CN114262688B - Method for preparing inhibitory interneurons retaining age characteristics from non-neural cell transformation - Google Patents

Method for preparing inhibitory interneurons retaining age characteristics from non-neural cell transformation Download PDF

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CN114262688B
CN114262688B CN202011623728.1A CN202011623728A CN114262688B CN 114262688 B CN114262688 B CN 114262688B CN 202011623728 A CN202011623728 A CN 202011623728A CN 114262688 B CN114262688 B CN 114262688B
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刘猛六
柳明杰
沈雁飞
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Ningbo Yisaiteng Biotechnology Co ltd
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Abstract

The present invention relates to a method for preparing inhibitory interneurons from non-neural cell transformations that preserve age and pathological characteristics of the stage of onset. The preparation method optimizes the key cell direct transgenic differentiation gene combination and the promoter for regulating the expression level, packages the viruses which can efficiently infect donor source cells of various ages, adopts a proper coating matrix to promote the adherent growth of the donor source cells and transformed nerve cells, utilizes an induced differentiation culture solution containing small molecular compounds and growth factors which can promote the transdifferentiation, transdifferentiates the differentiation culture solution for a plurality of times within 10-14 days to obtain a large number of inhibitory interneurons, and finally obtains the inhibitory interneurons with high purity through separation and purification.

Description

Method for preparing inhibitory interneurons retaining age characteristics from non-neural cell transformation
Technical Field
The present invention relates to the field of research and treatment of epilepsy, depression, schizophrenia, and more particularly, to a method related thereto for preparing inhibitory interneurons from non-neural cell transformations that preserve age and pathological characteristics of the onset stage.
Background
Epilepsy (epiepsy) is a common neurological disorder due to transient dysfunction of the brain caused by abnormal discharge of neurons of the brain (mainly the temporal lobe), and may cause systemic or local attacks such as sudden mental interruption, loss of consciousness, general cramping, vomiting, urinary incontinence, pale complexion, mydriasis, etc. in patients with attack time of several seconds to tens of seconds. Seizures can cause direct or indirect physical damage and cause great pain and severe mental stress to the patient.
The current phase epilepsy treatment method mainly comprises drug treatment and operation treatment. The research of epileptic drugs is slow, and the drugs which are widely used at present include phenobarbital (beginning to be used in 1912) and phenytoin sodium (beginning to be used in 1938), and the drugs have the defects of good effect, ineffective effect, large side effect and the like of more specific patients although the drugs are used up to now. Clinical surgical treatment is considered a relatively straightforward treatment, but is effective, but is at greater risk, sometimes resulting in serious deficits in the neurological function of the patient's operative field.
Epileptic onset is often associated with abnormalities in inhibitory interneurons (GABAergic interneuron, abbreviated GIN, supra). Abnormalities in inhibitory interneurons affect excitation of their radiating areas and an imbalance in inhibitory nerve signaling, thereby producing symptoms of epilepsy. Abnormalities in interneurons can also lead to clinical symptoms such as depression, schizophrenia, etc.
The adoption of the human-derived inhibitory interneurons in the pathogenesis stage as a cell pathology model has very important significance and considerable market demands for widely, deeply and effectively researching the pathogenesis of nervous system diseases such as epilepsy, depression and the like, screening and developing specific therapeutic drugs, even developing gene therapies and the like.
The obtaining of the nerve cells can be achieved by an animal nerve cell in vitro separation culture technology, but is mainly limited to embryo or primary murine cells, and has the problems of age mismatch, species mismatch, pathological feature mismatch and the like in various applications, and the inhibitory interneurons with higher purity are difficult to separate and obtain. Because of the specificity of the nervous system and the nerve cells (nerve cells cannot proliferate), the separation, preparation and culture of human-derived nerve cells by conventional technical methods often involve ethical and legal problems, and the sources and the number of uses are extremely limited.
Although the induced pluripotent stem cell (induced pluripotent stem cells, iPSC) technology proposed by nobel medicine or physiology winning mountain extension and extension et al in 2012 solves the difficult problem that humanized sub-neural cells cannot be obtained, the method of the iPSC induction technology is complex, the separation and identification process is complicated, the time for obtaining the cells is long (6-7 months), and the cells are reset to embryogenic characteristics in the preparation process, so that key pathological characteristics of the pathogenesis stage which are urgent for pathological model cells of diseases such as epilepsy are lost. Although iPSC induced subtype nerve cells have a large application prospect in the treatment field of neurodegenerative diseases such as AD, PD and the like, due to the limitation of modern technology and the repair characteristics of nerve cells, the application of the iPSC induced subtype nerve cells in repairing neurodegenerative disease injuries in a short period of time has a plurality of unresolved problems. In the fields of mechanism research and new drug screening, the application effect of iPSC-induced nerve cells is yet to be deeply evaluated and verified due to the loss of age in the onset period and key pathological characteristics. Relevant methods for preparing inhibitory interneurons based on iPSC technology can be found in the paper published by Liu et al (Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells, nature Protocols, 2013).
The direct transdifferentiation technology (direct cell reprogramming technology) is an emerging biotechnology that rapidly develops on the basis of iPSC technology, i.e., under specific culture conditions, uses specific gene combinations and the like to promote direct conversion between different cell types. Several specialized subtypes of neurons, including inhibitory interneurons, motor neurons, and dopaminergic neurons, have been directly transdifferentiated from a variety of somatic cells using this technology. The technology has the unique advantages that the generated functional nerve cells of the patient retain the age-related characteristics of the blast cells and the key pathological characteristics of the similar nerve cells in the patient body affected by the disease, so the technology has an attractive application prospect in the aspects of pathological mechanism research, new drug screening, drug research and development and gene therapy development of the disease at the disease stage. Correspondingly, normal human functional nerve cells generated by direct transdifferentiation retain normal physiological function characteristics of parent cells and can be used for toxicity analysis and test of medicines and the like.
However, the general disadvantages of the above techniques are low transformation efficiency, low isolation and purification yields, and difficulty in obtaining large amounts of subtype neurons of high purity. Taking inhibitory interneurons as an example, the direct transdifferentiation technique reported so far can only be used for transforming into inhibitory interneurons from mouse fibroblasts or human embryonic fibroblasts, etc., and the transformation efficiency and the product purity of the process are low. Taking Colasant et al (Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming, cell Stem Cell, 2015) as an example, when the donor-derived cells used are murine embryonic fibroblasts, the transdifferentiation efficiency is less than 15%; the transdifferentiation efficiency of adult mouse fibroblasts is reduced to 9.4%; when human embryonic fibroblasts are used for transformation, the transdifferentiation efficiency is also low (statistical results are not reported) from the stained image, and the extremely low transdifferentiation efficiency is difficult to meet the practical application requirements. In addition, the method of using the mouse embryo cells has a transformation efficiency of 80% according to the prior paper (Conversion of Fibroblasts to Parvalbumin Neurons by One Transcription Factor, ascl1, and the Chemical Compound Forskolin, J Biological Chemistry, 2016) by Shi et al, but the transformation efficiency of adult mouse cells is extremely low (not calculated herein) from the images herein, and the combination method of the genes and the compounds reported herein has been demonstrated to be low in transformation efficiency of human embryo fibroblasts and cannot be used for the transformation of skin cells of postnatal humans by the paper published earlier by the first inventor of the present technology (Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons,Nat Commun.2013;4:2183). See table 1 below for relevant prior art documents and technical details.
In summary, inhibitory interneurons with age at onset and pathological characteristics will be ideal cell models for studying diseases associated with abnormal conditions of inhibitory interneurons such as epilepsy and depression. Despite the great demand, there is no related art and product on the market that can efficiently and highly purity produce inhibitory interneurons in the onset phase of normal humans or patients. There is therefore a great need in the art to develop a method for preparing human inhibitory interneurons with high efficiency and purity.
Disclosure of Invention
In order to solve the technical problems, the invention aims to efficiently convert non-nerve cells such as skin fibroblasts of patients with abnormal diseases of inhibitory interneurons such as epilepsy or normal people into high-purity inhibitory interneurons which retain the age and pathological characteristics of the corresponding onset period by adopting a direct transdifferentiation technology. The functional cells obtained by the direct transdifferentiation technology can retain key pathological characteristics of donor-source cells, so that the functional cells have unique application prospects in the aspects of disease mechanism research and new drug screening research and development.
In accordance with the above objects, in a first aspect, the present invention provides a method for preparing an age-characteristic preserving inhibitory interneuron from non-neural cell transformation, comprising the steps of:
1) Constructing a viral vector containing a cell transgene combination, and carrying out viral packaging by transfecting cells;
2) Culturing donor-derived cells and infecting the cultured donor-derived cells with the packaged virus;
3) Inducing direct conversion and differentiation of the donor-derived cells into inhibitory interneurons in an induced differentiation medium; and
4) Isolating and purifying the resulting inhibitory interneurons,
wherein the cell transdifferentiation gene combination comprises a combination of: (1) at least one gene selected from NGN1, NGN2 and ASCL 1; (2) At least two genes selected from the group consisting of DLX1, DLX2, DLX5, DLX6, LHX5 and LHX 6; and optionally (3) at least one gene selected from SOX4 and SOX 11.
In a further embodiment, the gene is a homologous gene derived from any other species, such as human or mouse.
In further embodiments, the viral vector may be selected from a retroviral vector, a lentiviral vector, or an AAV viral vector, and the promoter in each vector that modulates the expression level may be selected from any one or more of CMV, CAG, EF a, PGK, TRE light, TRE 3G. Preferably, the promoter is selected from CMV.
In further embodiments, the genes may be linked by 2A sequences of different origin (e.g., T2A, E2A, P2A, F2A, etc.) or IRES sequences during construction of the viral vector, and optionally further incorporating green or red fluorescent reporter genes to facilitate determination of virus packaging quality and titer, observation of changes in cell morphology, determination of cell purity, and for subsequent analysis for various specific applications, etc.
In further embodiments, the donor-derived cells may be skin fibroblasts or other non-neural cells of normal humans or patients of any age, such as various stem cells, lung fibroblasts, foreskin fibroblasts, glial cells, and the like. Preferably, the donor-derived cells are skin fibroblasts of a normal human or patient.
In a further embodiment, the culture vessel such as a petri dish is pre-coated during the culturing of donor-derived cells with a suitable coating matrix, which may be selected from at least one of laminin (laminin), gelatin (gelatin), fibronectin (fibronectin), matrigel. Preferably, the coating matrix is Matrigel.
In a further embodiment, the induced differentiation medium contains a combination of a specific transdifferentiated small molecule compound comprising one or more, preferably two or more, of Forskolin (FSK), cAMP, dibutyryl cyclic adenosine monophosphate (DB-cAMP), RA, LDN-193189 (LDN), SB431542, and a growth factor; the growth factor comprises more than one of bFGF2, BDNF and GDNF. Preferably, the small molecule compound is forskolin and/or LDN-193189. Preferably, the growth factor is bFGF2.
In a further embodiment, the isolation and purification is performed by digesting and re-suspending the transdifferentiated cells into a single-cell suspension using digestion methods commonly used in the art (e.g., trypsin, etc.), and isolating the highly purified inhibitory interneurons using cell filters, flow cytometry, or differential adherence to gelatin-coated dishes, etc.
In a second aspect, the invention provides inhibitory interneurons retaining pathological features prepared using the preparation method of the first aspect.
In a third aspect, the present invention provides the use of an inhibitory interneuron as described in the second aspect to retain the disease characteristics of a patient in the establishment of a method for screening for drugs such as epilepsy, depression, etc.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Furthermore, it is to be understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a schematic diagram showing transfection efficiency of 293T cells after cotransfecting packaging plasmids and expression plasmids carrying green fluorescent protein and target genes during viral packaging. After about 45 hours of plasmid transfection in 293T cells, almost all cells expressed higher levels of green fluorescent reporter gene with transfection efficiencies above 90%. The white arrows in the figure show a few untransfected cells. The scale bar is shown as 50 μm. Abbreviations meaning (same below): GFP: green fluorescent protein; BF: bright field images; TFs: a transcription factor.
FIG. 2 is a microscopic schematic of human skin fibroblasts before and after infection with virus. In the skin fibroblasts of adult normal people, after virus infection for 48 hours, most cells show stronger green fluorescence after being cultured in specific induced transdifferentiation liquid for 2 days, which indicates that the expression level of transgene in GFP positive cells is higher, thereby being beneficial to subsequent efficient transdifferentiation into target neurons. The white arrows in the figure show a few uninfected viral or GFP weakly expressed skin fibroblasts. The scale bar is shown as 300 μm. Abbreviations meaning (same below): GIN: inhibitory interneurons.
FIG. 3 is a schematic representation of the induced generation of adult inhibitory interneurons after isolation and purification. During the 10 th to 14 th days after virus infection, most GFP positive cells are completely converted into neurons, and the purity of the neurons reaches more than 95% after the unconverted skin cells are separated and removed. The scale bar is shown as 300 μm.
FIG. 4 is a schematic diagram showing the effect of the coated substrate on conversion efficiency. 4 coating matrices, i.e., laminin (L), gelatin (G), fibronectin (F), matrigel (M), were selected, each coated overnight in different combinations in culture dishes, 5-10 20x fields were randomly selected per group on day 10 of transdifferentiation using a fluorescence microscope, the number of GFP-positive nerve cells in each field was counted, and then the relative conversion relative to the most numerous groups was calculated for comparative analysis. As can be seen, the experimental group (-LGFM) without coated matrix showed little inhibitory interneuron production, whereas the above 4 matrices were coated alone or in combination of ≡2 to achieve different degrees of conversion effect.
FIG. 5 is a schematic diagram showing the effect of a combination of small molecule compounds and growth factors on conversion efficiency. Small molecule compounds such as FSK, RA, LDN, SB431542 (SB) and growth factors such as bFGF2, BDNF and GDNF are selected and added into the induced differentiation culture solution according to different combinations, 5-10 20x fields are randomly selected for each group by a fluorescence microscope on day 12, the number of GFP positive nerve cells in each field is counted, and then the relative conversion rate relative to the most groups of the numbers is calculated for comparison analysis. As can be seen from the graph, the conversion efficiency is the highest when the above components are added into the induced differentiation culture solution, and the effect on the conversion efficiency is the greatest after FSK, LDN or bFGF2 is removed.
FIG. 6 is a schematic representation of the induced expression of the general neuronal signature protein Tuj1 by human inhibitory interneurons. Wherein more than 99% of GFP positive cells simultaneously and specifically express the general neuronal characteristic protein Tuj1. The scale bar is shown as 300 μm
FIG. 7 is a schematic representation of the induced expression of general neuronal signature protein MAP2 by human inhibitory interneurons. Wherein more than 99% of GFP and Tuj1 positive cells simultaneously and specifically express general neuron characteristic protein MAP2. The scale bar is shown as 50 μm.
FIG. 8 is a schematic representation of the induction of human inhibitory interneuron-expressed neuronal synaptoprotein SYN1. GFP and Tuj1 double positive cells are transforming human inhibitory interneurons that markedly express the neurite SYN1. The scale bar is shown as 20 μm.
FIG. 9 is a schematic representation of the human inhibitory interneuron production inhibitory neurotransmitter GABA induced. GFP and Tuj1 double positive cells are human inhibitory interneurons transformed to produce significantly the inhibitory neurotransmitter GABA. The scale bar is shown as 20 μm.
FIG. 10 is a schematic representation of the induced human inhibitory interneurons expressing the inhibitory interneuron-characteristic proteins GAD67 and VGAT. Wherein: a) The human inhibitory interneurons generated by the transformation specifically expressed GAD67, indicating that they already have in their cell bodies the key enzymes for GABA production; b) The human inhibitory interneurons generated by transformation specifically express VGAT, indicating that the cell bodies and neurites possess key proteins involved in the transport and release of the neurotransmitter GABA. GFP/Tuj1 double positive cells are human inhibitory interneurons generated by transformation. The scale bar is shown as 20 μm.
FIG. 11 shows the sequence of human NGN1 gene and protein. The NGN1 homologous genes of mice or other species have similar effects in the direct transdifferentiation production method of the present invention.
FIG. 12 shows the sequence of human NGN2 gene and protein. The mouse or other species of NGN2 homologous genes also have similar effects in the direct transdifferentiation production process of the present invention.
FIG. 13 shows the sequence of human ASCL1 gene and protein. The ASCL1 homologous genes of mice or other species have similar effects in the direct transdifferentiation preparation method of the present invention.
FIG. 14 shows the sequence of human SOX4 gene and protein. The SOX4 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 15 shows the sequence of human SOX11 gene and protein. The SOX11 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 16 shows the human DLX1 gene and protein sequence. The DLX1 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 17 shows the human DLX2 gene and protein sequence. The DLX2 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 18 shows the human DLX5 gene and protein sequence. The DLX5 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 19 shows the human DLX6 gene and protein sequence. The DLX6 homologous gene of mice or other species has similar effects in the direct transdifferentiation production method of the present invention.
FIG. 20 shows the sequence of human LHX5 gene and protein. The LHX5 homologous genes of mice or other species have similar effects in the direct transdifferentiation preparation method of the present invention.
FIG. 21 shows the sequence of human LHX6 gene and protein. The LHX6 homologous genes of mice or other species have similar effects in the direct transdifferentiation preparation method of the present invention.
Detailed Description
In order to prepare the inhibitory interneurons retaining pathological characteristics, the invention introduces key gene combinations capable of determining the fate of nerve cells and subtypes thereof into donor source cells through viruses according to the technical principle of direct transdifferentiation, and induces transformation under proper transformation culture conditions to generate functional inhibitory interneurons.
In particular, the present invention provides a method for preparing inhibitory interneurons retaining pathological features from non-neural cell transformations comprising the steps of:
1) Constructing a viral vector containing a cell transgene combination, and carrying out viral packaging by transfecting cells;
2) Culturing donor-derived cells and infecting the cultured donor-derived cells with the packaged virus;
3) Inducing direct conversion and differentiation of the donor-derived cells into inhibitory interneurons in an induced differentiation medium; and
4) Isolating and purifying the resulting inhibitory interneurons,
wherein the cell transdifferentiation gene combination comprises a combination of: (1) at least one gene selected from NGN1, NGN2 and ASCL 1; (2) At least two genes selected from the group consisting of DLX1, DLX2, DLX5, DLX6, LHX5 and LHX 6; and optionally (3) at least one gene selected from SOX4 and SOX 11.
Among them, NGN1, NGN2 and ASCL1 genes play a decisive role in differentiation of different types of nerve cells during development of the nervous system under the synergistic effect of SOX4 or SOX11, whereas six genes DLX1, DLX2, DLX5, DLX6, LHX5 and LHX6 play a decisive role in the specialization of specific subtype inhibitory interneurons. Various effective combinations of the above genes may induce the production of inhibitory interneurons according to the present invention. It is important to emphasize that SOX4 or SOX11, which exert a synergistic effect in the above-described gene combinations, can significantly improve the conversion efficiency to 70% or more, which is unexpected.
The above genes and their protein sequences can be seen in the nucleotide and amino acid sequence tables of the specification and figures 11-21 of the accompanying drawings.
For the preparation method, the actual preparation application can specifically comprise the following steps:
a. the above genes are respectively constructed into commercial or own retrovirus vectors, lentiviral vectors or AAV virus vectors, and the promoter for regulating the expression level in each vector can be any one or more of CMV, CAG, EF1 alpha, PGK, TRE light and TRE 3G. The genes can also be linked by a 2A sequence (e.g., T2A, E2A, P2A, F2A, etc.) or IRES sequence of different origin, and optionally further introduced with a green or red fluorescent reporter gene to facilitate determination of virus packaging quality and titer, observation of changes in cell morphology, determination of cell purity, and analysis for various subsequent specific applications.
b. The gene vector is respectively packed into corresponding retrovirus, slow virus or AAV virus through cell transfection, the titer of various viruses is measured, and the virus dosage of the infected donor source cells is determined, so that the infection rate of various viruses reaches 50-100%.
c. Donor-derived cells are seeded at an appropriate density in culture dishes pre-coated with at least one of laminin, gelatin, fibronectin, matrigel. The donor-derived cells may be skin fibroblasts or other non-neural cells of normal human or patient of any age, such as various stem cells, lung fibroblasts, foreskin fibroblasts, glial cells, etc. Preferably, the donor-derived cells are skin fibroblasts of a normal human or patient.
d. An appropriate amount of virus is added for infection and after a period of time (e.g., 24 hours) replaced with fresh source cell culture broth.
e. Determining a specific induced differentiation medium containing a small molecule compound that promotes transdifferentiation and a growth factor: to a DMEM/F12/Neurobasal (1:1:1 or 2:2:1) base solution containing 0.5% -2% B27 and 0.5% -2% N2, one or more of 1-20 mu M FSK,0.1-5 mu M RA,0.1-1 mu M LDN,1-10 mu M SB431542 and one or more of 1-200ng/mL bFGF2, BDNF and GDNF are added.
f. The differentiation inducing culture medium is replaced periodically (e.g., on days 3, 5, 7, 10; or daily; or at other different intervals) after infection of the donor-derived cells with the virus.
g. Inhibitory interneurons with fluorescence were isolated and purified on days 10-14, and total cells and nerve cells were counted separately using fluorescence microscopy to determine their purity.
h. And (3) inoculating the partially purified inhibitory interneurons into a culture dish which is coated in advance, continuously culturing with inhibitory interneuron cell culture solution for identification, and storing the rest of the cells in a frozen stock solution suitable for nerve cells for storage and transportation.
i. Characterization of the inhibitory interneurons prepared above may include, for example, the following:
A. Characteristic proteins expressed by general neurons: tuj1, MAP2, neuN, tau, etc.;
B. neuronal synapse proteins: sydapsin 1 (SYN 1);
C. inhibitory interneuron-specific proteins GABA, GAD67, VGAT, etc.
It should be noted that the above specific preparation method is only for better illustrating the present invention, and not limiting the scope of the present invention. The person skilled in the art can selectively adjust the order of some of the steps according to the actual preparation requirements and omit some of the steps. Furthermore, it will be apparent to those skilled in the art that reagents, time, concentration, and other parameters in the respective steps may be appropriately adjusted according to the actual circumstances.
From the above, the preparation method optimizes the key direct cell transdifferentiation gene combination and the promoter for regulating the expression level, packages viruses capable of efficiently infecting various donor cells, adopts a proper coating matrix to promote the adherent growth of the donor cells and transformed nerve cells, utilizes an induced differentiation culture solution containing small molecular compounds capable of promoting transdifferentiation and growth factors, transdifferentiates the differentiation culture solution for a plurality of times within 10-14 days to obtain a large number of inhibitory interneurons, and finally obtains the inhibitory interneurons with high purity through separation and purification.
The unique advantages of the preparation method of the invention are listed as follows:
1. the method is simple: after the donor-derived cells are infected with viruses, the induced differentiation culture solution is only required to be replaced for a plurality of times (for example, 4 times), and complicated operation is not required;
2. and (3) quick: a large number of target products can be obtained only by 10-14 days;
3. the cost is low: compared with the iPSC technology, the need for long-term daily replacement of expensive culture broth (the culture broth of iPSC is very expensive);
4. the conversion efficiency is high, and the conversion efficiency of various skin cells infected by virus (GFP positive) is as high as more than 90 percent;
5. the yield is high: millions of purified inhibitory interneurons can be obtained with only 1-2 100mm dishes;
6. the separation and purification are simple, the product purity is high, and the purity of the purified inhibitory interneuron cells can reach more than 90%;
7. the prepared inhibitory interneuron cell accords with the age and pathological characteristics of epileptic in the onset period;
8. the prepared inhibitory interneuron cells can be effectively frozen and recovered, the liquid nitrogen frozen and stored for more than half a year, and the recovery survival rate reaches 90%;
9. the obtained inhibitory interneuron cells can be cultured for a short period or a long period, and can be cultured for more than 3 months at maximum;
10. the prepared inhibitory interneuron cell is suitable for being used as a cytopathology model, researching pathogenesis, drug action mechanism, screening and developing drugs and developing gene therapy, or used for testing toxicity of compounds and the like;
11. The method can be used for quickly establishing an inhibitory interneuron cell library of large sample normal and patient population and is used for supporting large data analysis and accurate therapy development.
The invention will be further illustrated with reference to specific examples.
Examples
Example 1 rapid and efficient preparation of highly pure inhibitory interneurons from human dermal fibroblasts
1. Material
1) And (3) cells: 293T cells for viral packaging were purchased from the United states ATCC (CRL-3216); human skin fibroblasts were purchased from U.S. Sciencell (cat# 2320). 293T and human skin fibroblasts were cultured in high-sugar DMEM medium containing 10-20% FBS and 1 XP/S double antibody.
2) Instrument and reagent:
CO2 cell incubator (ESCO CLM-170B-8-CN); ultra clean bench (ESCO AC2-5S 1); fluorescence microscopy (Thermo EVOS M5000); ultra-low temperature refrigerator (sea DW-86L 388J); liquid nitrogen tank (sea YDS-175-216-F); a high-speed cryocentrifuge (ocean BY-R20 type); a normal temperature high speed centrifuge (Hunan instrument H1650-W);
B. an intelligent high-pressure steam sterilizer (Shanghai Shen An LDZM-80 KCS); SHELLAB drying oven (CE 3F-2) in the United states; electric heating digital display constant temperature water bath (Lichen HH-2);
BIO-RAD gradient PCR instrument (T100); BIO-RAD electrophoresis apparatus (PowerPac HC); chemiluminescent imaging systems (commute chemicope 6200 Touch); ultraviolet tapping analyzer (junyi JY 02); a water-proof incubator (Shanghai-constant GHP-9080); shaking incubator (ZQTY-70N, know Chu instrument);
Dmem, F12 (Hyclone); FBS, neurobasal, B27, N2, trypsin, DMSO (Invitrogen); gelatin, fibronectin, laminin, matrigel (BD); forskolin, RA, LDN-193189.HCl, SB431542 (Selleck); bFGF2, BDNF, GDNF (Peprotech); lipofectamine2000 (Invitrogen); PEI (Polysciences); a series of DNA restriction endonucleases (Neb), QIAGEN Plasmid Midi kit (100), zymoclean Gel DNA recovery kit, 60mm Petri dish, 100mm Petri dish, 24-MTP,48-MTP,96-MTP, cell cryopreservation tube (Corning); other chemical reagents (Sigma), and the like.
2. Preparation method
1) Constructing a plasmid: ASCL1, DLX2, LHX6 and SOX11 genes were constructed into the respective geneSlow downThe virus vector adopts CMV as a promoter for regulating the expression level of genes in the vector. Meanwhile, a green fluorescent reporter gene is introduced through an IRES sequence, so that the virus packaging quality and titer can be conveniently determined, the change of cell morphology can be observed, and the cell purity can be determined and used for various subsequent specific applications.
2) And (3) virus packaging: the plasmids carrying the genes are mixed with pRSV, pMDL and pVSV-G according to the common proportion in the field and a transfection reagent PEI, the mixture is added into 293T cells which are pre-planted before 24 hours for overnight transfection, fresh culture solution is replaced, supernatant containing viruses is collected after 24 hours and added into the fresh culture solution, the collection is repeated once after 24 hours, dead cells and fragments thereof are removed by a filter with 0.45 mu m after combining the two virus solutions, the final concentration of the condensed amine is 1-12 mu G/mL, a small amount of virus titer is measured, and the rest is stored in a refrigerator with the temperature of 4 ℃ for standby. Plasmid transfection efficiency can be obtained by randomly selecting 5-10 20x fields, counting the total number of cells and GFP-positive number of cells in each field, calculating the proportion of GFP-positive cells in each field, and taking the average value. Plasmid transfection efficiency in 293T cells was as high as 90% for about 45 hours, and high titers of virus could be ensured, see FIG. 1 of the drawings.
3) Skin fibroblast culture and infection: matrigel was diluted with DMEM or PBS at any ratio between 1:50 and 1:2000 and 0.1-20 μg/mL laminin was added to pre-coat the dishes overnight. The selection of the coating matrix is shown in figure 4 of the accompanying drawings. The skin fibroblasts were then pre-seeded in culture dishes at the appropriate density overnight. Adding a proper amount of virus carrying genes into a skin fibroblast culture solution, mixing uniformly, culturing in an incubator for overnight, directly sucking the virus-containing culture solution into a waste liquid bottle containing a disinfectant, and rapidly and carefully adding fresh skin fibroblast culture solution into a cell culture dish along the wall. The viral infection rate can be obtained by randomly selecting 5 to 10 20x fields, counting the total number of cells and the number of GFP-positive cells in each field, calculating the proportion of GFP-positive cells in each field, and then taking an average value. By adjusting the amount of virus in adult or aged skin fibroblasts, the rate of virus infection can be as high as 70% or more in about 48-96 hours, see figure 2 of the drawings, thereby ensuring that the virus-infected cells can be efficiently transformed into inhibitory interneurons.
4) Direct induced transdifferentiation 48 hours after infection, the culture broth was directly sucked into a waste liquid bottle containing a disinfectant, and an induced differentiation culture broth containing a small molecular compound and a growth factor for promoting transdifferentiation was carefully added along the wall in a cell culture dish, and the selection of the small molecular compound and the growth factor was shown in FIG. 5. Then changing liquid at intervals of 1-2 days. The composition of the induced differentiation culture solution is as follows: to a DMEM/F12/Neurobasal (1:1:1 or 2:2:1) base solution containing 0.5% -2% B27, 0.5% -2% N2, 1-20. Mu.M FSK, 0.1-5. Mu.M RA, 1-10. Mu.M SB431542 and 0.1-1. Mu.M LDN, and 1-200ng/mL bFGF2, BDNF and GDNF were added.
5) Isolation and purification of inhibitory interneurons: a large amount of transformed inhibitory interneurons can be seen around 10 days, and are more clearly observed under a fluorescence microscope. The separation and purification were performed using a cell filter and differential adherence to gelatin-coated dishes. The purified inhibitory interneurons were counted by a cell counter and a fluorescence microscope to obtain total cell numbers and GFP-positive neuronal cell numbers, respectively, and the purity was as high as 99% and the conversion efficiency was as high as 90% by repeating the calculation three times, see FIG. 3 of the accompanying drawings. A small amount of purified inhibitory interneurons were inoculated into a pre-coated petri dish and continued to be cultured with inhibitory interneuron cell culture medium for identification.
6) Freezing and transporting: according to the conventional cell cryopreservation method, 50 ten thousand or 100 ten thousand cells per tube of inhibitory interneurons are cryopreserved in a special cryopreservation solution suitable for nerve cells for storage and transportation.
3. Characterization of
At a suitable time (e.g., 15dpi, 20dpi, or any other time during incubation), the mouse cortical glial cells were fixed with 4% PFA at room temperature for 10-20 minutes, and then blocked with PBS buffer containing 0.1-0.2% Triton X-100 and 3-5% BSA for about 0.5-2 hours. The antibodies to the characteristic proteins to be analyzed are then diluted in the same buffer, added to the cells and incubated overnight at 4℃for 2-3 times, after washing for 5 minutes, the diluted corresponding secondary antibodies with fluorescein are added, incubated at room temperature for 0.5-1 hour, after washing for 2-3 times for 5 minutes, and then treated with a slide seal. The presence or absence of the expression of general neuronal characteristic proteins (Tuj 1 and MAP 2), the neurite protein (SYN 1), the inhibitory interneuron characteristic proteins (GABA, GAD67, VGAT) and the like were analyzed by using an EVOS fluorescence microscope or a confocal fluorescence microscope. The antibody sources and dilutions are shown in Table 2, and the analysis results are shown in FIG. 6 (Tuj 1), FIG. 7 (MAP 2), FIG. 8 (SYN 1), FIG. 9 (GABA) and FIG. 10 (GAD 67 and VGAT), respectively. The above results indicate that the inhibitory interneurons prepared by the technique of example 1 expressed not only the characteristic proteins of the general neurons, the neurite proteins, but also the characteristic proteins of the inhibitory interneurons specifically.
Example 2 rapid and efficient preparation of highly pure inhibitory interneurons from human embryonic lung fibroblasts
1. Material
Human embryonic lung fibroblasts MRC-5 were purchased from Qiao Xin boat biotechnology Co., ltd (ZQ 0006) in Shanghai. The rest of the materials are the same as in example 1 (slightly).
2. Preparation method
1) Constructing a plasmid: ASCL1, DLX1, LHX6 and SOX4 genes are respectively constructed into a retrovirus vector, and a CMV is adopted as a promoter for regulating the expression level of the genes in the vector. Meanwhile, a green fluorescent reporter gene is introduced through a T2A sequence, so that the virus packaging quality and titer can be conveniently determined, the change of cell morphology can be observed, and the cell purity can be determined and used for various subsequent specific applications.
2) And (3) virus packaging: mixing plasmids carrying the genes with pGP and pVSV-G according to the common proportion in the field, transfecting the plasmids with a transfection reagent Lipofectamine2000 overnight, adding the pre-seeded 293T cells before 24 hours, replacing fresh culture solution, collecting supernatant containing viruses after 24 hours, adding the fresh culture solution, repeating the collection once after 24 hours, combining the two virus solutions, filtering to remove dead cells and fragments thereof by a filter with the thickness of 0.45 mu m, adding condensed amine to the final concentration of 1-12 mu G/mL, measuring the virus titer by a small amount, and storing the rest in a refrigerator with the temperature of 4 ℃ for standby. Plasmid transfection efficiency can be obtained by randomly selecting 5-10 20x fields, counting the total number of cells and GFP-positive number of cells in each field, calculating the proportion of GFP-positive cells in each field, and taking the average value. Plasmid transfection efficiency in 293T cells was as high as 90% around 45 hours.
3) MRC-5 fibroblast culture and infection: the dishes were pre-coated overnight with 0.1-20. Mu.g/mL laminin and fibronectin. MRC-5 fibroblasts were then pre-seeded in culture dishes at appropriate densities overnight. Adding a proper amount of virus carrying genes into MRC-5 fibroblast culture solution, mixing uniformly, culturing in an incubator for overnight, directly sucking the virus-containing culture solution into a waste liquid bottle containing disinfectant, and rapidly and carefully adding fresh MRC-5 fibroblast culture solution into a cell culture dish along the wall. The viral infection rate can be obtained by randomly selecting 5 to 10 20x fields, counting the total number of cells and the number of GFP-positive cells in each field, calculating the proportion of GFP-positive cells in each field, and then taking an average value. By adjusting the amount of virus in MRC-5 fibroblasts, the virus infection rate can be as high as 70% in about 48-96 hours.
4) Directly inducing transdifferentiation, namely directly sucking the culture solution into a waste liquid bottle containing a disinfectant after 48 hours of infection, and carefully adding an induced differentiation culture solution containing a small molecular compound for promoting transdifferentiation and a growth factor along the wall of a cell culture dish. Then changing liquid at intervals of 1-2 days. The composition of the induced differentiation culture solution is as follows: to a DMEM/F12/Neurobasal (1:1:1 or 2:2:1) base solution containing 0.5% -2% B27, 0.5% -2% N2, 1-20. Mu.M FSK and 0.1-1. Mu.M LDN, and 1-200ng/mL GDNF were added.
5) Isolation and purification of inhibitory interneurons: a large amount of transformed inhibitory interneurons can be seen around 10 days, and are more clearly observed under a fluorescence microscope. The separation and purification were performed using a cell filter and differential adherence to gelatin-coated dishes. The purified inhibitory interneurons are counted by a cell counter and a fluorescence microscope respectively to obtain the total cell number and GFP positive nerve cell number, and the purity is up to 90% and the conversion efficiency is up to 90% by repeating the calculation for three times. A small amount of purified inhibitory interneurons were inoculated into a pre-coated petri dish and continued to be cultured with inhibitory interneuron cell culture medium for identification.
3. Characterization of
The presence or absence of the expression of general neuronal characteristic proteins (Tuj 1 and MAP 2), neuronal synaptic proteins (SYN 1), inhibitory interneuron characteristic proteins (GABA, GAD67, VGAT) and the like in the obtained inhibitory interneurons was analyzed by the same method as in example 1 using an EVOS fluorescence microscope or a confocal fluorescence microscope. The analysis results also show that the inhibitory interneurons prepared by the technique of example 2 express not only characteristic proteins of general neurons, but also characteristic proteins of inhibitory interneurons specifically.
Example 3 rapid and efficient preparation of highly pure inhibitory interneurons from human foreskin fibroblasts
1. Material
Human foreskin fibroblast BJ (CRL-2522) was purchased from ATCC, USA. The rest of the materials are the same as in example 1 (slightly).
2. Preparation method
1) Constructing a plasmid: the NGN1, ASCL1, DLX2, DLX6, LHX5 and SOX11 genes are respectively constructed into slow virus vectors, CMV is adopted as a promoter for regulating the expression level of the NGN1 and ASCL1 genes in the vectors, and PGK is adopted as a promoter for regulating the expression level of other genes. Meanwhile, a green fluorescent reporter gene is introduced before an ASCL1 gene stop codon through a T2A sequence so as to determine the virus packaging quality and titer, observe the change of cell morphology, determine the cell purity and be used for various subsequent specific applications.
2) And (3) virus packaging: the plasmids carrying the genes are mixed with pRSV, pMDLg and pVSV-G according to the common proportion in the field and a transfection reagent PEI, the mixture is added into 293T cells which are prespecified before 24 hours for overnight transfection, fresh culture solution is replaced, supernatant containing viruses is collected after 24 hours and added into the fresh culture solution, the collection is repeated once after 24 hours, dead cells and fragments thereof are removed by filtration through a 0.45 mu m filter after combining the two virus solutions, the final concentration of the condensed amine is 1-12 mu G/mL, a small amount of virus titer is measured, and the rest is stored in a refrigerator at 4 ℃ for standby. Plasmid transfection efficiency can be obtained by randomly selecting 5-10 20x fields, counting the total number of cells and GFP-positive number of cells in each field, calculating the proportion of GFP-positive cells in each field, and taking the average value. Plasmid transfection efficiency in 293T cells was as high as 90% around 45 hours.
3) BJ fibroblast culture and infection: matrigel was first diluted with DMEM or PBS at any ratio between 1:50 and 1:2000 and pre-coated onto dishes overnight. BJ fibroblasts were then pre-seeded in dishes at appropriate densities overnight. Adding a proper amount of virus carrying genes into BJ fibroblast culture solution, mixing uniformly, culturing in an incubator for overnight, directly sucking the virus-containing culture solution into a waste liquid bottle containing disinfectant, and rapidly and carefully adding fresh BJ fibroblast culture solution along the wall in a cell culture dish. The viral infection rate can be obtained by randomly selecting 5 to 10 20x fields, counting the total number of cells and the number of GFP-positive cells in each field, calculating the proportion of GFP-positive cells in each field, and then taking an average value. By adjusting the amount of virus in BJ fibroblasts, the virus infection rate can be as high as 70% in about 48-96 hours.
4) Directly inducing transdifferentiation, namely directly sucking the culture solution into a waste liquid bottle containing a disinfectant after 48 hours of infection, and carefully adding an induced differentiation culture solution containing a small molecular compound for promoting transdifferentiation and a growth factor along the wall of a cell culture dish. Then changing liquid at intervals of 1-2 days. The composition of the induced differentiation culture solution is as follows: to a DMEM/F12/Neurobasal (1:1:1 or 2:2:1) base solution containing 0.5% -2% B27, 0.5% -2% N2, 1-20. Mu.M FSK and 0.1-1. Mu.M LDN, and 1-200ng/mL bFGF2, BDNF and GDNF were added.
5) Isolation and purification of inhibitory interneurons: a large amount of transformed inhibitory interneurons can be seen around 10 days, and are more clearly observed under a fluorescence microscope. And (3) separating and purifying by using a cell filter and a flow cytometer. The purified inhibitory interneurons are counted by a cell counter and a fluorescence microscope respectively to obtain the total cell number and GFP positive nerve cell number, and the purity is up to 90% and the conversion efficiency is up to 90% by repeating the calculation for three times. A small amount of purified inhibitory interneurons were inoculated into a pre-coated petri dish and continued to be cultured with inhibitory interneuron cell culture medium for identification.
3. Characterization of
The presence or absence of the expression of general neuronal characteristic proteins (Tuj 1 and MAP 2), neuronal synaptic proteins (SYN 1), inhibitory interneuron characteristic proteins (GABA, GAD67, VGAT) and the like in the obtained inhibitory interneurons was analyzed by the same method as in example 1 using an EVOS fluorescence microscope or a confocal fluorescence microscope. The analysis results also show that the inhibitory interneurons prepared by the technique of example 3 express not only characteristic proteins of general neurons, but also characteristic proteins of inhibitory interneurons specifically.
Example 4 selection study of coated substrates
Typical cell culture dishes have no significant effect on the adherent growth of dermal fibroblasts without prior coating, but cells will fall off the glass medium after infection with virus, so appropriate coating conditions should be selected for the dishes used to promote adherent growth of cells during transdifferentiation. Importantly, the selection of the coating matrix is critical for the adherent growth and survival of the neural cells induced during transdifferentiation, an important premise for improving the transformation efficiency.
Thus, this example selects 4 kinds of fibroblast or nerve cell coating matrices: laminin (L), gelatin (G), fibronectin (F), matrigel (M) were added separately to dishes for coating overnight in different combinations, 5-10 20x fields were randomly selected on day 10 of transdifferentiation with a fluorescence microscope for each group, the number of GFP positive nerve cells in each field was counted, and then the relative conversion relative to the most numerous groups was calculated for comparative analysis. As can be seen from FIG. 4, the experimental group (-LGFM) without coated matrix had little inhibitory interneuron production, whereas the above 4 matrices were coated alone or in combination of ≡2 to achieve varying degrees of conversion. Matrigel, when coated alone, resulted in the highest conversion efficiency compared to other coated substrates. Thus, a suitable coating matrix may be selected during the actual manufacturing process with reference to cost, efficiency, and the like.
EXAMPLE 5 selection study of small molecule Compounds and growth factors in induced differentiation Medium
This example examined the effect of different small molecule compounds and growth factors on transdifferentiation efficiency. A transdifferentiation assay was performed as described in example 1, selecting small molecule compounds such as FSK, RA, LDN, SB431542 (SB) and growth factors such as bFGF2, BDNF, GDNF, and the like, adding the compounds to the induced differentiation culture broth in different combinations, randomly selecting 5-10 20X fields per group on day 10 using a fluorescence microscope, counting the number of GFP positive nerve cells in each field, and then calculating the relative conversion rate with respect to the most number groups for comparative analysis. As can be seen from FIG. 5, the conversion efficiency was the highest when ALL the above components (ALL) were added to the induced differentiation medium, and the effect on the conversion efficiency was the greatest after FSK, LDN or bFGF2 was removed. Therefore, in the actual preparation process, the proper small molecule compound and growth factor combination can be selected by referring to factors such as cost, efficiency and the like.
EXAMPLE 6 application of inhibitory interneurons in screening of New epileptic drugs
Firstly, a plurality of 96-well culture plates are pre-coated overnight according to the method, a tube of frozen nerve cells is taken out, carefully and quickly thawed and revived in a water bath, after inoculation according to proper density, the pre-prepared compound to be screened is added into each well (which should contain solvent contrast, positive contrast and the like) designed in advance, after light mixing, the mixture is put back into a cell incubator, and after proper culture time, qualitative and quantitative analysis is carried out according to a proper method, so that the effective compound is determined. The optimal active compounds may further employ inhibitory interneurons to study their mechanism of action, define primary action targets, optimize structures to improve drug formation, and exploit action targets to develop potential gene therapies, etc.
The above-described specific embodiments further describe in detail the objects, technical solutions and advantageous effects of the present invention, however, it should be understood that the above-described embodiments are not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Ningbo Yisaiteng biotechnology Co., ltd
<120> a method for preparing inhibitory interneurons retaining age characteristics from non-neural cell transformation
<160> 22
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<400> 1
atgccagccc gccttgagac ctgcatctcc gacctcgact gcgccagcag cagcggcagt 60
gacctatccg gcttcctcac cgacgaggaa gactgtgcca gactccaaca ggcagcctcc 120
gcttcggggc cgcccgcgcc ggcccgcagg ggcgcgccca atatctcccg ggcgtctgag 180
gttccagggg cacaggacga cgagcaggag aggcggcggc gccgcggccg gacgcgggtc 240
cgctccgagg cgctgctgca ctcgctgcgc aggagccggc gcgtcaaggc caacgatcgc 300
gagcgcaacc gcatgcacaa cttgaacgcg gccctggacg cactgcgcag cgtgctgccc 360
tcgttccccg acgacaccaa gctcaccaaa atcgagacgc tgcgcttcgc ctacaactac 420
atctgggctc tggccgagac actgcgcctg gcggatcaag ggctgcccgg aggcggtgcc 480
cgggagcgcc tcctgccgcc gcagtgcgtc ccctgcctgc ccggtccccc aagccccgcc 540
agcgacgcgg agtcctgggg ctcaggtgcc gccgccgcct ccccgctctc tgaccccagt 600
agcccagccg cctccgaaga cttcacctac cgccccggcg accctgtttt ctccttccca 660
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Met Pro Ala Arg Leu Glu Thr Cys Ile Ser Asp Leu Asp Cys Ala Ser
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Ser Ser Gly Ser Asp Leu Ser Gly Phe Leu Thr Asp Glu Glu Asp Cys
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Ala Arg Leu Gln Gln Ala Ala Ser Ala Ser Gly Pro Pro Ala Pro Ala
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Arg Arg Gly Ala Pro Asn Ile Ser Arg Ala Ser Glu Val Pro Gly Ala
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Gln Asp Asp Glu Gln Glu Arg Arg Arg Arg Arg Gly Arg Thr Arg Val
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Asp Ala Leu Arg Ser Val Leu Pro Ser Phe Pro Asp Asp Thr Lys Leu
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Ala Glu Thr Leu Arg Leu Ala Asp Gln Gly Leu Pro Gly Gly Gly Ala
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Arg Glu Arg Leu Leu Pro Pro Gln Cys Val Pro Cys Leu Pro Gly Pro
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Pro Ser Pro Ala Ser Asp Ala Glu Ser Trp Gly Ser Gly Ala Ala Ala
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Ala Ser Pro Leu Ser Asp Pro Ser Ser Pro Ala Ala Ser Glu Asp Phe
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Asp Leu Leu His Thr Thr Pro Cys Phe Ile Pro Tyr His
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<213> human NGN2 cDNA
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atgttcgtca aatccgagac cttggagttg aaggaggaag aggacgtgtt agtgctgctc 60
ggatcggcct cccccgcctt ggcggccctg accccgctgt catccagcgc cgacgaagaa 120
gaggaggagg agccgggcgc gtcaggcggg gcgcgtcggc agcgcggggc tgaggccggg 180
cagggggcgc ggggcggcgt ggctgcgggt gcggagggct gccggcccgc acggctgctg 240
ggtctggtac acgattgcaa acggcgccct tcccgggcgc gggccgtctc ccgaggcgcc 300
aagacggccg agacggtgca gcgcatcaag aagacccgta gactgaaggc caacaaccgc 360
gagcgaaacc gcatgcacaa cctcaacgcg gcactggacg cgctgcgcga ggtgctcccc 420
acgttccccg aggacgccaa gctcaccaag atcgagaccc tgcgcttcgc ccacaactac 480
atctgggcac tcaccgagac cctgcgcctg gcggatcact gcgggggcgg cggcgggggc 540
ctgccggggg cgctcttctc cgaggcagtg ttgctgagcc cgggaggcgc cagcgccgcc 600
ctgagcagca gcggagacag cccctcgccc gcctccacgt ggagttgcac caacagcccc 660
gcgccgtcct cctccgtgtc ctccaattcc acctccccct acagctgcac tttatcgccc 720
gccagcccgg ccgggtcaga catggactat tggcagcccc cacctcccga caagcaccgc 780
tatgcacctc acctccccat agccagggat tgtatctag 819
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<213> human NGN2 protein
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Met Phe Val Lys Ser Glu Thr Leu Glu Leu Lys Glu Glu Glu Asp Val
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Leu Ser Ser Ser Ala Asp Glu Glu Glu Glu Glu Glu Pro Gly Ala Ser
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Gly Gly Ala Arg Arg Gln Arg Gly Ala Glu Ala Gly Gln Gly Ala Arg
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Gly Gly Val Ala Ala Gly Ala Glu Gly Cys Arg Pro Ala Arg Leu Leu
65 70 75 80
Gly Leu Val His Asp Cys Lys Arg Arg Pro Ser Arg Ala Arg Ala Val
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Ser Arg Gly Ala Lys Thr Ala Glu Thr Val Gln Arg Ile Lys Lys Thr
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Arg Arg Leu Lys Ala Asn Asn Arg Glu Arg Asn Arg Met His Asn Leu
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Asn Ala Ala Leu Asp Ala Leu Arg Glu Val Leu Pro Thr Phe Pro Glu
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Asp Ala Lys Leu Thr Lys Ile Glu Thr Leu Arg Phe Ala His Asn Tyr
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Ile Trp Ala Leu Thr Glu Thr Leu Arg Leu Ala Asp His Cys Gly Gly
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Gly Gly Gly Gly Leu Pro Gly Ala Leu Phe Ser Glu Ala Val Leu Leu
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Ser Pro Gly Gly Ala Ser Ala Ala Leu Ser Ser Ser Gly Asp Ser Pro
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Ser Pro Ala Ser Thr Trp Ser Cys Thr Asn Ser Pro Ala Pro Ser Ser
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Ser Val Ser Ser Asn Ser Thr Ser Pro Tyr Ser Cys Thr Leu Ser Pro
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<213> human ASCL1 cDNA
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atggaaagct ctgccaagat ggagagcggc ggcgccggcc agcagcccca gccgcagccc 60
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gcagccgccg cagcggcagc gcagagcgcg cagcagcagc agcagcagca gcagcagcag 180
cagcaggcgc cgcagctgag accggcggcc gacggccagc cctcaggggg cggtcacaag 240
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gcgcgccgca acgagcgcga gcgcaaccgc gtcaagttgg tcaacctggg ctttgccacc 420
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<210> 6
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<213> human ASCL1 protein
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Ser Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala Pro
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Gln Leu Arg Pro Ala Ala Asp Gly Gln Pro Ser Gly Gly Gly His Lys
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Ser Ala Pro Lys Gln Val Lys Arg Gln Arg Ser Ser Ser Pro Glu Leu
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Met Arg Cys Lys Arg Arg Leu Asn Phe Ser Gly Phe Gly Tyr Ser Leu
100 105 110
Pro Gln Gln Gln Pro Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg
115 120 125
Asn Arg Val Lys Leu Val Asn Leu Gly Phe Ala Thr Leu Arg Glu His
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Val Pro Asn Gly Ala Ala Asn Lys Lys Met Ser Lys Val Glu Thr Leu
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Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Gln Leu Leu Asp Glu
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His Asp Ala Val Ser Ala Ala Phe Gln Ala Gly Val Leu Ser Pro Thr
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Ile Ser Pro Asn Tyr Ser Asn Asp Leu Asn Ser Met Ala Gly Ser Pro
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Val Ser Ser Tyr Ser Ser Asp Glu Gly Ser Tyr Asp Pro Leu Ser Pro
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<210> 7
<211> 1425
<212> DNA
<213> human SOX4 cDNA
<400> 7
atggtgcagc aaaccaacaa tgccgagaac acggaagcgc tgctggccgg cgagagctcg 60
gactcgggcg ccggcctcga gctgggaatc gcctcctccc ccacgcccgg ctccaccgcc 120
tccacgggcg gcaaggccga cgacccgagc tggtgcaaga ccccgagtgg gcacatcaag 180
cgacccatga acgccttcat ggtgtggtcg cagatcgagc ggcgcaagat catggagcag 240
tcgcccgaca tgcacaacgc cgagatctcc aagcggctgg gcaaacgctg gaagctgctc 300
aaagacagcg acaagatccc tttcattcga gaggcggagc ggctgcgcct caagcacatg 360
gctgactacc ccgactacaa gtaccggccc aggaagaagg tgaagtccgg caacgccaac 420
tccagctcct cggccgccgc ctcctccaag ccgggggaga agggagacaa ggtcggtggc 480
agtggcgggg gcggccatgg gggcggcggc ggcggcggga gcagcaacgc ggggggagga 540
ggcggcggtg cgagtggcgg cggcgccaac tccaaaccgg cgcagaaaaa gagctgcggc 600
tccaaagtgg cgggcggcgc gggcggtggg gttagcaaac cgcacgccaa gctcatcctg 660
gcaggcggcg gcggcggcgg gaaagcagcg gctgccgccg ccgcctcctt cgccgccgaa 720
caggcggggg ccgccgccct gctgcccctg ggcgccgccg ccgaccacca ctcgctgtac 780
aaggcgcgga ctcccagcgc ctcggcctcc gcctcctcgg cagcctcggc ctccgcagcg 840
ctcgcggccc cgggcaagca cctggcggag aagaaggtga agcgcgtcta cctgttcggc 900
ggcctgggca cgtcgtcgtc gcccgtgggc ggcgtgggcg cgggagccga ccccagcgac 960
cccctgggcc tgtacgagga ggagggcgcg ggctgctcgc ccgacgcgcc cagcctgagc 1020
ggccgcagca gcgccgcctc gtcccccgcc gccggccgct cgcccgccga ccaccgcggc 1080
tacgccagcc tgcgcgccgc ctcgcccgcc ccgtccagcg cgccctcgca cgcgtcctcc 1140
tcggcctcgt cccactcctc ctcttcctcc tcctcgggct cctcgtcctc cgacgacgag 1200
ttcgaagacg acctgctcga cctgaacccc agctcaaact ttgagagcat gtccctgggc 1260
agcttcagtt cgtcgtcggc gctcgaccgg gacctggatt ttaacttcga gcccggctcc 1320
ggctcgcact tcgagttccc ggactactgc acgcccgagg tgagcgagat gatctcggga 1380
gactggctcg agtccagcat ctccaacctg gttttcacct actaa 1425
<210> 8
<211> 474
<212> PRT
<213> human SOX4 protein
<400> 8
Met Val Gln Gln Thr Asn Asn Ala Glu Asn Thr Glu Ala Leu Leu Ala
1 5 10 15
Gly Glu Ser Ser Asp Ser Gly Ala Gly Leu Glu Leu Gly Ile Ala Ser
20 25 30
Ser Pro Thr Pro Gly Ser Thr Ala Ser Thr Gly Gly Lys Ala Asp Asp
35 40 45
Pro Ser Trp Cys Lys Thr Pro Ser Gly His Ile Lys Arg Pro Met Asn
50 55 60
Ala Phe Met Val Trp Ser Gln Ile Glu Arg Arg Lys Ile Met Glu Gln
65 70 75 80
Ser Pro Asp Met His Asn Ala Glu Ile Ser Lys Arg Leu Gly Lys Arg
85 90 95
Trp Lys Leu Leu Lys Asp Ser Asp Lys Ile Pro Phe Ile Arg Glu Ala
100 105 110
Glu Arg Leu Arg Leu Lys His Met Ala Asp Tyr Pro Asp Tyr Lys Tyr
115 120 125
Arg Pro Arg Lys Lys Val Lys Ser Gly Asn Ala Asn Ser Ser Ser Ser
130 135 140
Ala Ala Ala Ser Ser Lys Pro Gly Glu Lys Gly Asp Lys Val Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly His Gly Gly Gly Gly Gly Gly Gly Ser Ser Asn
165 170 175
Ala Gly Gly Gly Gly Gly Gly Ala Ser Gly Gly Gly Ala Asn Ser Lys
180 185 190
Pro Ala Gln Lys Lys Ser Cys Gly Ser Lys Val Ala Gly Gly Ala Gly
195 200 205
Gly Gly Val Ser Lys Pro His Ala Lys Leu Ile Leu Ala Gly Gly Gly
210 215 220
Gly Gly Gly Lys Ala Ala Ala Ala Ala Ala Ala Ser Phe Ala Ala Glu
225 230 235 240
Gln Ala Gly Ala Ala Ala Leu Leu Pro Leu Gly Ala Ala Ala Asp His
245 250 255
His Ser Leu Tyr Lys Ala Arg Thr Pro Ser Ala Ser Ala Ser Ala Ser
260 265 270
Ser Ala Ala Ser Ala Ser Ala Ala Leu Ala Ala Pro Gly Lys His Leu
275 280 285
Ala Glu Lys Lys Val Lys Arg Val Tyr Leu Phe Gly Gly Leu Gly Thr
290 295 300
Ser Ser Ser Pro Val Gly Gly Val Gly Ala Gly Ala Asp Pro Ser Asp
305 310 315 320
Pro Leu Gly Leu Tyr Glu Glu Glu Gly Ala Gly Cys Ser Pro Asp Ala
325 330 335
Pro Ser Leu Ser Gly Arg Ser Ser Ala Ala Ser Ser Pro Ala Ala Gly
340 345 350
Arg Ser Pro Ala Asp His Arg Gly Tyr Ala Ser Leu Arg Ala Ala Ser
355 360 365
Pro Ala Pro Ser Ser Ala Pro Ser His Ala Ser Ser Ser Ala Ser Ser
370 375 380
His Ser Ser Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser Asp Asp Glu
385 390 395 400
Phe Glu Asp Asp Leu Leu Asp Leu Asn Pro Ser Ser Asn Phe Glu Ser
405 410 415
Met Ser Leu Gly Ser Phe Ser Ser Ser Ser Ala Leu Asp Arg Asp Leu
420 425 430
Asp Phe Asn Phe Glu Pro Gly Ser Gly Ser His Phe Glu Phe Pro Asp
435 440 445
Tyr Cys Thr Pro Glu Val Ser Glu Met Ile Ser Gly Asp Trp Leu Glu
450 455 460
Ser Ser Ile Ser Asn Leu Val Phe Thr Tyr
465 470
<210> 9
<211> 1326
<212> DNA
<213> human SOX11 cDNA
<400> 9
atggtgcagc aggcggagag cttggaagcg gagagcaacc tgccccggga ggcgctggac 60
acggaggagg gcgaattcat ggcttgcagc ccggtggccc tggacgagag cgacccagac 120
tggtgcaaga cggcgtcggg ccacatcaag cggccgatga acgcgttcat ggtatggtcc 180
aagatcgaac gcaggaagat catggagcag tctccggaca tgcacaacgc cgagatctcc 240
aagaggctgg gcaagcgctg gaaaatgctg aaggacagcg agaagatccc gttcatccgg 300
gaggcggagc ggctgcggct caagcacatg gccgactacc ccgactacaa gtaccggccc 360
cggaaaaagc ccaaaatgga cccctcggcc aagcccagcg ccagccagag cccagagaag 420
agcgcggccg gcggcggcgg cgggagcgcg ggcggaggcg cgggcggtgc caagacctcc 480
aagggctcca gcaagaaatg cggcaagctc aaggcccccg cggccgcggg cgccaaggcg 540
ggcgcgggca aggcggccca gtccggggac tacgggggcg cgggcgacga ctacgtgctg 600
ggcagcctgc gcgtgagcgg ctcgggcggc ggcggcgcgg gcaagacggt caagtgcgtg 660
tttctggatg aggacgacga cgacgacgac gacgacgacg agctgcagct gcagatcaaa 720
caggagccgg acgaggagga cgaggaacca ccgcaccagc agctcctgca gccgccgggg 780
cagcagccgt cgcagctgct gagacgctac aacgtcgcca aagtgcccgc cagccctacg 840
ctgagcagct cggcggagtc ccccgaggga gcgagcctct acgacgaggt gcgggccggc 900
gcgacctcgg gcgccggggg cggcagccgc ctctactaca gcttcaagaa catcaccaag 960
cagcacccgc cgccgctcgc gcagcccgcg ctgtcgcccg cgtcctcgcg ctcggtgtcc 1020
acctcctcgt ccagcagcag cggcagcagc agcggcagca gcggcgagga cgccgacgac 1080
ctgatgttcg acctgagctt gaatttctct caaagcgcgc acagcgccag cgagcagcag 1140
ctggggggcg gcgcggcggc cgggaacctg tccctgtcgc tggtggataa ggatttggat 1200
tcgttcagcg agggcagcct gggctcccac ttcgagttcc ccgactactg cacgccggag 1260
ctgagcgaga tgatcgcggg ggactggctg gaggcgaact tctccgacct ggtgttcaca 1320
tattga 1326
<210> 10
<211> 441
<212> PRT
<213> human SOX11 protein
<400> 10
Met Val Gln Gln Ala Glu Ser Leu Glu Ala Glu Ser Asn Leu Pro Arg
1 5 10 15
Glu Ala Leu Asp Thr Glu Glu Gly Glu Phe Met Ala Cys Ser Pro Val
20 25 30
Ala Leu Asp Glu Ser Asp Pro Asp Trp Cys Lys Thr Ala Ser Gly His
35 40 45
Ile Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser Lys Ile Glu Arg
50 55 60
Arg Lys Ile Met Glu Gln Ser Pro Asp Met His Asn Ala Glu Ile Ser
65 70 75 80
Lys Arg Leu Gly Lys Arg Trp Lys Met Leu Lys Asp Ser Glu Lys Ile
85 90 95
Pro Phe Ile Arg Glu Ala Glu Arg Leu Arg Leu Lys His Met Ala Asp
100 105 110
Tyr Pro Asp Tyr Lys Tyr Arg Pro Arg Lys Lys Pro Lys Met Asp Pro
115 120 125
Ser Ala Lys Pro Ser Ala Ser Gln Ser Pro Glu Lys Ser Ala Ala Gly
130 135 140
Gly Gly Gly Gly Ser Ala Gly Gly Gly Ala Gly Gly Ala Lys Thr Ser
145 150 155 160
Lys Gly Ser Ser Lys Lys Cys Gly Lys Leu Lys Ala Pro Ala Ala Ala
165 170 175
Gly Ala Lys Ala Gly Ala Gly Lys Ala Ala Gln Ser Gly Asp Tyr Gly
180 185 190
Gly Ala Gly Asp Asp Tyr Val Leu Gly Ser Leu Arg Val Ser Gly Ser
195 200 205
Gly Gly Gly Gly Ala Gly Lys Thr Val Lys Cys Val Phe Leu Asp Glu
210 215 220
Asp Asp Asp Asp Asp Asp Asp Asp Asp Glu Leu Gln Leu Gln Ile Lys
225 230 235 240
Gln Glu Pro Asp Glu Glu Asp Glu Glu Pro Pro His Gln Gln Leu Leu
245 250 255
Gln Pro Pro Gly Gln Gln Pro Ser Gln Leu Leu Arg Arg Tyr Asn Val
260 265 270
Ala Lys Val Pro Ala Ser Pro Thr Leu Ser Ser Ser Ala Glu Ser Pro
275 280 285
Glu Gly Ala Ser Leu Tyr Asp Glu Val Arg Ala Gly Ala Thr Ser Gly
290 295 300
Ala Gly Gly Gly Ser Arg Leu Tyr Tyr Ser Phe Lys Asn Ile Thr Lys
305 310 315 320
Gln His Pro Pro Pro Leu Ala Gln Pro Ala Leu Ser Pro Ala Ser Ser
325 330 335
Arg Ser Val Ser Thr Ser Ser Ser Ser Ser Ser Gly Ser Ser Ser Gly
340 345 350
Ser Ser Gly Glu Asp Ala Asp Asp Leu Met Phe Asp Leu Ser Leu Asn
355 360 365
Phe Ser Gln Ser Ala His Ser Ala Ser Glu Gln Gln Leu Gly Gly Gly
370 375 380
Ala Ala Ala Gly Asn Leu Ser Leu Ser Leu Val Asp Lys Asp Leu Asp
385 390 395 400
Ser Phe Ser Glu Gly Ser Leu Gly Ser His Phe Glu Phe Pro Asp Tyr
405 410 415
Cys Thr Pro Glu Leu Ser Glu Met Ile Ala Gly Asp Trp Leu Glu Ala
420 425 430
Asn Phe Ser Asp Leu Val Phe Thr Tyr
435 440
<210> 11
<211> 768
<212> DNA
<213> human DLX1 cDNA
<400> 11
atgaccatga ccaccatgcc agaaagtctc aacagccccg tgtcgggcaa ggcggtgttt 60
atggagtttg ggccgcccaa ccagcaaatg tctccttctc ccatgtccca cgggcactac 120
tccatgcact gtttacactc ggcgggccat tcgcagcccg acggcgccta cagctcagcc 180
tcgtccttct cccgaccgct gggctacccc tacgtcaact cggtcagcag ccacgcatcc 240
agcccctaca tcagttcggt gcagtcctac ccgggcagcg ccagcctcgc ccagagccgc 300
ctggaggacc caggggcgga ctcggagaag agcacggtgg tggaaggcgg tgaagtgcgc 360
ttcaatggca agggaaaaaa gatccgtaaa cccaggacga tttattccag tttgcagttg 420
caggctttga accggaggtt ccagcaaact cagtacctag ctctgccgga gagggcggag 480
ctcgcggcct ctttgggact cacacagact caggtcaaga tctggttcca aaacaagcga 540
tccaagttca agaagctgat gaagcagggt ggggcggctc tggagggtag tgcgttggcc 600
aacggtcggg ccctgtctgc tggctcccca cccgtgccgc ccggctggaa ccctaactct 660
tcatccggga agggctcagg aggaaacgcg ggctcctata tccccagcta cacatcgtgg 720
tacccttcag cgcaccaaga agctatgcag caaccccaac ttatgtga 768
<210> 12
<211> 255
<212> PRT
<213> human DLX1 protein
<400> 12
Met Thr Met Thr Thr Met Pro Glu Ser Leu Asn Ser Pro Val Ser Gly
1 5 10 15
Lys Ala Val Phe Met Glu Phe Gly Pro Pro Asn Gln Gln Met Ser Pro
20 25 30
Ser Pro Met Ser His Gly His Tyr Ser Met His Cys Leu His Ser Ala
35 40 45
Gly His Ser Gln Pro Asp Gly Ala Tyr Ser Ser Ala Ser Ser Phe Ser
50 55 60
Arg Pro Leu Gly Tyr Pro Tyr Val Asn Ser Val Ser Ser His Ala Ser
65 70 75 80
Ser Pro Tyr Ile Ser Ser Val Gln Ser Tyr Pro Gly Ser Ala Ser Leu
85 90 95
Ala Gln Ser Arg Leu Glu Asp Pro Gly Ala Asp Ser Glu Lys Ser Thr
100 105 110
Val Val Glu Gly Gly Glu Val Arg Phe Asn Gly Lys Gly Lys Lys Ile
115 120 125
Arg Lys Pro Arg Thr Ile Tyr Ser Ser Leu Gln Leu Gln Ala Leu Asn
130 135 140
Arg Arg Phe Gln Gln Thr Gln Tyr Leu Ala Leu Pro Glu Arg Ala Glu
145 150 155 160
Leu Ala Ala Ser Leu Gly Leu Thr Gln Thr Gln Val Lys Ile Trp Phe
165 170 175
Gln Asn Lys Arg Ser Lys Phe Lys Lys Leu Met Lys Gln Gly Gly Ala
180 185 190
Ala Leu Glu Gly Ser Ala Leu Ala Asn Gly Arg Ala Leu Ser Ala Gly
195 200 205
Ser Pro Pro Val Pro Pro Gly Trp Asn Pro Asn Ser Ser Ser Gly Lys
210 215 220
Gly Ser Gly Gly Asn Ala Gly Ser Tyr Ile Pro Ser Tyr Thr Ser Trp
225 230 235 240
Tyr Pro Ser Ala His Gln Glu Ala Met Gln Gln Pro Gln Leu Met
245 250 255
<210> 13
<211> 987
<212> DNA
<213> human DLX2 cDNA
<400> 13
atgactggag tctttgacag tctagtggct gatatgcact cgacccagat cgccgcctcc 60
agcacgtacc accagcacca gcagcccccg agcggcggcg gcgccggccc gggtggcaac 120
agcagcagca gcagcagcct ccacaagccc caggagtcgc ccacccttcc ggtgtccacc 180
gccaccgaca gcagctacta caccaaccag cagcacccgg cgggcggcgg cggcggcggg 240
ggctcgccct acgcgcacat gggttcctac cagtaccaag ccagcggcct caacaacgtc 300
ccttactccg ccaagagcag ctatgacctg ggctacaccg ccgcctacac ctcctacgct 360
ccctatggaa ccagttcgtc cccagccaac aacgagcctg agaaggagga ccttgagcct 420
gaaattcgga tagtgaacgg gaagccaaag aaagtccgga aaccccgcac catctactcc 480
agtttccagc tggcggctct tcagcggcgt ttccaaaaga ctcaatactt ggccttgccg 540
gagcgagccg agctggcggc ctctctgggc ctcacccaga ctcaggtcaa aatctggttc 600
cagaaccgcc ggtccaagtt caagaagatg tggaaaagtg gtgagatccc ctcggagcag 660
caccctgggg ccagcgcttc tccaccttgt gcttcgccgc cagtctcagc gccggcctcc 720
tgggactttg gtgtgccgca gcggatggcg ggcggcggtg gtccgggcag tggcggcagc 780
ggcgccggca gctcgggctc cagcccgagc agcgcggcct cggcttttct gggcaactac 840
ccctggtacc accagacctc gggatccgcc tcacacctgc aggccacggc gccgctgctg 900
caccccactc agaccccgca gccgcatcac caccaccacc atcacggcgg cgggggcgcc 960
ccggtgagcg cggggacgat tttctga 987
<210> 14
<211> 328
<212> PRT
<213> human DLX2 protein
<400> 14
Met Thr Gly Val Phe Asp Ser Leu Val Ala Asp Met His Ser Thr Gln
1 5 10 15
Ile Ala Ala Ser Ser Thr Tyr His Gln His Gln Gln Pro Pro Ser Gly
20 25 30
Gly Gly Ala Gly Pro Gly Gly Asn Ser Ser Ser Ser Ser Ser Leu His
35 40 45
Lys Pro Gln Glu Ser Pro Thr Leu Pro Val Ser Thr Ala Thr Asp Ser
50 55 60
Ser Tyr Tyr Thr Asn Gln Gln His Pro Ala Gly Gly Gly Gly Gly Gly
65 70 75 80
Gly Ser Pro Tyr Ala His Met Gly Ser Tyr Gln Tyr Gln Ala Ser Gly
85 90 95
Leu Asn Asn Val Pro Tyr Ser Ala Lys Ser Ser Tyr Asp Leu Gly Tyr
100 105 110
Thr Ala Ala Tyr Thr Ser Tyr Ala Pro Tyr Gly Thr Ser Ser Ser Pro
115 120 125
Ala Asn Asn Glu Pro Glu Lys Glu Asp Leu Glu Pro Glu Ile Arg Ile
130 135 140
Val Asn Gly Lys Pro Lys Lys Val Arg Lys Pro Arg Thr Ile Tyr Ser
145 150 155 160
Ser Phe Gln Leu Ala Ala Leu Gln Arg Arg Phe Gln Lys Thr Gln Tyr
165 170 175
Leu Ala Leu Pro Glu Arg Ala Glu Leu Ala Ala Ser Leu Gly Leu Thr
180 185 190
Gln Thr Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Ser Lys Phe Lys
195 200 205
Lys Met Trp Lys Ser Gly Glu Ile Pro Ser Glu Gln His Pro Gly Ala
210 215 220
Ser Ala Ser Pro Pro Cys Ala Ser Pro Pro Val Ser Ala Pro Ala Ser
225 230 235 240
Trp Asp Phe Gly Val Pro Gln Arg Met Ala Gly Gly Gly Gly Pro Gly
245 250 255
Ser Gly Gly Ser Gly Ala Gly Ser Ser Gly Ser Ser Pro Ser Ser Ala
260 265 270
Ala Ser Ala Phe Leu Gly Asn Tyr Pro Trp Tyr His Gln Thr Ser Gly
275 280 285
Ser Ala Ser His Leu Gln Ala Thr Ala Pro Leu Leu His Pro Thr Gln
290 295 300
Thr Pro Gln Pro His His His His His His His Gly Gly Gly Gly Ala
305 310 315 320
Pro Val Ser Ala Gly Thr Ile Phe
325
<210> 15
<211> 870
<212> DNA
<213> human DLX5 cDNA
<400> 15
atgacaggag tgtttgacag aagggtcccc agcatccgat ccggcgactt ccaagctccg 60
ttccagacgt ccgcagctat gcaccatccg tctcaggaat cgccaacttt gcccgagtct 120
tcagctaccg attctgacta ctacagccct acggggggag ccccgcacgg ctactgctct 180
cctacctcgg cttcctatgg caaagctctc aacccctacc agtatcagta tcacggcgtg 240
aacggctccg ccgggagcta cccagccaaa gcttatgccg actatagcta cgctagctcc 300
taccaccagt acggcggcgc ctacaaccgc gtcccaagcg ccaccaacca gccagagaaa 360
gaagtgaccg agcccgaggt gagaatggtg aatggcaaac caaagaaagt tcgtaaaccc 420
aggactattt attccagctt tcagctggcc gcattacaga gaaggtttca gaagactcag 480
tacctcgcct tgccggaacg cgccgagctg gccgcctcgc tgggattgac acaaacacag 540
gtgaaaatct ggtttcagaa caaaagatcc aagatcaaga agatcatgaa aaacggggag 600
atgcccccgg agcacagtcc cagctccagc gacccaatgg cgtgtaactc gccgcagtct 660
ccagcggtgt gggagcccca gggctcgtcc cgctcgctca gccaccaccc tcatgcccac 720
cctccgacct ccaaccagtc cccagcgtcc agctacctgg agaactctgc atcctggtac 780
acaagtgcag ccagctcaat caattcccac ctgccgccgc cgggctcctt acagcacccg 840
ctggcgctgg cctccgggac actctattga 870
<210> 16
<211> 289
<212> PRT
<213> human DLX5 protein
<400> 16
Met Thr Gly Val Phe Asp Arg Arg Val Pro Ser Ile Arg Ser Gly Asp
1 5 10 15
Phe Gln Ala Pro Phe Gln Thr Ser Ala Ala Met His His Pro Ser Gln
20 25 30
Glu Ser Pro Thr Leu Pro Glu Ser Ser Ala Thr Asp Ser Asp Tyr Tyr
35 40 45
Ser Pro Thr Gly Gly Ala Pro His Gly Tyr Cys Ser Pro Thr Ser Ala
50 55 60
Ser Tyr Gly Lys Ala Leu Asn Pro Tyr Gln Tyr Gln Tyr His Gly Val
65 70 75 80
Asn Gly Ser Ala Gly Ser Tyr Pro Ala Lys Ala Tyr Ala Asp Tyr Ser
85 90 95
Tyr Ala Ser Ser Tyr His Gln Tyr Gly Gly Ala Tyr Asn Arg Val Pro
100 105 110
Ser Ala Thr Asn Gln Pro Glu Lys Glu Val Thr Glu Pro Glu Val Arg
115 120 125
Met Val Asn Gly Lys Pro Lys Lys Val Arg Lys Pro Arg Thr Ile Tyr
130 135 140
Ser Ser Phe Gln Leu Ala Ala Leu Gln Arg Arg Phe Gln Lys Thr Gln
145 150 155 160
Tyr Leu Ala Leu Pro Glu Arg Ala Glu Leu Ala Ala Ser Leu Gly Leu
165 170 175
Thr Gln Thr Gln Val Lys Ile Trp Phe Gln Asn Lys Arg Ser Lys Ile
180 185 190
Lys Lys Ile Met Lys Asn Gly Glu Met Pro Pro Glu His Ser Pro Ser
195 200 205
Ser Ser Asp Pro Met Ala Cys Asn Ser Pro Gln Ser Pro Ala Val Trp
210 215 220
Glu Pro Gln Gly Ser Ser Arg Ser Leu Ser His His Pro His Ala His
225 230 235 240
Pro Pro Thr Ser Asn Gln Ser Pro Ala Ser Ser Tyr Leu Glu Asn Ser
245 250 255
Ala Ser Trp Tyr Thr Ser Ala Ala Ser Ser Ile Asn Ser His Leu Pro
260 265 270
Pro Pro Gly Ser Leu Gln His Pro Leu Ala Leu Ala Ser Gly Thr Leu
275 280 285
Tyr
<210> 17
<211> 528
<212> DNA
<213> human DLX6 cDNA
<400> 17
atgagccact cgcagcacag cccttacctc cagtcctacc acaacagcag cgcagccgcc 60
cagacgcgag gggacgacac agatcaacaa aaaactacag tgattgaaaa cggggaaatc 120
aggttcaatg gaaaagggaa aaagattcgg aagcctcgga ccatttattc cagcctgcag 180
ctccaggctt taaaccatcg ctttcagcag acacagtatc tggcccttcc agagagagcc 240
gaactggcag cttccttagg actgacacaa acacaggtga agatatggtt tcagaacaaa 300
cgctctaagt ttaagaaact gctgaagcag ggcagtaatc ctcatgagag cgaccccctc 360
cagggctcgg cggccctgtc gccacgctcg ccagcgctgc ctccagtctg ggacgtttct 420
gcctcggcca agggtgtcag tatgcccccc aacagctaca tgcctggcta ttctcactgg 480
tactcctctc cacaccagga cacgatgcag agaccacaga tgatgtga 528
<210> 18
<211> 175
<212> PRT
<213> human DLX6 protein
<400> 18
Met Ser His Ser Gln His Ser Pro Tyr Leu Gln Ser Tyr His Asn Ser
1 5 10 15
Ser Ala Ala Ala Gln Thr Arg Gly Asp Asp Thr Asp Gln Gln Lys Thr
20 25 30
Thr Val Ile Glu Asn Gly Glu Ile Arg Phe Asn Gly Lys Gly Lys Lys
35 40 45
Ile Arg Lys Pro Arg Thr Ile Tyr Ser Ser Leu Gln Leu Gln Ala Leu
50 55 60
Asn His Arg Phe Gln Gln Thr Gln Tyr Leu Ala Leu Pro Glu Arg Ala
65 70 75 80
Glu Leu Ala Ala Ser Leu Gly Leu Thr Gln Thr Gln Val Lys Ile Trp
85 90 95
Phe Gln Asn Lys Arg Ser Lys Phe Lys Lys Leu Leu Lys Gln Gly Ser
100 105 110
Asn Pro His Glu Ser Asp Pro Leu Gln Gly Ser Ala Ala Leu Ser Pro
115 120 125
Arg Ser Pro Ala Leu Pro Pro Val Trp Asp Val Ser Ala Ser Ala Lys
130 135 140
Gly Val Ser Met Pro Pro Asn Ser Tyr Met Pro Gly Tyr Ser His Trp
145 150 155 160
Tyr Ser Ser Pro His Gln Asp Thr Met Gln Arg Pro Gln Met Met
165 170 175
<210> 19
<211> 1209
<212> DNA
<213> human LHX5 cDNA
<400> 19
atgatggtgc actgcgccgg ttgcgagcgg cccatcctcg accgctttct gctgaacgtg 60
ctggaccgcg cgtggcacat caaatgtgtt cagtgctgcg agtgcaaaac caacctctcg 120
gagaagtgct tctcgcgcga gggcaagctc tactgcaaaa atgacttttt caggcgcttt 180
ggcacgaaat gcgccggctg cgcgcaaggc atctcgccca gcgacctggt gcgcaaggcc 240
cggagcaaag tctttcacct caactgtttc acctgcatgg tgtgtaacaa gcagctgtcc 300
accggcgagg agctctacgt catcgacgag aacaagttcg tgtgcaaaga cgactacctg 360
agctcatcca gcctcaagga gggcagcctc aactcagtgt catcctgtac ggaccgcagt 420
ttgtccccgg acctccagga cgcactgcag gacgacccca aagagacgga caactcgacc 480
tcgtcggaca aggagacggc caacaacgag aacgaggagc agaactcggg caccaagcgg 540
cgcggccccc gcaccaccat caaggccaag cagctggaga cgctcaaggc tgccttcgcc 600
gccacgccca agcccacgcg ccacatccgc gagcagctgg cgcaggagac cggcctcaac 660
atgcgcgtca tccaggtgtg gtttcagaac cgacggtcca aagaacgccg gatgaaacag 720
ctgagcgccc taggcgcccg gaggcacgcc ttcttccgga gtccgcggcg catgcgtccg 780
ctgggcggcc gcttggacga gtctgagatg ttggggtcca ccccgtacac ctactacgga 840
gactaccaag gcgactacta cgcgccggga agcaactacg acttcttcgc gcacggcccg 900
ccttcgcagg cgcagtcccc ggccgactcc agcttcctgg cggcctctgg ccccggctcg 960
acgccgctgg gagcgctgga accgccgctc gccggcccgc acgccgcgga caaccccagg 1020
ttcaccgaca tgatctcgca cccggacaca ccgagccccg agccaggcct gccgggcacg 1080
ctgcacccca tgcccggcga ggtattcagc ggcgggccca gcccgccctt cccaatgagc 1140
ggcaccagcg gctacagcgg acccctgtcg catcccaacc ccgagctcaa cgaagccgcc 1200
gtgtggtaa 1209
<210> 20
<211> 402
<212> PRT
<213> human LHX5 protein
<400> 20
Met Met Val His Cys Ala Gly Cys Glu Arg Pro Ile Leu Asp Arg Phe
1 5 10 15
Leu Leu Asn Val Leu Asp Arg Ala Trp His Ile Lys Cys Val Gln Cys
20 25 30
Cys Glu Cys Lys Thr Asn Leu Ser Glu Lys Cys Phe Ser Arg Glu Gly
35 40 45
Lys Leu Tyr Cys Lys Asn Asp Phe Phe Arg Arg Phe Gly Thr Lys Cys
50 55 60
Ala Gly Cys Ala Gln Gly Ile Ser Pro Ser Asp Leu Val Arg Lys Ala
65 70 75 80
Arg Ser Lys Val Phe His Leu Asn Cys Phe Thr Cys Met Val Cys Asn
85 90 95
Lys Gln Leu Ser Thr Gly Glu Glu Leu Tyr Val Ile Asp Glu Asn Lys
100 105 110
Phe Val Cys Lys Asp Asp Tyr Leu Ser Ser Ser Ser Leu Lys Glu Gly
115 120 125
Ser Leu Asn Ser Val Ser Ser Cys Thr Asp Arg Ser Leu Ser Pro Asp
130 135 140
Leu Gln Asp Ala Leu Gln Asp Asp Pro Lys Glu Thr Asp Asn Ser Thr
145 150 155 160
Ser Ser Asp Lys Glu Thr Ala Asn Asn Glu Asn Glu Glu Gln Asn Ser
165 170 175
Gly Thr Lys Arg Arg Gly Pro Arg Thr Thr Ile Lys Ala Lys Gln Leu
180 185 190
Glu Thr Leu Lys Ala Ala Phe Ala Ala Thr Pro Lys Pro Thr Arg His
195 200 205
Ile Arg Glu Gln Leu Ala Gln Glu Thr Gly Leu Asn Met Arg Val Ile
210 215 220
Gln Val Trp Phe Gln Asn Arg Arg Ser Lys Glu Arg Arg Met Lys Gln
225 230 235 240
Leu Ser Ala Leu Gly Ala Arg Arg His Ala Phe Phe Arg Ser Pro Arg
245 250 255
Arg Met Arg Pro Leu Gly Gly Arg Leu Asp Glu Ser Glu Met Leu Gly
260 265 270
Ser Thr Pro Tyr Thr Tyr Tyr Gly Asp Tyr Gln Gly Asp Tyr Tyr Ala
275 280 285
Pro Gly Ser Asn Tyr Asp Phe Phe Ala His Gly Pro Pro Ser Gln Ala
290 295 300
Gln Ser Pro Ala Asp Ser Ser Phe Leu Ala Ala Ser Gly Pro Gly Ser
305 310 315 320
Thr Pro Leu Gly Ala Leu Glu Pro Pro Leu Ala Gly Pro His Ala Ala
325 330 335
Asp Asn Pro Arg Phe Thr Asp Met Ile Ser His Pro Asp Thr Pro Ser
340 345 350
Pro Glu Pro Gly Leu Pro Gly Thr Leu His Pro Met Pro Gly Glu Val
355 360 365
Phe Ser Gly Gly Pro Ser Pro Pro Phe Pro Met Ser Gly Thr Ser Gly
370 375 380
Tyr Ser Gly Pro Leu Ser His Pro Asn Pro Glu Leu Asn Glu Ala Ala
385 390 395 400
Val Trp
<210> 21
<211> 1092
<212> DNA
<213> human LHX6 cDNA
<400> 21
atggcccagc cagggtccgg ctgcaaagcg accacccgct gtcttgaagg gaccgcgccg 60
cccgccatgg ctcagtctga cgccgaggcc ctggcaggag ctctggacaa ggacgagggt 120
caggcctccc catgtacgcc cagcacgcca tctgtctgct caccgccctc tgccgcctcc 180
tccgtgccgt ctgcaggcaa gaacatctgc tccagctgcg gcctcgagat cctggaccga 240
tatctgctca aggtcaacaa cctcatctgg cacgtgcggt gcctcgagtg ctccgtgtgt 300
cgcacgtcgc tgaggcagca gaacagctgc tacatcaaga acaaggagat cttctgcaag 360
atggactact tcagccgatt cgggaccaag tgtgcccggt gcggccgaca gatctacgcc 420
agcgactggg tgcggagagc tcgcggcaac gcctaccacc tggcctgctt cgcctgcttc 480
tcgtgcaagc gccagctgtc cactggtgag gagttcggcc tggtcgagga gaaggtgctc 540
tgccgcatcc actacgacac catgattgag aacctcaaga gggccgccga gaacgggaac 600
ggcctcacgt tggagggggc agtgccctcg gaacaggaca gtcaacccaa gccggccaag 660
cgcgcgcgga cgtccttcac cgcggaacag ctgcaggtta tgcaggcgca gttcgcgcag 720
gacaacaacc ccgacgctca gacgctgcag aagctggcgg acatgacggg cctcagccgg 780
agagtcatcc aggtgtggtt tcaaaactgc cgggcgcgtc ataaaaagca cacgccgcaa 840
cacccagtgc cgccctcggg ggcgcccccg tcccgccttc cctccgccct gtccgacgac 900
atccactaca ccccgttcag cagccccgag cgggcgcgca tggtcaccct gcacggctac 960
attgagagtc aggtacagtg cgggcaggtg cactgccggc tgccttacac cgcacccccc 1020
gtccacctca aagccgatat ggatgggccg ctctccaacc ggggtgagaa ggtcatcctt 1080
tttcagtact aa 1092
<210> 22
<211> 363
<212> PRT
<213> human LHX6 protein
<400> 22
Met Ala Gln Pro Gly Ser Gly Cys Lys Ala Thr Thr Arg Cys Leu Glu
1 5 10 15
Gly Thr Ala Pro Pro Ala Met Ala Gln Ser Asp Ala Glu Ala Leu Ala
20 25 30
Gly Ala Leu Asp Lys Asp Glu Gly Gln Ala Ser Pro Cys Thr Pro Ser
35 40 45
Thr Pro Ser Val Cys Ser Pro Pro Ser Ala Ala Ser Ser Val Pro Ser
50 55 60
Ala Gly Lys Asn Ile Cys Ser Ser Cys Gly Leu Glu Ile Leu Asp Arg
65 70 75 80
Tyr Leu Leu Lys Val Asn Asn Leu Ile Trp His Val Arg Cys Leu Glu
85 90 95
Cys Ser Val Cys Arg Thr Ser Leu Arg Gln Gln Asn Ser Cys Tyr Ile
100 105 110
Lys Asn Lys Glu Ile Phe Cys Lys Met Asp Tyr Phe Ser Arg Phe Gly
115 120 125
Thr Lys Cys Ala Arg Cys Gly Arg Gln Ile Tyr Ala Ser Asp Trp Val
130 135 140
Arg Arg Ala Arg Gly Asn Ala Tyr His Leu Ala Cys Phe Ala Cys Phe
145 150 155 160
Ser Cys Lys Arg Gln Leu Ser Thr Gly Glu Glu Phe Gly Leu Val Glu
165 170 175
Glu Lys Val Leu Cys Arg Ile His Tyr Asp Thr Met Ile Glu Asn Leu
180 185 190
Lys Arg Ala Ala Glu Asn Gly Asn Gly Leu Thr Leu Glu Gly Ala Val
195 200 205
Pro Ser Glu Gln Asp Ser Gln Pro Lys Pro Ala Lys Arg Ala Arg Thr
210 215 220
Ser Phe Thr Ala Glu Gln Leu Gln Val Met Gln Ala Gln Phe Ala Gln
225 230 235 240
Asp Asn Asn Pro Asp Ala Gln Thr Leu Gln Lys Leu Ala Asp Met Thr
245 250 255
Gly Leu Ser Arg Arg Val Ile Gln Val Trp Phe Gln Asn Cys Arg Ala
260 265 270
Arg His Lys Lys His Thr Pro Gln His Pro Val Pro Pro Ser Gly Ala
275 280 285
Pro Pro Ser Arg Leu Pro Ser Ala Leu Ser Asp Asp Ile His Tyr Thr
290 295 300
Pro Phe Ser Ser Pro Glu Arg Ala Arg Met Val Thr Leu His Gly Tyr
305 310 315 320
Ile Glu Ser Gln Val Gln Cys Gly Gln Val His Cys Arg Leu Pro Tyr
325 330 335
Thr Ala Pro Pro Val His Leu Lys Ala Asp Met Asp Gly Pro Leu Ser
340 345 350
Asn Arg Gly Glu Lys Val Ile Leu Phe Gln Tyr
355 360

Claims (8)

1. A method of preparing inhibitory interneurons preserving age and pathological characteristics of the onset period from non-neuronal cell transformation comprising the steps of:
1) Constructing a viral vector comprising a combination of cellular transgene comprising a combination of the following genes, and packaging the virus by transfecting the cells: (1) at least one gene selected from NGN1, NGN2 and ASCL 1; (2) At least two genes selected from the group consisting of DLX1, DLX2, DLX5, DLX6, LHX5 and LHX 6; and optionally (3) at least one gene selected from SOX4 and SOX 11;
2) Culturing donor-derived cells, which are one of skin fibroblasts, lung fibroblasts, foreskin fibroblasts, and infecting the cultured donor-derived cells with the packaged virus;
3) Inducing direct conversion of the donor-derived cells into inhibitory interneurons in an induced differentiation medium containing a combination of transdifferentiated small molecule compounds comprising at least one of forskolin, cAMP, dibutyryl cyclic adenylate, RA, LDN, SB431542 and growth factors comprising at least one of bFGF2, BDNF, GDNF;
4) Isolating and purifying the resulting inhibitory interneurons.
2. The method of claim 1, wherein the gene is a homologous gene derived from a human, mouse or any other species.
3. The method of claim 1, wherein the viral vector is selected from a retroviral vector, a lentiviral vector, or an AAV viral vector; and/or the promoter regulating the expression level in the vector is selected from any one or more of CMV, CAG, EF1 alpha and PGK, TRETight, TRE G.
4. The method of claim 1, wherein the cellular transgene combination is linked by a 2A sequence or IRES sequence of different origin and a fluorescent reporter gene can be introduced during construction of the viral vector.
5. The method of claim 1, wherein the culture vessel is pre-coated with a coating matrix selected from at least one of laminin, gelatin, fibronectin, matrigel during the culturing of the donor-source cells.
6. The method of claim 1, wherein the isolating and purifying is by digesting and re-suspending the transdifferentiated cells into a single cell suspension, and isolating the inhibitory interneurons using a cell filter, flow cytometer, or differential adherence on gelatin-coated dishes.
7. The method of any one of claims 1-6, wherein inhibitory interneurons retaining age and pathological characteristics of the onset stage are produced.
8. Use of an inhibitory interneuron retaining age and pathological characteristics in the onset of disease as claimed in claim 7 for the establishment of a method for screening epileptic, depressive, schizophrenic drugs.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6087168A (en) * 1999-01-20 2000-07-11 Cedars Sinai Medical Center Conversion of non-neuronal cells into neurons: transdifferentiation of epidermal cells
KR20180062631A (en) * 2016-12-01 2018-06-11 서울대학교산학협력단 Composition for cell regeneration comprising cells that hypersecreate growth factors, and at least one of a neural stem cells, neurons and GABAergic neurons
WO2019107354A1 (en) * 2017-11-30 2019-06-06 アイ ピース, インコーポレイテッド Method for producing neural cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6087168A (en) * 1999-01-20 2000-07-11 Cedars Sinai Medical Center Conversion of non-neuronal cells into neurons: transdifferentiation of epidermal cells
KR20180062631A (en) * 2016-12-01 2018-06-11 서울대학교산학협력단 Composition for cell regeneration comprising cells that hypersecreate growth factors, and at least one of a neural stem cells, neurons and GABAergic neurons
WO2019107354A1 (en) * 2017-11-30 2019-06-06 アイ ピース, インコーポレイテッド Method for producing neural cells

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