CN109554342A - Method for obtaining spinal GABA energy interneuron by inducing pluripotent stem cell - Google Patents
Method for obtaining spinal GABA energy interneuron by inducing pluripotent stem cell Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
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Abstract
The invention relates to a method for obtaining spinal cord GABAergic interneurons by inducing pluripotent stem cells, which adopts a monolayer cell induction method and utilizes the pluripotent stem cells to perform induction differentiation so as to gradually form neuroepithelial cells, spinal cord neural precursor cells and spinal cord GABAergic interneurons. The method is rapid and efficient, and the clinical-grade spinal GABA energy interneuron can be obtained.
Description
Technical field
The present invention relates to pharmaceutical technology fields, refer specifically to pluripotent stem cell induction and obtain spinal cord GABA energy intrerneuron
Method.
Background technique
Spinal cord injury is common trauma, causes a series of movement and psychological problems, is common in 30 years old or less
Crowd.According to statistics, the whole world increases 250,000 to 500,000 Patients of Spinal newly every year, and China does not have national model also at present
Interior statistics is enclosed, but from the point of view of the statistics of each place property, the spinal cord injury disease incidence in China is much higher than American-European countries.According to the U.S.
The discovery of statistics in 2013, spinal cord injury influence the quality of life of nearly 1,000,000 people, and annual health care costs nearly 40,000,000,000 (Armour,
B.S.,Courtney-Long,E.A.,Fox,M.H.,Fredine,H.,and Cahill,A.(2016).Prevalence
and causes of paralysis-United States,2013.Am.J.Public Health 106,1855–1857)。
Wherein nearly 40%~50% patient is secondary in 1 year there is neurogenic pain, and is chronic ache in later transformation,
Influence patient recovery (Jay M Margolis, Paul Juneau, Alesia Sadosky, Joseph C Cappelleri,
Thomas N Bryce,Edward C Nieshoff.Health care utilization and expenditures
among Medicaid beneficiaries with neuropathic pain following spinal cord
injury.Journal of Pain Research.2014:7 379–387)。
Pain caused by cancer is most common and most painful one of the symptom of malignant tumor patient.About 70%~87% patient sends out in carcinoma
There is different degrees of pain during exhibition, and liver cancer, cancer of pancreas, Patients with Osteosarcoma are even more just to have pain in disease early stage.
Only China just has more than 100 ten thousand patients by the torment of cancer pain every year.Middle and advanced stage patient is up to 60% with the pain person of being chief complaint
~90%, seriously reduce the life quality of patient.Aggravation and intense stimulus due to pain directly affect the appetite of patient, sleep
It sleeps, psychologic status and therapeutic effect, malignant tumor patient can usually lose treatment confidence and existence desire (Zhang J.A
nalgesia mechanism and clinical application of acupuncture.Beijing:People's M
edical Publishing House.2007:522.Chinese.L i Q S,Qian H.Cancer complications
and its management.In:Tang Z Y.Modern oncology.Shanghai:Shanghai Medical
University Press.2000:556.Chinese).Simulate the classical model such as spinal cord injury and cancer of central nervous system injury
There is the missing and dysfunction of spinal levels GABA serotonergic neuron in pain model, so as to cause pain sensation conduction to varying degrees
" disinhibition " phenomenon of access, it is final to induce the Novel presentations such as hyperalgia and allodynia.
Clinically mainly there are drug therapy and physical factor treatment, but the whole body of drug therapy for the treatment of pain at present
Side effect is obvious, and the paracmasis of physical factor treatment is short, and ideal therapeutic effect is all not achieved, and needs to seek new treatment side
Method.In recent years, domestic and foreign scholars focus on the novel therapeutic of stem cell one after another, achieve major progress, but still remain many
Problem influences cell and plays in vivo for example, being implanted into intracorporal cell accurately cannot form Synaptic junction with host cell
Effect.The cell of targeted type is not moved into the research of pain caused by stem-cell therapy disease both at home and abroad at present
(Young S.Gwak*,Claire E.Hulsebosch；GABA and central neuropathic pain
following spinal cord injury；Neuropharmacology 60(2011)799e808).
Summary of the invention
In order to solve the problems in the existing technology, the invention proposes be height using pluripotent stem cell directed differentiation
The spinal cord GABA energy intrerneuron of purity, the pain caused for treating various diseases.
Pluripotent stem cell of the present invention induction obtain spinal cord GABA can the method for intrerneuron include:
Step (1) pluripotent stem cell breaks up under the action of CHIR99021, SB431542 and DMH1 obtains neural epithelium
Cell；
Cyclopamine is added in step (2) and retinoic acid carries out successive induction differentiation, obtains spinal nerve precursor；
Step (3) after digestion process, broken up under the action of cyclopamine and retinoic acid by continuation, obtains spinal cord GABA
It can intrerneuron；And
For step (4) after digestion process, the spinal cord GABA energy intrerneuron, which is carried out adhere-wall culture, makes its maturation.
Further, the molar concentration rate of described CHIR99021, SB431542 and DMH1 are 3:2:2.
Further, the molar concentration rate of the cyclopamine and retinoic acid is 5:1.
Further, the incubation time of the step (4) is 3~4 weeks.
Further, in step (1)~step (4) required cell culture fluid by the other culture medium of clinical grade and clinical grade
Other additive is prepared.
The present invention also provides preceding methods to prepare the purposes of gained spinal cord GABA energy intrerneuron in medicine preparation,
The drug is for treating pain.
The purpose of the present invention is to provide Liraglutides to treat the application in acute and chronic graft-rejection drug in preparation,
A kind of new drug is provided for the treatment of graft rejection.
Further, the pain is caused by spinal cord injury or malignant tumour.
Treatment the previous day, nerve ball to be transplanted is digested to individual cells, counted.It is outstanding that cell was made in the transplanting same day
Liquid, the cell suspension include Neurabasal, NEAA, B27, AA, N2, BDNF, GDNF, cAMP.
Pluripotent stem cell of the present invention induction obtain spinal cord GABA can intrerneuron can also be used to cell and combine control
It treats, for example cell transplantation joint electroacupuncture treatment, cell transplantation combine Neural stem cell therapy.
Beneficial effects of the present invention:
The method that pluripotent stem cell induction of the present invention obtains spinal cord GABA energy intrerneuron can be rapidly and efficiently
Ground obtains the spinal cord GABA energy intrerneuron of high-purity, and avoids the interference of extrinsic factor.
Detailed description of the invention
Fig. 1 is the flow chart that pluripotent stem cell is divided into spinal cord GABA energy intrerneuron in the embodiment of the present invention 1；
Fig. 2-a is that people's pluripotent stem cell breaks up to the Sox1 of the 7th day gained neuro-epithelial cell in the embodiment of the present invention 1
Immunohistochemistry figure, Fig. 2-b are that people's pluripotent stem cell breaks up the neuro-epithelial cell occurred to the 7th day in the embodiment of the present invention 1
Hoxa3 immunohistochemistry figure；
Fig. 3-a is that people's pluripotent stem cell breaks up to the 14th day gained spinal interneuron in the embodiment of the present invention 1
The OLIG2 immunofluorescence of body cell, Fig. 3-b are that people's pluripotent stem cell breaks up to the 14th day gained in the embodiment of the present invention 1
The Hoxb4 immunofluorescence of spinal interneuron precursor；
Fig. 4-a is that people's pluripotent stem cell breaks up to the 21st day intermediate mind of gained spinal cord GABA energy in the embodiment of the present invention 1
GABA immunofluorescence through member, Fig. 4-b are that people's pluripotent stem cell breaks up to the 21st day gained spinal cord in the embodiment of the present invention 1
The NF-200 immunofluorescence of GABA energy intrerneuron；Fig. 4-c be the embodiment of the present invention 1 in people's pluripotent stem cell break up to
The GAD65/67 immunofluorescence of 21st day gained spinal cord GABA energy intrerneuron；Fig. 4-d is that people is more in the embodiment of the present invention 1
Pluripotent stem cell breaks up to the Tuj1 immunofluorescence of the 21st day gained spinal cord GABA energy intrerneuron.
Specific embodiment
Below in conjunction with the attached drawing performance that the present invention will be described in detail, but they are not constituted a limitation of the invention, and are only made
Citing.By specific embodiment, the present invention is described in further detail simultaneously.
The method that pluripotent stem cell induction of the present invention obtains spinal cord GABA energy intrerneuron includes four steps:
The culture solution that the method that pluripotent stem cell induction of the present invention obtains spinal cord GABA energy intrerneuron uses
All non-animal derived property, obtained GABA can intrerneuron be that clinical grade is other, can be used for clinical research and future
Clinical treatment.
This method uses cell monolayer abductive approach, and the nerve of tubulose is obtained using CHIR99021, SB431542 and DMH1
After epithelial cell, cyclopamine (cyclopamine) is added and Retinoic Acid (retinoic acid) is induced, finally obtains ridge
The GABA energy intrerneuron in marrow source, the pain caused for treating various diseases.
Fig. 1 is the specific culture process of this method and the marker detection for cultivating each stage.In figure, D0, D7, D14 and D21
The 0th day, the 7th day, the 14th day and the 21st day of culture is respectively indicated, PSC represents pluripotent stem cell；NEP represents neural epithelium
Cell；SCNP represents spinal interneuron precursor；It is small that 3C represents tri- kinds of chemistry of CHIR99021, SB431542 and DMH1
Molecule；5C represents five kinds of small molecules of CHIR99021, SB431542, DMH1, cyclopamine and Retinoic Acid.
The preparation of the spinal cord GABA energy intrerneuron of 1 high-purity of embodiment
Recovery MEF (l cell)
Cryopreservation tube is taken out, melts (1-2min) in concussion in 37 degree of water-baths, the stopping when there is a small amount of ice cube residue, with shifting
Frozen stock solution is drawn in the 15mL centrifuge tube added with 5mLMEF culture medium by liquid rifle, and 1000 turns, 5min, centrifugation.Take out 0.1%
Gelatin spreads six overnight orifice plates, and with liquid-transfering gun sucked away gelatin, MEF is pressed 3x105/ hole is taped against on six orifice plates.
Ipsc passage
50mL ipsc culture medium is prepared, in addition 39mL DF-12,500 μ L NEAA, 500 μ L in 50mL centrifuge tube
Glutamax, 10mL KSR, 0.35 μ L beta -mercaptoethanol, 20 μ LbFGF, piping and druming mix.II digestive ferment of 1mg/mL dispase in
37 DEG C of digestion 3min stop digestion when observing ipsc clone's surrounding crimping under the microscope, and DF-12 2mL is washed twice, and 2mL is added
Ipsc culture medium blows down clone, and 1000 turns, cell is taped against on MEF feeder cells by 2min l centrifugation in suitable ratio.
D0, I stage, ipsc differentiation become neuro-epithelial cell
Culture medium is prepared, and 24.5mL DF-12,24.5mL Neurobasal are added in 50mL centrifuge tube, rear to be added
500 μ L NEAA, 500 μ L N2,100 μ L SB431542,150 μ L CHIR99021,100 μ L DMH1, piping and druming mix.Use liquid relief
Rifle draws original ipsc culture medium, and I stage of 2mL culture medium is added, liquid is changed every other day, until the 7th day.
Neuro-epithelial cell is divided into spinal cord precursor by D7, II stage
Culture medium is prepared, and 24.5mL DF-12,24.5mL Neurobasal are added in 50mL centrifuge tube, rear to be added
500μL NEAA、500μL N2、1mL B27、100μL SB431542、50μL CHIR99021、100μL DMH1、5μL RA、
25μL cyclopamine.It spreads MEF (repeating first step) and draws the former culture medium in six orifice plates with liquid-transfering gun, DF-12 washes two
Time, II stage culture medium is added, blows down clone with 1mL liquid-transfering gun, cell is taped against MEF feeder layer in suitable ratio (1:8)
On cell, every hole culture medium added to 2mL, partly changes liquid daily, until the 14th day.
D14, III stage, suspension stages
Culture medium is prepared, and 24.5mL DF-12,24.5mL Neurobasal are added in 50mL centrifuge tube, rear to be added
500μL NEAA,500μL N2,1mLB27,5μL RA,25μL cyclopamine.The former training in six orifice plates is drawn with liquid-transfering gun
Base is supported, and DF-12 is washed twice, and III stage culture medium is added, blows down clone with 1mL liquid-transfering gun, cell is hanged by proper ratio (2:1)
Supernatant liquid moves into T25 culture bottle, and culture medium adds to 10mL.Nerve ball is blown and beaten with 1mL liquid-transfering gun daily, prevents adhesion, every other day partly
Liquid is changed, until the 21st day.
D20
One 24 orifice plates are taken, every hole is put into sterile circle slide, and every hole addition 0.1mg/mL PLL to entire circle slide is spread
It is full, it is placed in 37 and spends night.Nerve ball is digested, the nerve ball suspension in T25 culture bottle is drawn to 15mL centrifuge tube with liquid-transfering gun,
1000 turns, 3min, supernatant is abandoned, accutaes2mL is added and is placed in 37 degree of digestion 5min, until nerve ball becomes smaller, 4mLDF-12 is added
Digestion is terminated, 1000 turns, 3min, abandons supernatant.5mL III stage culture medium is added to mix, moves into T25 culture bottle and continues to cultivate.
D21
It draws the PLL in 24 orifice plates, with sterile washing three times, dries 24 orifice plates (general 6H is advisable).It is 20ng/ by concentration
ML laminin is paved with entire circle slide, is placed in 37 degree of culture 2h.IV stage culture medium is prepared, with liquid-transfering gun by 48mL
Neurobasal is added in 50mL centrifuge tube, rear that 500 μ L NEAA, 500 μ L N2,1mLB27,10 μ L camp, 10 μ L are added
BDNF, 10 μ L GDNF, 10 μ L AA, piping and druming mix.With liquid-transfering gun by the nerve ball suspension in T25 culture bottle be drawn to 15mL from
Heart pipe, abandons supernatant by 1000 turns, 3min.100 μ L, IV stage culture medium is added, piping and druming mixes, and 100 μ L suspension are instilled 10cm
Culture dish middle is drawn with 10 μ L liquid-transfering guns in 24 orifice plates that nerve ball by proper density kind is entered to be covered with laminin.Merging 37
After spending incubator 2h, until microscopically observation nerve ball is adherent, IV stage culture medium to 500 μ L is added.Culture 3-4 weeks.
Embodiment 2 cultivates the detection of the marker in each stage
Sox1 and Hoxa3 immunohistochemical staining is carried out to the neuro-epithelial cell that differentiation occurs on the 7th day, is obtained such as Fig. 2 institute
The neuro-epithelial cell qualification figure shown, it is the mark of spinal cord cervical part of esophagus that wherein Sox1, which is the marker of neuro-epithelial cell, Hoxa3,
Object.
Similarly, OLIG2 and Hoxb4 is carried out to the spinal interneuron precursor that differentiation occurred to the 14th day to be immunized
Fluorescent staining obtains spinal interneuron precursor qualification figure as shown in Figure 3, and wherein OLIG2 is the mark of ventral side of spinal cord
Will object, Hoxb4 are the markers of spinal cord.
And the spinal cord GABA occurred to 21 days to differentiation can intrerneuron carry out GABA, NF-200, GAD65/67 and
Tuj1 immunofluorescence dyeing, then available spinal cord GABA as shown in Figure 4 can intrerneuron qualification figure, wherein GABA be
It is the special of GABA neuron that marker, the NF-200 of GABA neuron, which are the marker of neuron cytoskeletal, GAD65/67,
Property marker, Tuj1 be neuron marker.
The results show, the method can be obtained quickly and efficiently in high-purity spinal cord GABA energy through this embodiment
Between neuron.
Claims (7)
1. pluripotent stem cell induction obtain spinal cord GABA can intrerneuron method, it is characterised in that: it includes,
Step (1) pluripotent stem cell breaks up that obtain neural epithelium thin under the action of CHIR99021, SB431542 and DMH1
Born of the same parents；
Cyclopamine is added in step (2) and retinoic acid carries out successive induction differentiation, obtains spinal nerve precursor；
Step (3) after digestion process, broken up under the action of cyclopamine and retinoic acid by continuation, is obtained in spinal cord GABA energy
Between neuron；And
For step (4) after digestion process, the spinal cord GABA energy intrerneuron, which is carried out adhere-wall culture, makes its maturation.
2. the method that pluripotent stem cell induction according to claim 1 obtains spinal cord GABA energy intrerneuron, special
Sign is: the molar concentration of the CHIR99021 is 3 μM, the molar concentration of the SB431542 is 2 μM, and the DMH1's rubs
You have 2 μM of concentration.
3. the method that pluripotent stem cell induction according to claim 2 obtains spinal cord GABA energy intrerneuron, special
Sign is: the molar concentration of the cyclopamine is 5nM, and the molar concentration rate of the retinoic acid is 1nM.
4. the method that pluripotent stem cell induction according to claim 3 obtains spinal cord GABA energy intrerneuron, special
Sign is: step (4) the progress adhere-wall culture time is 3~4 weeks.
5. the induction of pluripotent stem cell described in any one of -4 obtains spinal cord GABA energy intrerneuron according to claim 1
Method, it is characterised in that: required cell culture fluid is by the other culture medium of clinical grade and clinical grade in step (1)~step (4)
Other additive is prepared.
6. the purposes of spinal cord GABA energy intrerneuron in medicine preparation, the drug is for treating pain, the spinal cord
GABA energy intrerneuron is induced by pluripotent stem cell of any of claims 1-5 to be obtained among spinal cord GABA energy
The method of neuron is prepared.
7. purposes according to claim 6, the pain is caused by spinal cord injury or malignant tumour.
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|---|---|---|---|---|
| WO2004087870A2 (en) * | 2003-03-25 | 2004-10-14 | The Johns Hopkins University | Neuronal cell lineages and methods of production thereof |
| WO2009024448A1 (en) * | 2007-08-20 | 2009-02-26 | Universite Libre De Bruxelles | Generation of neuronal cells from pluripotent stem cells |
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| WO2004087870A2 (en) * | 2003-03-25 | 2004-10-14 | The Johns Hopkins University | Neuronal cell lineages and methods of production thereof |
| WO2009024448A1 (en) * | 2007-08-20 | 2009-02-26 | Universite Libre De Bruxelles | Generation of neuronal cells from pluripotent stem cells |
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| INSIK HWANG等: "Intrathecal Transplantation of Embryonic Stem Cell-Derived Spinal GABAergic Neural Precursor Cells Attenuates Neuropathic Pain in a Spinal Cord Injury Rat Model", 《CELL TRANSPLANTATION》 * |
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| CN114703196A (en) * | 2022-05-16 | 2022-07-05 | 中国科学技术大学 | RNA composition for inducing neuron differentiation, differentiation method and application thereof |
| CN114703196B (en) * | 2022-05-16 | 2024-03-29 | 中国科学技术大学 | RNA composition for inducing neuron differentiation, differentiation method and application thereof |
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