CN111876443A

CN111876443A - Method for inducing reprogramming of somatic cells to neural cell model by using transdifferentiation technology

Info

Publication number: CN111876443A
Application number: CN202010795950.3A
Authority: CN
Inventors: 樊晋宇; 池金鹏; 赵亚琪; 程雪; 张诗薇
Original assignee: Shandong Tianchuan Precision Medical Technology Co Ltd
Current assignee: Shandong Tianchuan Precision Medical Technology Co Ltd
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-11-03

Abstract

The invention provides a method for inducing somatic cell reprogramming to a nerve cell model by utilizing a transdifferentiation technology, and particularly relates to a method for directly transdifferentiating skin fibroblasts of a schizophrenia patient into nerve cells by utilizing a nerve cell development key transcription factor combination. The invention has the advantages that somatic cells can be successfully transdifferentiated into nerve cells, the transdifferentiation efficiency is high, a relatively accurate cell model can be established, the neural development process of schizophrenia can be simulated, and a new possibility is provided for further researching the pathogenesis of schizophrenia.

Description

Method for inducing reprogramming of somatic cells to neural cell model by using transdifferentiation technology

Technical Field

The invention relates to the field of biotechnology, in particular to a method for inducing reprogramming of somatic cells to a neural cell model by utilizing a transdifferentiation technology.

Background

The central nervous system injury includes brain injury, spinal cord injury or neurodegenerative disease, etc., the treatment of central nervous system injury or degenerative disease is still a worldwide medical problem at present, the clinical main treatment methods for nerve injury include surgical removal of etiology, avoidance of secondary injury, protection of residual neuron function, and late rehabilitation treatment, etc., however, these methods have little effect on nerve regeneration, and the prognosis effect is still poor, which is related to adverse factors such as low neuronal regeneration function, existence of a barrier of scar tissue hyperplasia between the injured area and normal tissue, etc. Therefore, how to promote the regeneration of nerve cells and break through the regeneration barrier is an important focus of present neuroscientists and clinical researchers.

The current methods for promoting nerve cell regeneration mainly comprise:

1. by inducing traditional induced pluripotent stem cells (iPS), namely inducing somatic cells into stem cells and then transforming the stem cells into neurons or precursor cells, the central nervous system cells induced and differentiated by the iPS have autologous cell replacement therapy potential for various diseases. However, the use of iPS has the following problems: (1) a certain amount of iPS needs to be prepared in time, and a patient prepared for nerve transplantation can not wait for the process of long preparation time of the stem cells such as induction, differentiation, screening and the like in vitro; (2) the induction conversion rate of iPS cells is low, and the iPS cells need to be proliferated again, and the differentiation is uncertain in the process; (3) undifferentiated stem cells used for transplantation, if not differentiated in time, increase the risk of tumor formation in vivo.

2. Differentiation was directly induced by somatic cells in vitro. In recent years, a large number of scientific experiments show that part of cells can be directly induced into somatic cells through reprogramming of the cells by transcription factors under the mediation of vectors such as lentiviruses and retroviruses, and the time and uncertainty required by secondary induced differentiation are greatly reduced after the stem cell stage is passed. At present, researchers transfer two factors, namely Neurog2 and Dlx2, into astrocytes respectively, and prove that the astrocytes can be directly induced into mature neurons under the action of the two factors respectively and can form synaptic structures among the differentiated neurons. In addition, it has been found that fibroblasts have the ability to be directly transformed into cholinergic neurons under the combined influence of internal and external factors. After intensive research, it was found that adult skin fibroblasts can be directly and efficiently converted into high-purity motor neurons without passing through the induced pluripotent stem cell stage. However, researchers found that stem cell therapy was derived from cells of patients themselves during clinical transformation, but fibroblasts from patients with mental diseases (such as amyotrophic lateral sclerosis, schizophrenia, etc.) have poor viability after transformation compared with fibroblasts of normal people, and partial cell differentiation uncertainty always exists during the differentiation induction process of the fibroblasts, which all cause difficulty in practical application of stem cell therapy for neurological diseases.

Disclosure of Invention

In order to solve the problems of low transdifferentiation efficiency, high differentiation uncertainty, lack of mental disease models and the like in the process of transforming fibroblasts from patients with nervous system diseases into nerve cells in the prior art, the invention provides a method for inducing somatic cell reprogramming to a nerve cell model by using a transdifferentiation technology, and particularly relates to a method for directly transdifferentiating skin fibroblasts of patients with schizophrenia into nerve cells by using a nerve cell development key transcription factor combination.

More specifically, the method constructs a nerve induction related factor lentiviral plasmid, and amplifies ASCL1 and MYT1L fragments by a PCR method to connect the lentiviral vector with the plasmid. Recombinant plasmids pLVX-ASCL1 and pLVX-MYT1L are obtained by enzyme digestion, linkage, escherichia coli transformation and the like, and virus particles with good infection activity are combined to infect somatic cells to induce and form nerve-like cells.

In one embodiment, the method of the present invention comprises the steps of:

firstly, a nerve induction related factor lentivirus plasmid is constructed, a PCR method is used for amplification to obtain ASCL1 and MYT1L fragments, and the fragments are connected into a lentivirus vector pLVX-IRES-mCherry. And obtaining recombinant plasmids pLVX-ASCL1 and pLVX-MYT1L through enzyme digestion, chaining, escherichia coli transformation and the like, respectively combining the two recombinant plasmids serving as target plasmids with packaging plasmids pMD2.G and pSPAX2 to carry out lentivirus packaging to obtain viruses with infection activity, infecting skin fibroblasts of a patient with schizophrenia by using virus particle combinations with good infection activity, and further successfully inducing and cloning to obtain a nerve cell model.

The invention has the advantages that somatic cells can be successfully transdifferentiated into nerve cells, the transdifferentiation efficiency is high, a more accurate nerve disease cell model can be established, the nerve development process of schizophrenia can be simulated, and a new possibility is provided for further researching the pathogenesis of schizophrenia and the stem cell therapy thereof.

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 principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows a comparison of 15-day survival and transformation rates of cells induced by various groups of genes;

FIG. 2 nerve cell induction formation process;

FIG. 3 induction of nerve cell protein level detection marker gene expression.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

EXAMPLE 1 transdifferentiation of fibroblasts

Step 1, using cDNA reverse transcription of mRNA of healthy human whole blood as a template, designing primers according to sequence information in NCBI websites to amplify full-length genes of ASCL1, MYT1L, Scl1, Brn2 and Isx9, carrying out electrophoresis on PCR products by 8% agarose gel, cutting off bands with proper size for gel recovery, and carrying out double enzyme digestion on target bands recovered by the gel and a vector plasmid pLVX (purchased from Addgene). The double digestion system was allowed to react at 37 ℃ for 2-4 hours, and the plasmid was purified using a plasmid purification kit (purchased from OMEGA). The concentration of the purified plasmid was measured on a Nanodrop2000, the target fragment and the vector fragment were mixed at a ratio of 3:1, and ligation was performed overnight at 4 ℃. Transforming the connected plasmid through DH5 alpha competent cell, smearing on LB solid plate with ampicillin resistance, screening;

step 2, packaging lentivirus: transfection of 293T cells: 12-24 hours before transfection, 1 × 10⁶293T cells in a logarithmic growth phase are inoculated into a cell culture bottle of 75CM2 and cultured in a 5% CO2 incubator at 37 ℃, and the cells can be used for transfection when the confluence degree is 40% -70%. One hour prior to transfection, cells were replaced with fresh medium and plasmid transfection was performed using the calcium phosphate method. Mu.g of recombinant plasmid, 10. mu.g of each of the viral packaging plasmids pMD2.G and pSPAX2 (both from Clontech), was added to ultrapure water in a total volume of 450. mu.l, 50. mu.l of 2.5mol/L CaCl2 was added and mixed well, and then mixed dropwise with 500. mu.l of 2X BES, left at room temperature for 15-30min, added well to HEK293T cells, and then cultured in a 37 ℃ 5% CO2 incubator. After 8-16 hours, wash twice with PBS and replace with fresh medium. Collecting lentiviruses: collecting virus 48 hours after liquid change, adding culture solution containing slow virus into a 50ml centrifuge tube, centrifuging at 4 ℃ and 10000g for 15min to remove cell debris, adding supernatant into an ultrafiltration tube at 4 ℃ and 2700g, centrifuging for 30min, and concentrating virus solution to 600 mu l.

And 3, transferring the skin fibroblasts (donated by the psychiatric hospital in Henan province) of the schizophrenia patient to a cell culture bottle 24 hours before collecting the viruses, culturing in a 5% CO2 incubator at 37 ℃, and infecting when the cell confluency is 40% -70%. Before cell infection, fresh culture solution is replaced, and concentrated lentiviruses are respectively added into cells to be infected singly or in a 1:1 mixed mode (the total addition amount is 1.2 ml). After 36 hours, the medium was changed to a neural cell induction medium (DMEM/F12 medium supplemented with 1X N2 and B27, 20ng/ml β FGF, 20ng/ml EGF), and then observed every 2 days.

The experimental results are as follows:

the recombinant virus solutions are respectively added into the patient fibroblasts with the confluency of about 70-90% singly or in combination, and the cell survival rate and the transformation rate are shown in figure 1 after 15d observation. As shown in fig. 1, co-transfection of ASCL1 and MYT1L genes significantly improved the cell survival rate and transformation rate of fibroblasts reprogrammed to neural cells in schizophrenic patients. Based on this screening result, a group of cells co-transfected with ASCL1 and MYT1L genes was selected for the following observation and further experiments.

Morphology observation of co-transfected ASCL1 and MYT1L groups of cells: red fluorescence expression was observed after 24 hours. As shown in FIG. 2, 0Day is the cell state before infection, 5Day, the cell morphology begins to change, most cells gradually become slender, 10Day, the cells begin to have synapses and become nerve-like cells, 15Day, the cells can obviously be observed to grow slender synapses, most cells are transdifferentiated into nerve cells, and the differentiation efficiency reaches more than 80%.

Example 2 nerve cell identification

To identify whether the induced cells are nerve cells, MAP2 and GFAP two nerve marker genes are selected to carry out protein level identification on ASCL1 and MYT1L induced nerve cells by an immunofluorescence method. As shown in fig. 3: the mCherry is an exogenous gene inserted by cell expression and has red fluorescence; immunofluoresence is the detection result of immunofluorescence, and a secondary antibody is labeled by an FITC group and carries green fluorescence; DAPI is the result of staining the nuclei; merge is a composite image of three fluorescence pictures; the results showed that almost all induced cellular MAP2 and GFAP protein expression were positive.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method of inducing reprogramming of somatic cells to a neural cell model, the method comprising the steps of:

step 1, amplifying full-length genes of ASCL1 and MYT1L by using cDNA reverse transcribed by mRNA of healthy human whole blood as a template, performing electrophoresis on a PCR product by using 8% agarose gel, cutting off a band with a proper size for gel recovery, performing double enzyme digestion on a target band recovered by the gel and a vector plasmid pLVX, purifying, performing mixing and connection reaction according to the concentration ratio of a target fragment to a vector fragment of 3:1, transforming the connected plasmid by using DH5 alpha competent cells, coating the transformed plasmid on an LB solid plate with ampicillin resistance, and screening;

step 2, packaging lentivirus: transfection of 293T cells: taking 20 mu g of the recombinant plasmid obtained in the step 1, respectively taking 10 mu g of the virus packaging plasmids pMD2.G and pSPAX2, adding into ultrapure water to make the total volume be 450 mu L, and then adding 50 mu L of 2.5mol/L CaCl₂Mixing, mixing with 500 μ l 2X BES drop by drop, standing at room temperature for 15-30min, adding into HEK293T cell, culturing at 37 deg.C in 5% CO2 incubator for 8-16 hr, washing with PBS twice, and replacing with fresh culture solution; collecting lentiviruses: collecting virus 48 hours after liquid change, centrifuging culture solution containing slow virus, removing cell debris, centrifuging supernatant, and concentrating virus solution to 600 μ l;

and 3, transferring the skin fibroblasts of the schizophrenia patient to a cell culture bottle 24 hours before collecting the viruses, infecting the skin fibroblasts when the confluence degree of the cells is 40-70%, changing fresh culture solution before infecting the cells, mixing and adding 600 mu L of two kinds of concentrated slow viruses of ASCL1 and MYT1L into the cells to be infected, changing a nerve cell induction culture solution after 30-36 hours, and changing the solution every 2 days until the fibroblasts are differentiated into the nerve cells.

2. The method of claim 1, wherein the neural cell induction culture solution of step 3 is prepared by the following method: DMEM/F12 medium was supplemented with 1X N2 and B27, 20ng/ml β FGF, 20ng/ml EGF.

3. A method of modeling a disease in a neural cell, wherein the neural cell is produced by the method of claim 1.