diagnosis and classification of epilepsy, determining the location and extent of the epileptic focus, locating the origin of the epileptic seizure, and conducting a neurological physical examination and cognitive function assessment; T2: Targeted manipulation, under the guidance of stereotactic electroencephalography, targeting GABAergic interneurons to the core area of the epileptic focus; T3: Closed-loop intervention, combining antiepileptic drug dosage optimization, neurorehabilitation training, and rTMS stimulation; T4: Long-term follow-up, long-term monitoring of epileptic seizures, neurological function recovery, and adverse reactions, and dynamically adjusting the intervention plan.
[0012] Furthermore, in the T2 step, the transplantation route is percutaneous injection under stereotactic electroencephalography guidance. The transplantation site is the core area of the epileptic focus and a 1-2 cm periphery, injected at 3-5 points, with each point receiving 0.1-0.2 mL of cell solution at a concentration of [missing information]. .
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] (i) The present invention uses the patient’s own induced pluripotent stem cells as the source, avoiding immune rejection and improving the safety of transplantation. Furthermore, through steps S2 and S3, high-purity GABAergic inhibitory interneurons can be obtained in a short time. High-purity GABAergic neurons can more effectively supplement the damaged inhibitory neurons in the brain of epilepsy patients, rebuild the inhibitory balance of the neural network, and thus more effectively control epileptic seizures.
[0015] (ii) The GABAergic neuron transplantation prepared in this invention can significantly reduce the frequency of epileptic seizures. Furthermore, through closed-loop intervention and long-term follow-up, it can reduce the seizure frequency in patients with refractory epilepsy, demonstrating significant clinical efficacy. Attached Figure Description
[0016] Figure 1 This is a flowchart of the present invention. Detailed Implementation
[0017] To make the content of this invention easier to understand, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Identical components are represented by the same reference numerals. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, while the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.
[0018] like Figure 1As shown in this embodiment, a method for inducing differentiation of neural stem cells for refractory epilepsy and its clinical application is provided. The induction differentiation method includes the following steps:
[0019] S1: Cell source and preparation. Induced pluripotent stem cells (iPSCs) derived from the patient's own cells were used. These iPSCs were obtained from the patient's skin fibroblasts and other somatic cells through reprogramming. The reprogramming process involved collecting a small number of skin fibroblasts from patients with refractory epilepsy and inducing them into iPSCs in the laboratory using reprogramming factors Oct4, Sox2, Klf4, and c-Myc. Viral or non-viral vector transduction methods could be used during the reprogramming process to introduce these transcription factors into the fibroblasts. The transduced cells were then cultured in a stem cell culture medium containing basic fibroblast growth factor to form iPSC clones. Pluripotency markers were then detected in the obtained iPSCs. The ability of the induced pluripotent stem cells to differentiate into three germ layers in vitro was verified. The induced pluripotent stem cells were prepared by placing them in a matrix gel-coated culture dish and using a medium containing basic fibroblast growth factor. The culture medium in the dish was DMEM / F12 basal medium supplemented with N2 and B27, basic fibroblast growth factor, and penicillin / streptomycin antibiotics. The cells were cultured at 37-38°C, and their growth status was observed daily. The culture medium was changed periodically. When the cells reached 70% confluence, they were passaged using trypsin digestion to maintain the cells in the logarithmic growth phase. During passage, the cells underwent regular mycoplasma testing and karyotype analysis to ensure that no contamination or genetic mutations occurred during cell expansion.
[0020] S2: Construct an induced differentiation system to differentiate induced pluripotent stem cells into GABAergic inhibitory interneurons, and then differentiate the GABAergic inhibitory interneurons;
[0021] Specifically, it includes the following steps:
[0022] S2.1: Neural progenitor cell induction, involving the digestion of induced pluripotent stem cells from the culture dish. Cells were seeded at a density of 1,000 cells on culture plates pre-coated with matrix gel and cultured in induction medium containing recombinant human activin A and recombinant human basic fibroblast growth factor. The recombinant activin A plays a signal regulation role during cell differentiation, guiding cells towards neural progenitor cells and initiating cell differentiation. The recombinant human basic fibroblast growth factor promotes cell proliferation and maintains the undifferentiated state of cells, ensuring a sufficient number of cells to participate in subsequent differentiation.
[0023] S2.2: Neuronal differentiation induction. After collecting neurospheres, they are further expanded in a neural stem cell culture medium containing epidermal growth factor and basic fibroblast growth factor. After culturing for 7-9 days, retinoic acid is added to induce neural stem cells to differentiate into neurons. Retinoic acid is a classic neural inducing factor that can promote the differentiation of neural stem cells into neurons. Under the induction of retinoic acid, neural stem cells gradually grow protrusions and exhibit neuronal morphological characteristics.
[0024] S2.3: Directed differentiation of GABAergic interneurons. Based on S2.2, growth factors and small molecule compounds related to inhibitory neuron differentiation are added. These growth factors and small molecule compounds promote the directed differentiation of cells into GABAergic interneurons by regulating intracellular signal transduction pathways and gene expression patterns. This allows the cells to gradually acquire the typical characteristics and functions of GABAergic inhibitory interneurons, providing functionally adapted cell resources for subsequent treatment of refractory epilepsy.
[0025] S3: Cell purification and expansion. Immunomagnetic bead sorting was used to purify and enrich cells expressing GABAergic neuron markers. Specifically, magnetic beads labeled with anti-GAD67 antibody were used to separate GABAergic interneurons using magnetically activated cell sorting technology. The purified cells were then expanded in a neural stem cell culture medium containing a small amount of basic fibroblast growth factor to increase the cell number.
[0026] S4: Cell quality control. The purified cells undergo quality control, including cell shape and purity, cell viability and quantity, functional testing, tumorigenicity testing, and sterility and microbiological testing. Cell shape and purity are assessed by detecting the expression of cell markers using immunofluorescence staining to evaluate cell purity and differentiation level. Operators observe cell morphology under a microscope to ensure differentiated cells are mature neurons, with no obvious undifferentiated or malformed cells. Cell viability and quantity are assessed using trypan blue staining to determine cell survival rate, and the total number of cells is counted to ensure the prepared cell quantity. To meet clinical transplantation needs; the functional testing includes electrophysiological recording and neurotransmitter release analysis; the tumorigenicity test involves subcutaneously inoculating cells into immunodeficient mice and observing whether tumors form. This invention requires differentiated cells to not exhibit tumorigenicity in the above tests, further reducing the risk of tumor formation after transplantation; the sterility and microbial testing ensures that the cells are not contaminated by bacteria, fungi, or other microorganisms during preparation and storage, and simultaneously detects the endotoxin content in the cells to ensure that the endotoxin level meets clinical use standards; the detection methods for sterility and microbial testing are existing technologies and will not be elaborated upon here.
[0027] The clinical application method includes the following steps:
[0028] T1: Clinical assessment, including a detailed history of epilepsy and assessment of seizure type, confirmation of epilepsy diagnosis and classification, determination of the location and extent of the epileptic focus, localization of the origin of the seizures, and neurological physical examination and cognitive function assessment. Specifically, the neurological physical examination and cognitive function assessment involve a comprehensive neurological physical examination, including checking muscle strength, muscle tone, reflexes, and sensory function to assess the basic functional state of the nervous system. In addition, professional cognitive function assessment tools, such as the Mini-Mental State Examination and the Montreal Cognitive Assessment Scale, are used to quantitatively assess the patient's cognitive function, understanding the patient's performance in attention, memory, language ability, executive function, etc. This comprehensive assessment helps to fully understand the patient's condition, monitor changes in cognitive function during treatment, and adjust the treatment plan in a timely manner to avoid adverse effects on cognitive function.
[0029] T2: Targeted manipulation, under stereotactic EEG guidance, delivers GABAergic interneurons to the core region of the epileptic focus; the transplantation route is percutaneous injection under stereotactic EEG guidance, with the transplantation site being the core region of the epileptic focus and a 1-2 cm periphery, injected at 3-5 points, with an injection volume of 0.1-0.2 mL at each point, and a cell concentration of [missing information]. This ensures that cells are evenly distributed and do not put excessive pressure on local brain tissue. In actual operation, medical staff use a puncture needle to percutaneously puncture the predetermined target location under aseptic conditions, based on the detailed information provided by the stereotactic electroencephalogram, to ensure that the GABA interneurons can be accurately delivered to the core area of the epileptic focus, thereby maximizing their role in regulating nerve excitability and inhibiting epileptic seizures.
[0030] T3: Closed-loop intervention. After GABAergic interneuron transplantation, medical staff provide postoperative support to patients by combining antiepileptic drug dosage optimization, neurorehabilitation training, and rTMS stimulation. The rTMS stimulation generates an alternating magnetic field that penetrates the skull to stimulate the cerebral cortex, regulating neuronal excitability and neural plasticity. In the clinical treatment of refractory epilepsy, rTMS can improve neural circuit function by regulating the excitability of the cerebral cortex. It works synergistically with GABAergic interneuron transplantation and antiepileptic drug treatment to further control epileptic seizures and promote the recovery of neurological function.
[0031] T4: Long-term follow-up, monitoring of epileptic seizures, neurological function recovery, and adverse reactions, and dynamic adjustment of the intervention plan. If poor seizure control is found, the dosage of antiepileptic drugs can be further optimized or the rTMS stimulation parameters can be adjusted. If slow neurological function recovery occurs, the intensity and targeting of neurorehabilitation training can be strengthened. Through long-term follow-up and dynamic adjustment of the intervention plan, the continuity and stability of the treatment effect can be ensured, and the patient's condition and quality of life can be improved to the greatest extent.
[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for inducing differentiation and clinical application of neural stem cells in refractory epilepsy, characterized in that: The differentiation-inducing method includes the following steps: S1: Cell source and preparation: Induced pluripotent stem cells (iPSCs) derived from the patient are used. These iPSCs are obtained from the patient's skin fibroblasts and other somatic cells through reprogramming. The iPSCs are then placed in a matrix gel-coated culture dish and prepared using a culture medium containing basic fibroblast growth factor. S2: Construct an induced differentiation system to differentiate induced pluripotent stem cells into GABAergic inhibitory interneurons, and then differentiate the GABAergic inhibitory interneurons; S3: Cell purification and expansion, using immunomagnetic beads to purify and enrich cells expressing GABAergic neuron markers; S4: Cell quality control, which involves quality control of purified cells, including cell shape and purity, cell viability and quantity, functional testing, tumorigenicity testing, and sterility and microbial testing.
2. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to claim 1, characterized in that: In step S1, the reprogramming process involves collecting a small number of skin fibroblasts from patients with refractory epilepsy and inducing them into induced pluripotent stem cells in the laboratory using reprogramming factors Oct4, Sox2, Klf4, and c-Myc. During the reprogramming process, viral or non-viral vector transduction methods can be used to introduce the above transcription factors into fibroblasts. The transduced cells are then cultured in stem cell culture medium containing basic fibroblast growth factor to form induced pluripotent stem cell clones.
3. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to claim 2, characterized in that: The culture medium in the culture dish in step S1 is: DMEM / F12 basal medium, supplemented with N2 and B27 nutrient supplements, basic fibroblast growth factor, and penicillin / streptomycin dual antibiotics.
4. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to claim 3, characterized in that: Step S2 includes the following steps: S2.1: Neural progenitor cell induction: Induced pluripotent stem cells are digested from the culture dish and seeded at a predetermined density on a culture plate pre-coated with matrix gel, and cultured in an induction medium containing recombinant human activin A and recombinant human basic fibroblast growth factor; S2.2: Neuronal differentiation induction. After collecting the neurospheres, they were further expanded in a neural stem cell culture medium containing epidermal growth factor and basic fibroblast growth factor. After culturing for 7-9 days, retinoic acid was added to induce neural stem cells to differentiate into neurons. S2.3: Directed differentiation of GABAergic interneurons. Based on S2.2, growth factors and small molecule compounds related to inhibitory neuron differentiation are added to promote the directed differentiation of cells into GABAergic interneurons.
5. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to claim 4, characterized in that: In step S4, cell viability and number are detected using trypan blue staining to determine cell survival rate; the functional assays include electrophysiological recording and neurotransmitter release analysis.
6. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to any one of claims 1-5, characterized in that: The clinical application method includes the following steps: T1: Clinical assessment, including a detailed history of epilepsy and assessment of seizure type, diagnosis and classification of epilepsy, location and extent of epileptic focus, localization of the origin of epileptic seizures, and neurological physical examination and cognitive function assessment. T2: Targeted manipulation, under the guidance of stereotactic electroencephalography, GABAergic interneurons are targeted to the core area of the epileptic focus; T3: Closed-loop intervention, combining antiepileptic drug dosage optimization, neurorehabilitation training and rTMS stimulation; T4: Long-term follow-up, long-term monitoring of epileptic seizures, neurological function recovery and adverse reactions, and dynamic adjustment of intervention plan.
7. The method for inducing differentiation and clinical application of neural stem cells for refractory epilepsy according to claim 6, characterized in that: In the T2 step, the transplantation route is percutaneous injection under stereotactic electroencephalography (EEG) guidance. The transplantation site is the core area of the epileptic focus and a 1-2 cm periphery, injected at 3-5 points, with each point receiving 0.1-0.2 mL of cell solution at a concentration of [missing information]. .