CN110819593B - Directional-induction anti-apoptosis pluripotent stem cell, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a directional-induction anti-apoptosis pluripotent stem cell, a preparation method and application thereof, wherein a Tet-On system and an anti-apoptosis gene Bcl are transferred into a hiPSC by utilizing a gene modification technology, and the hiPSC which can be directionally induced into motor neurons is established. The obtained multipotent stem cells not only keep the advantages of wide sources and rapid proliferation of the hiPSC, but also ensure the controllability of the differentiation direction of the stem cells and the survival number of the transplanted cells in vivo, and solve the problems of the safety and the effectiveness of stem cell transplantation. Meanwhile, the scheme of the invention can lay a foundation for the treatment of ALS and other neurodegenerative diseases.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a directional-induction anti-apoptosis pluripotent stem cell, a preparation method and application thereof.
Background
Degenerative diseases are often accompanied by lesions or lesions of specific neurons, with amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS) patients mainly due to extensive death of motor neurons. None of the current treatments reverse nerve damage due to the nonrenewability of neurons. Stem cells can differentiate into corresponding nerve cells to replace damaged neurons and repair nerve loops, and thus can be applied to repair treatment of neurodegenerative diseases. As early as 2001, mazzini et al demonstrated a high safety and tolerability of mesenchymal stem cells in ALS patients in a first clinical trial [1] . Later, more and more clinical studies indicate that transplantation of mesenchymal stem cells can reduce the degeneration rate of motor neurons, regulate and control the secretion of cytokines, inhibit neuroinflammatory reactions, further improve the clinical symptoms of ALS patients and the survival quality of patients [2,3] . Although in spite ofThe mesenchymal stem cells are transplanted in clinical experiments with high safety and good tolerance, but have weak treatment effect, probably because the mesenchymal stem cells cannot be embedded in the spinal cord of a patient, are integrated and differentiated into motor neurons, and the aim of replacing injured neurons by the stem cells is difficult to realize.
Thus, some scientists have directly transplanted differentiated motor neurons into animals for research. Wherein Wichterle et al injected Embryonic Stem (ES) differentiated motor neurons into the spinal cord of chick embryos (15-17 d) found that the differentiated motor neurons survived in large amounts and prolonged the long processes to the muscles, forming neuromuscular junctions [4] . In addition, harper et al injected ES differentiated motor neurons into the lumbar spine of paraplegic rats, found that the differentiated motor neurons could extend axonal length, form neuromuscular junctions, and re-innervate paralyzed muscle tissue [5] . However, this method is only suitable for a static model such as paraplegia, and has no effect on ALS with continuously worsening and extremely poor prognosis [6] . In addition, the neuron differentiation process is complex, high in cost, long in time consumption (more than 1 month), low in efficiency (20% -50%), incapable of proliferation and severely limited in clinical application [7] 。
Induced pluripotent stem cells (Induced Pluripotent Stem Cell, iPSC) are breakthroughs with milestone significance in the field of stem cells in recent years. In particular, reprogramming somatic cells by ectopic expression of several critical nuclear transcription factors (e.g., c-MYC, KLF4, OCT3/4, SOX2, etc.) associated with maintaining ES pluripotency has increased a new approach to obtaining pluripotent stem cells consistent with the genetic background of the patient. The method is simple to operate, avoids ethical problems of using ES, is an ideal seed cell for tissue engineering, regenerative medicine, disease model and drug screening, and therefore has wide clinical application prospect.
iPSC proliferates rapidly, is widely available, has high differentiation potential, and is not ethically controversial relative to ES, but iPSC-based therapies still face two major challenges: on one hand, the iPSC has a tendency to form tumors, and the safety is extremely challenged; on the other hand, the transplanted cells are not easy to survive in vivo, and the differentiation direction is uncontrollable, so that the effectiveness of the transplanted cells is not guaranteed. And the directional differentiation of motor neurons is mostly limited to in vitro research at present, and whether the genetically modified iPSC can be directionally and efficiently differentiated into motor neurons in vivo and has a therapeutic effect on ALS is unknown.
Reference is made to:
[1]Mazzini L,Fagioli F,Boccaletti R,Mareschi K,Oliveri G,Oliveri C,Pastore I,Marasso R,Madon E.Stem cell therapy in amyotrophic lateral sclerosis:a methodological approach in humans[J].Amyotrophic Lateral Sclerosis and Other Motor Neuron Disorders,2003,4(3):158-161.
[2]Ciervo Y,Ning K,Jun X,Shaw P J,Mead R J.Advances,challenges and future directions for stem cell therapy in amyotrophic lateral sclerosis[J].Molecular neurodegeneration,2017,12(1):85.
[3]Petrou P,Gothelf Y,Argov Z,Gotkine M,Levy Y S,Kassis I, Vaknin-Dembinsky A,Ben-Hur T,Offen D,Abramsky O,Melamed E,Karussis D. Safety and clinical effects of mesenchymal stem cells secreting neurotrophic factor transplantation in patients with amyotrophic lateral sclerosis:results of phase 1/2and 2a clinical trials[J].JAMA neurology,2016,73(3):337-344.
[4]Wichterle H,Lieberam I,Porter J A,Jessell T M.Directed differentiation of embryonic stem cells into motor neurons[J].Cell,2002,110(3):385-397.
[5]Harper J M,Krishnan C,Darman J S,Deshpande D M,Peck S,Shats I, Backovic S,Rothstein J D,Kerr D A.Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats[J].Proceedings of the National Academy of Sciences,2004,101(18):7123-7128.
[6]Papadeas S T,Maragakis N J.Advances in stem cell research for Amyotrophic Lateral Sclerosis[J].Current opinion in biotechnology,2009,20(5): 545-551.
[7]Sances S,Bruijn L I,Chandran S,Eggan K,Ho R,Klim J R,Livesey M R, Lowry E,Macklis J D,Rushton D,Sadegh C,Sareen D,Wichterle H,Zhang S,Svendsen C N. Modeling ALS with motor neurons derived from human induced pluripotent stem cells[J].Nature neuroscience,2016,19(4):542.
disclosure of Invention
The first technical problem to be solved by the invention is that: provides a directional induced, anti-apoptotic pluripotent stem cell.
The second technical problem to be solved by the invention is that: a method for preparing the pluripotent stem cells is provided.
The third technical problem to be solved by the invention is that: the use of the pluripotent stem cells described above is provided.
In order to solve the first technical problem, the invention adopts the following scheme: a directional induced anti-apoptosis pluripotent stem cell is introduced with a Tet-On system and an anti-apoptosis gene Bcl.
The directional-induction anti-apoptosis pluripotent stem cells provided by the embodiment of the invention have at least the following beneficial effects: the multipotent stem cells can improve the retention rate of the cells in vivo when transplanted due to the transfer of anti-apoptosis gene Bcl; transferring into a Tet-On system, regulating and controlling the gene expression of Ngn2, isl1 and Lhx3, thereby directionally inducing the formation of motor neurons. The Tet-On system is a tetracycline adjustable system, and the Tet-On gene expression system becomes an ideal induction expression system due to the advantages of tight adjustment, high induction efficiency and the like, and the prepared pluripotent stem cells can be directionally induced and differentiated by being transferred into the Tet-On system. The multipotent stem cells prepared by the embodiment of the invention not only maintains the advantages of wide sources and rapid proliferation of hiPSC, but also ensures the controllability of the differentiation direction of the stem cells and the survival number of the transplanted cells in vivo, and solves the problems of safety and effectiveness of stem cell transplantation.
According to some embodiments of the invention, the Tet-On system includes a tetracycline response factor (Tetracycline Response Element, TRE) and genes of interest Ngn2, isl1, lhx3, which regulate Ngn2, isl1, and Lhx3 gene expression via the TRE.
In order to solve the second technical problem, the invention adopts the following scheme: the preparation method comprises the step of transferring a Tet-On system and an anti-apoptosis gene Bcl into a pluripotent stem cell by using a gene modification technology to obtain the directional-induction anti-apoptosis pluripotent stem cell.
According to some embodiments of the invention, the Tet-On system includes Ngn2, isl1, lhx3 genes and a TRE, which regulates Ngn2, isl1 and Lhx3 gene expression by the TRE.
According to some embodiments of the invention, the method of preparing pluripotent stem cells comprises the steps of:
cloning the Tet-On system and the anti-apoptosis gene Bcl to a vector, co-transfecting the vector and hiPSC, and obtaining the pluripotent stem cells transformed with the Tet-On system and the anti-apoptosis gene Bcl through resistance screening.
According to some embodiments of the invention, the co-transfection procedure uses a vector comprising two PB vectors and a transposase in a mass ratio of 2:2:1.
According to some embodiments of the invention, the resistance gene of the resistance selection is a BSD resistance gene, and the anti-apoptosis vector contains GFP markers therein, and GFP positive cells after BSD selection are cells transfected with the two vectors.
In order to solve the third technical problem, the invention adopts the following scheme: a preparation method of motor neuron comprises adding tetracycline drugs to induce the above directional induced and anti-apoptosis multipotent stem cells to differentiate into motor neuron, wherein the tetracycline drugs comprise at least one of tetracycline and tetracycline derivatives.
In order to solve the third technical problem, another technical scheme adopted by the invention is as follows: a pharmaceutical formulation for the treatment of neurodegenerative diseases comprising directionally inducible, anti-apoptotic pluripotent stem cells as described above.
According to some embodiments of the invention, the pharmaceutical formulation may induce differentiation into motor neurons in vivo, and the neurodegenerative disease is a motor neuron injury disease (e.g., ALS and other related diseases).
The application according to the embodiment of the invention has at least the following beneficial effects: human induced pluripotent stem cells (human induced pluripotent stem cells, hiPSC) are directly injected into a human body for the first time, and the in-vivo directional induction and differentiation of the hiPSC into motor neurons are realized under the condition of medicaments, so that the problems of safety and effectiveness of stem cell transplantation are solved. The scheme of the invention can provide a research basis for the treatment of ALS and other neurodegenerative diseases.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects, experimental results, and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a plasmid map of PB vector of example 1 of the present invention;
FIG. 2 is a fluorescence characterization diagram of the in vitro directed differentiation of pluripotent stem cells according to example 2 of the invention;
FIG. 3 is a diagram showing the detection of motor neuron electrophysiological activity according to example 2 of the present invention;
FIG. 4 is a graph showing the results of a passaging stress experiment in example 2 of the present invention;
FIG. 5 is a graph showing the results of in vivo imaging experiments of the directional differentiation of pluripotent stem cells according to example 3 of the invention;
FIG. 6 is a fluorescence characterization of the in vivo directed differentiation of pluripotent stem cells of example 3 of the invention.
Detailed Description
In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments.
The main idea of the invention is as follows: the invention provides a directional induction and anti-apoptosis pluripotent stem cell, which is transferred into a Tet-On system, and regulates and controls the expression of Ngn2, isl1 and Lhx3 genes through TRE, so that the cell can be directionally induced into motor neurons, and simultaneously transferred into an anti-apoptosis gene Bcl, thereby improving the in-vivo retention rate of transplanted cells.
The first embodiment of the invention is as follows: a directionally-inducible, anti-apoptotic pluripotent stem cell is obtained, which is specifically operated as follows:
1. construction of the Piggy-Bac (PB) vector:
(1) PB vector (1) for inducing motor neuron directed differentiation PB-Ngn2-Isl1-Lhx3 (NIL) -BSD the PB vector used in this example was donated by the Italy professor Alessandro Rosa to thank it, and this plasmid was introduced into tetracycline response factor (tetracycline response element, TRE) to regulate gene expression of Ngn2, isl1 and Lhx3, and contained Blasticidin (BSD) resistance, which could be used for cell screening.
TRE consists of 7 repeated Tet operator (TetO) sequences.
(2) To increase the in vivo retention of hiPSC we constructed a PB vector (2) PB-Bcl-luciferase-GFP, cloning the anti-apoptotic gene Bcl into vector (2) with the addition of the marker genes luciferase (luciferase) and green fluorescent protein (Green Fluorescent Protein, GFP), where GFP facilitates the selection of cell monoclonal under the microscope and luciferase facilitates the detection of survival of transplanted cells in vivo by in vivo imaging.
The Piggy-Bac (PB) transposon system is a currently commonly used tool for genetic engineering of mammals, transposons are DNA fragments that can be site-transformed on the genome of both prokaryotes and eukaryotes, by cleavage or replication at the original site, and by cyclization for insertion into other sites of the host genome with the aid of transposases. Compared with virus-mediated and traditional transposon subsystems, the PB system has the advantages of safety, high efficiency, large load capacity, high specificity and the like.
The PB vector (1 PB-Ngn2-Isl1-Lhx3 (NIL) -BSD and PB vector (2) PB-Bcl-luciferase-GFP in this example were prepared according to the procedures shown in FIG. 1, and the parameters such as the addition amount of each reagent were set conventionally.
Bcl (Gene ID 397536) Gene sequence SEQ ID No.1:
ATGGACTGGTGGAGCCCATCCCTATTATAAAAATGTCTCAGAGCAACCGGG AGCTGGTGGTTGACTTTCTCTCCTACAAGCTTTCCCAGAAAGGATACAGCT GGAGTCAGTTTACTGATGTGGAAGAGAACAGAACTGAGGCCCCAGAAGGG ACTGAATCAGAAGCGGAAACCCCTAGTGCCATCAATGGCAACCCATCCTGG CACCTGGCGGACAGCCCCGCGGTGAATGGAGCCACTGGCCACAGCAGCAG CTTGGATGCCCGGGAGGTGATCCCCATGGCTGCAGTGAAGCAAGCGCTGA GGGAGGCGGGCGATGAGTTTGAACTGAGGTACCGGAGGGCATTCAGTGAC CTGACGTCCCAGCTCCACATCACCCCAGGGACAGCGTATCAGAGCTTTGAG CAGGTAGTGAACGAACTCTTCCGGGATGGGGTGAACTGGGGTCGCATTGT GGCCTTTTTCTCCTTCGGTGGGGCACTGTGCGTGGAGAGCGTAGACAAGG AGATGCAGGTATTGGTGAGTCGGATCGCAACTTGGATGGCCACTTACCTGA ATGACCACCTAGAGCCTTGGATCCAGGAGAACGGCGGCTGGGACACTTTT GTGGAACTCTACGGAAACAATGCAGCAGCTGAGAGCCGGAAGGGCCAGG AGCGCTTCAACCGATGGTTCCTGACGGGCATGACTCTAGCTGGGGTGGTTC TGCTGGGTTCGCTCTTCAGTCGGAAATGA。
(3) The specific experimental operation is as follows:
1) And (3) PCR reaction: PCR amplification was performed using Bcl cDNA and pGL4.21 (from Promega corporation) as templates, corresponding primers;
2) Double enzyme cutting: the PB-CAG-EGFP plasmid was digested with EcoRI and HindIII;
3) Electrophoresis: the PCR amplification products and the enzyme digestion reaction are subjected to agarose gel electrophoresis, and a gel imaging system is used for observing whether the band size is correct.
4) Recovering the vector and gene fragment: after the PCR identification is correct, the DNA is recovered by a gel DNA recovery kit;
5) Homologous recombination: ligating the gene fragment to a vector by means of a homologous recombination kit;
6) Ligation product conversion: transformation with competent bacteria TOP10 and plating, placing in a 37℃incubator, and culturing overnight;
7) Small-scale extraction of recombinant plasmid: 6 monoclonals are selected to LB liquid medium with ampicillin resistance, shake culture is carried out for 8-10 hours at 37 ℃ by using a kit drawer plasmid;
8) Identification of recombinant plasmids: performing enzyme digestion and sequencing, and performing moderate extraction after the result is correct;
9) Plasmid extraction and preservation: plasmid DNA concentration was determined by extracting plasmid from plasmid of Tiangen corporation using the plasmid extraction kit.
2. Transfection and screening of hipscs:
(1) Transfection of hiPSC: hiPSC co-transfects a PB vector and a transposase, wherein the mass ratio of PB vector to transposase is PB-NIL-BSD: PB-Bcl-luciferase-GFP: piggyBac transposase =2:2:1.
(2) Screening of hipscs: after 3 days, 5000 transfected cells were inoculated into a 10-cm dish, and 10. Mu.g/mL of Blasticidin (BSD) was added for continuous screening until no significant cell death was observed, and GFP positive cell clone was generated under a fluorescence microscope, and then the positive cell clone was picked into a 24-well plate for continuous culture, thereby obtaining an inducible, stable, anti-apoptotic, high purity positive cell clone PB-NIL-BSD+Bcl-luciferase-GFP. Through resistance screening and obtaining the pluripotent stem cells transformed into a Tet-On system and an anti-apoptosis gene Bcl.
The Piggy-Bac (PB) vector needs to insert a target gene fragment into a host cell by the action of a transposase, so that a transposase with a certain concentration is added in the transfection process to complete fusion of an introduced gene and DNA of the host cell.
The initial hiPSC used in this example was taught by Pan Guangjin of the guangzhou biomedical and health institute. The hiPSC cell line was UH10, induced by cord blood cells. The hiPSC can be purchased from stem cell banks such as Shanghai cell banks of China academy of sciences or biological companies to realize the embodiment of the invention.
The second embodiment of the invention is as follows: an in vitro experiment of a directional induced, anti-apoptosis pluripotent stem cell, which comprises the following specific operations:
1. hiPSC-NIL-BSD+Bcl-luciferase-GFP in vitro directed differentiation and identification:
(1) Induction of motor neurons: the hiPSC-NIL-BSD+Bcl-luciferase+GFP directed differentiation was induced by the addition of doxycycline (DOX, 1. Mu.g/mL).
Doxycycline DOX is a derivative of tetracycline. The more commonly used inducer in Tet induction control systems is DOX, which requires less amount of DOX to fully activate or inhibit the expression of the gene of interest and has a longer half-life than tetracycline Tet. In the scheme, the tetracycline medicaments can be tetracycline or derivatives thereof, and the DOX induction effect is better. The Tet-On system activates transcription in the presence of tetracycline, and transcribed Ngn2, isl1, lhx3 transcription factors induce directed differentiation of cells into motor neurons.
(2) Characterization of related markers after induced differentiation: the motor neuron-specific marker HB9, and the mature motor neuron marker ChAT were characterized sequentially by immunofluorescence at 7d, 14d after continuous 7d induction of DOX, respectively.
The obtained immunofluorescence characterization result is shown in fig. 2, wherein hiPSC-NIL-bsd+bcl-luciferase+gfp is directionally differentiated into motor neurons under the induction of DOX, and the motor neuron marker HB9 and the mature motor neuron marker ChAT are expressed respectively, wherein bar=100 μm, in fig. 2, the upper graph is the 7 th day after the continuous induction for 7d, and the lower graph is the 14 th day immunofluorescence characterization result graph after the continuous induction for 7 d. The results showed that the pluripotent stem cells hiPSC-NIL-BSD+Bcl-luciferase+GFP were efficiently and directionally differentiated into motor neurons in vitro.
2. Identification of in vitro directed differentiation results:
(1) Generation of electrophysiological activity: the induced neurons were co-cultured with glial cells for 3 weeks, and after the neurite mass formation, the electrophysiological activity of the neurons was measured by patch clamp technique.
The results of the resulting electrophysiological activity are shown in fig. 3: after 3 weeks of hiPSC-NIL-BSD+Bcl-luciferase+GFP-induced motor neurons were co-cultured with glial cells, na was detected by patch clamp technique + 、K + 、Ca 2+ Ion current and action potential. The result shows that the motor neuron formed by induction has normal electrophysiological activity index.
(2) Neuronal anti-apoptotic activity assay: the hiPSC is used for respectively transfecting a recombinant vector 1 (PB-NIL-BSD) and a recombinant vector 1 heavy group vector 2 (PB-Bcl-luciferase-GFP), and two positive cell clones are obtained through antibiotic screening: one strain was stable, inducible hiPSC-NIL-BSD, and the other strain was stable, inducible and anti-apoptotic, hiPSC-NIL-BSD+Bcl-luciferase-GFP. The same number of glial cells were seeded in 24-well plates, and the next day the same number of hiPSC-NIL-BSD and hiPSC-NIL-BSD+Bcl-luciferase-GFP were re-seeded on the glial cells, and DOX (1. Mu.g/mL, 7 d) was added to induce differentiation into motor neurons. After passage, the survival condition of the motor neurons is observed and counted, and then the anti-apoptosis capability of Bcl is judged.
Studies have shown that induced neurons are quite sensitive to stress caused by the propagation, and the death rate of neurons exceeds 60% when the neurons are subjected to the propagation under the condition of being co-cultured with glial cells. Thus, bcl anti-apoptotic effects can be investigated by a model of passaging stress-accelerated cell death.
The results of the anti-apoptotic ability obtained are shown in FIG. 4: in the passaging stress experiments, the same number of cells were inoculated, and the number of neuronal survival induced by hiPSC-NIL-BSD+Bcl-luciferase-GFP after passaging was significantly greater than that induced by hiPSC-NIL-BSD, bar=100. Mu.m. The result shows that the anti-apoptosis capability of the pluripotent stem cells can be effectively improved by introducing Bcl anti-apoptosis genes.
The third embodiment of the invention is as follows: an in vivo experiment of a directional induced, anti-apoptosis pluripotent stem cell, which comprises the following specific operations:
1. hiPSC-NIL-BSD+Bcl-luciferase-GFP in vivo directed differentiation:
(1) hiPSC-NIL-BSD+Bcl-luciferase-GFP cell transplantation and in vivo induction: the hiPSC-NIL-BSD+Bcl-luciferase-GFP (1X 10) 6 cells were resuspended in 200. Mu.L of DMEM/F12: matrix=1:1) and subcutaneously injected into immunodeficient mice (Severe Combined Immunodeficiency, SCID) (5-7 weeks old), 3 mice in the same batch were intraperitoneally injected with DOX (50 mg/kg), followed by 7d of continuous injection for directional induction.
2. Identification of in vivo directed differentiation results:
(1) Survival of hipscs in vivo: the in vivo survival of hiPSC-NIL-BSD+Bcl-luciferase-GFP was determined by detecting the luciferase signal through in vivo imaging experiments.
The results of the in vivo survival obtained are shown in figure 5: FIG. 5 shows the results of 2 weeks after induction of three mice in the same batch, and in FIG. 5, the left panel shows the front view, the right panel shows the back view, and a large number of luciferase signals are detected in the mice after cell transplantation, indicating that the transplanted cells survived well in vivo.
(2) hiPSC differentiation and maturation levels in vivo: the grafts were removed and motor neuron markers (. Beta. -tubulin, chAT) were characterized by immunofluorescence techniques to determine the level of hiPSC-NIL-BSD+Bcl-luciferase-GFP differentiation and maturation in vivo.
The results of the immunofluorescence characterization obtained are shown in fig. 6: DOX (50 mg/kg,7 d) was injected intraperitoneally to induce hiPSC-NIL-BSD+Bcl-luciferase-GFP in vivo, after two weeks, the grafts were removed, frozen sections were taken and immunofluorescence was performed to label the transplanted cells with human nuclear antibodies (nucleic) which simultaneously expressed neuronal markers (. Beta. -tubulin, chAT), indicating that the transplanted cells differentiated into mature motor neurons, bar=100. Mu.m.
In summary, the beneficial effects of the invention are as follows: the Tet-On system and the anti-apoptosis gene are transferred into the hiPSC by utilizing a gene modification technology, so as to prepare the hiPSC which can be directionally induced and is anti-apoptotic. Transferring into a Tet-On system, regulating and controlling the gene expression of Ngn2, isl1 and Lhx3, and directionally inducing the formation of motor neurons; transfer into anti-apoptosis gene Bcl to raise the in vivo detention rate of transplanted cell. The obtained multipotent stem cells not only keep the advantages of wide sources and rapid proliferation of the hiPSC, but also ensure the controllability of the differentiation direction of the stem cells and the survival number of the transplanted cells in vivo, and solve the problems of the safety and the effectiveness of stem cell transplantation. Meanwhile, the scheme of the invention can lay a research foundation for the treatment of ALS and other neurodegenerative diseases.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Sequence listing
<110> university of five-Yi
Jiangmen Great Health International Innovation Research Institute
<120> directed induced, anti-apoptotic pluripotent stem cells, methods of making and uses thereof
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 734
<212> DNA
<213> Sus scrofa
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atggactggt ggagcccatc cctattataa aaatgtctca gagcaaccgg gagctggtgg 60
ttgactttct ctcctacaag ctttcccaga aaggatacag ctggagtcag tttactgatg 120
tggaagagaa cagaactgag gccccagaag ggactgaatc agaagcggaa acccctagtg 180
ccatcaatgg caacccatcc tggcacctgg cggacagccc cgcggtgaat ggagccactg 240
gccacagcag cagcttggat gcccgggagg tgatccccat ggctgcagtg aagcaagcgc 300
tgagggaggc gggcgatgag tttgaactga ggtaccggag ggcattcagt gacctgacgt 360
cccagctcca catcacccca gggacagcgt atcagagctt tgagcaggta gtgaacgaac 420
tcttccggga tggggtgaac tggggtcgca ttgtggcctt tttctccttc ggtggggcac 480
tgtgcgtgga gagcgtagac aaggagatgc aggtattggt gagtcggatc gcaacttgga 540
tggccactta cctgaatgac cacctagagc cttggatcca ggagaacggc ggctgggaca 600
cttttgtgga actctacgga aacaatgcag cagctgagag ccggaagggc caggagcgct 660
tcaaccgatg gttcctgacg ggcatgactc tagctggggt ggttctgctg ggttcgctct 720
tcagtcggaa atga 734
Claims (4)
1. A method for preparing a directional-induced anti-apoptotic pluripotent stem cell, comprising the steps of:
respectively cloning a Tet-On system and an anti-apoptosis gene Bcl onto a PB carrier to obtain a PB-Ngn2-Isl1-Lhx3 (NIL) -BSD carrier and a PB-Bcl-luciferase-GFP carrier, and then co-transfecting the PB-Ngn2-Isl1-Lhx3 (NIL) -BSD carrier, the PB-Bcl-luciferase-GFP carrier and a transposase into hiPSC, and obtaining a multipotent stem cell transformed into the Tet-On system and the anti-apoptosis gene Bcl through blasticidin resistance screening;
the Tet-On system comprises Ngn2, isl1, lhx3 genes and a TRE, and regulates and controls the expression of the Ngn2, isl1 and Lhx3 genes through the TRE;
the promoter of the PB-Bcl-luciferase-GFP vector is a CMV promoter;
the mass ratio of the PB-Ngn2-Isl1-Lhx3 (NIL) -BSD vector, the PB-Bcl-luciferase-GFP vector and the transposase is 2:2:1;
the nucleotide sequence of the anti-apoptosis gene Bcl is shown as SEQ ID No. 1.
2. The method of claim 1, wherein said PB-Bcl-luciferase-GFP vector comprises a GFP marker, and said GFP positive cells after blasticidin resistance selection are cells transfected with said PB-Ngn2-Isl1-Lhx3 (NIL) -BSD vector and said PB-Bcl-luciferase-GFP vector.
3. A method for preparing a motor neuron, wherein doxycycline is added to induce directional induction and differentiation of the directionally-inducible, anti-apoptotic pluripotent stem cells prepared by the preparation method according to claim 1 or 2 into the motor neuron.
4. A pharmaceutical formulation for the treatment of neurodegenerative diseases, comprising directionally-inducible, anti-apoptotic pluripotent stem cells prepared by the method of claim 1 or 2;
the pharmaceutical preparation can induce differentiation into motor neurons in vivo, and the neurodegenerative disease is motor neuron injury disease.
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