Method for inducing human spinal astrocytes to be converted into motor neurons

Abstract

The invention discloses a method for inducing human spinal astrocytes to be converted into motor neurons, which comprises the following steps of: co-culturing a lentiviral vector carrying a gene Ascl1, a lentiviral vector carrying a gene Myt1l, a lentiviral vector carrying a gene Pou3f2, a lentiviral vector carrying a gene Isl1 and human spinal astrocytes; therefore, human spinal astrocytes are induced to be converted into motor neurons. The four-factor cocktail composition provided by the invention can successfully convert human spinal astrocytes into motor neurons in vitro, and provides a new strategy and theoretical basis for SCI repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for inducing human spinal cord astrocytes to transform into motor neurons. Background Art

[0002] Spinal cord injury (SCI) is a central nervous system injury disease with a high disability rate and serious consequences. It seriously impairs the patient's motor function and often leads to paralysis below the injury site. It is currently recognized that there are three major difficulties in the treatment of SCI: first, rapidly activated reactive glial cells, especially astrocytes, will proliferate in large numbers to form dense glial scar tissue, so that the axons, which grow relatively slowly, encounter insurmountable mechanical barriers and chemical barriers formed by inhibitory factors secreted by them when extending through the injury site; second, some damaged neurons will be lost due to necrosis or apoptosis, and the small amount of remaining neural stem cells alone cannot fully replenish the lost neurons, and the regeneration capacity of the damaged neurons that have not been lost is also very limited; third, a variety of regeneration inhibitors and inflammatory factors will appear at the injury site, forming a chemical microenvironment that is not conducive to axon regeneration. These unfavorable factors coordinate with each other, ultimately making it difficult to repair tissues and reconstruct functions in SCI.

[0003] In terms of replenishing the number of neurons, most studies use seed cells, such as embryonic stem cells, neural stem cells, stromal stem cells, and induced pluripotent stem cells (iPSCs), which are induced to transdifferentiate into a certain number of neurons in vivo. Although embryonic stem cells and neural stem cells have strong differentiation ability, and the latter are directed stem cells that differentiate into neural tissue cells, they are difficult to obtain; stromal stem cells are convenient to obtain and suitable for autologous transplantation, but they need to differentiate across germ layers, so the proportion of functional neurons obtained is not high. iPSCs use a series of cocktail transcription factors to infect somatic cells and induce their reprogramming to obtain embryonic stem cells. This type of cell has attracted much attention due to its advantages such as convenient material source, self-transplantation, and strong differentiation ability; but there are also some areas that need improvement. First, the entire induction process of iPSCs takes a long time and there are many uncertainties in its differentiation. Secondly, when iPSCs are transplanted in vivo, the residual undifferentiated cells have the potential risk of forming teratomas.

[0004] When nerve tissue is damaged, astrocytes will proliferate in large numbers and show progenitor cell characteristics, indicating that they have a high degree of plasticity in cell transdifferentiation. If we can reduce the reactive astrocytes that proliferate in large numbers and have negative effects after SCI, and try to transform reactive astrocytes into motor neurons with corresponding functions, we can achieve the goal of both properly eliminating glial scars and replenishing neurons lost due to SCI, while improving the microenvironment for axon regeneration. This should be a good way to solve the difficulties of repair and regeneration after SCI, and contribute a new way to realize the concept of SCI cell therapy and personalized medicine research and development. Summary of the invention

[0005] The purpose of the present invention is to provide a method for inducing human spinal cord astrocytes to transform into motor neurons, which method comprises co-culturing human spinal cord astrocytes with a lentiviral vector carrying the gene Ascl1, a lentiviral vector carrying the gene Myt1l, a lentiviral vector carrying the gene Pou3f2 and a lentiviral vector carrying the gene Isl1, thereby inducing the human spinal cord astrocytes to transform into motor neurons.

[0006] Furthermore, the lentiviral vector is pLenti-EF1a-GFAP-EGFP-CMV-MCS.

[0007] Furthermore, the co-culture is carried out using an induction medium, and the induction medium includes a DMEM / F12 medium and a Neurobasal medium.

[0008] Furthermore, the induction medium also contains insulin, sodium selenite, putrescine, progesterone, ChIR99021, SB431542, dibutyryl cyclic adenosine monophosphate, neurotrophic factor-3, brain-derived neurotrophic factor, ciliary ganglion neurotrophic factor, glial cell-derived neurotrophic factor, fetal bovine serum and B27.

[0009] Preferably, the volume ratio of DMEM / F12 medium and Neurobasal medium in the induction medium is 1:1, and the concentrations of insulin, sodium selenite, putrescine, progesterone, ChIR99021, SB431542, dibutyryl adenosine cyclophosphate, neurotrophic factor-3, brain-derived neurotrophic factor, ciliary ganglion neurotrophic factor, glial cell-derived neurotrophic factor, fetal bovine serum and B27 are: 25 μg / mL insulin, 30 nM sodium selenite, 100 nM putrescine, 20 nM progesterone, 1 μΜ ChIR99021, 10 μΜ SB431542, 1 mM dibutyryl adenosine cyclophosphate, 10 ng / mL neurotrophic factor-3, 10 ng / mL brain-derived neurotrophic factor, 10 ng / mL ciliary ganglion neurotrophic factor, 10 ng / mL glial cell-derived neurotrophic factor, 2% fetal bovine serum and 0.4% B27.

[0010] More preferably, the number of viruses infected per cell in the induction medium is 50 MOI.

[0011] The present invention is designed to induce the conversion of human spinal cord astrocytes into motor neuron cells by using a combination of four transcription factors, which is a 4-factor cocktail combination consisting of Ascl1, Myt1l, Pou3f2 and Isl1. In the initial stage of reprogramming, the 4-factor cocktail combination effectively induces a series of intermediate plastic states similar to neural progenitor-like cells by causing the loss of astrocyte identity, and then induces the occurrence of motor neuron progenitor-like cells; as the reprogramming process is prolonged, the 4-factor cocktail combination ultimately promotes the conversion of reprogrammed cells to the motor neuron lineage.

[0012] The 4-factor cocktail combination provided by the present invention can successfully convert human spinal cord astrocytes into motor neurons in vitro; the motor neurons induced by the 4-factor cocktail combination may be transplanted into the SCI model for SCI repair, which brings great hope for the repair of SCI; the motor neurons derived from the resident astrocytes in situ in the damaged spinal cord may be able to repair SCI, providing a new strategy and theoretical basis for the repair of SCI. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Results of GFAP cell immunofluorescence staining of human spinal cord astrocytes (scale bar = 40 μm).

[0014] Figure 2In the figure, A is the immunofluorescence staining result of MAP2, a neuron-specific marker, in human spinal cord astrocytes after 4 weeks of treatment with 4F cocktail combination (scale bar = 40 μm); B is the statistical analysis of the proportion of MAP2-positive cells in GFP-positive cells after 4 weeks of treatment with 4F cocktail combination; C is the immunofluorescence staining result of ChAT, a motor neuron-specific marker, in human spinal cord astrocytes after 5 weeks of treatment with 4F cocktail combination (scale bar = 40 μm); D is the statistical analysis of the proportion of MAP2 and ChAT double-positive cells in GFP-positive cells after 5 weeks of treatment with 4F cocktail combination. nd: not detected.

[0015] Figure 3 In the figure, AD is the results of real-time RT-PCR reaction to detect the mRNA levels of specific genes in astrocytes (A), neural progenitor cells (B, C) and motor neuron progenitor cells (D) at the initial stage of transdifferentiation; EN is the results of real-time RT-PCR reaction to detect the mRNA levels of neuron-related genes (EH) and motor neuron-related genes (IN) during transdifferentiation. *P<0.05, **P<0.01 vs 0 w. DETAILED DESCRIPTION

[0016] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1

[0019] 1. Human spinal cord astrocyte cell culture The specific process includes the following: (1-1) Human spinal cord astrocytes (Catalog Number: 1820) were purchased from ScienCell Research Laboratories. The culture medium was DMEM / F12 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin to prepare a complete culture medium. 75 cm 2The culture flask was cultured overnight, and the poly-lysine in the flask was discarded the next day. The flask was rinsed twice with sterile water, and the excess liquid was removed with a dropper. 15 mL of complete culture medium was added to the culture flask for later use. The frozen cryopreservation tube containing human spinal cord astrocytes was placed in a 37°C water bath, and the cryopreservation tube was held and rotated until the frozen liquid was completely thawed. The cryopreservation tube was removed from the water bath in time and wiped clean with 70% ethanol. Carefully remove the bottle cap, gently resuspend the liquid in the cryopreservation tube and transfer it to the culture flask containing complete culture medium. The culture flask was gently shaken to evenly distribute the cells. The culture flask was placed in a CO2 incubator, and new complete culture medium was replaced after 18 hours. The culture medium was replaced every three days until the cell density in the culture flask reached 70%. When the cell density reached 70%, the culture medium was replaced every two days until the cell density in the culture flask reached 90%.

[0020] (1-2) When the cell density in the culture flask reaches 90%, cell subculture is performed. The culture medium is discarded, and the cells are rinsed twice with DPBS. 3 mL of 0.25% trypsin is added to digest the human spinal cord astrocytes until the cell bodies are observed to be round under the microscope. 3 mL of complete culture medium is added to terminate the digestion, and the cells are blown up with a pipette. The cell suspension is transferred to a 15 mL centrifuge tube, centrifuged at 157 × g for 6 minutes, and the supernatant is discarded. 5 mL of complete culture medium is added for subculture.

[0021] 2. Four genes were selected based on the key genes in the induction and development of motor neurons: Ascl1, Myt1l, Pou3f2 and Isl1. These four genes were cloned and inserted into the lentiviral vector of pLenti-EF1a-GFAP-EGFP-CMV-MCS.

[0022] The specific process includes the following: (2-1) To construct lentiviral vectors, Ascl1 (Gene ID: 429), Myt1l (Gene ID: 23040), Pou3f2 (Gene ID: 5454), and Isl1 (Gene ID: 3670) genes were subcloned and inserted into the pLenti-EF1a-GFAP-EGFP-CMV-MCS lentiviral vector (constructed by OBiO Biotechnology). The expression of these genes is controlled by the human glial fibrillary acidic protein (GFAP) promoter, and the lentiviral vector encodes enhanced green fluorescent protein (EGFP). Empty virus was used as a control group.

[0023] (2-2) The final virus product concentration is as follows: pLenti-EF1a-GFAP-EGFP-CMV-Ascl1-3FLAG (virus titer 2.17×10 8 TU / mL) pLenti-EF1a-GFAP-EGFP-CMV-Myt1l-3FLAG (virus titer 2.76×10 8 TU / mL) pLenti-EF1a-GFAP-EGFP-CMV-Pou3f2-3FLAG (virus titer 4.14×10 8 TU / mL) pLenti-EF1a-GFAP-EGFP-CMV-Isl1-3FLAG (virus titer 4.29×10 8 TU / mL) pLenti-EF1a-GFAP-EGFP-CMV-3FLAG (virus titer 1.45×10 9 TU / mL).

[0024] 3. Add the lentivirus carrying Ascl1, Myt1l, Pou3f2 and Isl1 to a culture medium containing insulin, sodium selenite, putrescine, progesterone, ChIR99021, SB431542, dibutyryl cyclic adenosine monophosphate, neurotrophin-3, brain-derived neurotrophic factor, ciliary ganglion neurotrophic factor, glial cell-derived neurotrophic factor, fetal bovine serum and B27 (the volume ratio of DMEM / F12 culture medium and Neurobasal culture medium is 1:1) to establish a 4-factor cocktail combination containing Ascl1, Myt1l, Pou3f2 and Isl1; then use this cocktail combination to induce transdifferentiation of human astrocytes in vitro.

[0025] The specific process includes the following: (3-1) Take out the cell slide (biosharp) and place it in a 24-well cell culture plate.

[0026] (3-2) The human spinal cord astrocytes cultured in step (1) were seeded in a 24-well plate at a density of 5 × 10 4 The cells were seeded in a 24-well plate containing a cell slide and cultured overnight in a 5% CO2 incubator.

[0027] (3-3) On the second day, prepare the induction medium: mix equal volumes of DMEM / F12 and Neurobasal medium, and add 25 μg / mL insulin, 30 nM sodium selenite, 100 nM putrescine, 20 nM progesterone, 1 μΜ ChIR99021, 10 μΜ SB431542, 1 mM dibutyryl adenosine cyclophosphate, 10 ng / mL neurotrophin-3, 10 ng / mL brain-derived neurotrophic factor, 10 ng / mL ciliary ganglion neurotrophic factor, 10 ng / mL glial cell-derived neurotrophic factor, 2% fetal bovine serum and 0.4% B27; add the 4-factor virus to the above-mentioned induction medium, and the number of viruses infected per single cell is 50, to prepare the 4-factor cocktail combination induction medium.

[0028] (3-4) Replace the culture medium in the 24-well plate with the 4-factor cocktail combination induction culture medium and place the plate in a 5% CO2 incubator for continued culture.

[0029] (3-5) After 2 days, replace the culture medium in the 24-well plate with induction medium and place the plate in a 5% CO2 incubator for continued culture, with the medium changed every 4 days.

[0030] 4. Use cell immunofluorescence staining technology, real-time fluorescence quantitative PCR and statistical methods to analyze the transdifferentiation of human spinal cord astrocytes into motor neurons.

[0031] Among them, all data were expressed as mean ± standard error for statistical analysis. GraphPadPrism 6.01 software (San Diego) was used for statistical analysis. The normality of the data was tested by Shapiro-Wilk test. Statistical analysis was performed using t-test or one-way analysis of variance followed by Tukey post hoc test, and p < 0.05 indicated statistical significance.

[0032] Cell immunofluorescence staining technology specifically includes the following processes: (4-1-1) Take out the cell culture plate, remove the culture medium in the culture plate, add pre-warmed PBS to rinse three times, remove the PBS, add pre-warmed 4% paraformaldehyde, and fix for 8 min.

[0033] (4-1-2) Aspirate off 4% paraformaldehyde and add 250 μl pre-warmed PBS to each well and rinse three times for 10 min each time.

[0034] (4-1-3) Aspirate PBS and add 250 μl of PBS containing 1% Triton X-100 to each well to permeabilize the membrane and incubate for 15 min.

[0035] (4-1-4) Aspirate the permeabilization solution and add 250 μl of pre-warmed PBS to each well for 3 rinses, 10 min each time. Add 250 μl of PBS containing 3% BSA to each well for blocking and incubate at room temperature for 30 min.

[0036] (4-1-5) Aspirate the blocking solution and add 250 μl of PBS containing primary antibody and 3% BSA to each well. Incubate at 4°C for 18 h.

[0037] (4-1-6) Remove the primary antibody and add 250 μl PBS to each well for three washes, each for 10 min.

[0038] (4-1-7) Aspirate PBS and add 250 μl of PBS containing secondary antibody to each well. Incubate in a dark box at room temperature for 2 h.

[0039] (4-1-8) Remove the secondary antibody under light-protected conditions and add 250 μl PBS to each well and rinse three times, each time for 10 min.

[0040] (4-1-9) Aspirate PBS in a dark environment, add 250 μl PBS containing 1 / 1000 Hoechst to each well, incubate at 4°C for 30 min, aspirate PBS containing Hoechst, and add 250 μl PBS to rinse three times, 10 min each time.

[0041] (4-1-10) Add 1-2 drops of mounting solution containing fluorescence quencher on the adhesive slide, cover the slide with a cover glass, and store away from light.

[0042] (4-1-11) Observe and take photos under a laser confocal microscope.

[0043] The process of real-time fluorescence quantitative PCR includes the following steps: (4-2-1) Take out the cell culture plate, remove the culture medium, and rinse the cells once with PBS; place the culture plate on ice and use the UUBIO Cell RNA Rapid Extraction Kit (CoUbio) to extract the RNA of the reprogrammed cells.

[0044] (4-2-2) Convert RNA transcripts into cDNA using a reverse transcription kit (Thermo Fisher Scientific).

[0045] (4-2-3) cDNA was used for qPCR amplification. Real-time quantitative RT-PCR detection was performed on a Bio-Rad CFX96 real-time PCR instrument using the QuantiNova SYBR Green PCR Kit (Qiagen).

[0046] (4-2-4) The relative level of the target gene was normalized using the Gapdh expression level.

[0047] Human spinal cord astrocytes were stained with the astrocyte marker GFAP. Figure 1 As shown, human spinal cord astrocytes stained positive for GFAP.

[0048] After 4 weeks of induction with the 4-factor cocktail combination, cells reprogrammed with the 4-factor cocktail showed neurite outgrowth and smaller cell bodies compared with the vehicle group ( Figure 2 Middle A), quantitatively, approximately 15% of cells showed double positive staining for GFP and MAP2 ( Figure 2 Middle B). After 5 weeks of induction, about 38% of GFP+ cells were positive for MAP2 staining, and about 8% of GFP+ cells were positive for ChAT staining ( Figure 2 Middle C, Figure 2 Middle D).

[0049] Real-time quantitative RT-PCR was used to study the mRNA expression levels of astrocyte-specific marker Gfap, neural progenitor cell markers Sox2 and Ncam1, and motor neuron progenitor cell marker Olig2 in the early stage of human spinal cord astrocyte reprogramming mediated by the 4-factor cocktail combination. The results showed that as early as 3 days after reprogramming, the expression of Gfap in the 4-factor cocktail-reprogrammed cells was significantly inhibited ( Figure 3 At the same time, the mRNA expression of Sox2 and Ncam1 in the 4-factor cocktail-reprogrammed cells increased significantly, reaching a peak on the third day of reprogramming, and then decreased to a level similar to that before reprogramming after 7 days of reprogramming ( Figure 3 In addition, the mRNA expression of Olig2 in cells reprogrammed with the 4-factor cocktail was upregulated, reaching a peak on the 5th day of reprogramming, and then decreased to a level similar to that before reprogramming ( Figure 3 D). These results suggest that cells reprogrammed by the 4-factor cocktail may have the characteristics of neural progenitor cells and further tend to transform into motor neuron progenitor cells in the early stages of the reprogramming process. To determine whether these reprogrammed cells have typical characteristics of neurons, the mRNA expression of neuron-specific markers Map2, Syn1, Tubb3, and Nf-h was measured. The results showed that the mRNA expression of Map2, Syn1, Tubb3, and Nf-h in the 4-factor cocktail reprogrammed cells gradually increased throughout the reprogramming process ( Figure 3In order to further verify that the 4-factor cocktail combination mediates the transformation of human spinal cord astrocytes into motor neurons, real-time RT-PCR was also used to determine the expression changes of motor neuron-related genes during the reprogramming process. The results showed that after reprogramming, the mRNA expression levels of motor neuron-related genes Isl1, Hb9, Lhx1, Lhx3, Nkx 6.1, and Smn1 gradually increased ( Figure 3 IN).

[0050] The above results indicate that the 4-factor cocktail combination reprogramming method can initially induce human spinal cord astrocytes to transition to an intermediate state similar to neural progenitor cells and motor neuron progenitor cells; in addition, as the reprogramming time is prolonged, the 4-factor cocktail combination can ultimately induce the transformation of human spinal cord astrocytes into motor neurons.

Claims

1. A method for inducing human spinal cord astrocytes to transform into motor neurons, characterized in that: The lentiviral vectors carrying the gene Ascl1, the lentiviral vectors carrying the gene Myt1l, the lentiviral vectors carrying the gene Pou3f2 and the lentiviral vectors carrying the gene Isl1 were co-cultured with human spinal cord astrocytes to induce the human spinal cord astrocytes to transform into motor neurons.

2. The method according to claim 1, characterized in that The lentiviral vector is pLenti-EF1a-GFAP-EGFP-CMV-MCS.

3. The method according to claim 1, characterized in that The co-culture is carried out using an induction medium, which includes a DMEM / F12 medium and a Neurobasal medium.

4. The method according to claim 3, characterized in that The induction medium also contains insulin, sodium selenite, putrescine, progesterone, ChIR99021, SB431542, dibutyryl cyclic adenosine monophosphate, neurotrophic factor-3, brain-derived neurotrophic factor, ciliary ganglion neurotrophic factor, glial cell-derived neurotrophic factor, fetal bovine serum and B27.

5. The method according to claim 4, characterized in that The volume ratio of DMEM / F12 medium and Neurobasal medium in the induction medium is 1:1, and the concentrations of insulin, sodium selenite, putrescine, progesterone, ChIR99021, SB431542, dibutyryl cyclic adenosine monophosphate, neurotrophic factor-3, brain-derived neurotrophic factor, ciliary ganglion neurotrophic factor, glial cell-derived neurotrophic factor, fetal bovine serum and B27 are: 25 μg / mL insulin, 30 nM sodium selenite, 100 nM putrescine, 20 nM progesterone, 1 μΜ ChIR99021, 10 μΜ SB431542, 1 mM dibutyryl cyclic adenosine monophosphate, 10 ng / mL neurotrophic factor-3, 10 ng / mL brain-derived neurotrophic factor, 10 ng / mL ciliary ganglion neurotrophic factor, 10 ng / mL glial cell-derived neurotrophic factor, 2% fetal bovine serum and 0.4% B27.

6. The method according to claim 5, characterized in that The number of viruses infected in a single cell in the induction medium is 50 MOI.

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