A method for highly efficiently inducing the reprogramming of human cells into functional neurons by overexpressing the SMAD3 gene

Abstract

The present invention provides a use of overexpressing the SMAD3 gene in inducing the reprogramming of human cells into functional neurons. Under the condition of overexpressing a single gene SMAD3, neuron cells with a TUJ1 positive rate of 60% can be obtained in only about 5 days of induction time. The induction time is greatly shortened and the induction efficiency is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for highly efficiently inducing reprogramming of human cells into functional neurons by overexpressing the SMAD3 gene. Background Art

[0002] Brain diseases, paralysis, etc. caused by nerve injuries severely restrict people's normal life and mobility. The non-renewability of neurons determines the difficulty of treating and recovering from such diseases at present. The current treatment for the above diseases still remains at the level of drug conservative treatment and exercise rehabilitation. Therefore, regenerating neurons by using exogenous transcription factors or small molecule compounds is a method for treating diseases caused by nerve injuries at the root.

[0003] Currently, due to the increasingly severe trend of the aging world population, the number of patients with neurodegenerative diseases caused by it has also increased significantly. It is particularly important to find more genes that regulate neuron fate and have a wider range of effects, so as to serve as targets for future traditional drug diagnosis and treatment and develop more effective "gene drugs". At present, for exogenous transcription factors packaged by adenovirus, it has been successfully used to significantly improve the treatment of neurodegenerative diseases, blindness caused by ocular neuron lesions and other related diseases in model animals such as mice. Due to its own characteristics such as not integrating into the host gene, adenovirus is becoming an excellent and currently widely used gene vector. Therefore, in order to better treat nerve-related diseases, developing more extensive and effective targets is a very important scientific research task.

[0004] By using methods such as overexpression (Overexpress), knockout or knockdown (Knockout / Knockdown) of exogenous transcription factors and combinations of small molecule compounds, terminally differentiated somatic cells have been successfully reprogrammed into neurons in many species such as humans and mice. Since 2011, when Pang ZP et al. induced human fibroblasts into neurons by overexpressing four genes, Brn2, Ascl1, Myt1l, and Ngn2, the research on inducing neurons by exogenous transcription factors has become increasingly mature.

[0005] However, the currently discovered gene targets have defects such as being scattered, unclear mechanism of action, low efficiency, long induction time, too long CDS region of key genes to obtain, and the need to simultaneously regulate multiple gene targets to obtain neurons, which will greatly affect the popularization and application of future clinical treatment. Contents of the Invention

[0006] In view of this, the present invention aims to provide a method for highly efficiently inducing the reprogramming of human cells into functional neurons by overexpressing the SMAD3 gene. By overexpressing this key gene, human cells can be induced into neurons, which well avoids the above-mentioned disadvantages, has a clearer mechanism of action, and when used in combination with small molecule compounds, can further improve the induction efficiency.

[0007] The present invention first provides the use of overexpressing the SMAD3 gene in inducing the reprogramming of human cells into functional neurons.

[0008] Preferably, the somatic cell is one of human skin fibroblasts, human follicular granulosa cells, and human astrocytes.

[0009] The induction medium for inducing the reprogramming of human cells into functional neurons can preferably be N2B27, which includes Knockout DMEM / F12, N2 (100×), Neurobasal, B27 (50×), Glutamine (100×); and the volume ratio of the components is 99:1:97:2:1.

[0010] The induction efficiency can be further improved in combination with any of the following single small molecule compounds, PKA / CREB activators (Forskolin / Colforsin / 8-Bromo-cAMP / Dibutyryl-cAMP (Bucladesine) / cAMP and its analogs), ACVR1 (ALK2) inhibitors (LDN-193189 / LDN-193189-2HCl / K02288 / LDN-212854 / LDN-214117 / ML347 / DMH1 / ), JNK inhibitors (SP600125 / Resveratrol / JNK-IN-8 / JNK-InhibitorVIII / DB07268 / IQ-1S / Bentamapimod (AS602801) / Tanzisertib (CC-930) / BI-78D3 / JNKInhibitorIX / UrolithinB / LoureirinB / LoureirinB / Falcarindiol / CucurbitacinIIb / Mulberroside A / Trans-ZeatinAstragaloside IV), P38 inhibitors (SB203580 / Doramapimod (BIRB796) / SB202190 (FHPI) / Ralimetinib dimesylate / VX-702 / PH-797804 / VX-745 / TAK-715 / PD169316 / TA-02 / SD0006 / Pamapimod / BMS-582949 / SB239063 / Losmapimod (GW856553X) / Skepinone-L / SEA0400 / AUDA / PraeruptorinA / MulberrosideA / UM-164 / Trans-Zeatin / 3'-Hydroxypterostilbene / Pexmetinib (ARRY-614)), AMPK inhibitors (Dorsomorphin / Dorsomorphin (CompoundC) / Dorsomorphin (CompoundC)2HCl / WZ4003 / ON123300 / HTH-01-015 / Doxorubicin (Adriamycin)HCl / GSK690693 / XMD-17-51), cAMP.

[0011] The present invention also provides a method for inducing the reprogramming of human cells into functional neurons, which includes the following steps: (1) delivering the overexpressed SMAD3 gene into somatic cells; (2) inducing in N2B27 medium or a small molecule compound induction medium.

[0012] Preferably, the method for delivering the overexpressed SMAD3 gene into somatic cells includes, but is not limited to, one of electroporation, liposome method, virus infection method (lentivirus, adeno-associated virus); preferably, infecting recipient cells with the lentivirus or adenovirus overexpressing SMAD3.

[0013] Preferably, the N2B27 medium includes Knockout DMEM / F12, N2 (100×), Neurobasal, B27 (50×), Glutamine (100×); and the volume ratio of the components is 99:1:97:2:1; preferably, the small molecule compound induction medium is the N2B27 medium supplemented with any one of the following small molecule compounds, PKA / CREB activator, ACVR1 (ALK2) inhibitor, JNK inhibitor, P38 inhibitor, AMPK inhibitor, and cAMP.

[0014] It further includes step (3) of culturing successively in a neuron maturation medium and a neuron medium.

[0015] Preferably, the neuron maturation medium is DMEM / F12:Neurobasal = 1:1 (volume ratio), 0.5% N2 by volume fraction, 1% B27 by volume fraction, 100 μM cAMP, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, 1% penicillin or streptomycin by volume fraction.

[0016] Preferably, the neuron medium is DMEM / F12:Neurobasal = 1:1 (volume ratio), 0.5% N2 by volume fraction, 1% B27 by volume fraction, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, 1% penicillin or streptomycin by volume fraction.

[0017] The adenovirus overexpressing SMAD3 can be obtained by the following method:

[0018] 1) Gene cloning: Design amplification primers by homologous recombination, amplify the CDS region of SMAD3 in human fibroblasts, and obtain the complete CDS sequence of the human SMAD3 gene.

[0019] 2) Restriction digestion, ligation, transformation, and identification: First, the overexpression plasmid vector was digested using double restriction enzymes to convert the circular overexpression plasmid vector into a linear plasmid vector. Through a homologous recombination kit, the digested linear overexpression plasmid vector was ligated to the SMAD3 cloning fragment. Subsequently, it was transformed into competent cells, and using the property of competent cells to amplify massively at 37°C, a sufficient amount of recombinant overexpression plasmid vector with SMAD3 ligated to the overexpression vector was obtained. The positive recombinant plasmid vector with successful ligation was detected by agarose gel electrophoresis and sent to the company for Sanger sequencing to detect base mutations.

[0020] 3) Package the plasmid matching the CDS region of SMAD3 into an adenovirus to obtain an adenovirus overexpressing SMAD3. Then infect the recipient cells and observe the fluorescence rate after 48 h.

[0021] Induction process:

[0022] 1) Using a 60 mm culture dish as a standard, the seeding density of human skin fibroblasts (BJ) was 5×10 5 , and after seeding, high-glucose DMEM + 10% (v / v) FBS (fibroblast medium / FM) was added and cultured in an incubator with 5% carbon dioxide, 95% humidity, and 37°C. When the cell confluence reached 30%, the above-packaged adenovirus (MOI = 20) was added.

[0023] 2) To ensure that the recombinant overexpression plasmid has been successfully transferred into the recipient cells, observe the fluorescence efficiency under a fluorescence microscope 48 h after induction culture. Subsequently, change its medium to the above neuron induction medium. When obvious synapses appear in the induced cells and the cell body density increases, change the medium to the neuron maturation medium. When the neurons exhibit more complex synapses, change the medium to the neuron medium for long-term culture.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1) Under the condition of overexpressing a single gene SMAD3, the present invention can obtain neuron cells with a TUJ1 positive rate of 60% in only about 5 days of induction time. Compared with the previous work, the induction time is greatly shortened and the induction efficiency is greatly improved.

[0026] 2) The present invention further enriches the mechanism of somatic cell induction into neurons, and further clarifies the molecular regulation pathway and specific regulatory genes of the entire reprogramming process of somatic cell transdifferentiation into neurons. This mechanism has not been clearly described by anyone.

[0027] 3) The present invention has a short induction time and high induction efficiency, and is expected to be applied to the treatment of human nerve injury and degenerative diseases, providing a safer and more efficient cell therapy means for the treatment of neurodegenerative diseases. Since neurons do not have the ability to divide and proliferate, we can use the technology of the present invention to induce fibroblasts or astrocytes with the ability to divide and proliferate in vivo and in vitro, and continuously obtain a large number of functional neurons in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 is the time course of inducing the reprogramming of human skin fibroblasts into neurons by overexpressing SMAD3;

[0030] Figure 2 is the morphological diagram of inducing the reprogramming of human skin fibroblasts into neurons by overexpressing SMAD3;

[0031] Figure 3 The immunofluorescence results show that overexpressing SMAD3 induces the reprogramming of human skin fibroblasts into neurons (CiNCs). These neuronal cells express the neuronal marker proteins TUJ1 and MAP2;

[0032] Figure 4 The immunofluorescence results show that the small molecule compound LDN193189 is added during the process of overexpressing SMAD3 to induce the reprogramming of human skin fibroblasts into neurons (CiNCs). These neuronal cells express the neuronal marker proteins TUJ1 and MAP2 with higher efficiency compared to the overexpressing SMAD3 group;

[0033] Figure 5 The results of quantitative PCR show that during the process of overexpressing SMAD3 to induce the reprogramming of human skin fibroblasts into neurons (CiNCs), the neuronal marker genes are significantly up-regulated, while the expression of fibroblast marker genes is significantly down-regulated. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0035] The present invention will be described in detail below with reference to the drawings and in combination with the embodiments.

[0036] Using this method of induced transdifferentiation, human skin fibroblasts (BJ) have been successfully transformed into functional neurons.

[0037] Culture medium:

[0038] 1. Components of neuron induction medium: Basal medium (N2B27): 200 mL system: Knockout DMEM / F12: 99 mL, N2 (100×): 1 mL, Neurobasal: 97 mL, B27 (50×): 2 mL, Glutamine (100×): 1 mL

[0039] 2. Components of neuron maturation medium: DMEM / F12:Neurobasal = 1:1 (volume ratio), 0.5% N2 by volume fraction, 1% B27 by volume fraction, 100 μM cAMP, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, 1% penicillin / streptomycin by volume fraction.

[0040] 3. Components of neuron medium: DMEM / F12:Neurobasal = 1:1 (volume ratio), 0.5% N2 by volume fraction, 1% B27 by volume fraction, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, 1% penicillin / streptomycin by volume fraction.

[0041] The overall experimental induction is as Figure 1 shown.

[0042] The specific operations are as follows:

[0043] 1. Inoculate human skin fibroblasts into a 60 mm culture dish at a density of 5×10 5 . After inoculation, add high - glucose DMEM + 10% FBS by volume fraction (fibroblast medium / FM) and place it in an incubator with 5% carbon dioxide, 95% humidity, and 37°C for culture. Observe 24 hours after inoculation. When the cell confluence reaches 30%, inoculate the packaged SMAD3 over - expression adenovirus with the parameter of MOI = 20.

[0044] 2. Observe the fluorescence efficiency under a fluorescence microscope 48 h after culture, and change its medium to neuron induction medium. After about 2 - 5 d, synapses are significantly observed in the induced cells, and the cell body density increases, and the neuron - specific proteins TUJ1 and MAP2 are positive. As Figure 2 . 3 shown. Change the medium to neuron maturation medium.

[0045] 3. When more complex synapses are shown in neurons about 5 - 10 d after over - expressing SMAD3, change the medium to neuron medium for long - term culture.

[0046] To prove the cell characteristics of the overexpression cell line, the neuron cells induced by overexpression of SMAD3 were detected for neuron marker proteins and genes. The specific steps are as follows:

[0047] Fix the human fibroblasts overexpressing the empty plasmid vector and the SMAD3 plasmid vector in the culture plate with 4% paraformaldehyde (PFA) by mass fraction at room temperature for 30 min; wash three times with the blocking solution, 5 min each time; then permeabilize the cells with 1% Triton X-100 by volume fraction at room temperature for 15 min; wash three times again with the blocking solution; subsequently, add 5% donkey serum by volume fraction to block non-specific sites and block for 2 h at room temperature; wash three times with TBP (Tritonx-BSA-PBS), 5 min each time; dilute and add the primary antibodies (neuron marker proteins TUJ1, MAP2) in proportion and incubate overnight at 4 °C; the next day, place the culture plate at room temperature and rewarm for 20 min, then wash 3 times with TBP, 5 min each time, add the secondary antibody (Mouse-488; Rabbit-555) and Hoechst mixed solution in the dark and incubate at room temperature for 1 h; wash 3 times with the TBP solution, and then the fluorescence microscopy observation and photographing experiment can be carried out. The immunofluorescence staining results showed ( Figure 3 ), the cells overexpressing SMAD3 expressed the neuron marker antigens TUJ1 and MAP2, while human skin fibroblasts did not express them.

[0048] During the process of overexpressing SMAD3 to induce the reprogramming of human skin fibroblasts into neurons (CiNCs), the small molecule compound LDN193189 (0.5 μM - 250 μM) was added to the neuron induction medium. The expression of the neuron marker proteins TUJ1 and MAP2 in these neuron cells was more efficient compared to the overexpression SMAD3 group. The immunofluorescence staining results are shown as Figure 4 , where the concentration of the small molecule compound LDN193189 was 5 μM.

[0049] Detect the expression of neuron marker genes by quantitative PCR (qPCR). The specific operation steps are as follows:

[0050] (1) Extract the RNA of the cell lines overexpressing the empty plasmid vector and the plasmid vector overexpressing SMAD3 using the TRIZOL method: Discard the culture medium, wash three times with PBS, add 1 ml of pre-cooled TRIZOL, and lyse on ice for 5 min; add 200 μL of chloroform, shake vigorously for 15 s, and place on ice for 5 min; centrifuge at 12,000 r / min at 4 °C for 15 min; transfer the upper aqueous phase to pre-cooled isopropanol, invert and mix well, and place on ice for 5 min; centrifuge at 12,000 r / min at 4 °C for 10 min; discard the supernatant, add 1 mL of pre-cooled 75% ethanol (volume fraction), gently flick the bottom of the tube with your fingertips to suspend the RNA, wash the RNA and the tube wall thoroughly, centrifuge at 7,500 r / min at 4 °C for 8 min; discard the supernatant. When the precipitate becomes semi-transparent, add an appropriate amount of DEPC water to completely dissolve the RNA, take 1 μL for purity and integrity detection, and perform reverse transcription on the rest or freeze it in an -80 °C refrigerator.

[0051] (2) Preparation of cDNA template. Use the Vazyme R223-01 synthesis kit and follow the instructions.

[0052] (3) Fluorescent quantitative PCR. Use the Vazyme Q711-02 / 03 reagent and follow the instructions. The qPCR results show ( Figure 5 ), compared with human skin fibroblasts, the cells overexpressing SMAD3 highly express the neuron-related marker genes TUJ1, DCX, and MAP2, while the expression levels of the fibroblast marker genes COL1A1 and CTGF are significantly downregulated.

Claims

1. Use of overexpressing the SMAD3 gene in inducing the reprogramming of human cells into functional neurons; the somatic cells are human skin fibroblasts.

2. The use according to claim 1, characterized in that, The induction medium for inducing the reprogramming of human cells into functional neurons is N2B27, which includes Knockout DMEM / F12, N2 (100×), Neurobasal, B27 (50×), Glutamine (100×); and the volume ratio of the components is 99:1:97:2:

1.

3. The use according to claim 1, characterized in that, The small molecule compound induction medium is used to induce the reprogramming of human cells into functional neurons. The small molecule compound induction medium is N2B27 added with any one of the following small molecule compounds: PKA / CREB activator, ACVR1 (ALK2) inhibitor, JNK inhibitor, P38 inhibitor, AMPK inhibitor, and cAMP.

4. A method for inducing the reprogramming of human cells into functional neurons, characterized in that, It includes the following steps: (1) Deliver the overexpressed SMAD3 gene into the somatic cells; (2) Induce in the N2B27 medium or the small molecule compound induction medium, and the somatic cells are human skin fibroblasts.

5. The method according to claim 4, wherein: The method of delivering the overexpressed SMAD3 gene into the somatic cells includes one of electroporation, liposome method, and virus infection method.

6. The method according to claim 4, characterized in that: Infect the recipient cells with the lentivirus or adenovirus overexpressing SMAD3.

7. The method according to claim 4, wherein: The N2B27 medium includes Knockout DMEM / F12, N2 (100×), Neurobasal, B27 (50×), Glutamine (100×); and the volume ratio of the components is 99:1:97:2:

1.

8. The method according to claim 4, characterized in that: The small molecule compound induction medium is the N2B27 medium added with any one of the following small molecule compounds: PKA / CREB activator, ACVR1 (ALK2) inhibitor, JNK inhibitor, P38 inhibitor, AMPK inhibitor, and cAMP.

9. The method according to claim 4, wherein: It further includes step (3) of culturing successively in the neuron maturation medium and the neuron medium.

10. The method according to claim 9, wherein: In the neuron maturation medium, the volume ratio of DMEM / F12 to Neurobasal is 1:1, 0.5% N2 by volume fraction, 1% B27 by volume fraction, 100 μM cAMP, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, and 1% penicillin or streptomycin by volume fraction.

11. The method according to claim 9, characterized in that: In the neuron medium, the volume ratio of DMEM / F12 to Neurobasal is 1:1, 0.5% N2 by volume fraction, 1% B27 by volume fraction, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, and 1% penicillin or streptomycin by volume fraction.

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