Method for promoting maturation of neurons and application of mature neurons

Abstract

The invention discloses a method for promoting maturation of neurons and application of mature neurons, and relates to the technical field of biology. The method for promoting neuronal maturation comprises the step of overexpressing a transcription factor NEUROD2 in the process of inducing and differentiating nerve cells by human pluripotent stem cells. Experiments prove that overexpression of the transcription factor NEUROD2 can promote high-level expression of nerve maturation related markers (CAMK2A, MYT1L, NEFH, STX1A, PSD95 or RIM), increase expression of synaptic related proteins (including DLG4 and RIM1) and ion channel related proteins (including SCN2A, SCN2B, KCNK9 and KCNA1), promote maturation of electrophysiological functions and accelerate the maturation speed of differentiated nerve cells. The mature neurons prepared by the method for promoting maturation of the neurons are higher in maturity, and can be applied to preparation of cell drugs for preventing, improving or treating nervous system diseases or construction of nerve-related disease in-vitro models with better simulation degree.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for promoting neuron maturation and the application of mature neurons. Background Art

[0002] Stem cell technology, also known as regenerative medicine technology, refers to the process of isolating, culturing in vitro, and inducing stem cells to breed new, normal, or even younger cells, tissues, or organs in vitro, and ultimately achieve the treatment of clinical diseases through the transplantation of cell tissues or organs. Stem cell technology is the research basis for developmental research, disease modeling, and regenerative medicine, and has opened up new directions for the study of human diseases. Human pluripotent stem cells (hPSCs) have the ability to differentiate into neurons. Compared with mouse models, hPSCs directed differentiation technology provides a pathological and physiological model that is closer to humans for the study of neurodegenerative diseases. At the same time, the transplantation of differentiated neural cells also provides new ideas for the treatment of neurological diseases.

[0003] There are two main methods for in vitro neural differentiation in existing technology: one is environmentally induced stepwise cell differentiation, which simulates the differentiation process of developmental biology and regulates WNT, TGF-β and other known signal pathways in the developmental process through small molecules. This method takes a long time to differentiate (neural differentiation can be seen after 2-3 weeks); the other differentiation method is forward programming induced by transcription factors (TFs), which can see neural differentiation in a short time (4-7 days).

[0004] In addition, human neurodegenerative diseases are closely related to aging and usually occur at a stage of high neural maturity; however, neural cells induced and differentiated in vitro are similar to neural cells before and after fetal birth, and their neural maturity is low. The results of previous in vitro induced differentiation studies showed that although TFs-induced differentiation shortened the cell differentiation time of hPSCs, the process from neural differentiation to neural maturation was not accelerated. Therefore, the long maturation time of neurons in vitro has, to a certain extent, limited their application in disease research and drug development. Summary of the invention

[0005] In order to solve the above problems, the present application provides a method for promoting neuron maturation and the application of mature neurons based on the existing technology.

[0006] In a first aspect, the present application provides a method for promoting neuron maturation, comprising overexpressing the transcription factor NEUROD2 in cells; the CDS base sequence of NEUROD2 is shown in SEQ ID NO.1; and the above cells are human pluripotent stem cells.

[0007] Furthermore, the above-mentioned transcription factors can promote the expression of neural maturation factors, and the above-mentioned neural maturation factors include at least one of CAMK2A, MYT1L, NEFH, STX1A, PSD95 or RIM.

[0008] Furthermore, the above-mentioned transcription factors include homologs, analogs or variants thereof.

[0009] Furthermore, the above method specifically comprises the following steps:

[0010] (1) Overexpression of transcription factors NEUROG1, NEUROG2, and NEUROD1 in human pluripotent stem cells using mRNA, lentivirus, or dox-inducible system to differentiate human pluripotent stem cells into glutamatergic neurons;

[0011] (2) Based on the differentiated glutamatergic neurons, the transcription factor NEUROD2 is overexpressed through mRNA or lentivirus system to obtain more mature glutamatergic neurons.

[0012] In a second aspect, the present application provides a mature neuron prepared by the above-mentioned method for promoting neuron maturation.

[0013] Furthermore, the mature neurons are used to prepare drugs for preventing, improving or treating nervous system diseases.

[0014] Furthermore, the above-mentioned mature neurons are used to construct an in vitro model of the nervous system, conduct pathological research on nervous system-related diseases or develop nervous system-related drugs.

[0015] In some embodiments, the research of the above-mentioned nerve-related diseases includes neurogenesis and nerve regeneration.

[0016] In some embodiments, the development of the above-mentioned nerve-related drugs includes nerve cell transplantation and nerve cell therapy.

[0017] In a third aspect, the present application provides a drug for preventing, ameliorating or treating nervous system diseases, comprising the above-mentioned mature neurons and pharmaceutically acceptable excipients.

[0018] The present application can promote the high-level expression of neural maturation factors such as CAMK2A, MYT1L, NEFH, STX1A, PSD95 or RIM, as well as the increased expression of synapse-related proteins (including DLG4 and RIM1) and ion channel-related proteins (including SCN2A, SCN2B, KCNK9 and KCNA1) in human pluripotent stem cells in vitro by overexpressing the transcription factor NEUROD2, thereby accelerating the maturation of differentiated neurons and promoting the maturation of electrophysiological functions; RNA sequencing results and immunofluorescence results show that the expression of postsynaptic and presynaptic membrane potential proteins DLG4 and RIM1 is increased, and the efficiency of in vitro neural differentiation and maturation is greatly improved.

[0019] The mature neurons prepared by the method of promoting neuron maturation of the present application are more mature and show greater potential in simulating a variety of human neurodegenerative diseases. On the one hand, they can be used to prepare cellular drugs for preventing, improving or treating nervous system diseases, providing new drug options for patients with nervous system diseases; on the other hand, they can be used to construct in vitro models, providing a better disease model screening platform for the development of related drugs, and conducting pathological research on nervous system diseases or the development of nervous system-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Two methods were used to induce neural differentiation in vitro;

[0022] Figure 2 Sampling at different time points for single-cell sequencing;

[0023] Figure 3 For rate analysis of the two differentiation methods;

[0024] Figure 4 Analyze GRN for GENIE3;

[0025] Figure 5 For transcription factor screening;

[0026] Figure 6 The role of overexpression of NEUROD2 in promoting neural maturation was further verified by qPCR, immunofluorescence, electrophysiology, RNA seq, etc. DETAILED DESCRIPTION

[0027] The following will be combined with specific embodiments to clearly and completely describe the technical solution 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 in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0028] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, the reagents involved in the examples of this application are all commercially available products and can be purchased through commercial channels.

[0029] The term "overexpression" means that the content (e.g., expression level) of a transcription factor in a cell exceeds that of an initial cell (a cell into which the exogenous gene has not been transferred); for example, compared to the initial cell, the content is 20% higher, preferably 50% higher; more preferably 100% higher, such as 200%, 300%, 500% or higher. One case of "overexpression" is when a gene encoding an exogenous transcription factor is transferred into a cell and expressed.

[0030] The CDS base sequence of the transcription factor NEUROD2 in this application is shown in Table 1 below:

[0031] Table 1 Transcription factors, sequence numbers and CDS base sequences

[0032]

[0033]

[0034] The term "transcription factor" refers to a polypeptide having a sequence as shown in Table 1 above in the present application. Homologs of transcription factors refer to variant forms having the same function as the polypeptides having the sequences shown in Table 1. These variant forms include, but are not limited to: deletion, insertion and / or substitution of one or more (such as 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition or deletion of one or several (such as within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of polypeptides having sequences as shown in Table 1 below. The variant forms have a premise that they retain the function of the transcription factors shown in Table 1 of the full length.

[0035] The difference between the analogs of transcription factors and natural transcription factors can be differences in amino acid sequence, differences in modification forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids).

[0036] The variant of the transcription factor refers to a variant that hybridizes with the transcription factor encoding gene in Table 1 above and has at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% identity between the two sequences. The present application particularly relates to polynucleotides that can hybridize with the transcription factor polynucleotides described in the present application under stringent conditions.

[0037] According to the present application, the transcription factor or variants or derivatives of its encoding gene are easily obtained and used by those skilled in the art.

[0038] Gene sequences encoding transcription factors can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this application, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain relevant sequences.

[0039] Experimental example: Screening and validation of transcription factor NEUROD2

[0040] This experimental example selected two methods for in vitro neural differentiation: forward programming induced by pioneer transcription factor (3N_TF) and stepwise cell differentiation induced by environment (Dual-Smad).

[0041] First, NEUROG1, NEUROG2 and NEUROD1 mRNAs were synthesized, and these transcription factor mRNAs were transfected into hPSCs cells with lipostem reagent (transfected 3 times), and cultured in a neural differentiation medium (or a cell line with overexpression of NEUROG1, NEUROG2 and NEUROD1 was constructed, and overexpression of NEUROG1, NEUROG2 and NEUROD1 in hPSCs was activated under dox induction, and then cultured in a neural differentiation medium) (called 3N_TF induction). In contrast, a common neural differentiation method was used to induce neuronal differentiation by inhibiting the Dual smad signaling pathway (including TGFβ and BMP) (called Dual-Smad).

[0042] A comparative analysis of these two differentiation methods at the single-cell level is as follows:

[0043] like Figure 1 As shown in the figure, according to the analysis results of single cells, 3N-TF induced neural differentiation significantly faster, showing obvious neural morphology on the fourth day, while dual-Smad differentiation began to gradually show neural morphological cells on day 13. There was a significant difference in the rate of neural differentiation induced by the two differentiation methods. Therefore, we selected different time points for single cell sequencing, such as Figure 2 As shown. Single cell results show that the two differentiation methods have different pathways, and 3N_TF shows a faster differentiation rate, as shown Figure 3 As shown (the redder the color, the faster the rate). Figure 4 As shown, GENIE3 analysis was used to select the subnetwork gene list related to promoting the late stage of neural maturation; Figure 5 As shown, the gene sequence of NEUROD2 was constructed into a plasmid (the corresponding gene sequence is shown in Table 1), and the mRNA corresponding to NEUROD2 was synthesized, or a lentivirus for overexpression of NEUROD2 was constructed for screening by overexpression. Through qPCR detection, it was found that the transcription factor NEUROD2 can increase the expression of neuromaturation-related markers CAMK2A and MYT1L. Subsequently, we detected the expression of other neuromaturation-related markers NEFH, STX1A, PSD95 and RIM, such as Figure 6 As shown, the role of NEUROD2 in promoting neural maturation was further verified by qPCR, immunofluorescence, electrophysiology, etc. Figure 6 -a's qPCR test results showed that NEUROD2 promoted the increase of genes related to neural maturation (CAMK2A, MYT1L, DLG4, RIM1, STX1A, SYN1); Figure 6 -b and Figure 6-c Immunofluorescence results showed that NEUROD2 promoted the expression of synapse-related proteins DLG4 and RIM1; Figure 6 -d and Figure 6 -e Electrophysiological detection results showed that NEUROD2 promoted the enhancement of postsynaptic membrane potential (increased amplitude and faster frequency); (f) RNAseq results showed that NEUROD2 promoted the increase of genes related to neural maturation (neural synaptic development, sodium and potassium ion channels, postsynaptic membrane, presynaptic membrane and other related genes)

[0044] From the above, it can be seen that the present application can promote the high-level expression of neural maturation factors such as CAMK2A, MYT1L, NEFH, STX1A, PSD95 or RIM, as well as the increased expression of synaptic-related proteins (including DLG4 and RIM1) and ion channel-related proteins (including SCN2A, SCN2B, KCNK9 and KCNA1) by overexpressing the transcription factor NEUROD2 in human pluripotent stem cells in vitro, thereby accelerating the maturation rate of differentiated nerve cells, greatly improving the efficiency of in vitro neural differentiation and maturation, and obtaining more mature neurons. The prepared mature neurons can be used to prepare cell drugs for the prevention, improvement or treatment of nervous system diseases, providing new drug options for patients with nervous system diseases; on the other hand, they can be used to construct in vitro models for pathological studies of nervous system-related diseases or the development of nervous system-related drugs.

[0045] The present application is further described above with the aid of specific embodiments, but it should be understood that the specific description here should not be construed as limiting the essence and scope of the present application, and various modifications made to the above embodiments by ordinary technicians in this field after reading this specification are all within the scope of protection of this application.

Claims

1. A method for promoting neuron maturation, characterized in that: The method for promoting neuronal maturation comprises overexpressing the transcription factor NEUROD2 in cells; The CDS base sequence of NEUROD2 is shown in SEQ ID NO.1; The cells are human pluripotent stem cells.

2. The method according to claim 1, characterized in that The transcription factors include homologues, analogs or variants thereof.

3. The method for promoting neuron maturation according to claim 1, characterized in that: The method specifically comprises the following steps: (1) Overexpression of transcription factors NEUROG1, NEUROG2, and NEUROD1 in human pluripotent stem cells using mRNA, lentivirus, or dox-inducible system to differentiate human pluripotent stem cells into glutamatergic neurons; (2) Based on the differentiated glutamatergic neurons, the transcription factor NEUROD2 is overexpressed through mRNA, lentivirus or dox-inducible induction system to obtain more mature glutamatergic neurons.

4. A mature neuron, characterized in that The method is prepared by the promotion method according to any one of claims 1 to 3.

5. The use of mature neurons according to claim 4, characterized in that: Used for preparing medicines to prevent, improve or treat nervous system diseases.

6. The use of mature neurons according to claim 4, characterized in that: Used to construct in vitro models of the nervous system, conduct pathological research on nervous system diseases or develop nervous system-related drugs.

7. The use of mature neurons according to claim 6, characterized in that: The research on nerve-related diseases includes neurogenesis and nerve regeneration.

8. The use of mature neurons according to claim 6, characterized in that: The development of the nerve-related drugs includes nerve cell transplantation and nerve cell therapy.

9. A drug for preventing, improving or treating a nervous system disease, characterized in that: The method comprises the mature neuron according to claim 4, and pharmaceutically acceptable excipients.

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