Preparation method and application of transplantable cell strain for treating Parkinson's disease

Abstract

The invention discloses a preparation method of a transplantable cell strain for treating Parkinson's disease, which comprises the following steps: injecting pluripotent stem cells into animal body tissues, and inducing through a tissue microenvironment. According to the method, the uniqueness of in-vivo tissues is utilized, so that the stem cells are finally proliferated and differentiated into the nerve-like precursor cells capable of being transplanted for treating the Parkinson's disease, a large number of transplantable cells can be efficiently, conveniently and safely obtained through the method, and the method is used for solving the problem that the source and the number of cells are limited in previous cell transplantation. And a new possibility is provided for treatment of nerve injury diseases.

Description

Technical Field

[0001] The invention relates to a preparation method of a transplantable cell line for treating Parkinson's disease and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Parkinson's disease is a common neurodegenerative disease. It is mainly caused by the degeneration and death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain, which leads to a significant decrease in the level of dopamine (DA) in the striatum, causing a series of motor and non-motor symptoms. Its typical motor symptoms include resting tremor, muscle rigidity, bradykinesia and postural balance disorders. These symptoms will gradually worsen with the progression of the disease, seriously affecting the patient's quality of life. Cell transplantation for Parkinson's disease is a treatment method that is being studied and developed. It mainly includes inducing stem cells (such as embryonic stem cells, induced pluripotent stem cells, etc.) to differentiate into dopaminergic neurons or neural stem cells, and then transplanting them into the striatum area of ​​the patient's brain, or transplanting tissues containing dopaminergic neurons (such as fetal midbrain tissue) to supplement damaged dopaminergic neurons and improve the symptoms of Parkinson's disease.

[0003] However, whether it is neural stem cells, dopaminergic neurons or other cells for transplantation, it is a challenge to obtain a sufficient number of cells for treatment. For example, the number of neural stem cells isolated from human tissues is often limited, and during in vitro culture, the proliferation rate of cells may not meet the needs of clinical treatment. Currently, the main cells that can be used for cell transplantation are neural stem cells, mesenchymal stem cells, embryonic stem cells, olfactory ensheathing cells, myogenic stem cells, Schwann cells, etc. Various stem cells have differentiation potential and can differentiate into dopamine neurons to replace necrotic cells and restore the dopamine secretion function of the striatum.

[0004] At present, the most promising stem cell transplantation pretreatments in clinical practice require high culture conditions and expensive culture additives, and the time period is long, and it is difficult to obtain a large number of cells at one time. However, our invention implants stem cells into animal tissues for in vivo proliferation induction culture, which takes a relatively short time and can obtain a large number of cells at the same time. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a transplantable cell line for treating Parkinson's disease.

[0006] The technical solution adopted by the present invention is:

[0007] A method for preparing a transplantable cell line for treating Parkinson's disease, wherein pluripotent stem cells are injected into tissues in an animal body and induced through tissue microenvironment.

[0008] Preferably, the pluripotent stem cells are neural stem cells, induced pluripotent stem cells, embryonic stem cells, umbilical cord blood stem cells, olfactory ensheathing cells, myogenic stem cells, Schwann cells, human embryonic kidney epithelial cells HEK-293T or mesenchymal stem cells.

[0009] Preferably, the animal in vivo tissue is spleen, spinal cord, brain, testicle, kidney or liver.

[0010] Preferably, when the pluripotent stem cells are induced pluripotent stem cells, the in vivo induction time is 1-2 weeks; when the pluripotent stem cells are HEK-293T, the in vivo induction time is 2-5 days.

[0011] The invention also discloses a transplantable cell strain prepared by the method for preparing the transplantable cell strain for treating Parkinson's disease.

[0012] The invention also discloses the application of the transplantable cell strain in preparing medicine for treating Parkinson's disease.

[0013] The present invention also discloses a method for preparing a cell suspension for treating Parkinson's disease, the steps of which include:

[0014] (1) After the pluripotent stem cells are expanded, the supernatant is discarded by centrifugation, and the cells are resuspended into a cell suspension using a biological scaffold material;

[0015] (2) Injecting the cell suspension into the animal's tissues and feeding the animal for a period of time;

[0016] (3) The tissues in the animal body are peeled off, homogenized, separated and purified, and the obtained cells are mixed with independently extracted auxiliary cells, biological scaffold materials, and physiological saline to obtain a cell suspension for treating Parkinson's disease.

[0017] Preferably, the biological scaffold material is collagen, gelatin or matrigel.

[0018] Preferably, the animal in vivo tissue refers to spleen, spinal cord, brain, testis, kidney or liver, and the auxiliary cells refer to glial cells, primary neurons, astrocytes, mesenchymal stem cells, testicular supporting cells, testicular interstitial cells, a testicular tissue cell mixture, embryonic stem cells, induced pluripotent stem cells, olfactory ensheathing cells, myogenic stem cells, Schwann cells, umbilical cord blood stem cells or neural stem cells.

[0019] The present invention also discloses an application of a cell suspension in preparing a medicine for treating Parkinson's disease, wherein the cell suspension is prepared according to the above-mentioned preparation method.

[0020] The present invention utilizes the uniqueness of in vivo tissues to allow a type of stem cells to ultimately proliferate and differentiate into neural progenitor cells that can be transplanted to treat Parkinson's disease. This method can efficiently, conveniently and safely obtain a large number of transplantable cells, which can be used to solve the problems of limited cell sources and numbers in previous cell transplants, and provides a new possibility for the treatment of neurological injury-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 is a cell morphology diagram of iPSCs under different culture conditions in the present invention.

[0022] Figure 2 The photographs are of sections in which in vivo tissues at different time points are embedded in paraffin and sliced ​​for hematoxylin-eosin staining after iPSC cells are implanted in the present invention.

[0023] Figure 3 These are the evaluation results of apomorphine-induced circling behavior in each group of PD rats before and after cell transplantation treatment in the present invention.

[0024] Figure 4 These are the H&E staining results of each group of PD rats before and after cell transplantation therapy in the present invention. Specific implementation methods

[0026] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0027] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0028] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0029] Example 1

[0030] 1. Preparation of cells for implantation into mouse testicular tissue

[0031] (1) Preparation of HEK-293T cell suspension:

[0032] After HEK-293T cells are expanded to an appropriate number, the supernatant is discarded by centrifugation and resuspended into a cell suspension using a biological scaffold material, ready to be implanted into the testicular tissue.

[0033] (2) Preparation of induced pluripotent stem cell (iPSC) cell suspension

[0034] iPSCs are expanded to an appropriate number using basal medium, and the cells are not differentiated and in good condition, such as Figure 1 As shown in A, the culture medium with complete nutrition is used to cultivate until the ball state is formed. Figure 1 As shown in B, the supernatant is discarded after centrifugation, and the cell suspension is resuspended with biological scaffold material to prepare for implantation into testicular tissue.

[0035] 2. Implantation of cell suspension into mouse testicular tissue

[0036] After the mice were anesthetized, the cell suspension was injected into one testicle of the mice using a microsyringe. The mice were then observed and returned to their cages after they recovered to normal.

[0037] Subsequently, the condition of the mice was observed at regular intervals, and testicular tissue was collected on the 2nd, 5th, 7th, and 14th days after the cells were implanted into the testicles. The testicles of 5 mice were collected at each time point to observe whether there were any changes in their morphology and volume. The tissue samples were fixed for 24 hours, embedded in paraffin, and pathologically sectioned and stained with hematoxylin-eosin to observe the pathological changes inside the testicular tissue.

[0038] 3. In vivo growth of cell suspension after implantation into mouse testicular tissue

[0039] (1) Growth of HEK-293T cells after testicular implantation

[0040] After HEK-293T cells were implanted into the testis, the testicular tissue was collected at 2 days, 5 days, 7 days, and 14 days according to the cell growth cycle. The tissue was embedded in paraffin, sliced, and stained with hematoxylin-eosin and immunofluorescence. The results showed that the cells were in a proliferative state at 2 days and 5 days, and the proliferation marker Ki67 was expressed in large quantities. At 7 days, Ki67 was basically not expressed, and the cell morphology was also in a differentiated state. Testicular tissue cells at 2, 5, and 7 days were extracted and separated, the testicular tissue was peeled off, cut into pieces, and digested with digestive fluid, centrifuged at 1000 rpm for 5 minutes, and the cells were resuspended in stem cell basal medium, sieved twice with a 70-micron mesh to remove adhesion tissue in the testis, and then the cells were resuspended in stem cell basal medium and cultured in culture flasks. After 24 hours, the medium was changed to remove other impurity cells, and the culture was continued for 7 days. The medium was changed every 2 days to ensure that only neural precursor cells were contained in the culture flask. The cells were collected and identified, and specific markers tyrosine hydroxylase (TH) and nestin (Nestin) were fluorescently stained. It was found that Nestin was highly positively expressed and TH was expressed in small amounts. Real-time quantitative PCR (qRT-PCR) detected that the mRNA level of LMX1A was increased. Therefore, the cells are currently in the state of dopamine neural precursor cells. Therefore, we selected cells induced in vivo for 2-5 days for cell transplantation. 2-5 days after HEK-293T cells were implanted into the mouse testis, the testicular tissue was peeled off and dopamine neural precursor cells were extracted as described above. The cells were collected and mixed with independently extracted Leydig cells at a ratio of 1:1 and resuspended in matrix gel to obtain a cell suspension that can be used for Parkinson's disease treatment.

[0041] The proliferation rate of cells extracted after 2 or 5 days of induction of proliferation in vivo was calculated, as shown in Tables 1 and 2. Due to the large individual differences among mice, the proliferation rate was about 20%-60%.

[0042] The proliferation rate was calculated as follows:

[0043] Proliferation rate = (number of cells after proliferation - number of cells implanted) / number of cells implanted

[0044] The proliferation rate of cells on day 2 is shown in Table 1 below:

[0045] Table 1. Proliferation rate of HEK-293T cells induced to proliferate for 2 days in vivo (based on implantation of 2x10 6 Cell count)

[0046]

[0047] The proliferation rate of cells after 5 days is shown in Table 2 below:

[0048] Table 2. Proliferation rate of HEK-293T cells induced to proliferate for 5 days in vivo

[0049]

[0050] This example confirms that HEK-293T cells can proliferate and differentiate in the testis, and cells after proliferation induction can be directly obtained for the treatment of Parkinson's disease. In addition, only one testicle of the mouse is taken each time, which does little harm to the mouse's body.

[0051] (2) Growth of iPSCs after testicular implantation

[0052] After iPSCs were implanted into the testes, testicular tissues were collected at 0, 1, 2, and 4 weeks according to the cell growth cycle. Paraffin embedding, sectioning, and hematoxylin-eosin staining were performed ( Figure 2 ) and immunofluorescence staining. The results showed that the cells were in a proliferative state at 1 and 2 weeks, and expressed a large amount of the proliferation marker Ki67. At 4 weeks, Ki67 was basically not expressed, and the cell morphology was also in a differentiated state. Extraction of 1-2 week cells for identification showed that the cell state at this time was consistent with the differentiation stage of HEK-293T at 2-5 days.

[0053] Therefore, we selected cells induced in vivo for 1-2 weeks for cell transplantation. After 1-2 weeks of iPSC implantation into mouse testis, the same extraction and purification steps as above were performed for HEK-293T implantation into mouse testis. The obtained cells were mixed with independently extracted testicular interstitial cells in a 1:1 ratio and resuspended in matrix gel to obtain a cell suspension that can be used for Parkinson's disease treatment.

[0054] The proliferation rate of cells extracted after 1 or 2 weeks of in vivo proliferation induction was calculated, as shown in Tables 3 and 4. Due to the large individual differences among mice, the proliferation rate was about 30%-60%.

[0055] The proliferation rate of cells in 1 week is shown in Table 3 below:

[0056] Table 3. Proliferation rate of iPSC cells induced to proliferate in vivo for 1 week (based on implantation of 2x10 6 Cell count)

[0057]

[0058] The proliferation rate of cells at 2 weeks is shown in Table 4 below:

[0059] Table 4. Proliferation rate of iPSC cells induced to proliferate for 2 weeks in vivo

[0060]

[0061]

[0062] This example confirms that iPSCs can proliferate and differentiate in the testis, and cells after proliferation induction can be directly obtained for the treatment of Parkinson's disease. In addition, only one testicle of the mouse is taken each time, which does little harm to the mouse's body.

[0063] Example 2

[0064] 1. Preparation of 6-OHDA-induced disease model in PD rats Select 200-250 g Sprague Dawley female rats and follow the steps below to prepare and evaluate the model.

[0065] Step 1: Construction of PD rat model. During the operation, 2% sodium pentobarbital solution was used for anesthesia by intraperitoneal injection at 40 mg / kg of rat body weight. The head of the anesthetized rat was fixed on the stereotaxic instrument, and the position of the head of the rat was adjusted so that the bregma and lambda were at the same level. The skin of the rat's head was disinfected, and then the skin was cut open at the midline of the top of the head to expose the skull. A dental drill, a small skull drill or other tools was used to carefully drill a small hole in the right striatum brain area (such as the coordinates of the striatum: 0.5 mm in front of the bregma, 3.0 mm outside, and 5.0 mm below the skull surface), and a microsyringe containing 6-OHDA solution (e.g., a concentration of 4 μg / μL) was slowly inserted into the predetermined depth of the brain area through the injection device of the stereotaxic instrument. The injection speed is generally 0.5-1 μL / min, and the total injection volume is about 4-8 μL. After the injection is completed, the syringe is kept in the brain for 5-10 minutes to prevent the drug from refluxing, and then the syringe is slowly withdrawn. After the injection is completed, the wound is rinsed with saline and the scalp is sutured.

[0066] 2. Cell transplantation for PD rat model and evaluation of therapeutic effects

[0067] Step 1: Transplantation Treatment Grouping

[0068] 40 SPF female Sprague-Dawley rats weighing 200-250g were used in the experiment. The model had four groups, as shown in Table 5, including normal control group, sham operation group, Parkinson's group, and cell therapy group, with 10 rats in each group. The normal control group was not treated; the sham operation group had a hole drilled in the right skull, and a micro-syringe (without 6-OHDA solution) was inserted into the depth of the brain region at the position of the striatum through the injection device of the stereotaxic instrument. After the injection was completed, the 6-OHDA solution in the micro-syringe was injected into the same depth of the brain region as the sham operation group. Rotational behavior was performed 2 weeks after modeling. Model rats with behavioral scores of more than 7 revolutions per minute were considered to have successfully modeled and were classified as the Parkinson's group; the cell therapy group selected PD rats with successful modeling, and the prepared appropriate amount of cell suspension was transplanted into the same brain depth as the Parkinson's rat modeling. The status of rats in each group was observed regularly, and behavioral scores were performed to determine their recovery.

[0069] Table 5 Rat model experimental groups

[0070] Normal control group Sham operation group Parkinson's disease group Cell therapy group Skull drilling no yes yes yes Injection of 6-OHDA no no yes yes Implantation of cells no no no yes

[0071] Step 2: Behavioral evaluation of cell transplantation therapy

[0072] Evaluation of apomorphine-induced rotation behavior: Apomorphine (0.5 mg / Kg) was injected intraperitoneally 2 weeks after cell transplantation. The rats were monitored for 12 consecutive weeks. Each rat was monitored for 30 minutes each time. The total number of rotations of the rats toward the side opposite to the lesion within 30 minutes was recorded. After the monitoring was completed, the data were statistically analyzed. Figure 3 shown.

[0073] The rats in the normal control group had basically no rotation behavior within 8 weeks; the behavioral scores of the rats in the sham operation group were 3-4 revolutions per minute within two weeks, and basically returned to normal after 4 weeks; within 2 weeks, the behavioral scores of the Parkinson's group and the cell therapy group were no different from those at week 0, and motor function began to recover to varying degrees after 2 weeks. The rats in the Parkinson's group recovered slightly at week 4, but the behavioral scores still remained above 7 revolutions per minute, and there was no improvement until week 8; the cell therapy group began to recover continuously after 2 weeks, recovered slightly at week 4, and the behavioral scores began to reach below 7 revolutions per minute, and continued to recover for 6-8 weeks, with a good treatment effect.

[0074] The behavioral recovery of the cell suspension derived from HEK-293T cells and that of the cell suspension derived from iPSCs was basically the same, indicating that cell therapy is effective.

[0075] Step 3: Slice staining: Take samples after 8 weeks of behavioral evaluation. After the animals are anesthetized, they are perfused with saline and 4% PFA. After perfusion, the brain is taken and fixed with 4% PFA at 4°C overnight. Embed the tissue in paraffin and slice it; place the tissue slides in the slice box in order, mark the tissue name, date, etc., and store them in a -20°C refrigerator; perform H&E staining on the slices, take pictures under a microscope, and obtain the staining results, such as Figure 4 shown.

[0076] By analyzing the staining results, in the H&E stained sections of normal brain tissue, the neuronal cell bodies were round or polygonal, the nuclei were large and round, located in the center of the cells, the nucleoli were obvious, and the chromatin was evenly distributed. The overall tissue structure was clear and the cells were arranged in an orderly manner; the sham operation group was the same as the normal brain tissue; the brain tissue H&E staining of the Parkinson's group showed a significant decrease in the number of neurons in the substantia nigra, a decrease in cell density, and the occurrence of neuronal vacuolization and nuclear condensation; in the two cell treatment groups, H&E staining showed that the morphology of neurons in the substantia nigra region tended to be normal, the nuclear structure was relatively intact, and the cytoplasmic fullness increased, indicating that neuroinflammation was controlled.

[0077] Combining the above cases, the cell suspension derived from HEK-293T cells and the cell suspension derived from iPSC showed good recovery effects after being transplanted into PD rats for 8 weeks of treatment.

[0078] Comparative Example 1

[0079] The process of inducing iPSCs to differentiate into dopamine neural progenitor cells in vitro:

[0080] Day 1-2: Induction medium 1 was used. This stage mainly pre-treated the cells to prepare for subsequent differentiation. The cell morphology had not yet changed significantly. The components of induction medium 1 included 85% knockout DMEM, 15% knockout serum replacement, 1x GlutaMAX, 1x MEM non-essential amino acids, 10mM 2-mercaptoethanol, 100nM LDN193189, and 10μM SB431542.

[0081] Day 2-3: Change to induction medium 2, the cells start the differentiation process, the morphology gradually changes, the cells become flatter, and the cell-cell connections become tight. Induction medium 2 is based on induction medium 1, with the addition of 100nM SAG hydrochloride, 2μm puromorphamine, and 100ng / ml human FGF-8a.

[0082] Day 4-5: Continue to use Induction Medium 3, the cells further differentiate, the morphological changes are more significant, the cell body becomes smaller, and some characteristics of neural progenitor cells begin to appear, such as the growth of cell processes. Induction Medium 3 has the same ingredients as Induction Medium 2.

[0083] Day 6-7: Using induction medium 4, the neural progenitor cell characteristics of the cells are more obvious, the cell processes become longer and more numerous, and a preliminary neural network-like structure is formed between the cells. The composition of induction medium 4 is relatively complex, including 63.2% knockout DMEM, 11.6% knockout serum replacement, 24.8% Neurobasal medium, 0.13% N-2supplement, 0.26% B-27supplement, 1x GlutaMAX, 1x MEM non-essential amino acids, 10μm 2-mercaptoethanol, 100nM LDN193189, 3μm CHIR99021, 100nM SAG hydrochloride, 2μm promomorphamine, and 100ng / ml human FGF-8a.

[0084] Day 8-9: Change to induction medium 5, the cells continue to differentiate and mature, the expression of neural progenitor cell markers gradually increases, and the morphology and function of the cells are closer to dopamine neural progenitor cells. Induction medium 5 contains 42.3% knockout DMEM, 7.6% knockout serum replacement, 49.3% Neurobasal medium, 0.26% N-2supplement, 0.5% B-27supplement, 1x GlutaMAX, 1x MEM non-essential amino acids, 10μm 2-mercaptoethanol, 100nM LDN193189, 3μm CHIR99021.

[0085] Day 10-14: After day 10, the specific differentiation of cells into dopamine neural precursor cells can be gradually observed. Around day 14, the formation of dopamine neural precursor cells is confirmed by detecting specific markers such as tyrosine hydroxylase.

[0086] The above process of inducing iPSC to differentiate into dopamine neural precursor cells in vitro requires the addition of small molecule compounds and growth factors at different induction stages, etc. The differentiation cycle is at least 14 days, which is relatively long compared with the method described in the above Example 1, and is about 10 days longer than HEK-293T. In addition, the proportion of induced dopamine neural precursor cells is about 10%-40%, which is relatively lower than the proportion of cells obtained by the method described in Example 1. Therefore, inducing iPSC to differentiate into dopamine neural precursor cells in vivo has more advantages.

[0087] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a transplantable cell line for treating Parkinson's disease, characterized in that: Pluripotent stem cells are injected into animal tissues and induced through the tissue microenvironment.

2. The preparation method according to claim 1, characterized in that: The pluripotent stem cells are neural stem cells, induced pluripotent stem cells, embryonic stem cells, umbilical cord blood stem cells, olfactory ensheathing cells, myogenic stem cells, Schwann cells, human embryonic kidney epithelial cells HEK-293T or mesenchymal stem cells.

3. The preparation method according to claim 2, characterized in that: The animal body tissue refers to spleen, spinal cord, brain, testicle, kidney or liver.

4. The preparation method according to claim 2, characterized in that: When the pluripotent stem cells are induced pluripotent stem cells, the in vivo induction time is 1-2 weeks; when the pluripotent stem cells are HEK-293T, the in vivo induction time is 2-5 days.

5. The transplantable cell line obtained by the method for preparing a transplantable cell line for treating Parkinson's disease according to any one of claims 1 to 4.

6. Use of the transplantable cell line according to claim 5 in the preparation of a drug for treating Parkinson's disease.

7. A method for preparing a cell suspension for treating Parkinson's disease, characterized in that: The steps include: (1) After the pluripotent stem cells are expanded, the supernatant is discarded by centrifugation, and the cells are resuspended into a cell suspension using a biological scaffold material; (2) Injecting the cell suspension into the animal's tissues and feeding the animal for a period of time; (3) The tissues in the animal body are peeled off, homogenized, separated and purified, and the obtained cells are mixed with independently extracted auxiliary cells, biological scaffold materials, and physiological saline to obtain a cell suspension that can treat Parkinson's disease.

8. The preparation method according to claim 7, characterized in that: The biological scaffold material refers to collagen, gelatin or matrix glue.

9. The preparation method according to claim 8, characterized in that: The animal in vivo tissue refers to the spleen, spinal cord, brain, testis, kidney or liver, and the auxiliary cells refer to glial cells, primary neurons, astrocytes, mesenchymal stem cells, testicular supporting cells, testicular interstitial cells, a mixture of testicular tissue cells, embryonic stem cells, induced pluripotent stem cells, olfactory ensheathing cells, myogenic stem cells, Schwann cells, umbilical cord blood stem cells or neural stem cells.

10. Use of a cell suspension in preparing a drug for treating Parkinson's disease, wherein the cell suspension is a cell suspension prepared by the preparation method according to any one of claims 7 to 9.

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