CN111484977A

CN111484977A - Method for reprogramming to generate functional noradrenergic neuron

Info

Publication number: CN111484977A
Application number: CN201910072527.8A
Authority: CN
Inventors: 程乐平; 李三兰; 饶志萍; 石玉含; 刘月光; 袁嘉成
Original assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2020-08-04
Anticipated expiration: 2039-01-25
Also published as: CN111484977B

Abstract

The invention relates to a method for reprogramming to generate functional noradrenergic neurons. The method of the invention induces differentiated somatic cells in vitro into functional noradrenergic neurons by overexpressing several transcription factors in non-neuronal cells. The noradrenergic neurons produced by the method of the present invention can survive transplantation into the body and can be used for the development of preventive, ameliorative or therapeutic drugs for various nervous system diseases.

Description

Method for reprogramming to generate functional noradrenergic neuron

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for reprogramming to generate functional noradrenergic neurons.

Background

Noradrenergic (NA) neurons are an important class of neuromodulator neurons. Such neurons are involved in the regulation of various physiological activities such as arousal, attention, emotion, and sensation (e.g., pain sensation), and dysfunction thereof is also associated with many nervous system diseases. Research reports indicate that NA neurons are largely lost in the brain of patients with both Parkinson's Disease (PD) and Alzheimer's Disease (AD) as neurodegenerative diseases. The replacement of NA neurons that die in the brain of PD or AD patients with healthy NA neurons would be a promising approach to treat these diseases.

The emerging direct reprogramming technique of one terminally differentiated cell directly into another has made a series of important advances in the directed induction of neurons in recent years. Neurons obtained by direct transdifferentiation mainly include dopaminergic neurons, motor neurons, striatal spiny neurons, peripheral excitatory sensory neurons, serotonin neurons, forebrain inhibitory gabaergic neurons, and the like. The availability of these different types of neuronal cells provides an important source of cells for cell replacement therapy for related diseases. However, the in vitro derivation of noradrenergic neurons by direct reprogramming has not been reported in the art.

The process of direct reprogramming does not go through the stem cell proliferation phase and the process of induction is simpler and shorter in cycle than the process of stem cell induced neural differentiation. Therefore, if NA neurons could be obtained by direct reprogramming in vitro, it would be of great significance for the cell therapy of neurological diseases such as neurodegenerative diseases.

Disclosure of Invention

The invention aims to provide a method for reprogramming to generate functional noradrenergic neurons.

In a first aspect of the invention there is provided a method of producing noradrenergic neurons comprising expressing (including recombinantly expressing or overexpressing) in non-neuronal cells exogenous transcription factors Ascl1, Phox2b, Gata3, Hand2 and one or both of AP-2 α and Nurr1, wherein the Ascl1, Phox2b, Gata3, Hand2, AP-2 α or Nurr1 further comprises a homologue, analogue or variant thereof.

In a preferred embodiment, exogenous transcription factors of the following are also expressed in the cell: phox2a, or a homologue, analogue or variant thereof.

In another preferred embodiment, the non-neuronal cell is a somatic cell.

In another preferred embodiment, the somatic cells include (but may not be limited to): fibroblasts (including embryonic fibroblasts), astrocytes, epithelial cells, blood cells, cells of tissue or organ origin.

In another preferred embodiment, the cell is a mammalian cell; more preferably, the cell is of human origin.

In another preferred embodiment, the method for expressing the exogenous transcription factor in a non-neuronal cell is: (a) providing a cell; (b) transferring the transcription factor into the cell of (a) by using an expression vector or transferring the transcription factor into the cell of (a) by protein transfection; (c) culturing the cells obtained in (b).

In another preferred embodiment, the expression vector is a viral vector or a non-viral vector, including but not limited to: lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, non-viral plasmids.

In another preferred embodiment, the homologue or the analogue of Gata3 is Gata2, or the homologue or the analogue of AP-2 α is AP-2 β.

In another aspect of the invention, there is provided a norepinephrine neuron or a culture thereof obtained by any one of the methods described above.

In a preferred embodiment, the noradrenergic neurons have one or more of the following properties: 1) release of norepinephrine; 2) expressing marker molecules Dbh, Th of noradrenergic neurons; 3) the marker molecules Ddc, Vmat2, Slc6a2, Galanin, Npy, Htr3a, Ret, which express noradrenergic neurons; 4) marker molecules Th, Dbh, Vmat2, ddc, Net, Map2, Npy, Gal, Ret expressing noradrenergic neurons; 5) the marker molecules Synapsin I, Ddc, Vmat2, Net, Galanin and Npy for expressing noradrenergic neurons; 6) possesses the basic electrophysiological characteristics of neurons, including: membrane impedance, resting membrane potential, and the ability to generate an action potential under depolarizing stimuli, and to record sodium current generated by the opening of sodium channels, and to accept inputs from other cells and generate outputs; 7) capable of establishing synaptic connections with and governing the activity of target cells or target organs.

In another aspect of the invention, there is provided the use of the noradrenergic neuron or culture thereof for: preparing a medicament for preventing, improving or treating nervous system diseases (mainly including mental diseases and neurodegenerative diseases, such as nerve injury, autism, depression, schizophrenia, drug addiction, Parkinson's disease, Alzheimer's disease, spinal cord injury and the like); or as an in vitro model, carrying out simulation of nervous system diseases and screening of medicines thereof; or for preparing compositions for in vivo cell transplantation.

In another preferred embodiment, the study of nervous system diseases and drugs thereof comprises: study of drug transport, drug metabolism, nervous system formation; or testing the toxicity of the neuron, screening the toxic substance of the neuron, and screening the substance for regulating the function of the neuron.

In another aspect of the present invention, there is provided a composition comprising: the noradrenergic neuron or the culture thereof, and a pharmaceutically acceptable carrier.

In another aspect of the invention there is provided a combination of transcription factors for use in the production of noradrenergic neurons comprising Ascl1, Phox2b, Gata3, Hand2 and one or both of AP-2 α and Nurr1, wherein the Ascl1, Phox2b, Gata3, Hand2, AP-2 α or Nurr1 further comprises a homologue, analogue or variant thereof, preferably wherein the transcription factor is Phox2a or a homologue, analogue or variant thereof.

In another aspect of the invention, there is provided the use of the transcription factor combination for introducing into a non-neuronal cell to induce it into a noradrenergic neuron.

In another aspect of the present invention, there is provided a kit for producing noradrenergic neurons, which contains the transcription factor combination, or a gene, an expression cassette or an expression vector encoding the transcription factor.

In another preferred embodiment, the kit further comprises one or more selected from the group consisting of: a cell for overexpressing a transcription factor or a homolog, analog, or variant thereof; an expression vector expressing a transcription factor or a homolog, analog or variant thereof; a transformation or transfection reagent; a cell culture medium; a growth factor or supplement comprising: b-27, PS, BDNF, GDNF or FSK.

In another preferred embodiment, the cell culture medium includes, but is not limited to: DMEM, MEM, RPMI, neuronalba sal or Fischer; preferably, said DMEM is selected from: DMEM/F12, Advanced DMEM/F12.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, construction of lentiviral vectors and lentiviral packaging.

FIG. 2, purity characterization of isolated primary cells.

FIG. 3, virus infection of primary cells for induction and factor screening in high performance liquid. A. A schematic of the operational flow; B. analysis of the action of each of the 8 candidate transcription factors; C. further reducing the induction effect after partial transcription factors; D. induction effects of different combinations of transcription factors.

FIG. 4, immunocytochemical staining (A) and real-time fluorescent quantitative PCR (B) identify induced cells.

FIG. 5, single cell patch clamp recordings identify induced cells.

FIG. 6, induced noradrenergic neurons co-cultured with cardiomyocytes.

FIG. 7, induction with the cognate transcription factor Gata2 of the transcription factor Gata3, resulted in similar effects to that of Gata 3.

FIG. 8 shows that the similar effect (A) and the similar statistical chart (B) to those of AP-2 α can be obtained by induction with the family-related transcription factor AP-2 β of the transcription factor AP-2 α.

FIGS. 9A-I, mouse embryonic fibroblasts were also able to be induced to become functional norepinephrine neurons.

FIGS. 10A-I, transplantation of induced noradrenergic neurons.

FIGS. 11A-I, human foreskin fibroblasts were also able to be induced to become functional noradrenergic neurons.

Detailed Description

The inventor has extensively studied, through expressing several transcription factors in non-neuron cell (preferably somatic cell), the differentiated somatic cell is induced into functional Noradrenergic (NA) neuron in vitro, the transcription factor combination includes Ascl1, Phox2b, Phox2a, AP-2 α, Gata3, Hand2 and Nurr1 combination and 1-2 transcription factor combination on the basis of the above.

Term(s) for

As used herein, unless otherwise indicated, the term "cell" or "cell for overexpressing a transcription factor" refers to a wide variety of non-neuronal cells, provided that they are capable of transdifferentiating into noradrenergic neurons upon transfer of the transcription factor. Preferably, the cell is a mammalian somatic cell; for example, the cell is an astrocyte, an epithelial cell, a fibroblast, a skin cell, a blood cell, a nerve cell, an embryonic cell, a tissue or organ derived cell. The genomic sequences of somatic cells are identical and their major components are substantially identical, despite the different somatic cell types, relative to the same individual, which makes it possible for a variety of somatic cells to be used in the present application, provided that they are capable of producing noradrenergic neurons upon transfer of the transcription factors described. In a preferred embodiment of the present invention, the cell is an astrocyte or a fibroblast.

As used herein, the term "overexpression" refers to the level of transcription factor in cells (e.g., expression level) that exceeds the level in the original cells (cells into which the foreign gene has not been introduced); as compared to the starting cells, it is 20% higher, preferably 50% higher; more preferably more than 100%, such as more than 200%, 300%. 500% or more. One case of "overexpression" is the transfer of a gene encoding an exogenous transcription factor into a cell and expression of the gene.

As used herein, a "mammal" is an animal of the Mammalia class (Mammalia) of the phylum Chordata (Chordata) vertebrate subphyla (Vertebrata). The mammals of the present invention include humans, and also non-human mammals. Examples of the non-human mammal include mice, rats, rabbits, dogs, rabbits, monkeys, pigs, cows, sheep, horses, and the like. Whether non-human mammals or humans, are very close in terms of genomic composition, individual development, metabolic patterns, organ anatomy, disease pathogenesis, and the like. During evolution, several key cellular functions or regulatory pathways are conserved between different species, such as signaling pathways for cell proliferation, apoptosis, and the like, are essentially identical in mammals. The cellular senescence pathway is also a conserved regulatory mechanism.

Transcription factor and use

The invention applies transcription factor combinations comprising Ascl1, Phox2b, Phox2a, AP-2 α, Gata3, Hand2 and Nurr1 and transcription factor combinations reduced by 1-2 on the basis, wherein the transcription factors and GenBank accession numbers thereof are shown in Table 1.

TABLE 1

Name of Gene	GenBank accession number
		Ascl1	NM_008553.4
Phox2b	NM_008888.3
		Phox2a	NM_008887.2
AP-2α	NM_011547.4
		Gata3	NM_008091.3
Hand2	NM_010402.4
		Nurr1	NM_001139509.1
Gata2	NM_008090
		AP-2β	NM_009334.3

Therefore, the invention provides a transcription factor combination which comprises Ascl1, Phox2b, Gata3 and Hand2, and one or two selected from AP-2 α and Nurr1, preferably, the transcription factor combination also comprises a transcription factor Phox2 a.

As a preferred mode of the invention, the transcription factor combination comprises five transcription factors, namely Ascl1, Phox2b, Gata3, Hand2 and AP-2 α, or Ascl1, Phox2b, Gata3, Hand2 and Nurr 1.

In the present invention, the term "transcription factor" refers to a polypeptide having a sequence shown in table 1. The term also includes variants having the same function as the polypeptides of the sequences shown in table 1. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (e.g., 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition or deletion of one or several (e.g., up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the polypeptides of the sequences shown in table 1. The variants have the premise that they retain the function of the full-length transcription factor shown in Table 1.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and the like. The invention also provides other polypeptides, such as fusion proteins comprising a polypeptide having a sequence as set forth in table 1, or a fragment thereof. In addition to almost full-length polypeptides, the invention also encompasses soluble fragments of the polypeptides having the sequences shown in Table 1, so long as they retain the function of the full-length transcription factors shown in Table 1.

The induced variants may be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagenizing agent, site-directed mutagenesis, or other known molecular biological techniques, and analogs also include analogs having residues other than the natural L-amino acid (e.g., the D-amino acid), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).

The gene encoding the transcription factor may be a polynucleotide comprising a sequence encoding the transcription factor, or may further comprise additional coding and/or non-coding sequences.

The present invention also relates to variants that hybridize to the above-described transcription factor-encoding gene and have 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, and particularly to polynucleotides that hybridize under stringent conditions to the transcription factor polynucleotides of the present invention, where "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60 ℃, or (2) hybridization with the addition of a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃, or the like, or (3) hybridization only if the identity between the two sequences is at least 90% or more, more preferably 95% or more.

The transcription factor or the variant or derivative of the coding gene thereof is easily obtained and applied by the technicians in the field under the suggestion of the invention.

The gene sequence encoding the transcription factor can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates.

Based on the novel findings of the present invention, there is also provided a kit containing the raw materials or reagents for producing the noradrenergic neurons of the present invention.

As an embodiment of the present invention, there is provided a kit for transdifferentiation to produce noradrenergic neurons, the kit comprising the combination of transcription factors described above.

As one embodiment of the present invention, a kit for transdifferentiation to produce noradrenergic neurons is provided, which contains one or more expression vectors (e.g., viruses) that can express the transcription factors of the above transcription factor combinations, either separately or simultaneously.

More preferably, the kit further comprises one or more selected from the group consisting of: a cell for overexpressing a transcription factor or a homolog, analog, or variant thereof; an expression vector expressing a transcription factor or a homolog, analog or variant thereof; a transformation or transfection reagent; a cell culture medium; a growth factor or supplement comprising: b-27, PS, BDNF, GDNF or FSK. More preferably, the kit further comprises an instruction for use.

Culture method

The present inventors have conducted intensive research and optimization on the transdifferentiation of noradrenergic neurons aiming at these problems, successfully obtained a population containing a high proportion of noradrenergic neurons, and confirmed the authenticity of the obtained neuronal cells by analysis.

The invention discloses a method for inducing the transdifferentiation of non-neuronal cells into noradrenergic neurons, which comprises expressing transcription factors of Ascl1, Phox2b, Gata3 and Hand2 and one or two of AP-2 α and Nurr1 in the non-neuronal cells, and culturing the cells, thereby the non-neuronal cells are transdifferentiated into the noradrenergic neurons.

Methods for overexpressing a foreign gene (in the present invention, a transcription factor) in a cell are well known to those skilled in the art. The polynucleotide sequence encoding the transcription factor may be inserted into a recombinant expression vector, or the transcription factor may also be transferred into cells by protein transfection. The term "recombinant expression vector" refers to a virus (e.g., lentivirus, adenovirus, retrovirus), bacterial plasmid, bacteriophage, yeast plasmid, or other vector well known in the art. Any plasmid or vector can be used as long as it can replicate and stably express in a host. Vectors comprising a polynucleotide sequence encoding a transcription factor, together with appropriate promoter or control sequences, may be used to transform cells so that they are capable of expressing the transcription factor.

As described above, the gene encoding the transcription factor can be introduced into a cell, so that the transcription factor is overexpressed in the cell. Alternatively, the transcription factor protein may be introduced into the cell by expressing the transcription factor protein exogenously and then co-culturing the cell with the transcription factor protein. An alternative approach is for example: the transcription factor protein is fused with a cell penetrating peptide, and the cell penetrating peptide mediates the entry into the cell. The cell penetrating peptide refers to a polypeptide with cell penetrating effect, and the polypeptide itself or fusion protein of the polypeptide and other proteins can enter cells through cell membranes. The cell penetrating peptide comprises: transactivator (TAT), Pennetratin, a peptide based on a signal sequence, pVEC, Transportan, Amphipilicic model peptide, Arg9, and the like.

By applying the culture method, the non-neuronal cells can be trans-differentiated into the noradrenergic neurons through culture and induction of a two-dimensional or three-dimensional culture system.

The noradrenergic neuron obtained by the method can be frozen, recovered, passaged and maintained and cultured for a long time. Furthermore, it is to be understood that the starting cells in the methods of the invention may be isolated primary cells of the organism, or may be cells that are established lines.

Cultured noradrenergic neurons and compositions

Based on the novel findings of the present invention, there is provided a noradrenergic neuron culture obtained by the method of the present invention or a purified noradrenergic neuron isolated from the noradrenergic neuron culture.

Methods for enriching or isolating purified cells from cell cultures are also well known to those skilled in the art, e.g., enrichment can be based on specific morphological characteristics of noradrenergic neurons; or selection for collection based on the particular protein or molecular marker expressed by the noradrenergic neurons (e.g., using a specific antibody or ligand).

The noradrenergic neurons cultured by the invention have a variety of uses. Including but not limited to: for preparing a medicament for preventing, improving or treating nervous system diseases (including mental diseases and neurodegenerative diseases, such as nerve injury, autism, depression, schizophrenia, drug addiction, Parkinson's disease, Alzheimer's disease, spinal cord injury and the like); for use as an in vitro model for the study of neurological diseases and their drugs (e.g., for screening small drug molecules acting on norepinephrine neuronal receptors and transporters); compositions for in vivo cell transplantation are prepared for in vivo studies (e.g., injection into the brain via AAV vector-loaded means), and tested for the ability to induce glial cells in situ in the brain to produce functional NA neurons and for nerve regeneration and functional recovery. Wherein, the research on the nervous system diseases and the medicines thereof comprises but is not limited to: study of drug transport, drug metabolism, nervous system formation; or testing the toxicity of the neuron, screening the toxic substance of the neuron, and screening the substance for regulating the function of the neuron.

If desired, the noradrenergic neurons cultured according to the invention can be further used for genetic engineering recombination to form recombinant cells, for example, to confer further functions or characteristics to the cells, to introduce foreign gene expression cassettes into the cells, to knock out or edit genes in the genome of the cells, and the like.

The present invention also provides a composition (medicament) comprising an effective amount of the noradrenergic neuron (e.g., 1 × 10)⁴-1×10¹²Preferably 1 × 10⁵-1×10¹⁰One); and a pharmaceutically acceptable carrier. Comprising an effective amount of the noradrenergic neurons together with a pharmaceutically acceptable carrier. The composition has no visible toxicity and side effects on animals.

The "effective amount" refers to an amount that is functional or active in humans and/or animals and acceptable to humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.

The invention also provides a kit comprising a cultured noradrenergic neuron of the invention, or a composition or culture comprising the noradrenergic neuron. Preferably, the kit further comprises instructions for use, thereby facilitating the study or clinical use of the kit by a person skilled in the art.

In a specific embodiment of the invention, a potent transcription factor combination for reprogramming to noradrenergic neurons was found by a multifaceted screen. Through various identification, the reprogramming produced noradrenergic neuron is confirmed to be a functional noradrenergic neuron. The detection combining various technical means such as cell biology, biochemistry, electrophysiology and the like shows that: the induced noradrenergic neurons are relatively close to endogenous noradrenergic neurons in vivo. Furthermore, the possibility of reprogramming produced noradrenergic neurons for nerve regeneration and repair was explored by transplantation experiments. Induced noradrenergic neurons were transplanted into the lateral ventricles of neonatal mice and found to survive.

The prior art for generating noradrenergic neurons is mainly induced by pluripotent stem cells, which have a long induction period (one month or more), relatively complicated induction conditions and may involve more ethical problems, especially using human embryonic stem cells. The method adopts a direct reprogramming mode, and has the advantages of short period (the NA neuron with mature function can be induced in three weeks), simple operation (the NA neuron can be obtained by packaging the transcription factor obtained by screening the method into virus for one-time infection), stable efficiency (the functional NA neuron can be stably induced as long as the virus titer and purity are ensured), and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 construction of Lentiviral vectors and Lentiviral packaging

A schematic of the construction of lentiviral vectors and the operational flow of lentiviral packaging is shown in FIG. 1. The cDNA of the candidate transcription factor is cloned to a lentiviral vector FUGW (vector capable of expressing green fluorescent protein), and the sequencing confirms that the cloned cDNA is correct. The resulting viral vector carrying the transcription factor was transformed into E.coli for large scale amplification and virus-packaged in 293T cells co-transfected with two additional viral packaging helper plasmids pCMV-dR8.91(Delta 8.9) and pCMV-VSV-G. And (3) concentrating the lentivirus supernatant by an ultracentrifugation mode, dissolving the obtained virus precipitate, and subpackaging at-80 ℃ for later use.

Example 2 preparation of Primary cells and purity characterization

Burying the experimental mouse in crushed ice for about 5min to freeze and dizzy, and quickly soaking the experimental mouse in 75% alcohol for sterilizing the whole body. The mouse heads were excised and immediately transferred to a 10cm petri dish containing ice-cold dissecting fluid, and the skin of the heads was excised. The separated head was placed on a dissecting scope and the skull removed, the whole brain dissected out and transferred to a new 10cm petri dish containing dissecting fluid. The cerebellum and the cerebrum are removed under the dissecting mirror, the midbrain is reserved, and the dorsal midbrain tissue can be separated by cutting along the medullary canal to two sides. After carefully removing the meninges with a sharp-tipped forceps, the midbrain dorsal head tissue was placed in a new 3.5cm petri dish, washed three times with the dissecting solution in sequence, and finally the tissue was transferred to a clean 3.5cm petri dish. Excess buffer was aspirated off with a micropipette, and brain tissue was subsequently minced to approximately 1mm with a Venus scissors³And (5) small blocks. 6ml of digest (average 1ml per midbrain) was added and the brain tissue was transferred to a 15ml centrifuge tube and digested at 37 ℃ for 15 min. The tubes were mixed by inversion every 5min during this period. The digestion was stopped by adding 6ml of stop solution (equal to the digestion solution), the tissue mass (DNase I was used to prevent thickening, final concentration 5U/ml) was blown about 20 times, and the released cells were passed through a 40 μm cell sieve to remove large tissue masses that were not completely digested. Centrifuging the obtained cell suspension at 1100rpm for 5min, discarding supernatant, adding 10ml of preheated colloid culture solution at 37 deg.C, adding into 10cm culture dish, and adding 5% CO at 37 deg.C₂Culturing for 20min (this step is to further purify the glial cells by utilizing the characteristic that fibroblasts and glial cells have differential adherence). After the differential adherence is finished, cell supernatant in a 10cm culture dish is sucked (the adhered cells are discarded), the cell supernatant is planted into two 25mm culture bottles, the cells are shaken evenly, 5% CO2 is cultured at 37 ℃, and the solution is changed after 3 days. The liquid was changed the next day and every three days thereafter. After 7-9 days of culture, the cells are found to be full, the cells can be placed in a shaker at 37 ℃ (3 layers are wrapped by a fresh-keeping bag to prevent pollution) and shaken at 220rpm for about 20h to remove the oligodendrocytes. After shaking, the cells were washed three more times with pre-warmed DMEM (containing 1% PS), one time with PBS and trypsinized to passage to 2-3 10cm dishes. And continuously culturing for about three days to be used for induction or cryopreservation.

The isolated mouse astrocytes were characterized by immunohistochemistry, and most of the cells were able to express the astrocyte marker molecule, GFAP, as shown in FIG. 2.

Example 3 Virus infection of Primary cells for Induction and factor screening

The inventor considers more factors in the early stage of the experiment and carries out early stage screening on the factors.

Cell culture glass discs were coated with poly-D-lysine (10. mu.g/ml) overnight at 37 ℃ the day before induction and placed in 24-well plates. On the day of induction, coated glass sheets were washed three times with ultrapure water and dried in a super clean bench, and then coated with laminin (10. mu.g/ml) at 37 ℃ for 2-4 h. Cultured glial cells or fibroblasts are then seeded onto 24-well plates at a density per glass slideHole 0.75 × 10⁵A cell. After the cells are plated for 2h, virus infection can be carried out, and the virus dosage is calculated in advance (the infection multiple of each virus is about 10 times of the cell dosage, and can be calculated according to the cell density and the virus titer). After 12-16h of infection, the liquid is completely changed into induction culture liquid (DMEM/F-12, 2% B-27, 1% PS, 10 μm FSK), and after 48h, the liquid is again completely changed into induction culture liquid. And half-amount liquid change is carried out every 3 days, and the culture solution for half-amount liquid change is as follows: DMEM/F-12, 2% B-27, 1% PS, 20ng/ml BDNF, 20ng/ml GDNF, 10 μm FSK. After 2-3 weeks of induction, cells induced by different transcription factor combinations were stimulated with 54mM high potassium cerebrospinal fluid, and the collected supernatants were treated with perchloric acid and loaded in HPLC. According to the result of the high performance liquid chromatography, the levels of noradrenaline released by cells induced by different transcription factor combinations are analyzed and used as important indexes for screening the transcription factor combinations. The schematic diagram of the operation flow is shown in fig. 3A.

In the early stage of the experiment, 8 factors (F8) were determined as the basis for further study through repeated experimental comparison, and they were Ascl1, Tlx3(T3), Phox2B (2B), Phox2A (2A), AP-2 α, Gata3(G3), Hand2(H2) and Nurr1(Nr1), wherein GenBank accession number of Tlx3 was NM-019916.2.

The analysis results showed that several transcription factors other than the transcription factor T3 in the 8 candidate transcription factor libraries were important for inducing the release of norepinephrine, as shown in FIG. 3B.

The present inventors also verified the induction effect after further reducing a part of transcription factors. As a result, it was found that the effect of using only three factors Ascl1, Phox2B (2B), Gata3(G3) (abbreviated as F3 combination) was not significantly different from that of the blank control group (FUGW), i.e., the production of noradrenergic neurons could not be induced, as shown in fig. 3C.

The results found that the production of noradrenergic neurons was hardly induced by only five factors, namely, Ascl1, Phox2B (2B), Phox2A (2A), AP-2 α, Tlx3(T3) (F5 combination), as compared with the blank control (FUGW), as shown in FIG. 3D.

Addition of Gata3 to the F5 combination produced a small number of noradrenergic neurons, but the induction efficiency was significantly lower than the F8 combination (F5 combination plus Gata3(G3), Hand2(H2), and Nurr1), as shown in figure 3D.

The induction efficiency of the F5 combination was also significantly lower than that of the F8 combination, as shown in FIG. 3D, in addition to Hand 2. The simultaneous addition of Gata3 and Hand2 based on the F5 combination resulted in some noradrenergic neurons, but the induction efficiency was still significantly lower than the F8 combination, as shown in fig. 3D.

Example 4 immunocytochemical staining and real-time fluorescent quantitative PCR identification of induced cells

The resulting combination of factors (Ascl1, Phox2b, Phox2A, AP-2 α, Gata3, Hand2, Nurr1) was screened (induction method is the same as in example 3) to induce (using CRISPR/Cas9 in combination with homologous recombination technique, p2A-mCherry was fused with the nucleotide sequence encoding the last amino acid of Dbh gene, and the induced cells were detected by immunocytochemical staining after 2-3 weeks of astrocytes of mouse Dbh-2A-mCherry knocked in 2A-mCherry by homologous recombination at gene Dbh site, specifically by fixing cells at 4% paraformaldehyde at room temperature for 15min, punching at 0.2% Triton X-100 for 10min 1 × PBS 2 times, blocking for 5 min.4% BSA at room temperature for 1h each time, then adding corresponding primary anti-alkaline detergent (PBS) for the second night (12-16 min), punching for 10min, and after further observation of fluorescence labeling with fluorescent hydroxylase (PBS), the fluorescence labeling of cells, and staining the fluorescence labeling the cells at room temperature, and after 2-3 min, the fluorescence labeling of the fluorescence labeling, the fluorescence labeling of cells, the fluorescence labeling, the fluorescence, the.

In addition, RNA from the induced cells was extracted and inverted to cDNA, followed by quantitative fluorescence PCR. It was found that the induced cells expressed mRNA of other marker molecules of NA neurons, such as Ddc, Vmat2, Slc6a2, Galanin, Npy, Htr3a, and mRNA of Ret gene, in addition to Dbh, Th. Compared with the control group infected with FUGW, the NA neurons induced by the infected transcription factor are obviously up-regulated by the marker molecules and are close to the NA neurons endogenous to the mice, as shown in figure 4B.

Example 5 recording of induced cells by Single cell Patch Clamp

The astrocytes of the Dbh-2A-mCherry mice were induced (in the same manner as in example 3) by using the factor combinations (Ascl1, Phox2b, Phox2A, AP-2 α, Gata3, Hand2 and Nurr1) obtained by screening, and the induced mCherry expression-positive cells were recorded by using the single-cell patch clamp technique when the induction was carried out for 3-4 weeks.

The recorded results indicate that induced NA neurons can generate sodium currents (fig. 5A) and can burst action potentials under the effect of depolarizing currents (fig. 5B); in addition, induced NA neurons were also able to generate spontaneous excitatory post-synaptic currents (fig. 5C).

These results demonstrate that the induced NA neurons possess the basic electrophysiological properties of neurons and are able to accept inputs from other cells.

Example 6 Co-culture of induced NA neurons with myocardium

NA neurons (iNAs) induced from astrocytes of Dbh-2A-mCherry mice were co-cultured with neonatal Mouse Ventricular Myocytes (MVM), and observation by immunoelectron microscopy revealed that the induced NA neurons could form synaptic connections with ventricular myocytes, as shown in FIG. 6. The black particles indicated by red arrows are enhanced immunocolloidal gold particles obtained using anti-DsRed antibody, which represents the marker molecule DBH induced by NA neuron expression. Significant aligned myocardial fibers, Z bands (indicated by black arrows), and more mitochondria (M) were observed in cardiomyocytes (fig. 6 left). The right half of fig. 6 is an enlargement of the yellow box on the left, and it can be seen that vesicles (V, indicated by white arrows) are contained within the terminal induced NA neurons, and that the induced NA neurons form structures (indicated by white small triangles) resembling synaptic compact bands with ventricular myocytes (MVMs).

Example 7 Induction of the transcription factor Gata3 analogue Gata2 in place of Gata3 produces a similar effect to Gata3

The present inventors also demonstrated the role of transcription factors of the same family of analogs in inducing NA neurons, as shown in fig. 7, F5 in combination (Ascl1, Phox2b, Phox2A, AP-2 α, Tlx3) plus Gata3(G3) induced mCherry (a marker molecule Dbh representing NA neurons) and positive TH expression noradrenergic neurons by astrocytes of Dbh-2A-mCherry mice compared to the control group FUGW.

The combination of F5 plus Gata2(G2) also induced astrocytes to produce mCherry, a marker molecule DBH for NA neurons, and noradrenergic neurons positive for TH expression, as shown in figure 7. This example demonstrates that Gata2 can be substituted for Gata3 in combination with other transcription factors to induce the production of noradrenergic neurons.

Example 8 Induction of AP-2 β, an analog of the transcription factor AP-2 α, in place of AP-2 α, resulted in similar effects to AP-2 α

The present inventors have also demonstrated the role of the cognate analog of the transcription factor AP-2 α, AP-2 β, in inducing NA neurons, as shown in FIG. 8A, a preferred seven-factor combination comprising AP-2 α (Ascl1, Phox2b, Phox2A, AP-2 α, Gata3, Hand2, Nurr1) is able to induce astrocytes in Dbh-2A-mChery mice to produce large amounts of mCherry and positive TH-expressing noradrenergic neurons.

The same induction was performed after the substitution of AP-2 α in the seven-factor combination with AP-2 β, and the results showed that the seven-factor combination containing AP-2 β also induced astrocytes to produce large numbers of mCheerry and noradrenergic neurons positive for TH expression, there was no significant difference in the efficiency of astrocytes to produce noradrenergic neurons with the combination F7(AP-2 α) and F7(AP-2 β), as shown in the statistical chart of FIG. 8B.

This example demonstrates that AP-2 β can be substituted for AP-2 α in combination with other transcription factors to induce noradrenergic neuronal production.

Example 9 mouse embryonic fibroblasts were induced to produce functional noradrenergic neurons

The present inventors also induced by using Dbh-2A-mCherry mouse embryos fibroblast cells of 14.5 days as starting cells, as shown in FIG. 9A. the transcription factors used were Ascl1, Phox2b, Phox2A, AP-2 α, Gata3, Hand2, Nurr1, i.e., F7 combination.

Induction results as shown in fig. 9B, the F7 combination induced fibroblasts to produce mCherry and TH positive noradrenergic neurons with high efficiency, approximately 42%, compared to the control group, FUGW. These induced noradrenergic neurons also expressed mature neurons and other marker molecules for noradrenergic neurons such as Synapsin I, DDC, VMAT2, NET, Galanin, NPY, as shown in figure 9C. And the noradrenergic neurons produced by the fibroblasts were able to release noradrenaline, as shown in figure 9D. At the same time, the fibroblast-produced noradrenergic neurons possess the basic electrophysiological characteristics of mature neurons, including: when a depolarization voltage is applied, a sodium current can be generated (fig. 9E and 9F), and an action potential can be generated by the depolarization current (fig. 9G); in addition, fibroblast-induced production of noradrenergic neurons was also able to produce postsynaptic hyperpolarizing potentials (fig. 9H) and spontaneous excitatory postsynaptic currents (fig. 9I).

This example demonstrates that the transcription factor combinations screened by the present invention are capable of inducing mouse embryonic fibroblasts into functional noradrenergic neurons.

Example 10 transplantation of induced NA neurons

As shown in FIG. 10A, neuronal-like cells were induced 2 weeks after the combination of Dbh-2A-mCherry mouse astrocytes by digestion of F7 with Accutase, and the supernatant was removed by centrifugation to give a cell concentration density of about 2 × 10⁵cells/. mu.l, 2. mu.l per mouse transplant, 4 × 10 in total⁵A cell. The transplanted site is lateral ventricle of newborn mouse.

The immunohistochemistry results indicated that induced mCherry positive NA neurons could survive near the neonatal mouse ventricle (fig. 10B) and at the mouse cortical rim (fig. 10C) and express the mature neuronal marker molecule NeuN (fig. 10D) and NA neuronal marker molecule TH (fig. 10E).

Further experimental results from brain slice electrophysiological recordings revealed that mCherry-positive induced NA neurons recorded (figure 10F) were able to generate sodium currents given a depolarizing voltage (figure 10G) and burst action potentials under the influence of depolarizing currents (figure 10H).

In addition, transplanted induced noradrenergic neurons were also able to generate spontaneous excitatory postsynaptic potentials (fig. 10I).

The above studies demonstrate that noradrenergic neurons produced by the present invention are capable of surviving transplantation into the ventricles of neonatal mice and have essential electrophysiological functions.

Example 11 human cells can be induced to functional noradrenergic neurons

The present inventors also performed experiments using human-derived cells. In this example, the human-derived cells are foreskin fibroblasts of asian male children. The transcription factor combination used was F7 in combination with the addition of the factor Bcl2l1 with anti-apoptotic effect.

As shown in FIG. 11A, the transcription factor combination can induce human foreskin fibroblasts to express the marker molecules TH and DBH of noradrenergic neurons, with an induction efficiency of about 14% (FIG. 11B).

In addition, human foreskin fibroblast-induced neurons also expressed the marker molecule SYN1 of mature neurons as well as the monoamine vesicle transporter VMAT2 (fig. 11C).

The results of fluorescent quantitative PCR showed that the F7 combination induced neurons expressed not only TH, DBH, VMAT2, but also DDC, NET, MAP2, NPY, GA L, RET compared to the control virus induced cells (fig. 11D).

High performance liquid chromatography results showed that neurons induced by human foreskin fibroblasts were able to release norepinephrine under high potassium stimulation (fig. 11E). And electrophysiological experimental results showed that induced DBH positive cells (fig. 11F) were able to produce sodium currents (fig. 11G), action potentials (fig. 11H) and spontaneous excitatory postsynaptic currents (fig. 11I).

The above studies indicate that human cells can also be induced to produce functional noradrenergic neurons by the superior transcription factor combinations screened by the present invention.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method of producing noradrenergic neurons, comprising expressing in a non-neuronal cell exogenous transcription factors:

ascl1, Phox2b, Gata3, Hand 2; and

one or two selected from AP-2 α and Nurr 1;

wherein the Ascl1, Phox2b, Gata3, Hand2, AP-2 α or Nurr1 further comprises homologues, analogues or variants thereof.

2. The method of claim 1, wherein the following exogenous transcription factors are also expressed in the cell: phox2a, or a homologue, analogue or variant thereof.

3. The method of claim 1, wherein the non-neuronal cell is a somatic cell.

4. The method of claim 3, wherein said somatic cells comprise: fibroblasts, astrocytes, epithelial cells, blood cells, cells of tissue or organ origin.

5. The method of claim 1, wherein the exogenous transcription factor is expressed in a non-neuronal cell by:

(a) providing a cell;

(b) transferring the transcription factor into the cell of (a) by using an expression vector or transferring the transcription factor into the cell of (a) by protein transfection;

(c) culturing the cells obtained in (b).

6. The method of claim 1, wherein the homologue or analogue of Gata3 is Gata2, or the homologue or analogue of AP-2 α is AP-2 β.

7. A noradrenergic neuron, or a culture thereof, obtained by the method of any one of claims 1-6.

8. The noradrenergic neuron or the culture thereof of claim 7, wherein the noradrenergic neuron has one or more of the following properties:

1) release of norepinephrine;

2) expressing marker molecules Dbh, Th of noradrenergic neurons;

3) the marker molecules Ddc, Vmat2, Slc6a2, Galanin, Npy, Htr3a, Ret, which express noradrenergic neurons;

4) marker molecules Th, Dbh, Vmat2, ddc, Net, Map2, Npy, Gal, Ret expressing noradrenergic neurons;

5) the marker molecules Synapsin I, Ddc, Vmat2, Net, Galanin and Npy for expressing noradrenergic neurons;

6) possesses the basic electrophysiological characteristics of neurons, including: membrane impedance, resting membrane potential, and the ability to generate an action potential under depolarizing stimuli, and to record sodium current generated by the opening of sodium channels, and to accept inputs from other cells and generate outputs;

7) capable of establishing synaptic connections with and governing the activity of target cells or target organs.

9. Use of the noradrenergic neurons or cultures thereof of claim 7 or 8 for:

preparing a medicament for preventing, improving or treating nervous system diseases; or

As an in vitro model, simulating nervous system diseases and screening medicines thereof; or

Compositions for in vivo cell transplantation are prepared.

10. A composition, comprising: the noradrenergic neuron or culture thereof of claim 7 or 8, and a pharmaceutically acceptable carrier.

11. A combination of transcription factors for the production of noradrenergic neurons; the following transcription factors are included:

ascl1, Phox2b, Gata3, Hand 2; and

one or two selected from AP-2 α and Nurr 1;

12. The transcription factor combination of claim 11, further comprising a transcription factor: phox2a, or a homologue, analogue or variant thereof.

13. Use of a combination of transcription factors according to claim 11 or 12 for introducing into a non-neuronal cell to induce it into a noradrenergic neuron.

14. The use of claim 13, wherein said non-neuronal cell is a somatic cell; preferably, the somatic cells comprise: fibroblasts, astrocytes, epithelial cells, blood cells, cells of tissue or organ origin.

15. A kit for producing noradrenergic neurons, which contains the transcription factor combination according to claim 11 or 12, or a gene, an expression cassette or an expression vector encoding said transcription factor.

16. The kit of claim 15, further comprising one or more selected from the group consisting of:

a cell for overexpressing a transcription factor or a homolog, analog, or variant thereof;

an expression vector expressing a transcription factor or a homolog, analog or variant thereof;

a transformation or transfection reagent;

a cell culture medium;

a growth factor or supplement comprising: b-27, PS, BDNF, GDNF or FSK.