CN109554342B

CN109554342B - Method for obtaining spinal GABA energy interneuron by inducing pluripotent stem cell

Info

Publication number: CN109554342B
Application number: CN201811237971.2A
Authority: CN
Inventors: 陈红; 王伟
Original assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Current assignee: Wuhan Hongchen Innovation Biotechnology Co ltd
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2021-09-21
Anticipated expiration: 2038-10-23
Also published as: CN109554342A

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

Method for obtaining spinal GABA energy interneuron by inducing pluripotent stem cell

Technical Field

The invention relates to the technical field of medicines, in particular to a method for obtaining GABA (gamma-aminobutyric acid) energy interneurons of spinal cords by inducing pluripotent stem cells.

Background

Spinal cord injury is a common traumatic disease causing a series of motor and psychological problems, common in people under 30 years of age. According to statistics, 250,000 to 500,000 spinal cord injury patients are newly increased every year around the world, China has no statistics on the national scale at present, but the incidence rate of spinal cord injury in China is far higher than that in European and American countries from statistics on various aspects. Spinal cord injuries affect the quality of life of nearly 100 million people as statistically found in U.S. 2013, and the annual medical costs are nearly 400 billion (armor, b.s., Courtney-Long, e.a., Fox, m.h., Fredine, h., and Cahill, a. (2016) (Prevalence and cases of clinical-United States,2013.am.j. public Health 106, 1855-. Approximately 40% to 50% of patients develop neuropathic Pain within 1 year and later convert to chronic Pain, affecting patient recovery (Jay M Margolis, Paul Juneau, Alesia Sadosky, Joseph C Cappeleri, Thomas N Bryce, Edward C Nieshoff. health care administration and expeditions among medical id beneficiences with a neuropathic Pain following surgery in bone study. journal of Pain research.2014: 7379. 387).

Cancer pain is one of the most common and painful symptoms for patients with malignant tumors. About 70-87% of patients have different pain during the development of cancer, while liver cancer, pancreatic cancer and osteosarcoma patients have pain at early stage of disease. More than 100 million patients suffer from cancer pain each year in china alone. The pain of the middle and late stage patients is as high as 60-90%, and the life quality of the patients is seriously reduced. Malignant patients often lose treatment confidence and survival desire due to the exacerbation of pain and the direct impact of intense stimuli on the patient 'S appetite, sleep, psychological condition and treatment efficacy (Zhang J.A nalgesia mechanisms and clinical application of acupunture. Beijing: peoples' M Medical Publishing House.2007:522.Chinese. L.Q S., Qian H. cancer complexes and its management. in: Tang Z. Y. model interaction. Shanghai: Shanghai Medical coverage Press.2000:556. Chinese). The classical models simulating central nerve injury such as spinal cord injury and cancer pain models have deletion and dysfunction of GABAergic neurons at the spinal cord level to different degrees, so that the phenomenon of 'disinhibition' of pain sense conduction paths is caused, and abnormal expressions such as hyperalgesia, allodynia and the like are finally induced.

At present, the clinical treatment of pain mainly comprises drug treatment and physical factor treatment, but the systemic side effect of the drug treatment is obvious, the remission period of the physical factor treatment is short, the ideal treatment effect cannot be achieved, and a new treatment method needs to be found. In recent years, there have been great progress made by researchers at home and abroad focusing on novel treatments for stem cells, but there still remain many problems, for example, the cells transplanted into the body cannot accurately form synaptic connection with host cells, and thus the cells are influenced to play a role in the body. In the current research on treating pain caused by diseases by stem cells at home and abroad, cells of a targeted type (Young S. Gwak, Claire E. Hulsebosch; GABA and central neuropathic pain in welling bone mineral input; Neuropharmacology 60(2011)799e808) are not transplanted.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for directionally differentiating pluripotent stem cells into high-purity spinal GABAergic interneurons by utilizing the pluripotent stem cells, which is used for treating pains caused by various diseases.

The method for obtaining the GABAergic interneuron of the spinal cord by inducing the pluripotent stem cells comprises the following steps:

differentiating the pluripotent stem cells to obtain neuroepithelial cells under the action of CHIR99021, SB431542 and DMH 1;

adding cyclopamine and retinoic acid to perform continuous induction differentiation to obtain spinal cord nerve precursor cells;

after digestion treatment, continuously differentiating under the action of cyclopamine and retinoic acid to obtain GABA (gamma-aminobutyric acid) interneurons of spinal cords; and the number of the first and second groups,

and (4) after digestion treatment, carrying out adherent culture on the GABAergic interneurons of the spinal cord to ensure that the GABAergic interneurons of the spinal cord are mature.

Further, the molar concentration ratio of CHIR99021, SB431542 and DMH1 is 3:2: 2.

Further, the molar concentration ratio of cyclopamine to retinoic acid is 5: 1.

Further, the culture time in the step (4) is 3-4 weeks.

Furthermore, the cell culture solution required in the steps (1) to (4) is prepared from a clinical-grade culture medium and a clinical-grade additive.

The invention also provides the application of the spinal GABAergic interneurons prepared by the method in preparing a medicament for treating pain.

The invention aims to provide application of liraglutide in preparing a medicine for treating acute and chronic transplant rejection, and provides a new medicine for treating transplant rejection.

Further, the pain is caused by spinal cord injury or malignancy.

The day before treatment, the neurospheres to be transplanted were digested into single cells and counted. Cell suspensions were made on the day of transplantation, containing neurobasal, NEAA, B27, AA, N2, BDNF, GDNF, cAMP.

The spinal GABAergic interneurons obtained by inducing the pluripotent stem cells can also be used for cell combination therapy, such as cell transplantation combined with electric needle therapy and cell transplantation combined with magnetic stimulation therapy.

The invention has the beneficial effects that:

the method for obtaining the spinal GABA energy interneurons by inducing the pluripotent stem cells can quickly and efficiently obtain the spinal GABA energy interneurons with high purity, and avoids the interference of exogenous factors.

Drawings

FIG. 1 is a flow chart of the differentiation of pluripotent stem cells into GABAergic interneurons of spinal cord in example 1 of the present invention;

FIG. 2-a is a Sox1 immunohistochemistry chart of neuroepithelial cells obtained by differentiation of human pluripotent stem cells to day 7 in example 1 of the present invention, and FIG. 2-b is a Hoxa3 immunohistochemistry chart of neuroepithelial cells obtained by differentiation of human pluripotent stem cells to day 7 in example 1 of the present invention;

FIG. 3-a is an OLIG2 immunofluorescence map of spinal cord interneuron precursor cells obtained from differentiation of human pluripotent stem cells to day 14 in example 1 of the present invention, and FIG. 3-b is a Hoxb4 immunofluorescence map of spinal cord interneuron precursor cells obtained from differentiation of human pluripotent stem cells to day 14 in example 1 of the present invention;

FIG. 4-a is a GABA immunofluorescence plot of GABAergic interneurons in spinal cord obtained from differentiation of human pluripotent stem cells to day 21 in example 1 of the present invention, and FIG. 4-b is an NF-200 immunofluorescence plot of GABAergic interneurons in spinal cord obtained from differentiation of human pluripotent stem cells to day 21 in example 1 of the present invention; FIG. 4-c is a GAD65/67 immunofluorescence plot of GABAergic interneurons in spinal cord obtained from differentiation of human pluripotent stem cells to day 21 in example 1 of the present invention; FIG. 4-d is a Tuj1 immunofluorescence plot of GABAergic interneurons in spinal cord obtained from differentiation of human pluripotent stem cells to day 21 in example 1 of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the drawings, but the embodiments are not intended to limit the present invention and are merely examples. While the invention will be described in further detail by means of specific embodiments.

The method for obtaining the GABAergic interneuron of the spinal cord by inducing the pluripotent stem cells comprises the following four steps:

the culture solution used by the method for obtaining the GABAergic interneurons of the spinal cord by inducing the pluripotent stem cells is completely animal-origin-free, and the obtained GABAergic interneurons are clinical-grade and can be used for clinical research and future clinical treatment.

The method adopts a monolayer cell induction method, tubular neuroepithelial cells are obtained by using CHIR99021, SB431542 and DMH1, cyclopamine (cyclopamine) and retinic Acid (Retinoic Acid) are added for induction, and GABA (gamma-aminobutyric Acid) interneuron derived from spinal cord is finally obtained and is used for treating pain caused by various diseases.

FIG. 1 shows the specific culture process and marker detection at each stage of the culture. In the figure, D0, D7, D14 and D21 indicate day 0, day 7, day 14 and day 21 of culture, respectively, and PSC represents pluripotent stem cells; NEP stands for neuroepithelial cells; SCNP represents spinal cord interneuron precursor cells; 3C represents three chemical small molecules of CHIR99021, SB431542 and DMH 1; 5C represents five small molecules of CHIR99021, SB431542, DMH1, cyclopamine and Retinoic Acid.

EXAMPLE 1 preparation of high purity spinal GABAergic interneurons

Resuscitation MEF (mouse fibroblast)

Taking out the frozen tube, shaking and melting in a 37-degree water bath (1-2min), stopping when a small amount of ice cakes are left, sucking the frozen liquid into a 15mL centrifuge tube added with 5mL MEF culture medium by using a liquid transfer gun, rotating for 1000 min, and centrifuging. 0.1% gelatin was removed and spread on a six-well plate overnight, the remaining gelatin was aspirated with a pipette gun, and the MEF was pressed at 3 × 10⁵The hole is paved on a six-hole plate.

Passage of ipsc

Prepare 50mL ipsc medium, add 39mL DF-12, 500. mu.L NEAA, 500. mu.L Glutamax, 10mL KSR, 0.35. mu.L beta-mercaptoethanol, 20. mu.L FGF into 50mL centrifuge tube, blow and mix well. Digesting with 1mg/mL dispase II digestive enzyme at 37 deg.C for 3min, observing under microscope to stop digestion when curling around ipsc clone, washing twice with DF-122 mL, adding 2mL ipsc culture medium to blow down clone, centrifuging at 1000 rpm for 2min, and spreading cells onto MEF feeder layer cells according to appropriate proportion.

D0, stage I, differentiation of ipsc into neuroepithelial cells

Preparing culture medium, adding 24.5mL DF-12 and 24.5mL Neurobasal into a 50mL centrifuge tube, adding 500. mu.L NEAA, 500. mu.L L N2, 100. mu.L SB431542, 150. mu.L CHIR99021 and 100. mu.L DMH1, and blowing and mixing uniformly. The original ipsc medium was aspirated by pipette, 2mL of stage i medium was added, and the medium was changed every other day until day 7.

Stage D7, II differentiation of neuroepithelial cells into spinal cord precursor cells

The medium was prepared by adding 24.5mL DF-12 and 24.5mL Neurobasal to a 50mL centrifuge tube, followed by 500. mu.L NEAA, 500. mu. L N2, 1mL B27, 100. mu.L SB431542, 50. mu.L CHIR99021, 100. mu.L DMH1, 5. mu.L RA, and 25. mu.L cyclopamine. MEF (repeat first step) draw stock medium from six well plates with pipette, DF-12 wash twice, add stage II medium, blow down clones with 1mL pipette, spread cells on MEF feeder cells at the appropriate ratio (1:8) to 2mL per well, half change daily until day 14.

D14, stage III, suspension stage

The medium was prepared by adding 24.5mL DF-12 and 24.5mL Neurobasal to a 50mL centrifuge tube, followed by 500. mu.L NEAA, 500. mu. L N2, 1mLB27, 5. mu.L RA, and 25. mu.L cyclopamine. The original medium in the six-well plate was aspirated by pipette, washed twice with DF-12, added to stage III medium, cloned by pipetting 1mL, the cell suspension was pipetted into a T25 flask at the appropriate ratio (2:1) and the medium was added to 10 mL. The neurospheres were blown with 1mL pipette each day to prevent adhesion, and the fluid was changed half day after day until day 21.

D20

A24-well plate was taken and placed into sterile round slides per well, and 0.1mg/mL PLL was added per well until the whole round slide was filled and left at 37 degrees overnight. Digesting neurospheres, sucking neurosphere suspension in a T25 culture flask to a 15mL centrifuge tube by using a pipette gun, rotating for 1000 minutes and 3 minutes, removing supernatant, adding accumtates 2mL, placing at 37 ℃ for digestion for 5 minutes until neurospheres become small, adding 4mLDF-12 to terminate digestion, rotating for 1000 minutes and 3 minutes, and removing supernatant. 5mL of stage III medium was added and mixed, and transferred to a T25 flask for further culture.

D21

The PLL in the 24-well plate is sucked up, washed three times with sterile water, and the 24-well plate is air-dried (generally 6H is preferred). The whole round slide was filled with lamin at a concentration of 20ng/mL and incubated at 37 ℃ for 2 h. Preparing a culture medium at the IV stage, adding 48mL of Neurobasal into a 50mL centrifuge tube by using a pipette gun, then adding 500 mu L of NEAA, 500 mu L N2, 1mLB27, 10 mu L of camp, 10 mu L of BDNF, 10 mu L of GDNF and 10 mu L of AA, and uniformly blowing and stirring. The neurosphere suspension in the T25 flask was pipetted into a 15mL centrifuge tube using a pipette gun, 1000 rpm for 3min, and the supernatant was discarded. Adding 100 μ L IV stage culture medium, blowing, mixing, dripping 100 μ L suspension into the middle of 10cm culture dish, sucking neurospheres with 10 μ L pipette gun, and inoculating into a 24-well plate with laminin. After placing in a 37 ℃ incubator for 2h, observing the adherence of neurospheres under a microscope, and adding IV stage culture medium to 500 mu L. Culturing for 3-4 weeks.

Example 2 detection of markers at various stages of culture

The immunohistochemical staining of Sox1 and Hoxa3 was performed on the neuroepithelial cells appearing on day 7 of differentiation, resulting in the neuroepithelial cell identification chart shown in fig. 2, in which Sox1 is a marker of the neuroepithelial cells and Hoxa3 is a marker of the cervical segment of the spinal cord.

Similarly, OLIG2 and Hoxb4 immunofluorescent staining of spinal cord interneuron precursor cells that differentiated to day 14 gave a profile of spinal cord interneuron precursor cell identification as shown in FIG. 3, where OLIG2 is a marker of the ventral side of the spinal cord and Hoxb4 is a marker of the spinal cord.

And performing immunofluorescence staining on GABA, NF-200, GAD65/67 and Tuj1 on the GABAergic interneurons of the spinal cord which differentiate to appear up to 21 days to obtain the identification chart of the GABAergic interneurons of the spinal cord as shown in figure 4, wherein GABA is a marker of the GABA neurons, NF-200 is a marker of the cytoskeleton of the neurons, GAD65/67 is a specific marker of the GABA neurons, and Tuj1 is a marker of the neurons.

Experimental results prove that the method can quickly and efficiently obtain high-purity spinal GABAergic interneurons.

Claims

1. The method for obtaining the GABA (gamma-aminobutyric acid) interneuron of spinal cord by inducing the multifunctional stem cells is characterized by comprising the following steps of: which comprises the steps of (a) preparing a mixture of,

step (1) day 0 to day 7: differentiating the induced multifunctional stem cells under the action of CHIR99021, SB431542 and DMH1 to obtain neuroepithelial cells;

step (2) day 7 to day 14: adding cyclopamine and retinoic acid for continuous induction differentiation to obtain spinal nerve precursor cells; the specific process is as follows:

preparing a culture medium at the stage II, namely adding 24.5mL of DF-12 and 24.5mL of Neurobasal into a 50mL centrifuge tube, and then adding 500 mu L of NEAA, 500 mu L N2, 1mL of B27, 100 mu L of SB431542, 50 mu L of CHIR99021, 100 mu L of DMH1, 5 mu L of RA and 25 mu L of cyclopamine;

paving MEF, sucking the original culture medium in a six-hole plate by using a pipette gun, washing twice by DF-12, adding a II-stage culture medium, blowing down the clone by using a 1mL pipette gun, paving cells on MEF feeder layer cells according to the proportion of 1:8, adding 2mL of culture medium in each hole, and changing the medium for half a day until the 14 th day;

step (3) day 14 to day 21: after digestion treatment, the spinal cord GABA energy interneuron is obtained by continuously differentiating under the action of cyclopamine and retinoic acid, and the specific process is as follows:

preparing a III-stage culture medium, namely adding 24.5mL of DF-12 and 24.5mL of Neurobasal into a 50mL centrifuge tube, and then adding 500 mu L of NEAA, 500 mu L N2, 1mLB27, 5 mu L of RA and 25 mu L of cyclopamine;

sucking the original culture medium in a six-hole plate by using a pipette gun, washing with DF-12 twice, adding a culture medium in stage III, blowing down the clone by using a 1mL pipette gun, transferring the cell suspension into a T25 culture bottle according to the ratio of 2:1, adding the culture medium to 10mL, blowing and beating neurospheres by using the 1mL pipette gun every day to prevent adhesion, and changing the culture medium half every other day until the 21 st day;

and the number of the first and second groups,

after digestion treatment, carrying out adherent culture on the GABAergic interneurons of the spinal cord to ensure that the GABAergic interneurons of the spinal cord are mature; the adherent culture time is 3-4 weeks;

the molar concentration of cyclopamine is 5nM, and the molar concentration ratio of retinoic acid is 1 nM.

2. The method of inducing the acquisition of gabaergic spinal interneurons in induced pluripotent stem cells according to claim 1, wherein: the molar concentration of CHIR99021 is 3 μ M, the molar concentration of SB431542 is 2 μ M, and the molar concentration of DMH1 is 2 μ M.

3. The method for obtaining spinal GABAergic interneurons induced by induced pluripotent stem cells as claimed in any one of claims 1 to 2, wherein: the cell culture solution required in the step (1) to the step (4) is prepared from a clinical-grade culture medium and a clinical-grade additive.