CN116162591A - Method for efficiently inducing human cells to reprogram into neuron cells in vitro - Google Patents
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Abstract
The invention provides a method for efficiently inducing human cells to reprogram into neuron cells in vitro, which inhibits the expression of JNK loci through small molecular compounds, gene interference and gene knockout. The induction small molecular compound is single and safe, has short induction time, high induction efficiency, definite induction action site and gene and clear molecular regulation mechanism, can be applied to clinical treatment of human neurodegenerative diseases, and provides a safer and more efficient treatment means for the treatment of the neurodegenerative diseases. Because the neurons have no division proliferation capacity, the patent can be used for inducing the fibroblasts with division proliferation capacity in vitro and in vivo, and a large number of induced neurons can be obtained in vitro and in vivo continuously.
Description
Technical Field
The invention belongs to the field of stem cells, and particularly relates to a method for efficiently inducing human cells to reprogram into neuron cells in vitro.
Background
Reprogramming terminally differentiated somatic cells into neurons has been achieved in a variety of species such as humans, mice, and the like, using transfection of exogenous transcription factors, and induction methods such as small molecule compound combinations. The 2015 Pei Gang group reports the reprogramming of fibroblasts into neurons in alzheimer's patients using a combination of 7 small molecule compounds. Human astrocytes were induced into neurons by Brueckner B et al 2005 using a combination of 6 small molecule compounds. These small molecule compounds inhibit the expression of non-neuronal genes, promote the expression of neuronal specific genes, and finally obtain mature functional neurons by prolonged culture of induced cells.
Although the existing methods for inducing neurons have a plurality of types, the methods have the defects of overlong induction time (20-30 days), low induction efficiency (10-30%), excessively complex method, high potential biosafety risk and the like. In terms of application of cell therapy, the prior art can greatly reduce the effect of clinical therapy due to excessive induction factors, overlong induction time and low efficiency, and increase potential side effects of therapy and worry about biosafety. In terms of mechanism, the prior art uses the combined action of a plurality of small molecule compounds, the signal paths of the action are staggered, the action sites are complex, and how the transformation of somatic cells into neuronal fate is determined cannot be accurately revealed. No technology is currently available to elucidate the mechanism by which somatic cells can be reprogrammed to neuronal cells using a single small molecule compound, nor is it fully elucidate the mechanism by which somatic cells are reprogrammed to neurons.
Whereas the induction system of the present invention allows reprogramming somatic cells to TUJ1 positive neuronal cells (up to about 80% positive rate) within two days using only a single small molecule compound or the regulatory effect of a single gene. More importantly, the methods of small molecule compound combinations used by the predecessor have failed to elucidate the mechanism of cell fate conversion. Our findings clearly illustrate the molecular regulatory pathways of reprogramming human cells to neuronal cells, confirming that the regulatory effects of a single small molecule compound or a single gene can induce reprogramming human cells to neuronal cells. In addition, no one has reported the key regulatory sites of reprogramming mentioned in the present invention.
Disclosure of Invention
In view of this, the present invention is directed to a method for efficiently inducing reprogramming of human cells into neuronal cells in vitro, so as to overcome the defects of the prior art, and determine that the regulation effect of a single small molecule compound or a single gene can induce reprogramming of human cells into neuronal cells.
A method for efficiently inducing human cells to reprogram into neuron cells increases cAMP concentration or up-regulates expression of any site of PKA and CREB or inhibits expression of any site of AMPK, ALK2, ALK3, P38 and JNK.
Preferably, small molecule compounds, including one or more of cAMP activators, cAMP analogs, PKA activators, CREB activators, AMPK inhibitors, ALK2 inhibitors, ALK3 inhibitors, P38 inhibitors, JNK inhibitors, are used, preferably, to increase the concentration of cAMP or to up-regulate the expression of any site of PKA, CREB, or to inhibit the expression of any site of AMPK, ALK2, ALK3, P38, JNK, or small molecule compounds, gene interference, gene knockout or gene overexpression.
The human cells are induced to reprogram into various action sites of neuron cells and small molecule compounds acting on the sites, wherein the action sites comprise cAMP (increasing concentration), PKA (activating/up-regulating expression), CREB (activating/up-regulating expression), ALK2/3 (inhibiting/down-regulating expression), JNK (inhibiting/down-regulating expression), P38 (inhibiting/down-regulating expression) and AMPK (inhibiting/down-regulating expression). Any single small molecule compound including, but not limited to, cAMP/PKA/CREB activator (Forskolin/Colforsin/8-Bromo-cAMP/Dibutyryl-cAMP (Bucladesine)/cAMP and the like), ALK2/3 inhibitor (LDN-193189/LDN-193189-2 HCl/K02288/LDN-212854/LDN-214117/ML347/DMH 1/and the like), JNK inhibitor (SP 600125/Resveratrol/JNK-IN-8/JNK-InbitorVIII/DB 07268/IQ-1S/bentamap imod (AS 602801)/Tanzisertib (CC-930)/BI-78D 3/JNK InbitorIX/Uithin B/Loureireib/falcarindi/Culcipariib/Mulboroside A/Trans-ZeatinAstragaloside IV and the like, P38 inhibitors (SB 203580/Doramapimod (BIRB 796)/SB 202190 (FHPI)/Ralimetinib dimesylate/VX-702/PH-797804/VX-745/TAK-715/PD169316/TA-02/SD0006/Pamapimod/BMS-582949/SB 239063/Losmaapimod (GW 856553X)/Sepinone-L/SEA 0400/AUDA/Praeroup torina/Mulberroside A/UM-164/Trans-Zeatin/3' -hydrooxymatrilbene/P exmetinib (ARRY-614) and the like), AMPK inhibitors (Dorsomophin/WZ 4003/ON123300/HTH-01-015/GSK690693/XMD-17-51/Dorsomorphin (CompoundC)/Dorsomorphin (CompoundC) 2HCl/Doxorubicin (Adriamycin) HCl, etc.) can induce human skin fibroblasts, human follicular granulosa cells, human astrocytes, and the like to reprogram functional neurons.
A second object of the present invention is to provide an induction medium for efficiently inducing reprogramming of human cells into neuronal cells, comprising a base fluid, KSR, and a small molecule compound, preferably, the small molecule compound comprises one or more of cAMP activator, cAMP analog (e.g. DBcAMP, 8-Cl-cAMP, etc.), PKA activator, CREB activator, AMPK inhibitor, ALK2 inhibitor, ALK3 inhibitor, P38 inhibitor, JNK inhibitor.
Preferably, the small molecule compound comprises Forskolin, 8-Bromo-cAMP, LDN193189, cAMP analog, SP600125, SB203580, dorsomorphin, and in the final medium, the respective concentrations are 0 to 100 μm, 0 to 500 μm, 0 to 25 μm, 0 to 10mM, 0 to 10 μm, 0 to 5 μm, 0 to 100 μm, respectively, at different concentrations of 0; preferably, the respective concentrations are 5 to 20. Mu.M, 5 to 50. Mu.M, 0.5 to 5mM, 0.5 to 5. Mu.M, 0.1 to 2.5. Mu.M, 0.5 to 20. Mu.M, respectively, in this order; more preferably, the respective concentrations are 10. Mu.M, 50. Mu.M, 2.5. Mu.M, 1mM, 1. Mu.M, 0.5. Mu.M, 10. Mu.M, respectively, in order.
Preferably, the volume ratio of the base liquid to the KSR is 80:20; preferably, the base liquid is N2B27, including KnockoutDMEM/F12, N2 (100×), neurobasal, B27 (50×), glutamine (100×); and the volume ratio of the two is 99:1:97:2:1.
A third object of the present invention is to provide the use of the induction medium described above for inducing reprogramming of somatic cells into neuronal cells in vitro and in vivo.
A fourth object of the present invention is to provide a method for in vitro induction of somatic reprogramming into neuronal cells using an induction medium, comprising the steps of:
1) Inoculating somatic cells into a culture dish, adding high-sugar DMEM+10% FBS after inoculation, placing the culture dish in an incubator with 5% carbon dioxide and humidity of 95% and 37 ℃, replacing the induction culture medium according to any one of claims 3-5 after overnight culture, and obtaining induction neurons (CiNCs) after 48h of induction culture;
2) The medium is replaced by a neuron maturation medium, the induced neurons are further matured continuously, and the neurons are replaced by the neuron maturation medium after 72 hours, so that the neurons can be cultured for a long time.
Preferably, the components of the neuronal maturation medium include DMEM/F12 and Neurobasal at a volume ratio of 1:1, 0.5% N2 (volume percent), 1% B27 (volume percent), 100 μM cAMP,20ng/mL bFGF,20ng/mL BDNF,20ng/mL GDNF,20ng/mL NT3, 100U/mL penicillin and 0.1mg/mL streptomycin.
Preferably, the composition of the neuronal medium comprises DMEM/F12 in a volume ratio of 1:1: neurobasal,0.5% N2 (volume percent), 1% B27 (volume percent), 20ng/mL bFGF,20ng/mL BDNF,20ng/mL GDNF,20ng/mL NT3, 100U/mL penicillin, and 0.1mg/mL streptomycin.
The somatic cells are derived from human, monkey or mouse; the somatic cells are skin fibroblasts, follicular granulosa cells or astrocytes.
Compared with the prior art, the method for efficiently inducing the reprogramming of the human cells into the neuron cells has the following beneficial effects:
(1) The patent fills a gap in the use of a single small molecule compound to induce reprogramming of terminally differentiated human cells into functional neurons.
(2) The patent fills a gap in regulating the expression of a single gene (PKA, CREB, JNK) to induce the reprogramming of terminally differentiated human cells into functional neurons.
(3) The present patent provides neuronal cells with a TUJ1 positive rate of about 80% with a single small molecule compound, and with an induction time of only about two days. Compared with the work of the former, the induction time is greatly shortened, and the induction efficiency is greatly improved.
(4) The patent uses the characteristics of clear action path and clear action site of single small molecular compound, and clarifies that the molecular regulation path of the whole reprogramming process of somatic cell reprogramming into neuron is cAMP-PKA-CREB-JNK and its key regulation genes are PKA, CREB and JNK. This mechanism has not been explicitly elucidated.
(5) The induction small molecular compound is single and safe, has short induction time, high induction efficiency and clear mechanism, can be applied to clinical treatment of human neurodegenerative diseases, and provides a safer and more efficient treatment means for the treatment of the neurodegenerative diseases. The patent can be used for inducing fibroblasts and astrocytes in vitro and in vivo to continuously obtain a large number of induced neurons in vitro and in vivo, so that the purpose of clinically treating neurodegenerative diseases is achieved by using regenerated induced neurons.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a time pathway of example 1 for a single small molecule compound Forskolin to induce reprogramming of human skin fibroblasts into neurons;
FIG. 2 is a graph showing the morphological change of the single small molecule compound Forskolin induced reprogramming of human skin fibroblasts into neurons of example 1;
FIG. 3 is the immunofluorescence results of example 1 for a single small molecule compound Forskolin to induce reprogramming of human skin fibroblasts into neurons;
FIG. 4 is a graph showing the results of quantitative PCR of the single small molecule compound Forskolin induced reprogramming of human skin fibroblasts into neurons of example 1;
FIG. 5 is a graph showing the effect of a small molecule compound, gene overexpression or gene knockout induction method on a single distinct site on a reprogramming control pathway.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to the following examples and drawings.
A method for efficiently inducing human cells to reprogram into neuron cells comprises the following steps: the concentration of cAMP is increased or the expression of any site of PKA and CREB is up-regulated or the expression of any site of AMPK, ALK2, ALK3, P38 and JNK is inhibited by using a small molecular compound, gene interference, gene knockout or gene overexpression, wherein the small molecular compound comprises one or more than two of cAMP activator, cAMP analogue, PKA activator, CREB activator, AMPK inhibitor, ALK2 inhibitor, ALK3 inhibitor, P38 inhibitor and JNK inhibitor.
1. Small molecule compound induction medium components that regulate any site of the neuronal cell reprogramming molecular pathway (cAMP, PKA, CREB, AMPK, ALK, ALK3, P38, JNK) (increase cAMP concentration or up-regulate expression of PKA, CREB or inhibit expression of AMPK, ALK2, ALK3, P38, JNK):
base liquid (N2B 27): 200mL system:
induction medium: 100mL system:
N2B2780mL
KSR (serum replacement) 20mL
The names and concentrations of the small molecule compounds are shown in tables 1 and 2.
Table 1 Induction concentration and efficiency of representative small molecule Compounds at each site of action and Gene validation of the site of action
TABLE 2 list of small molecule Compounds at each site of action
2. Neuron maturation medium composition: DMEM/F12: neurobasal=1:1 (volume ratio), 0.5% n2 (volume percent), 1% b27 (volume percent), 100 μΜ cAMP,20ng/mL bFGF,20ng/mL BDNF,20ng/mL lgdnf,20ng/mL NT3, 100U/mL penicillin and 0.1mg/mL streptomycin.
3. Neuron culture medium composition: DMEM/F12: neurobasal=1:1 (volume ratio), 0.5% n2 (volume percent), 1% b27 (volume percent), 20ng/mL bFGF,20ng/mL BDNF,20ng/mL GDNF,20ng/mL NT3, 100U/mL penicillin and 0.1mg/mL streptomycin.
4. The induction process comprises the following steps:
1. the cell inoculation density of human body is 5 multiplied by 10 by taking a 60mm culture dish as a standard 5 After inoculation, high-sugar dmem+10% fbs (fibroblast medium/FM) was added, and the mixture was placed in an incubator with 5% carbon dioxide, 95% humidity and 37 ℃ for overnight culture, and the above-mentioned neuron induction medium was replaced, and induced neurons (CiNCs) were obtained after 48 hours of induction.
2. The induced neuron obtained after 48h of induction culture is replaced by the neuron maturation medium, the further maturation of the induced neuron is further promoted, and the neuron can be cultivated for a long time after 72h of culture by replacing the neuron with the neuron medium again.
Example 1
Human skin fibroblasts (BJs) have been successfully reprogrammed into functional neurons using this method of inducing reprogramming.
The overall experimental induction is shown in figure 1.
The specific operation is as follows:
human skin fibroblasts were cultured at 5X 10 5 Is inoculated into a 60mm dish, and the neuron induction medium (N2B27+KSR+10. Mu.M Forskolin) is replaced within 24 hours of inoculation, and placed at 37℃with 5% CO 2 The incubator was incubated for 48 hours. The morphological changes of the cells during induction are shown in FIG. 2, and induced neurons (CiNCs) with a TUJ1 positive rate of 80% were obtained 48 hours after induction.
After 48 hours of induction, the neuronal maturation medium was replaced to further promote the maturation of CiNCs, and after 72 hours, the neuronal medium was replaced to allow for long-term culture.
We performed immunofluorescence detection of neuronal labeled antigen on human skin fibroblasts and cells induced by neuronal induction culture (N2B27+10. Mu.M Forskolin) for 48 hours. The method comprises the following specific steps: 4% Paraformaldehyde (PFA) cells of human skin fibroblasts, F-48h in a room temperature fixed culture plate for 30min; the blocking solution is washed for three times, and each time is washed for 5min; then adding 1% TritonX-100 permeabilized cells, and standing at room temperature for 15min; the blocking solution is washed for three times again; then adding 5% donkey serum to block the nonspecific site, and blocking for 2 hours at room temperature; washing with TBP (Triton X-BSA-PBS) three times for 5min each; adding a primary antibody, and standing at 4 ℃ for incubation overnight; the next day, the culture plate is placed under the condition of room temperature and rewarmed for 20min, then is washed by TBP for 3 times, each time is 5min, secondary antibody and Hoechst mixed solution are added in a dark place, and the culture plate is incubated for 1h at room temperature; and (3) washing the TBP solution for 3 times, and performing fluorescent microscope observation photographing experiments. Immunofluorescent staining results showed (see FIG. 3) that neuronal cells (F-48 h) were induced to express neuronal marker antigens, TUJ1, MAP2, whereas human skin fibroblasts were not.
Quantitative PCR (qPCR) detects the expression of the neuronal marker genes. The specific operation steps are as follows: (1) total RNA extraction. Discarding the culture medium, washing with PBS for three times, and adding 1ml of precooled TRIZOL ice for 5min for cleavage; 200 mu L of chloroform is added, and the mixture is vigorously shaken for 15s and placed on ice for 5min;12000r/min, centrifuging at 4 ℃ for 15min; transferring the upper water phase into precooled isopropanol, mixing, and standing on ice for 5min;12000r/min, centrifuging at 4deg.C for 10min; discarding the supernatant, adding 1mL of pre-cooled 75% ethanol, flicking the bottom of the tube with a fingertip to suspend the RNA, fully washing the RNA and the tube wall, centrifuging at 4 ℃ for 8min, and washing the RNA and the tube wall; removing supernatant, adding proper amount of DEPC water to completely dissolve RNA when the precipitate is semitransparent, taking 1 μl for purity and integrity detection, and performing reverse transcription or freezing the rest in-80deg.C refrigerator. (2) preparation of cDNA template. The Vazyme R223-01 synthesis kit was used in accordance with instructions. (3) fluorescent quantitative PCR. The procedure was followed using the Vazyme Q711-02/03 reagent. qPCR results show (FIG. 4) that compared with human skin fibroblasts (F-0 h), the expression levels of neuron-associated marker genes neuroD1, tubulin, ascl1 and the like are significantly reduced by inducing the neuronal cells (F-48 h) to highly express the fibroblast marker gene Col1A1 and the like.
Example 2
The difference from example 1 is that the neuron induction medium used is n2b27+ksr+10mcamp. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 3
The difference from example 1 is that the neuron induction medium used is N2B27+KSR+10μM 8-Bromo-cAMP. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 4
The difference from example 1 is that the neuron induction medium used is N2B27+KSR+10. Mu.M Dorsomorphin. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 5
The difference from example 1 is that the neuron induction medium used is n2b27+ksr+5 μm LDN193189. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 6
The difference from example 1 is that the neuron induction medium used is n2b27+ksr+5μ MSB203580. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 7
The difference from example 1 is that the neuron induction medium used is n2b27+ksr+10mu MSP600125. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 8
First, an overexpression recombinant plasmid (pLVX-PKA-IRES) of PKA gene was constructed. Extracting RNA from cells with high expression of a target gene (PKA) by adopting a Trizol method, designing a primer after reverse transcription, and amplifying the complete coding region sequence of the PKA gene by using cDNA as a template through PCR. Subsequently, the PCR product is subjected to agarose electrophoresis, and PKA gene fragments are cut off correspondinglyAnd (3) recycling the glue. And (3) after the gel is recovered, measuring the DNA concentration to obtain the target gene fragment. Double enzyme digestion is carried out on the over-expression empty vector (pLVX-IRES) plasmid by using restriction enzyme, agarose electrophoresis and gel recovery are carried out on the plasmid after enzyme digestion is completed, the concentration of gel recovery products is measured, and homologous recombination is carried out on the gel recovery products and PKA gene fragments. Then the recombinant product is transformed into E.coli T1 competent cells in ampicillin (Amp + ) Is spread on LB solid plate medium and placed in a 37 ℃ incubator to cause culture for 12-16 h. Picking up the monoclonal antibody in the presence of Amp + Continuous culture in LB liquid medium. And (3) performing colony PCR identification on the bacterial liquid, selecting the bacterial liquid with correct identification, extracting plasmids by using a plasmid extraction kit, and performing further enzyme digestion identification. The plasmids identified as correct were sent to the company for sequencing. And finally, carrying out amplification culture on bacterial liquid corresponding to the plasmid with correct sequencing, and extracting recombinant over-expression plasmid (pLVX-PKA-IRES) by using an endotoxin removal plasmid extraction kit.
Viral packaging and cell infection are then performed to induce neuronal transdifferentiation. Resuscitating HEK-293T cells at 37deg.C with 5% CO 2 In the incubator, when the cell confluence reaches 50% -60%, adopting a liposome transfection method to dilute Lipofectamine by using a serum-free DMEM culture medium TM 3000, and mixing thoroughly. DNA was diluted with serum-free DMEM medium (recombinant over-expression plasmid pLVX-PKA-IRES, viral packaging plasmid NRF and viral envelope plasmid VSVG=5:3:2), and P3000 was added after preparation of a DNA premix TM The reagent is fully and evenly mixed. In diluted Lipofectamine TM Diluted DNA was added to the 3000 reagent (ratio Lip3000: dna=2.5:1). Incubating at room temperature for 10min, co-transfecting HEK-293T cells with the DNA-liposome complex, culturing at 37 ℃ and 5% CO2, collecting virus supernatant after 48-72h, centrifuging at 4 ℃ and 2000r/min for 10min, and filtering with a 0.45 μm filter. The filtered virus solution and the culture medium are directly infected with human skin fibroblasts (5×10) according to a mixing ratio of 1:1 5 ) After 2 days, the medium (N2B27+KSR) was changed, and the culture was continued in a 5% CO2 incubator at 37℃for 2 days. Then, the culture medium is replaced by a neuron maturation medium to be continued for 3 days, so that the CiNCs maturation is further promoted. Finally, the culture medium is replaced by a neuron culture medium, so that long-time culture can be performed.
And finally, verifying the induced neurons. We performed immunofluorescence detection of neuronal labeled antigens on the neuronal cells induced as described above. The method comprises the following specific steps: 4% Paraformaldehyde (PFA) cells of human skin fibroblasts, F-48h in a room temperature fixed culture plate for 30min; the blocking solution is washed for three times, and each time is washed for 5min; then adding 1% TritonX-100 permeabilized cells, and standing at room temperature for 15min; the blocking solution is washed for three times again; then adding 5% donkey serum to block the nonspecific site, and blocking for 2 hours at room temperature; washing with TBP (Triton X-BSA-PBS) three times for 5min each; adding a primary antibody, and standing at 4 ℃ for incubation overnight; the next day, the culture plate is placed under the condition of room temperature and rewarmed for 20min, then is washed by TBP for 3 times, each time is 5min, secondary antibody and Hoechst mixed solution are added in a dark place, and the culture plate is incubated for 1h at room temperature; and (3) washing the TBP solution for 3 times, and performing fluorescent microscope observation photographing experiments. Immunofluorescent staining results showed (see fig. 5), that neuronal cells were induced to express neuronal marker antigens, TUJ1, MAP2 and NeuN.
Example 9
The difference from example 8 is that the overexpressed gene is CREB. Immunofluorescence assay results showed that the induced neuronal cells expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see fig. 5).
Example 10
Firstly, constructing a JNK gene knockout recombinant plasmid (U6-sgJNK-EF 1a-Cas9-FLAG-P2A-P uro). JNK gene exons were selected, targeted sgJNK was designed using MIT Zhang Feng laboratory sgRNA design tools, and the sticky ends of restriction enzymes were added to the sgJNK primers to facilitate ligation with empty vector (U6-sgRNA-EF 1a-Cas 9-FLAG-P2A-puro). The primer dry powder synthesized by the company is diluted to the working concentration by ddH2O, and an annealing system is configured for annealing. Linearizing empty vector by restriction endonuclease followed by insertion of sgJNK, ligation followed by transformation of competent escherichia coli, and transformation of the empty vector with ampicillin (Amp + ) Is spread on LB solid plate medium and placed in a 37 ℃ incubator to cause culture for 12-16 h. The monoclonal is picked up and cultured continuously in LB liquid medium containing Amp+. Bacterial liquid is subjected to colony PCR identification, and plasmid extraction kit for bacterial liquid with correct identification is selectedThe plasmids were extracted and further digested and identified, and the plasmids identified as correct were sent to the company for sequencing. And finally, performing amplification culture on bacterial liquid corresponding to the plasmid with correct sequencing, and extracting the plasmid containing the sgJNK expression original by using an endotoxel removal extraction kit for transfecting cells.
Packaging and cell infection of JNK knockout virus were then performed. Resuscitating HEK-293T cells at 37deg.C with 5% CO 2 In the incubator, when the cell confluence reaches 50% -60%, adopting a liposome transfection method to dilute Lipofectamine by using a serum-free DMEM culture medium TM 3000, and mixing thoroughly. DNA was diluted with serum-free DMEM medium (Cas 9 plasmid, virus packaging plasmid psPAX2 and pmdg2=5:3:2), prepared as a DNA premix followed by addition of P3000 TM The reagent is fully and evenly mixed. In diluted Lipofectamine TM Diluted DNA was added to the 3000 reagent (ratio Lip3000: dna=2.5:1). Incubating at room temperature for 10min, co-transfecting HEK-293T cells with the DNA-liposome complex, culturing at 37 ℃ and 5% CO2, collecting virus supernatant after 48-72h, centrifuging at 4 ℃ and 2000r/min for 10min, and filtering with a 0.45 μm filter. The filtered virus solution and the culture medium are directly infected with human skin fibroblasts (5×10) according to a mixing ratio of 1:1 5 ) After 2 days, the medium (N2B27+KSR) was changed, and the culture was continued in a 5% CO2 incubator at 37℃for 2 days. Then, the culture medium is replaced by a neuron maturation medium to be continued for 3 days, so that the CiNCs maturation is further promoted. Finally, the culture medium is replaced by a neuron culture medium, so that long-time culture can be performed.
And finally, verifying the induced neurons. We performed immunofluorescence detection of neuronal labeled antigens on the neuronal cells induced as described above. The method comprises the following specific steps: 4% Paraformaldehyde (PFA) cells of human skin fibroblasts, F-48h in a room temperature fixed culture plate for 30min; the blocking solution is washed for three times, and each time is washed for 5min; then adding 1% TritonX-100 permeabilized cells, and standing at room temperature for 15min; the blocking solution is washed for three times again; then adding 5% donkey serum to block the nonspecific site, and blocking for 2 hours at room temperature; washing with TBP (Triton X-BSA-PBS) three times for 5min each; adding a primary antibody, and standing at 4 ℃ for incubation overnight; the next day, the culture plate is placed under the condition of room temperature and rewarmed for 20min, then is washed by TBP for 3 times, each time is 5min, secondary antibody and Hoechst mixed solution are added in a dark place, and the culture plate is incubated for 1h at room temperature; and (3) washing the TBP solution for 3 times, and performing fluorescent microscope observation photographing experiments. Immunofluorescent staining results showed (see fig. 5), that neuronal cells were induced to express neuronal marker antigens, TUJ1, MAP2 and NeuN.
The results of these examples demonstrate that either a single small molecule compound or a gene knockout/overexpression control signal pathway (cAMP, PKA, CREB, AMPK, ALK, ALK3, P38, JNK) can induce efficient reprogramming of human cells into neuronal cells.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A method for efficiently inducing human cells to reprogram into neuron cells in vitro, which is characterized in that: inhibiting expression of JNK site, the somatic cells are skin fibroblasts.
2. The method according to claim 1, characterized in that: expression of JNK sites is inhibited using small molecule compounds, gene interference, gene knockout, preferably, small molecule compounds include JNK inhibitors.
3. The method according to claim 1, characterized in that: in vitro efficient induction of somatic reprogramming into neuronal cells is performed using an induction medium comprising a base fluid, KSR, and small molecule compounds, preferably, small molecule compounds comprising JNK inhibitors.
4. A method according to claim 3, characterized in that: the small molecule compound includes SP600125 and has a concentration of 0 to 10. Mu.M, preferably 0.5 to 5. Mu.M in the final medium.
5. The method according to claim 4, wherein: the concentration of the small molecule compound was 1. Mu.M.
6. A method according to claim 3, characterized in that: the volume ratio of the base solution to the KSR is 80:20.
7. The method according to claim 6, wherein: the base liquid is N2B27, including Knockout DMEM/F12, N2 (100×), neurobasal, B27 (50×), and Glutamine (100×); and the volume ratio of the two is 99:1:97:2:1.
8. A method of inducing somatic reprogramming into neuronal cells in vitro using an induction medium, characterized in that: the method comprises the following steps:
1) Inoculating somatic cells into a culture dish, adding high-sugar DMEM+10% FBS after inoculation, placing the culture dish in an incubator with 5% carbon dioxide and humidity of 95%, and at 37 ℃, and replacing an induction culture medium after overnight culture, wherein the induction culture medium comprises a base solution, KSR and small molecular compounds, the small molecular compounds comprise SP600125, the concentration of the small molecular compounds in the final culture medium is 0-10 mu M, the volume ratio of the base solution to the KSR is 80:20, the base solution is N2B27, and the base solution comprises Knockout DMEM/F12, N2 (100×), neurobasal, B27 (50×) and Glutamine (100×); and the volume ratio of the two is 99:1:97:2:1, and induced neurons (CiNCs) are obtained after 48h of induced culture;
2) The medium is replaced by a neuron maturation medium, the induced neurons are further matured continuously, and the neurons are replaced by the neuron maturation medium after 72 hours, so that the neurons can be cultured for a long time.
9. The method according to claim 8, wherein: the components of the neuron maturation medium include DMEM/F12 and Neurobasal at a volume ratio of 1:1, 0.5% N2 (volume percent), 1% B27 (volume percent), 100 μM cAMP,20ng/mL bFGF,20ng/mL BDNF,20ng/mL GDNF,20ng/mL NT3, 100U/mL penicillin and 0.1mg/mL streptomycin.
10. The method according to claim 9, wherein: the components of the neuron culture medium include DMEM/F12 in a volume ratio of 1:1: neurobasal,0.5% N2 (volume percent), 1% B27 (volume percent), 20ng/mL bFGF,20ng/mL BDNF,20ng/mL GDNF,20ng/mL NT3, 100U/mL penicillin, and 0.1mg/mL streptomycin.
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