A composition and method of reprogramming astrocytes into functional neurons

Abstract

The present application relates to the technical field of biotechnology, and particularly discloses a composition and a method for reprogramming astrocytes into functional neurons. The composition for reprogramming astrocytes into functional neurons comprises a compound PT109B. The composition for reprogramming astrocytes into functional neurons provided in the present application can reprogram astrocytes into functional neurons, and effectively solves the problems of a large number of small molecules and complicated steps in the current chemical reprogramming technology.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more particularly to a composition and method for reprogramming astrocytes into functional neurons. Background Technology

[0002] Neurodegenerative diseases (NDDs) constitute the most significant component of disabling diseases worldwide. In recent years, Parkinson's disease (PD), the second most prevalent NDD, has seen its annual patient numbers increase at a rate exceeding that of Alzheimer's disease (AD), the most prevalent NDD. The main clinical features of PD include resting tremor, rigidity, and bradykinesia. The primary cause of motor dysfunction in PD patients is the massive death or dysfunction of dopaminergic neurons (DANs) in the substantia nigra and striatum. Currently, the main treatments for PD can only alleviate motor function and non-motor symptoms, and cannot effectively reverse the massive loss of DANs in the brain. Furthermore, long-term use of dopamine-based drugs may cause adverse reactions such as sleep disturbances, nausea, and vomiting. Therefore, finding an anti-PD drug that can effectively increase the number of DANs with fewer side effects has become a pressing scientific problem for scientists.

[0003] The development of regenerative medicine has provided a new avenue for the treatment of Parkinson's disease (PD). Astrocytes (As) have the potential to be transformed into neurons. Through gene editing, As can be converted into neurons (DANs), which may then be used to treat PD. However, the tumorigenicity and immunogenicity of gene therapy limit its practical application in PD treatment. The rise of the concept of chemical reprogramming technology has effectively compensated for the shortcomings of gene therapy. Studies have found that As can be reprogrammed into neurons using combinations of multiple small molecules. However, current chemical reprogramming technology also has drawbacks, such as the excessive number of molecules in the small molecule composition, complex application steps, and difficulty in meeting pharmacokinetic requirements. These shortcomings limit the application of small molecule compounds developed based on chemical reprogramming technology. Therefore, existing technologies still need improvement. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a composition and method for reprogramming astrocytes into functional neurons, aiming to solve the problems of excessive number of small molecule compositions and complex application steps in existing methods for reprogramming astrocytes into functional neurons.

[0005] The technical solution of this application is as follows:

[0006] A first aspect of this application provides a composition for reprogramming astrocytes into functional neurons, the composition comprising compound PT109B.

[0007] Optionally, the composition may further include thyroid hormones.

[0008] Optionally, the composition further includes at least one of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and fibroblast growth factor.

[0009] Optionally, the composition further includes a neuron-inducing culture medium.

[0010] Optionally, the neuron induction medium is selected from one of the following: DMEM / F12-based medium, serum-free induction medium, and PSC neuron induction medium.

[0011] A second aspect of this application provides a method for reprogramming astrocytes into functional neurons, using the composition of the first aspect of this application for reprogramming astrocytes into functional neurons.

[0012] Optionally, the method for reprogramming astrocytes into functional neurons includes the steps of:

[0013] Culture astrocytes until they mature, achieving a confluence of 60%-80%.

[0014] Mature astrocytes were cultured in a culture medium containing the composition for reprogramming astrocytes into functional neurons to obtain the functional neurons.

[0015] Optionally, the step of further culturing the astrocytes in a culture medium containing the composition for reprogramming the astrocytes into functional neurons includes the following steps:

[0016] The astrocytes were cultured for 1-3 days in a first culture medium containing the composition for reprogramming astrocytes into functional neurons.

[0017] The astrocytes were then cultured in a second culture medium containing the composition for reprogramming the astrocytes into functional neurons for 14-90 days.

[0018] The first culture medium includes the composition for reprogramming astrocytes into functional neurons and DMEM high-glucose culture medium, wherein the volume ratio of the composition for reprogramming astrocytes into functional neurons to the DMEM high-glucose culture medium is 1:1.

[0019] The second culture medium includes the composition for reprogramming astrocytes into functional neurons.

[0020] Optionally, the concentration of compound PT109B in the composition for reprogramming astrocytes into functional neurons is 5-20 μM;

[0021] When the composition for reprogramming astrocytes into functional neurons includes thyroid hormones, the concentration of the thyroid hormones is 5-500 nM.

[0022] Optionally, the functional neuron includes at least one of an electrophysiologically active neuron and a neuron with dopamine-releasing function.

[0023] The beneficial effects of this application are as follows: The composition for reprogramming astrocytes into functional neurons provided in this application can reprogram astrocytes into functional neurons, effectively reducing the number of small molecule compounds used in chemical reprogramming technology, enabling the reprogramming of astrocytes to be achieved within two molecules. This not only effectively solves the drawbacks of the large number of small molecules and complicated steps in current chemical reprogramming technology, but also helps to explore the key mechanisms of astrocyte transformation, providing a theoretical basis for the subsequent development of drugs for Parkinson's disease. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0025] Figure 1 This is a flowchart illustrating a method for reprogramming astrocytes into functional neurons, as provided in an embodiment of this application.

[0026] Figure 2 The following diagram illustrates the results of reprogramming astrocytes into induced dopaminergic neurons using compound PT109B, as provided in the embodiments of this application:

[0027] A shows the immunofluorescence detection of dopaminergic neuron markers (tyrosine hydroxylase, TH, and dopamine transporter, DAT); B shows the quantitative analysis of dopaminergic neuron markers (tyrosine hydroxylase, TH, and dopamine transporter, DAT); C shows the immunofluorescence detection of dopaminergic neuron markers (receptor-associated factor 1, Nurr1, and tyrosine hydroxylase, TH); D shows the quantitative analysis of dopaminergic neuron markers (receptor-associated factor 1, Nurr1, and tyrosine hydroxylase, TH); E shows the immunofluorescence detection of synaptic markers (synapticin 1 and tyrosine hydroxylase, TH); F shows the quantitative analysis of synaptic markers (synapticin 1 and tyrosine hydroxylase, TH).

[0028] Figure 3 Electrophysiological activity analysis diagram of astrocytes reprogrammed into induced dopaminergic neurons using compound PT109B, provided for embodiments of this application:

[0029] A shows a morphological comparison of cells; B shows the detection of cell action potentials; C shows representative trajectories of sodium and potassium currents inducing neurons; D shows a quantitative analysis of the proportion of converted induced neurons with single action potential firing at different time points; E and F show statistical analysis of the amplitude of induced neuronal action potentials; G shows the peak sodium current (INa). + Analysis of the developmental increase over time; H is the peak potassium current (IK). + Analysis of developmental increases over time; I represents the statistical analysis results of the resting potentials of induced neurons;

[0030] Figure 4 Electrophysiological activity analysis diagram of reprogramming astrocytes into induced dopaminergic neurons using compounds PT109B and T3, provided for embodiments of this application:

[0031] A shows a representative image of induced neuron-like cells recorded using patch clamp; B shows a representative curve of repetitive action potentials recorded in PT109B and T3-induced neuron-like cells 30 days after initial drug treatment; C shows a representative curve of sodium (Na+) and potassium (K+) currents recorded in PT109B and T3-induced neurons after 30 days; D shows a representative curve of spontaneous synaptic events in neurons 30 days after transformation.

[0032] Figure 5 The fluorescence intensity detection diagram of Na-K-ATPase in astrocytes reprogrammed into induced dopaminergic neurons using compounds PT109B and T3, provided in the embodiments of this application. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0034] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0035] This application provides a composition for reprogramming astrocytes into functional neurons, comprising compound PT109B.

[0036] Compound PT109B, chemical name 5-(1,2-dithiolan-3-yl)-N-((1r,4r)-4-(isoquinolin-5-ylamino)cyclohexyl)

[0037] ntanamide, chemical formula C 23 H 31 N3OS2, with a molecular weight of 429.64, has the following chemical structural formula:

[0038]

[0039] In some embodiments, the composition for reprogramming astrocytes into functional neurons also includes thyroid hormones.

[0040] Thyroid hormone (Triiothyronine, T3), chemical name 3,3',5-Triiodo-L-thyronine, chemical formula C 15 H 12 I3NO4 has a molecular weight of 650.97 and its specific chemical structure is as follows:

[0041]

[0042] The reprogramming of astrocytes into functional neurons can be achieved using compositions containing compound PT109B or both PT109B and T3. This method requires fewer small-molecule compounds and involves a simpler procedure, overcoming the problems of existing chemical reprogramming techniques that require a large number of small-molecule compounds and involve complex steps.

[0043] In some embodiments, the composition for reprogramming astrocytes into functional neurons includes at least one of brain-derived neurotrophic factor (BDNF), glial cell-line-derived neurotrophic factor (GDNF), and fibroblast growth factors (b-FGF).

[0044] BDNF, GDNF, and β-FGF are three important endogenous neurotrophic factors that play different roles in the nervous system. Supplementation with these three neurotrophic factors plays a crucial role in neuronal survival, differentiation, and functional maintenance. BDNF has biological effects such as preventing neuronal death due to injury, improving the pathological state of neurons, and promoting the regeneration and differentiation of damaged neurons. It is also essential for the survival and normal physiological function of mature neurons in the central and peripheral nervous systems. GDNF and β-FGF play important roles in the survival and differentiation of various neurons during development.

[0045] In some embodiments, compositions for reprogramming astrocytes into functional neurons include a neuron-inducing medium. A neuron-inducing medium is a culture medium used to convert stem cells or precursor cells into neurons. The medium typically contains various growth factors and nutrients to promote neuronal differentiation and maturation.

[0046] In some embodiments, the neuron induction medium is selected from one of DMEM / F12-based medium, serum-free induction medium, and PSC neuron induction medium. Preferably, the main components of the neuron induction medium include 50 mL Neurobasl medium, 1% N2 supplement, 1% B27 supplement, 1% Glutamax, and 1% penicillin and streptomycin mixture.

[0047] Neurobasal medium is a basal medium specifically designed for neuron culture. It provides suitable nutrients for neuronal growth, such as amino acids and vitamins, and its physicochemical properties, such as osmotic pressure and pH, are also suitable for neuronal survival and development. N2 supplement is an additive mainly containing transferrin, insulin, progesterone, putrescine, and sodium selenite, providing essential nutrients and growth factors for neuronal growth. B27 supplement is a complex additive containing antioxidants, fatty acids, vitamins, and other components. Glutamax is an additive containing L-glutamine. Bismuth subsalicylate (BDS) is a mixture of penicillin and streptomycin, typically containing 100 U / mL penicillin and 100 μg / mL streptomycin, primarily to prevent bacterial contamination. Penicillin inhibits bacterial cell wall synthesis, while streptomycin mainly interferes with bacterial protein synthesis. The combined use of these two effectively prevents the growth and reproduction of most common bacteria in the culture medium, thus protecting the purity of the neuronal cell culture system and ensuring that neurons can grow in a relatively sterile environment.

[0048] Please see Figure 1 This application also provides a method for reprogramming astrocytes into functional neurons, using the composition for reprogramming astrocytes into functional neurons as described above in this application.

[0049] In some embodiments, the astrocytes are preferably primary SD rat astrocytes. Primary SD rat cells generally refer to cells obtained directly from Sprague-Dawley (SD) rats. SD rats are a commonly used laboratory animal strain, characterized by rapid growth, high reproductive capacity, good adaptability to the environment, and relatively docile temperament.

[0050] In some implementations, the method of reprogramming astrocytes into functional neurons specifically includes the steps of:

[0051] S1. Culture astrocytes until they mature, achieving a cell confluence of 60%-80%.

[0052] In some implementations, specific steps may include: extracting astrocytes from the brains of newborn SD rats and culturing them for 5-7 days; when immunofluorescence detection shows that more than 98% of the cells express the astrocyte markers GFAP or S100β, they are considered mature astrocytes; culturing continues until the confluence of astrocytes on the cell plate reaches 60%-80%. Cell confluence in cell culture refers to the proportion of cells that adhere to and connect with each other on the culture surface in a culture vessel such as a cell culture dish or flask, usually expressed as a percentage. For example, the confluence of astrocytes on a cell plate may be 60%, 65%, 70%, 75%, or 80%, etc.

[0053] In some implementations, specifically, the medium used to culture astrocytes to maturity can be DMEM high-glucose medium (Dulbecco's Modified Eagle Medium). DMEM high-glucose medium is a synthetic medium widely used in cell culture.

[0054] S2. Replace the culture medium for astrocytes with a culture medium containing a composition that reprograms astrocytes into functional neurons and continue culturing to obtain functional neurons.

[0055] Specifically, the culture medium in the culture medium of the astrocytes obtained in S1 was aspirated and neuron-inducing culture medium containing the compound PT109B was added.

[0056] In some embodiments, the concentration of compound PT109B in the neuron induction medium containing compound PT109B is 5-20 μM, for example, the concentration of compound PT109B in the neuron induction medium containing compound PT109B is 5 μM, 10 μM, 15 μM or 20 μM, etc.

[0057] In some embodiments, the neuron-inducing medium containing compound PT109B includes 10 μM of compound PT109B, 10 ng / mL BDNF, 10 ng / mL GDNF, 10 ng / mL b-FGF, 50 mL Neurobasl medium, 1% N2 supplement, 1% B27 supplement, 1% Glutamax and 1% penicillin-dextrose antibody.

[0058] In some embodiments, the neuron-inducing medium containing compound PT109B also includes thyroid hormone at a concentration of 5-500 nM. The concentrations of thyroid hormone in the neuron-inducing medium containing compound PT109B are 5 nM, 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, or 500 nM, etc.

[0059] In some embodiments, the neuron-inducing medium containing compound PT109B includes 10 μM of compound PT109B, 50 nM thyroid hormone, 10 ng / mL BDNF, 10 ng / mL GDNF, 10 ng / mL b-FGF, 50 mL Neurobasl medium, 1% N2 supplement, 1% B27 supplement, 1% Glutamax and 1% penicillin-dextrose antibody.

[0060] In some implementations, step S2 may further include the following steps:

[0061] S21. Discard the culture medium from the culture medium of the astrocytes obtained in S1, and add the first culture medium containing neuron induction medium containing compound PT109B, and culture for 1-3 days.

[0062] The first culture medium containing the compound PT109B includes the neuron induction medium containing the compound PT109B and DMEM high glucose medium as described above, with a volume ratio of 1:1.

[0063] S21. Discard the culture medium from the culture medium of the astrocytes obtained in S21, and add a second culture medium containing neuron induction medium containing compound PT109B, and culture for 14-90 days.

[0064] The second culture medium containing the neuron induction medium of compound PT109B is 100% the neuron induction medium containing compound PT109B as described above.

[0065] In some implementations, step S2 may further include the step:

[0066] The culture medium from the astrocytes obtained in S1 was aspirated, and a first culture medium containing neuronal induction medium with compound PT109B was added. The cells were cultured for 1-3 days. The first culture medium containing neuronal induction medium with compound PT109B consisted of the neuronal induction medium containing compound PT109B as described above and DMEM high-glucose medium, with a volume ratio of 1:1. After 1-3 days of culture, the medium with a higher proportion of neuronal induction medium containing compound PT109B was replaced. The proportion of neuronal induction medium containing compound PT109B was gradually increased over time, eventually being replaced with 100% neuronal induction medium, i.e., the second culture medium described above.

[0067] In some embodiments, the functional neuron includes at least one of an electrophysiologically active neuron and a neuron with dopamine-releasing function.

[0068] The following specific examples will provide further details.

[0069] Unless otherwise specified, all reagents and instruments used in the examples are commercially available products. Unless otherwise specified, all experimental methods used in the examples are conventional experimental methods or techniques in the art.

[0070] Example 1

[0071] This embodiment performs a biological evaluation of PT109B reprogramming astrocytes into neurons.

[0072] Experimental methods:

[0073] (1) According to the required concentration, add PT109B (10 μM), BDNF (10 ng / mL), GDNF (10 ng / mL), β-FGF (10 ng / mL), 1% N2 supplement, 1% B27 supplement, 1% Glutamax, and 1% penicillin antibiotics to 50 mL of Neurobasl medium to prepare a neuron induction medium containing compound PT109B. Prepare DMEM high-glucose medium.

[0074] (2) Primary SD rat astrocytes were cultured in DMEM high glucose medium for 5-7 days. If more than 98% of the cells expressed the astrocyte markers GFAP or S100β, they were considered to be mature astrocytes. The cells were cultured until the degree of confluence was 60-80%.

[0075] (3) Continue culturing the astrocytes from (2). On the first day of culture, discard the original culture medium and add a mixture of neuron-inducing medium containing compound PT109B and DMEM high-glucose medium at a volume ratio of 1:1 to the culture dish containing the astrocytes, replacing the original culture medium. Continue culturing for 3 days. After 3 days, discard the original culture medium and add 100% neuron-inducing medium containing compound PT109B to the culture dish containing the astrocytes. Continue culturing for 90 days to obtain functional neurons. Other culture conditions are the same as in (2). During this period, observe and record the cell status daily, and change the culture medium every 2-3 days according to the cell status.

[0076] (4) Use cell immunofluorescence and whole-cell patch-clamp techniques to detect the characteristics of functional neurons.

[0077] Following the methods in (1)-(3), astrocytes from primary SD rats were treated with PT109B for one month and then subjected to immunofluorescence analysis.

[0078] Following the methods in (1)-(3), primary SD rat astrocytes were treated with PT109B for 1 month, 2 months, and 3 months, respectively, and then their cell electrophysiology was detected.

[0079] The detection methods for cell electrophysiology include the following steps:

[0080] ① Prepare the relevant solutions for detecting cell electrophysiology. The solution formula is as follows:

[0081] The artificial cerebrospinal fluid for cell incubation consisted of 122 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 12.5 mM D-glucose, 2 mM CaCl2, 1 mM MgSO4, 1 mM MgCl2, and 5 mM HEPES.

[0082] Electrode internal solution formulation: 128mM K-gluconate, 10mM NaCl, 2mM MgCl2, 0.5mM EGTA, 10mM MEPES, 0.4mM Na2GTP and 4mM Na2ATP.

[0083] ② After preparing the relevant solutions, the cultured astrocyte smears were first incubated in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid for 30 minutes, and then transferred to a recording chamber perfused with artificial cerebrospinal fluid for recording at room temperature (25-30℃). Using a HEKAEPC10 dual patch-clamp amplifier (HEKA Elektronik, German), the glass electrode (5-8 MOhm) was filled with potassium-containing electrode solution, and whole-cell patch-clamping was performed on randomly selected cells at a sampling rate of 10 kHz, with filtering at 2 kHz. After data acquisition, the acquired data were analyzed offline using Clampfit 11 software (MDS Analytical Technologies). For voltage-clamp experiments, the voltage was maintained at -70 mV.

[0084] Experimental results:

[0085] The test results of cell immunofluorescence technology are as follows Figure 2 As shown, compared with the control group (CT group), ***P<0.001. N=5-6, scale bar=200 micrometers. From Figure 2 As can be seen from A, B, C, and D, in the cells of group PT109B, TH... + / DAT + and TH + / Nurr1 + The cell morphology is similar to that of neurons, but their numbers are significantly increased, and TH + DAT + Nurr1+ The proportion of these cells was approximately 20% of the total cells, suggesting that PT109B can reprogram astrocytes into inducible dopamine neurons. Further examination of the neuronal synapse marker Synap1 revealed increased Synap1 signaling (approximately 80%) in cells treated with PT109B, and the presence of synapse-like structures. Additionally, a small percentage of cells simultaneously expressed TH and Synap1 (e.g., TH). Figure 2 China E and Figure 2 As shown in Figure F, synapse-like structures have grown in the PT109B-reprogrammed inducible dopamine neurons. The significantly increased dopamine content in PT109B-induced neurons further suggests that PT109B can reprogram astrocytes into functional inducible dopamine neurons.

[0086] Test results of whole-cell patch-clamp technique for detecting the characteristics of functional neurons, as follows: Figure 3 As shown, from Figure 3 As can be seen, compared to astrocytes (As), the cell morphology of the PT109B group was similar to that of neurons. Figure 3 In the A group, no significant action potentials or sodium-potassium (Na-K) currents were observed. However, after one month of PT109B treatment, induced single action potentials appeared in A, with lower Na-K current amplitudes. Compared to the one-month group, the PT109B-treated two-month group showed a slight increase in the induction of single action potentials and Na-K current amplitudes, but the difference was negligible. Meanwhile, the PT109B-cultured three-month group showed significantly enhanced induced action potentials and Na-K current amplitudes compared to other groups, but sustained action potentials were not yet observed. Figure 3 (B, C, D). The amplitude of the action potential generated by neurons induced by PT109B was significantly greater than that of As ( Figure 3 (E and F). Meanwhile, compared to As, PT109B significantly increased the amplitude of sodium current and decreased the amplitude of potassium current in neurons treated for different durations, and significantly decreased cell capacitance. Figure 3 G and H). And the cells were allowed to survive in vitro for more than 3 months, but all recorded action potentials were single action potentials (G and H). Figure 3 Middle I).

[0087] Example 2

[0088] This embodiment performs a biological assessment of PT109B and T3 reprogramming of astrocytes into neurons.

[0089] Experimental methods:

[0090] (1) According to the required concentration, add PT109B (10 μM), T3 (50 nM), BDNF (10 ng / mL), GDNF (10 ng / mL), β-FGF (10 ng / mL), 1% N2 supplement, 1% B27 supplement, 1% Glutamax, and 1% penicillin antibiotics to 50 mL of Neurobasl medium to prepare a neuron induction medium containing compound PT109B. Prepare DMEM high-glucose medium.

[0091] (2) Primary SD rat astrocytes were cultured in DMEM high glucose medium for 5-7 days. If more than 98% of the cells expressed the astrocyte markers GFAP or S100β, they were considered to be mature astrocytes. The cells were cultured until the confluence was 60%-80%.

[0092] (3) Continue culturing the astrocytes from (2). On the first day of culture, discard the original culture medium and add a mixture of neuron-inducing medium containing compounds PT109B and T3 and DMEM high-glucose medium at a volume ratio of 1:1 to the culture dish containing the astrocytes, replacing the original culture medium. Continue culturing for 3 days. After 3 days, discard the original culture medium and add 100% neuron-inducing medium containing compounds PT109B and T3 to the culture dish containing the astrocytes. Continue culturing for 90 days to obtain functional neurons. Observe and record the cell status daily during this period, and change the culture medium every 2-3 days according to the cell status.

[0093] (4) Use cell immunofluorescence and whole-cell patch-clamp techniques to detect the characteristics of functional neurons.

[0094] The detection methods for cell electrophysiology include the following steps:

[0095] ① Prepare the relevant solutions for detecting cell electrophysiology. The solution formula is as follows:

[0096] The artificial cerebrospinal fluid for cell incubation consisted of 122 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 12.5 mM D-glucose, 2 mM CaCl2, 1 mM MgSO4, 1 mM MgCl2, and 5 mM HEPES.

[0097] Electrode internal solution formulation: 128mM K-gluconate, 10mM NaCl, 2mM MgCl2, 0.5mM EGTA, 10mM MEPES, 0.4mM Na2GTP and 4mM Na2ATP.

[0098] ② After preparing the relevant solutions, the cultured astrocyte smears were first incubated in oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid for 30 minutes, and then transferred to a recording chamber perfused with artificial cerebrospinal fluid for recording at room temperature (25-30℃). Using a HEKAEPC10 dual patch-clamp amplifier (HEKA Elektronik, German), the glass electrode (5-8 MOhm) was filled with potassium-containing electrode solution, and whole-cell patch-clamping was performed on randomly selected cells at a sampling rate of 10 kHz, with filtering at 2 kHz. After data acquisition, the acquired data were analyzed offline using Clampfit 11 software (MDS Analytical Technologies). For voltage-clamp experiments, the voltage was maintained at -70 mV.

[0099] Experimental results:

[0100] The results of cell electrophysiological testing are as follows: Figure 4 As shown, the results indicate that adding T3 to the PT109B induction system resulted in structures resembling neuronal synapses in the cells. Figure 4 (A). Further investigation revealed that after 30 days of combined treatment with PT109B and T3, the proportion of cells with continuous action potentials significantly increased. Figure 4 (B) Simultaneously, bipolar or tripolar neurons with smaller cell bodies exhibit significantly increased sodium current amplitude, and some cells possess excitatory postsynaptic electrophysiological characteristics. Figure 4 (C). On the other hand, continuous action potentials can be detected when T3 is applied to As in conjunction with PT109B. Figure 4 (D).

[0101] Immunofluorescence detection results as follows Figure 5 As shown, from Figure 5 As can be seen, T3 does not affect the reprogramming efficiency of PT109B and enhances the fluorescence intensity of Na-K-ATPase, which indicates that T3 can enhance the electrophysiological activity of neurons induced by PT109B without affecting the reprogramming effect of PT109B. This suggests that the combination of PT109B and T3 can reprogram astrocytes into electrophysiologically active neurons.

[0102] In summary, the composition for reprogramming astrocytes into functional neurons provided in this application can effectively reduce the number of small molecule compounds used in chemical reprogramming technology, enabling astrocyte reprogramming to be achieved with no more than two molecules. This not only effectively solves the drawbacks of the large number of small molecules and complex steps in current chemical reprogramming technology, but also facilitates the exploration of key mechanisms of astrocyte transformation, providing a theoretical basis for the subsequent development of drugs for Parkinson's disease. Furthermore, the development of the combined use method with T3 transforms the single-action potential of induced dopaminergic neurons reprogrammed with PT109B alone into multiple action potentials, making the induced dopaminergic neurons more physiologically functional in vivo.

[0103] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A composition for reprogramming astrocytes into functional neurons, characterized in that, The composition comprises 10 μM compound PT109B, 50 nM thyroid hormone, 10 ng / mL brain-derived neurotrophic factor, 10 ng / mL glial cell-derived neurotrophic factor, 10 ng / mL fibroblast growth factor, 1% N2 supplement, 1% B27 supplement, 1% Glutamax, 1% penicillin-dextrose antibody and Neurobasl medium.

2. A method for reprogramming astrocytes into functional neurons, characterized in that, Including the following steps: Astrocytes were cultured until mature, achieving a confluence of 60%-80%. Mature astrocytes are cultured in a culture medium containing the composition of claim 1 for reprogramming astrocytes into functional neurons to obtain the functional neurons. The continued cultivation includes the following steps: The astrocytes were cultured for 1-3 days in a first culture medium containing the composition for reprogramming astrocytes into functional neurons. The astrocytes were then cultured in a second culture medium containing the composition for reprogramming the astrocytes into functional neurons for 14-90 days. The first culture medium includes the composition for reprogramming astrocytes into functional neurons and DMEM high-glucose medium, wherein the volume ratio of the composition for reprogramming astrocytes into functional neurons to the DMEM high-glucose medium is 1:

1. The second culture medium includes the composition for reprogramming astrocytes into functional neurons.

3. The method for reprogramming astrocytes into functional neurons according to claim 2, characterized in that, The functional neurons have continuous action potentials and / or dopamine release capabilities.

Images