Composition and method for reprogramming astrocytes into functional neurons

Abstract

The invention relates to the technical field of biology, and particularly discloses a composition and a method for reprogramming astrocytes into functional neurons. Wherein the composition for reprogramming the astrocytes into the functional neurons comprises a compound PT109B. According to the composition for reprogramming the astrocytes into the functional neurons, the astrocytes can be reprogrammed into the functional neurons, and the problems that in the current chemical reprogramming technology, the number of used small molecules is large, and the steps are complex are effectively solved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a composition and method for reprogramming astrocytes into functional neurons. Background Art

[0002] Neurodegenerative diseases (NDDs) are the most important component of disabling diseases in the world. In recent years, the annual growth rate of Parkinson's disease (PD), which ranks second in prevalence among NDDs, has exceeded that of Alzheimer's disease (AD), which has the highest prevalence among NDDs. The main clinical features of PD include resting tremor, muscle rigidity, bradykinesia, etc. The main cause of motor dysfunction in PD patients is the massive death or dysfunction of dopaminergic neurons (DANs) in the substantia nigra and striatum. At present, the main treatment drugs for PD can only relieve the motor function and non-motor symptoms of PD patients, and cannot effectively reverse the massive loss of DANs in the brain of PD patients; at the same time, long-term use of dopamine drugs may cause adverse reactions such as sleep disorders, nausea, and vomiting. Therefore, finding an anti-PD drug that can effectively increase the number of DANs with low side effects has become a scientific problem that scientists need to solve urgently.

[0003] The development of regenerative medicine has provided a new approach for the treatment of PD. Astrocytes (As) have the potential to transform into neurons. Through gene editing methods, As can be converted into DANs, which may be used to treat PD. Nevertheless, the tumorigenicity and immunogenicity of gene therapy limit its practical application in the treatment of PD. The rise of the concept of chemical reprogramming technology has made up for the shortcomings of gene therapy. Studies have found that As can be reprogrammed into neurons using a variety of small molecule combinations. However, the current chemical reprogramming technology also has shortcomings such as too many molecules in the small molecule composition, complicated application steps, and pharmacokinetic characteristics that are difficult to meet the requirements. The above defects have led to the application of small molecule compounds developed based on chemical reprogramming technology. Limited. Therefore, the existing technology still needs to be improved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a composition and method for reprogramming astrocytes into functional neurons, aiming to solve the problem that there are too many small molecule compositions for reprogramming astrocytes into functional neurons and the application steps are complicated.

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

[0006] In a first aspect of the present application, a composition for reprogramming astrocytes into functional neurons is provided, wherein the composition comprises compound PT109B.

[0007] Optionally, the composition further comprises thyroid hormone.

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

[0009] Optionally, the composition further comprises a neuron induction medium.

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

[0011] The second aspect of the present application provides a method for reprogramming astrocytes into functional neurons, wherein the composition for reprogramming astrocytes into functional neurons of the first aspect of the present application is used to reprogram astrocytes into functional neurons.

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

[0013] Cultivate astrocytes until they are mature and reach a confluency of 60%-80%;

[0014] The mature astrocytes are placed in a culture medium containing the composition for reprogramming astrocytes into functional neurons and continue to be cultured to obtain the functional neurons.

[0015] Optionally, the step of placing the astrocytes in a culture medium containing the composition for reprogramming astrocytes into functional neurons and continuing to culture the astrocytes comprises the steps of:

[0016] placing the astrocytes in a first culture medium containing the composition for reprogramming astrocytes into functional neurons for 1-3 days;

[0017] placing the astrocytes in a second culture medium containing the composition for reprogramming 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, and 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 comprises 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 hormone, the concentration of the thyroid hormone is 5-500 nM.

[0022] Optionally, the functional neurons include at least one of neurons with electrophysiological activity and neurons with the function of releasing dopamine.

[0023] Beneficial effects of the present application: The composition for reprogramming astrocytes into functional neurons provided in the present application can reprogram astrocytes into functional neurons, effectively reducing the number of small molecule compounds used in chemical reprogramming technology, so that the reprogramming of astrocytes can be achieved within two molecules, which can not only effectively solve the disadvantages of the large number of small molecules and complicated steps in the current chemical reprogramming technology, but also is conducive to exploring the key mechanism of astrocyte transformation, and providing a theoretical basis for the subsequent development of Parkinson's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0025] Figure 1 A schematic diagram of a process for reprogramming astrocytes into functional neurons provided in an embodiment of the present application;

[0026] Figure 2 The results of the analysis of the use of compound PT109B to reprogram astrocytes into induced dopaminergic neurons provided in the examples of this application are as follows:

[0027] A is the immunofluorescence detection of dopaminergic neuron markers (tyrosine hydroxylase, TH and dopamine transporter, DAT); B is the quantitative analysis of dopaminergic neuron markers (tyrosine hydroxylase, TH and dopamine transporter, DAT); C is the immunofluorescence detection of dopaminergic neuron markers (receptor-associated factor 1, Nurr1 and tyrosine hydroxylase, TH); D is the quantitative analysis of dopaminergic neuron markers (receptor-associated factor 1, Nurr1 and tyrosine hydroxylase, TH); E is the immunofluorescence detection of synapse-related markers (synaptic protein 1, Synap1) and dopaminergic neuron markers (tyrosine hydroxylase, TH); F is the quantitative analysis of synapse-related markers (synaptic protein 1, Synap1) and dopaminergic neuron markers (tyrosine hydroxylase, TH);

[0028] Figure 3 Electrophysiological activity analysis diagram of reprogramming astrocytes into induced dopaminergic neurons using compound PT109B provided in the examples of the present application:

[0029] A is a comparison of cell morphology; B is the detection of cell action potential; C is the representative trajectory of sodium current and potassium current of induced neurons; D is the quantitative analysis of the proportion of conversion-induced neurons with single action potential at different time points; E and F are statistical analysis of the amplitude of action potential of induced neurons; G is the peak sodium current (INa + ) is the analysis of developmental increase over time; H is the peak potassium current (IK + ) is the analysis of developmental increase over time; I is the statistical analysis result of resting potential of induced neurons;

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

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

[0032] Figure 5 This is a fluorescence intensity detection diagram of Na-K-ATPase in the reprogramming of astrocytes into induced dopaminergic neurons using compounds PT109B and T3 provided in the examples of the present application. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0034] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0035] The embodiments of the present application provide a composition for reprogramming astrocytes into functional neurons, including 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 is C 23 H 31 N 3 OS 2 , molecular mass is 429.64, and the specific chemical structure is as follows:

[0038]

[0039] In some embodiments, the composition for reprogramming astrocytes into functional neurons further comprises thyroid hormone.

[0040] Thyroid hormone (Triiothyronine, T3), chemical name is 3,3',5-Triiodo-L-thyronine, chemical formula is C 15 H 12 I 3 NO 4 , molecular mass is 650.97, the specific chemical structure is as follows:

[0041]

[0042] The composition for reprogramming astrocytes into functional neurons containing compound PT109B or compound PT109B and T3 can achieve reprogramming of astrocytes into functional neurons, with a small number of small molecule compounds used and simple steps, thus solving the problem of a large number of small molecule compounds and complicated steps used in the prior art chemical reprogramming technology.

[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 factor (b-FGF).

[0044] BDNF, GDNF, and b-FGF are three important endogenous neurotrophic factors that play different roles in the nervous system. The addition of BDNF, GDNF, and b-FGF plays an important role in the survival, differentiation, and functional maintenance of neurons. BDNF can prevent neurons from being damaged and dying, improve the pathological state of neurons, promote the regeneration and differentiation of damaged neurons, and is also necessary for the survival and normal physiological functions of mature central and peripheral nervous system neurons. GDNF and b-FGF play an important role in the survival and differentiation of various neurons during the developmental period.

[0045] In some embodiments, the composition for reprogramming astrocytes into functional neurons includes a neuron induction medium. Neuron induction medium is a medium for converting stem cells or precursor cells into neurons. The medium generally contains a variety of growth factors and nutrients to promote the differentiation and maturation of neurons.

[0046] In some embodiments, the neuron induction medium is selected from one of a DMEM / F12-based medium, a serum-free induction medium, and a PSC neural 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% double antibody (a mixture of penicillin and streptomycin).

[0047] Among them, Neurobasal medium is a basic medium specially designed for neuron culture. It can provide suitable nutrients for the growth of neurons, such as basic nutrients such as amino acids and vitamins, and its physical and chemical properties such as osmotic pressure and pH are also suitable for the survival and development of neurons. N2 supplement is an additive that mainly contains transferrin, insulin, progesterone, putrescine and sodium selenite, which can provide some nutrients and growth factors necessary for neuron growth. B27 supplement is a complex additive that contains antioxidants, fatty acids, vitamins and other ingredients. Glutamax is an additive containing L-glutamine. Double antibiotics are a mixture of penicillin and streptomycin, usually containing 100U / mL penicillin and 100μg / mL streptomycin, mainly to prevent bacterial contamination. Penicillin can inhibit the synthesis of bacterial cell walls, while streptomycin mainly interferes with the synthesis of bacterial proteins. The combined use of the two can effectively prevent most common bacteria from growing and reproducing in the culture medium, thereby protecting the purity of the neuronal cell culture system and ensuring that neurons can grow in a relatively sterile environment.

[0048] See also Figure 1 The embodiments of the present application also provide a method for reprogramming astrocytes into functional neurons, using the composition for reprogramming astrocytes into functional neurons as described above in the embodiments of the present application to reprogram astrocytes into functional neurons.

[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 experimental animal strain with the advantages of fast growth, strong fertility, good adaptability to the environment, and relatively docile temperament.

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

[0051] S1. Cultivate astrocytes until they are mature and the confluence of the astrocytes is 60%-80%.

[0052] In some embodiments, the specific steps may include: extracting astrocytes from the brain of newborn SD rats and culturing them for 5-7 days, using immunofluorescence detection to detect that more than 98% of the cells express the astrocyte marker GFAP or S100β, which is considered to be mature astrocytes, and continuing to culture until the fusion degree of astrocytes on the cell plate is 60%-80%. The cell fusion degree in cell culture refers to the proportion of cells that grow adherently and connect to each other in a culture container such as a cell culture dish or culture bottle, occupying the culture surface, usually expressed as a percentage. For example, the fusion degree of astrocytes on the cell plate is 60%, 65%, 70%, 75% or 80%, etc.

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

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

[0055] Specifically, the medium in the culture medium of astrocytes obtained in S1 was discarded by aspiration, and a neuron induction medium containing 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 induction medium containing compound PT109B includes 10 μM 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% double antibody.

[0058] In some embodiments, the neuron induction medium containing compound PT109B further includes thyroid hormone, and the concentration of thyroid hormone is 5-500 nM. The concentration of thyroid hormone in the neuron induction medium containing compound PT109B is 5 nM, 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM or 500 nM, etc.

[0059] In some embodiments, the neuronal induction medium containing compound PT109B includes 10 μM 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% double antibody.

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

[0061] S21. Aspirate and discard the culture medium in the astrocyte culture medium obtained in S1, and add the first culture medium containing the compound PT109B, which is a neuron induction medium, and culture for 1-3 days.

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

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

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

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

[0066] The culture medium in the culture fluid of astrocytes obtained in S1 is discarded, and a first culture medium containing a neuron induction medium of compound PT109B is added and cultured for 1-3 days. The first culture medium containing a neuron induction medium of compound PT109B includes a neuron induction medium containing compound PT109B as described above and a DMEM high-glucose medium, and the volume ratio of the two is 1:1. After 1-3 days of culture, the culture medium containing a higher proportion of the neuron induction medium containing compound PT109B is replaced, and the proportion of the neuron induction medium containing compound PT109B is gradually increased as time goes by, and finally replaced with 100% neuron induction medium, that is, the second culture medium mentioned above.

[0067] In some embodiments, the functional neurons include at least one of neurons with electrophysiological activity and neurons with the function of releasing dopamine.

[0068] The following is further described by means of specific examples.

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

[0070] Example 1

[0071] This example conducts a biological evaluation of PT109B in reprogramming astrocytes into neurons.

[0072] Experimental methods:

[0073] (1) According to the required concentration, PT109B (10 μM), BDNF (10 ng / mL), GDNF (10 ng / mL), b-FGF (10 ng / mL), 1% N2 supplement, 1% B27 supplement, 1% Glutamax and 1% double antibody were added to 50 mL Neurobasl culture medium to prepare a neuron induction culture medium containing compound PT109B. DMEM high glucose culture medium was prepared.

[0074] (2) Primary SD rat astrocytes were cultured in DMEM high-glucose medium for 5-7 days. When more than 98% of the cells expressed astrocyte markers GFAP or S100β using immunofluorescence detection, mature astrocytes were considered to have been cultured. The cells were cultured until the cell confluence reached 60-80%.

[0075] (3) The astrocytes in (2) were cultured continuously. On the first day of culture, the original culture medium was discarded, and the neuron induction culture medium containing compound PT109B and DMEM high glucose culture medium were mixed evenly in a volume ratio of 1:1 and added to the culture dish for culturing astrocytes, replacing the original culture medium, and continuing to culture for 3 days. After 3 days, the original culture medium was discarded, and 100% neuron induction culture medium containing compound PT109B was added to the culture dish for culturing astrocytes, and culture was continued for 90 days to obtain functional neurons. The other culture conditions were the same as in (2). During this period, the cell status was observed and recorded every day, and the culture medium was replaced every 2-3 days according to the cell status.

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

[0077] Primary SD rat astrocytes were treated with PT109B for 1 month according to the methods (1)-(3) and then subjected to immunofluorescence analysis.

[0078] According to the methods (1)-(3), primary SD rat astrocytes were treated with PT109B for 1 month, 2 months, and 3 months, and then the cell electrophysiological tests were performed.

[0079] The cell electrophysiological detection method comprises the following steps:

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

[0081] Artificial cerebrospinal fluid for incubating cells: 122mM NaCl, 2.5mM KCl, 1.2mM NaH 2 PO 4 , 24 mM NaHCO 3 , 12.5 mM D-glucose, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM MgCl 2 and 5 mM HEPES.

[0082] Electrode solution formula: 128mM K-gluconate, 10mM NaCl, 2mM MgCl 2 , 0.5mM EGTA, 10mM HEPES, 0.4mM Na 2 GTP and 4 mM Na 2 ATP.

[0083] ② After preparing the relevant solutions, first place the cultured astrocyte slides in an oxygen-containing medium (95% O 2 and 5% CO 2 ) in artificial cerebrospinal fluid for 30 minutes and transferred to a recording chamber perfused with artificial cerebrospinal fluid for recording at room temperature (25-30°C). Using a HEKA EPC10 dual patch clamp amplifier (HEKA Elektronik, German), glass electrodes (5-8 MOhm) were filled with potassium-containing electrode solution and whole-cell patch clamps were performed on randomly selected cells at a sampling rate of 10 kHz and filtered at 2 kHz. After data acquisition, the collected data were analyzed offline using Clampfit 11 software (MDS Analytical Technologies). For voltage clamp experiments, the voltage was kept at -70 mV.

[0084] Experimental results:

[0085] The test results of cell immunofluorescence technology are as follows Figure 2 As shown, ***P<0.001 compared with the control group (CT group). N=5-6, scale bar=200 μm. Figure 2 As can be seen in A, B, C, and D, in the cells of the PT109B group, TH+ / DAT + and TH + / Nurr1 + The cell morphology was similar to that of neurons, the number increased significantly, and TH + , DAT + , Nurr1 + The proportion of PT109B to total cells is about 20%, suggesting that PT109B can reprogram astrocytes into induced dopamine neurons. Further testing of Synap1, a marker of neuronal synapses, showed that the Synap1 signal increased in cells treated with PT109B (about 80%), and there were structures similar to neuronal synapses in the cells. At the same time, a small number of cells expressed both TH and Synap1 (such as Figure 2 ZhongE and Figure 2 (as shown in F), indicating that synaptic-like structures have grown in the induced dopamine neurons reprogrammed by PT109B. The dopamine content in neurons induced by PT109B increased significantly, further suggesting that PT109B can reprogram astrocytes into functional induced dopamine neurons.

[0086] The whole-cell patch clamp technique was used to detect the characteristics of functional neurons. Figure 3 As shown, from Figure 3 It can be seen that compared with astrocytes (As), the cell morphology of the PT109B group is similar to that of neurons ( Figure 3 A), among which, no obvious action potential or sodium-potassium current was generated in the astrocyte group. However, after 1 month of PT109B treatment, induced single action potentials appeared in As, and the amplitude of sodium-potassium current was low. Compared with the 1-month group, the induced single action potential and sodium-potassium current amplitude of cells in the 2-month PT109B treatment group increased slightly, but the difference was negligible; at the same time, the induced action potential and sodium-potassium current amplitude of the PT109B cultured 3-month group were significantly enhanced compared with other groups, but no sustained action potential appeared ( Figure 3 B, C, and D). The action potential amplitude of neurons induced by PT109B was significantly greater than that of As ( Figure 3 At the same time, compared with As, the sodium current amplitude in neurons treated with PT109B for different time periods increased significantly, the potassium current amplitude decreased, and the cell capacitance decreased significantly ( Figure 3 G and H). The cells were kept alive in vitro for more than 3 months, but the action potentials recorded were all single action potentials ( Figure 3 Middle I).

[0087] Example 2

[0088] This example conducts a biological evaluation of PT109B and T3 in reprogramming astrocytes into neurons.

[0089] Experimental methods:

[0090] (1) PT109B (10 μM), T3 (50 nM), BDNF (10 ng / mL), GDNF (10 ng / mL), b-FGF (10 ng / mL), 1% N2 supplement, 1% B27 supplement, 1% Glutamax and 1% double antibody were added to 50 mL Neurobasl culture medium according to the required concentration to prepare a neuron induction culture medium containing compound PT109B. DMEM high glucose culture medium was prepared.

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

[0092] (3) The astrocytes in (2) were cultured continuously. On the first day of culture, the original culture medium was discarded, and the neuron induction culture medium containing compound PT109B and T3 was mixed with DMEM high glucose culture medium at a volume ratio of 1:1 and then added to the culture dish for culturing astrocytes, replacing the original culture medium, and continuing to culture for 3 days. After 3 days, the original culture medium was discarded, and 100% neuron induction culture medium containing compound PT109B and T3 was added to the culture dish for culturing astrocytes, and culture was continued for 90 days to obtain functional neurons. During this period, the cell status was observed and recorded every day, and the culture medium was replaced every 2-3 days according to the cell status.

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

[0094] The cell electrophysiological detection method comprises the following steps:

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

[0096] Artificial cerebrospinal fluid for incubating cells: 122mM NaCl, 2.5mM KCl, 1.2mM NaH 2 PO 4 , 24 mM NaHCO 3 , 12.5 mM D-glucose, 2 mM CaCl 2 , 1 mM MgSO 4 , 1 mM MgCl 2and 5 mM HEPES.

[0097] Electrode solution formula: 128mM K-gluconate, 10mM NaCl, 2mM MgCl 2 , 0.5mM EGTA, 10mM HEPES, 0.4mM Na 2 GTP and 4 mM Na 2 ATP.

[0098] ② After preparing the relevant solutions, first place the cultured astrocyte slides in an oxygen-containing medium (95% O 2 and 5% CO 2 ) in artificial cerebrospinal fluid for 30 minutes and transferred to a recording chamber perfused with artificial cerebrospinal fluid for recording at room temperature (25-30°C). Using a HEKA EPC10 dual patch clamp amplifier (HEKA Elektronik, German), glass electrodes (5-8 MOhm) were filled with potassium-containing electrode solution and whole-cell patch clamps were performed on randomly selected cells at a sampling rate of 10 kHz and filtered at 2 kHz. After data acquisition, the collected data were analyzed offline using Clampfit 11 software (MDS Analytical Technologies). For voltage clamp experiments, the voltage was kept at -70 mV.

[0099] Experimental results:

[0100] The results of cell electrophysiological tests were as follows Figure 4 As shown, the results showed that when T3 was added to the induction system of PT109B, there were structures similar to neuronal synapses in the cells ( Figure 4 Middle A). It was further found that after PT109B and T3 were combined for 30 days, the proportion of cells with continuous action potentials increased significantly ( Figure 4 Middle B). At the same time, the sodium current amplitude of bipolar or tripolar neurons with small cell bodies increased significantly, and some cells had excitatory postsynaptic electrophysiological characteristics ( Figure 4 On the other hand, continuous action potentials can be detected after T3 and PT109B act on As ( Figure 4 (middle D).

[0101] Immunofluorescence test results Figure 5 As shown, from Figure 5 It can be seen that T3 does not affect the reprogramming efficiency of PT109B and enhances the fluorescence intensity of Na-K-ATPase, which further indicates that T3 can enhance the electrophysiological activity of neurons induced by compound PT109B without affecting the reprogramming effect of compound PT109B, indicating that compound PT109B combined with T3 can reprogram astrocytes into neurons with electrophysiological activity.

[0102] In summary, the composition for reprogramming astrocytes into functional neurons provided in the present application can reprogram astrocytes into functional neurons, effectively reducing the number of small molecule compounds used in chemical reprogramming technology, so that astrocyte reprogramming can be achieved within two molecules, which can not only effectively solve the disadvantages of the large number of small molecules and complicated steps in the current chemical reprogramming technology, but also is conducive to exploring the key mechanism of astrocyte transformation, and providing a theoretical basis for the subsequent development of Parkinson's disease drugs. At the same time, the development of a combined use method with T3 can convert the single action potential of the induced dopaminergic neurons reprogrammed by PT109B alone into multiple action potentials, so that the induced dopaminergic neurons have more physiological functions in vivo.

[0103] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A composition for reprogramming astrocytes into functional neurons, characterized in that: The composition includes compound PT109B.

2. The composition for reprogramming astrocytes into functional neurons according to claim 1, characterized in that: The composition also includes thyroid hormone.

3. The composition for reprogramming astrocytes into functional neurons according to claim 1, characterized in that: The composition also includes at least one of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and fibroblast growth factor.

4. The composition for reprogramming astrocytes into functional neurons according to claim 1, characterized in that: The composition also includes a neuronal induction medium.

5. The composition for reprogramming astrocytes into functional neurons according to claim 4, characterized in that: The neuron induction medium is selected from a DMEM / F12-based medium, a serum-free induction medium, and a PSC neural induction medium.

6. A method for reprogramming astrocytes into functional neurons, characterized in that: Astrocytes are reprogrammed into functional neurons using the composition for reprogramming astrocytes into functional neurons according to any one of claims 1 to 5.

7. The method for reprogramming astrocytes into functional neurons according to claim 6, characterized in that: Includes steps: Cultivate astrocytes until they are mature and reach a confluency of 60%-80%; The mature astrocytes are placed in a culture medium containing the composition for reprogramming astrocytes into functional neurons and continue to be cultured to obtain the functional neurons.

8. The method for reprogramming astrocytes into functional neurons according to claim 7, characterized in that: The step of placing the astrocytes in a culture medium containing the composition for reprogramming astrocytes into functional neurons for continued culture comprises the steps of: placing the astrocytes in a first culture medium containing the composition for reprogramming astrocytes into functional neurons for 1-3 days; placing the astrocytes in a second culture medium containing the composition for reprogramming 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 culture medium, and the volume ratio of the composition for reprogramming astrocytes into functional neurons to the DMEM high-glucose culture medium is 1:1; The second culture medium comprises the composition for reprogramming astrocytes into functional neurons.

9. The method for reprogramming astrocytes into functional neurons according to claim 8, characterized in that: The concentration of the compound PT109B in the composition for reprogramming astrocytes into functional neurons is 5-20 μM; When the composition for reprogramming astrocytes into functional neurons includes thyroid hormone, the concentration of the thyroid hormone is 5-500 nM.

10. The method for reprogramming astrocytes into functional neurons according to claim 6, characterized in that: The functional neurons include at least one of neurons with electrophysiological activity and neurons with the function of releasing dopamine.

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