Method for inducing batch differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells based on 3D culture

Abstract

The present invention relates to a method for inducing the differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells in batches based on 3D culture, the method comprising the following steps: (1) obtaining human nasal mucosa mesenchymal stem cells using human nasal polyps as a tissue source; (2) 3D culturing the human nasal mucosa mesenchymal stem cells and inducing their differentiation into neural stem cells in batches. The present invention provides a method for inducing the differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells, and achieves batch production based on 3D culture. Therefore, the present invention provides a cell preparation method for treating neurodegenerative diseases such as Parkinson's disease caused by neuronal loss.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology technology and relates to a method for inducing the differentiation of human nasal mucosal mesenchymal stem cells into neural stem cells in batches based on 3D culture. The present invention relates to neural stem cells and their preparation methods and batch production. This study solves the problem that the source of neural stem cells in medical applications is affected by ethics and is relatively difficult to obtain, while improving the problem of heterogeneity and low yield of induced neural stem cells, which can be used for the prevention and treatment of Parkinson's disease and other nervous system diseases. Background Art

[0002] Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, are highly disabling and ultimately fatal diseases that affect millions of people worldwide, with increasing incidence and prevalence. However, there is currently no curative therapy available to stop or reverse their progression. Therefore, effective treatments are urgently needed. Neural stem cell transplantation has been studied as a potential treatment because it has the potential to act as "seed" cells within the central nervous system, can self-renew and generate neurons and glial cells during the development of the mammalian central nervous system, and ameliorate neural damage through different mechanisms. Therefore, how to obtain a large number of neural stem cells with stable performance is a prerequisite for affecting the treatment effect.

[0003] Wang Dongmei et al. pointed out in their discussion on the safety and ethical issues of mesenchymal stem cell therapy that the cells used to induce neural stem cells (such as human embryonic stem cells and human induced pluripotent stem cells) often involve ethical and safety issues (Wang Dongmei. Discussion on the safety and ethical issues of mesenchymal stem cell therapy [J]. Journal of Tissue Engineering and Reconstructive Surgery, 2014, 10(5):4.). At the same time, Xing Qu et al.'s study showed that different induction differentiation methods have a great influence on the production of neural stem cells (Xing Qu, Ma Shanshan, Wang Xinxin, et al. Optimization of the neural differentiation microenvironment of human umbilical cord mesenchymal stem cells [J]. Journal of Zhengzhou University: Medical Edition, 2016(4):6.). The cells used for induction differentiation of neural stem cells use a cell culture dish to pass down the cells one by one. The number of cells induced at a time is small, and the inconsistency of multiple induction generations makes it difficult to produce them in a stable batch. Therefore, it is urgent to find a cell source that is easy to obtain and a method for stable batch induction differentiation.

[0004] Human nasal mucosal mesenchymal stem cells can be obtained through the culture of autologous nasal mucosal tissue. The material is easy to obtain and can be transplanted after autologous cell induction, which can avoid the ethical issues involved in embryonic stem cells or human fetal brain-derived neural stem cells / progenitor cells. At the same time, compared with other common types of mesenchymal stem cells (such as bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells), studies have shown that human nasal mucosa mesenchymal stem cells have stronger proliferation ability and are easier to induce differentiation into neural stem cells, making them a better source of stem cells for the treatment of neurological diseases (Afizadeh, Rafieh Bagher, Zohreh Kamrava, Seyed Kamran Falah, Masoumeh Hamidabadi, Hatef Ghasemi Boroujeni, Mahdi Eskandarian Mohammadi, Fatemeh Khodaverdi, Sepideh Zare-Sadeghi, Arash Olya, Arta Komeili, Ali. Differentiation of human mesenchymal stem cells (MSC) to dopaminergic neurons: A comparison between Wharton's Jelly and olfactory mucosa as sources of MSCs [J]. Journal of chemical neuroanatomy, 2019, 96.).

[0005] At the same time, in order to overcome the problem of low yield of neural stem cells, our research adopts 3D culture to induce differentiation of human nasal mucosal mesenchymal stem cells into neural stem cells. The present invention provides a method for inducing batch differentiation of human nasal mucosal mesenchymal stem cells into neural stem cells based on 3D culture. Stem cells cultured in cell culture plates need to be frequently subcultured due to space limitations of culture dishes, which can easily lead to cell aging. The 3D culture system uses 3D microcarriers as carriers for cell growth, which can not only simulate the native 3D cell microenvironment in the body, but also reduce the number of subcultures by greatly increasing the cell growth space, avoiding cell aging, reducing cell heterogeneity, and obtaining a large number of more homogeneous and stable neural stem cells, thereby achieving the purpose of stable batch acquisition of neural stem cells. Summary of the invention

[0006] The first content of the present invention is to provide primary culture of human nasal mucosa mesenchymal stem cells.

[0007] The second aspect of the present invention is to provide a method for inducing differentiation of neural stem cells, the method comprising:

[0008] 1. Human nasal mucosa mesenchymal stem cells are induced to differentiate into neural stem cells;

[0009] 2. The induced neural stem cells were identified for multidirectional differentiation.

[0010] The third content of the present invention is the mass production of human nasal mucosa mesenchymal stem cells induced to differentiate into neural stem cells based on 3D culture.

[0011] The present invention adopts the following technical solutions:

[0012] A method for inducing batch differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells based on 3D culture, the method comprising the following steps:

[0013] (1) Human nasal polyps were used as a tissue source to obtain human nasal mucosal mesenchymal stem cells;

[0014] (2) The human nasal mucosa mesenchymal stem cells are cultured in 3D and induced to differentiate into neural stem cells in batches.

[0015] Preferably, the differentiation induction process is performed using a 3D culture method.

[0016] Furthermore, in step (1), the method of using human nasal polyps as a tissue source to obtain human nasal mucosal mesenchymal stem cells includes: washing the nasal polyps, removing mucus and blood, cutting the nasal polyps into multiple 1-2 mm tissue blocks, placing them in a culture dish, and infiltrating each tissue block with a small amount of complete culture medium; after 2 hours, completely immersing the tissue blocks in the culture dish in complete culture medium, placing them in an incubator at 36-37° C. for 10-12 hours, and then replacing the culture medium every two days; after another 6-7 days, cells begin to grow out of the tissue blocks and invade the culture dish, and when the confluence reaches 80-90%, the cells are digested with a trypsin-EDTA solution and centrifuged to collect the cells, thereby obtaining P0 generation human nasal mucosal mesenchymal stem cells.

[0017] Furthermore, in step (1), the preparation method of the complete culture medium is as follows: penicillin, streptomycin, amphotericin B and fetal bovine serum are added to DMEM / F12 to prepare the complete culture medium, wherein the fetal bovine serum concentration is 9-10% by volume, the penicillin concentration is 90 IU / ml-100 IU / ml, the streptomycin concentration is 90 IU / ml-100 IU / ml, and the amphotericin B concentration is 0.23-0.25 μg / ml.

[0018] Furthermore, in step (2), the human nasal mucosa mesenchymal stem cells are subjected to 3D culture, and batch induction differentiation into neural stem cells includes: inoculating the P0 generation human nasal mucosa mesenchymal stem cells obtained in step (1) into a 3D bioreactor containing a neural basal culture medium and microcarriers for dynamic culture, and inducing differentiation of the P0 generation human nasal mucosa mesenchymal stem cells with the neural basal culture medium to obtain neural stem cells.

[0019] Preferably, the speed of the agitator of the 3D bioreactor is set to 33-35 rpm / min for uniform stirring for 5-6 minutes on the first day and paused for 2-3 hours, the speed of the agitator is set to a constant speed of 40-45 rpm / min on the second day, and the speed of the agitator is set to a constant speed of 50-55 rpm / min from the third to the fourteenth day. The cells are cultured at 36.5-37° C. in an incubator with 5 volume % CO2 and a residual of 95 volume % air.

[0020] Furthermore, in step (2), the amount of the neural basal medium added is 50-200 ml, the amount of the microcarrier added is 5-10 pieces (20 mg / piece), and the cell seeding density is 4.5×10 5 ~5×10 5 cells / microcarrier sheet.

[0021] Furthermore, in step (2), the neural basal medium contains 1% by volume GlutaMAX TM (200 mM L-alanyl-L-glutamine dipeptide), 2% volume ratio 50×B-27 (50×neuronal cell culture serum-free supplement) and 10 ng / mL bFGF (fibroblast growth factor).

[0022] Furthermore, the method further comprises step (3), conducting multidirectional differentiation identification on the neural stem cells obtained in step (2).

[0023] Furthermore, the step (3) comprises: culturing the neural stem cells obtained in step (2) in a neuronal differentiation medium for 21 to 23 days to differentiate into neurons and glial cells, and then identifying the glial cell marker GFAP and the neuron marker TH.

[0024] Furthermore, in the step (3), the method for preparing the neuronal differentiation medium is as follows: DMEM / F12 medium and neural basal medium are mixed in a volume ratio of 1:1 to obtain a mixture, and fetal bovine serum (Fetal Bovine Serum, FBS) (Gibco, 10091148), N2 supplement Gibco, 17502001), B27 supplement (Gibco, 17504044) and 100× penicillin / streptomycin / glutamine (Solebol, P1400) are added to the mixture.

[0025] Furthermore, in the step (3), in the neuronal differentiation medium, the concentration of fetal bovine serum is 9-10% by volume, the concentration of N2 supplement is 1-1.5% by volume, the concentration of B27 supplement is 2-2.5% by volume, the concentration of penicillin is 90 IU / ml-100 IU / ml, and the concentration of streptomycin is 90 IU / ml-100 IU / ml.

[0026] The beneficial effects of the present invention are:

[0027] The method described in the present invention avoids the ethical issues arising from induced sources such as embryonic stem cells, and the cell source of the present invention is more easily differentiated into neural stem cells; the method of the present invention makes up for the shortcomings of poor quality and low quantity of induced differentiated neural stem cells, and realizes the batch stable expansion of induced differentiated neural stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The morphology of neural stem cells differentiated from human nasal mucosal mesenchymal stem cells in Example 1. The neural stem cells shown in the figure are spherical and suspended in the culture medium. The scale bar is 200 μm.

[0029] Figure 2 . Cell viability detection of 3D cultured human nasal mucosal mesenchymal stem cells in Example 2 (green: living cells; red: dead cells). The cells shown in the figure are well attached to the carrier, and more than 90% are green living cells. Scale bar 200μm.

[0030] Figure 3 . In Example 3, the batch expansion of the induced differentiated neural stem cells, the cells shown in the figure proliferated rapidly in the 3D culture system, achieving more than 10-fold growth on the fifth day.

[0031] Figure 4 . Identification of the induced differentiated neural stem cell marker Nestin in Example 4. The cells shown in the figure express the neural stem cell marker Nestin protein. The scale bar is 50 μm.

[0032] Figure 5 . Identification of multidirectional differentiation of induced differentiated neural stem cells in Example 5, the cells shown express the neural cell marker TH and the astrocyte marker GFAP protein, scale bar 50 μm. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are limited to explaining the present invention, and the protection scope of the present invention should include the entire contents of the claims, and through the description of the following embodiments, those skilled in the art can fully implement the entire contents of the claims of the present invention.

[0034] The method and effects of the present invention are further illustrated by examples below.

[0035] Example 1 Preparation of human nasal mucosal mesenchymal stem cells

[0036] Coat a 6-well plate with 500 μl 0.1 mg / ml poly-L-lysine for 12 hours, wash with sterile ultrapure water for more than 5 times, dry, and irradiate with ultraviolet light for half an hour. Take out the nasal polyps obtained by surgery and place them on ice. Use 5 ml tissue rinsing solution (the preparation method of tissue rinsing solution is as follows: add penicillin, streptomycin and amphotericin B to sterile PBS (Keygen Biotechnology, KGB5001) to obtain the tissue rinsing solution. In the tissue rinsing solution, the concentration of penicillin is 100 IU / ml, the concentration of streptomycin is 100 IU / ml, and the concentration of amphotericin B is 0.23 μg / ml) to rinse the tissue sample 10 times, wash the blood clots, dirt and mucus on the specimen, and gently remove / absorb the mucus and blood on the specimen with a 3 ml pipette or a 1 ml gun. Infiltrate the 6-well plate with culture medium, cut the tissue block into a size of about 1.5 mm*1.5 mm*1.5 mm, and evenly spread it on the coated 6-well plate. Place in an incubator and invert to dry. After 2 hours, add complete culture medium to each tissue block (in this embodiment, the preparation method of complete culture medium is as follows: 100× penicillin / streptomycin / glutamine (Solebol, P1400), amphotericin B (Solebol, A8252) and fetal bovine serum (Fetal Bovine Serum, FBS) (Gibco, 10091148) are added to DMEM / F12 (BI, 01-172-1ACS) to prepare the complete culture medium. Add complete culture medium (complete culture medium: fetal bovine serum concentration is 10% volume ratio, penicillin concentration is 100 IU / ml, streptomycin concentration is 100IU / ml, amphotericin B concentration is 0.25μg / ml) 1 drop, then add 500μl complete medium overnight (12h). Then put the tissue into complete medium, place it in an incubator at 37°C overnight (12h), and then change the medium every two days. After another week, cells began to grow out of the tissue block and invade the 6-well plate. When 90% confluence was reached, trypsin-EDTA digestion solution (Keygen Bio, KGY0012) was used to digest for 1 minute to separate the cells, and the cells were collected by centrifugation at 1000rpm for 5 minutes to obtain P0 generation human nasal mucosal mesenchymal stem cells.

[0037] Example 2 Batch induction differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells based on 3D culture

[0038] The 3DFloTrixminiSpin bioreactor (Huakan Bio, miniSpinS1) is used for scalable microcarrier-based 3D dynamic culture of human nasal mucosal mesenchymal stem cells. The P0 generation human nasal mucosal mesenchymal stem cells obtained in Example 1 are inoculated into a 3DFloTrixminiSpin bioreactor containing a neurobasal medium and microcarriers for dynamic culture. The amount of neurobasal medium added is 200 ml. The amount of microcarrier added is 10 pieces (20 mg / piece), and the cell seeding density is 5×105 cells / microcarrier piece. The neurobasal medium (Gibco, 21103049) contains 1% by volume GlutaMAX TM , 2% volume ratio 50×B-27 and 10ng / mL bFGF. The speed of the agitator of the miniSpin bioreactor was set to 35rpm / min for 5 minutes and pause for 2 hours on the first day, the speed of the agitator was constant at 40rpm / min on the second day, and the speed of the agitator was constant at 50rpm / min from the third day to the 14th day. The cells were cultured at 37°C in an incubator with 5% volume CO2-95% volume air. The P0 generation human nasal mucosal mesenchymal stem cells used neural basal medium (Gibco, 21103049) containing 1% volume ratio of GlutaMAX TM (glutamine supplement, Gibco, 35050061), 2% volume ratio 50×B-27 (50×neuronal cell culture serum-free supplement, Gibco, 17504044) and 10ng / mLbFGF (fibroblast growth factor, near shore protein, C046) were used to induce differentiation to obtain neural stem cells. On the third day, cell viability was identified (a small amount of cells were taken out, incubated with live and dead cell dye (purchased from Huakan Bio) for 15 minutes, rinsed with PBS and observed under a fluorescence microscope, live cells were stained green, and dead cells were stained red). The results are as follows Figure 2 As shown. On the fifth day, the number of cell expansion can reach 10-20 times the initial cell number. Human nasal mucosal mesenchymal stem cells grow on the surface of microcarriers and are distributed in serum-free medium by rotating wall container bioreactors and stirring in rotating bottles. This method can overcome many limitations associated with 2D culture systems. First, dynamic conditions may help nutrients move to the spheroids and waste away from the spheroids, promoting cell viability. Secondly, 2D cultured stem cells are prone to aging due to the number of passages, while 3D culture can avoid cell aging. At the same time, the heterogeneity of cells can be reduced to obtain more uniform and stable results. Finally, this method can recapitulate the natural 3D cell microenvironment in vivo and maintain the stemness of human nasal mucosal mesenchymal stem cells. The three-dimensional dynamic system can achieve high cell viability and high expansion rate.

[0039] Morphological observation: The neural stem cells differentiated from human nasal mucosal mesenchymal stem cells were observed using an optical microscope, and the cell morphology was recorded by taking photos. The results are shown in Figure 1 The scale bar is 200 μm. The number of cells in the 3D cultured nasal mucosal mesenchymal stem cells during the differentiation process into neural stem cells was recorded. Figure 3 shown.

[0040] Immunofluorescence detection of neural stem cell marker proteins: Identification of induced differentiated neural stem cells was performed two weeks later. The cells were fixed with paraformaldehyde, permeabilized with 0.2wt% TritonX-100 solution (Solebo, T8200) (TritonX-100 was added to 5wt% BSA (Sigma, A1933) to prepare 0.2wt% TritonX-100 solution.), and blocked with 1wt% BSA at 25°C for 1h. The samples were incubated with the primary antibody (anti-Nestin antibody (proteintech, 19483-1-AP) diluted 1:50) at 4°C for 24 hours. IgGH&LAlexa was diluted 1 / 500 647 (Abcam, ab150115) was used as the secondary antibody. Figure 4 shown.

[0041] The results showed that the neuron-like cells obtained by culturing human nasal mucosal mesenchymal stem cells had the morphological characteristics of neural stem cells and the expression of neural stem cell marker proteins (Nestin marker positive), and the process of induced differentiation of 3D cultured human nasal mucosal mesenchymal stem cells into neural stem cells achieved batch consistency amplification.

[0042] Example 3 Identification of multidirectional differentiation of induced neural stem cells

[0043] Next, the neural stem cells obtained in Example 2 were differentiated into neurons and glial cells: the neural stem cells obtained in Example 2 were cultured in a neuronal differentiation medium (the preparation method of the neuronal differentiation medium is as follows: DMEM / F12 medium (Gibco) and neural basal medium (Gibco) were mixed in a volume ratio of 1:1 to obtain a mixture, and fetal bovine serum (Fetal Bovine Serum, FBS) (Gibco, 10091148), N2 supplement Gibco, 17502001), B27 supplement (Gibco, 17504044) and 100× penicillin / streptomycin / glutamine (Solebao, P1400) were added to the mixture. In the neuronal differentiation medium, the concentration of fetal bovine serum was 10% by volume, the concentration of N2 supplement was 1.5% by volume, the concentration of B27 supplement was 2.5% by volume, the concentration of penicillin was 100 IU / ml, and the concentration of streptomycin was 100 IU / ml. After three weeks of culture, the cells were differentiated into neurons and glial cells. Then the identification of glial cell marker GFAP and neuronal marker TH was performed. The cells were fixed with paraformaldehyde, permeabilized with 0.2wt% TritonX-100 (Solebo, T8200) solution (TritonX-100 was added to 5wt% BSA (Sigma, A1933) to prepare 0.2wt% TritonX-100 solution), and blocked with 1wt% BSA at 25°C for 1h. The samples were incubated with primary antibodies (anti-GFAP antibody (proteintech, 16825-1-AP) diluted 1:100, anti-TH antibody (Abcam, ab137869) diluted 1:200) at 4°C for 24 hours. IgGH&LAlexa was diluted 1 / 500 647 was used as the secondary antibody. Figure 5 shown.

[0044] The results showed that the cultured neuron-like cells could undergo multidirectional differentiation and express neuronal and glial cell marker proteins (GFAP and TH markers were positive). The cells induced to differentiate from human nasal mucosal mesenchymal stem cells in 3D culture were indeed neural stem cells.

[0045] The present invention does not elaborate on some of the common technologies of those skilled in the art. The above-described embodiments are only descriptions of preferred implementations of the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementations described. Without departing from the design spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for inducing the differentiation of human nasal mucosa mesenchymal stem cells into neural stem cells in batches based on 3D culture, characterized in that: The method comprises the following steps: (1) Human nasal polyps were used as a tissue source to obtain human nasal mucosal mesenchymal stem cells; (2) 3D culturing the human nasal mucosal mesenchymal stem cells and inducing their differentiation into neural stem cells in batches; In step (2), the human nasal mucosa mesenchymal stem cells are subjected to 3D culture, and batch induction differentiation into neural stem cells comprises: inoculating the P0 generation human nasal mucosa mesenchymal stem cells obtained in step (1) into a 3D bioreactor containing a neural basal culture medium and microcarriers for dynamic culture, wherein the P0 generation human nasal mucosa mesenchymal stem cells are induced to differentiate with the neural basal culture medium to obtain neural stem cells; setting the speed of the agitator of the 3D bioreactor to 33-35 rpm / min for uniform stirring for 5-6 minutes on the first day and pausing for 2-3 hours, setting the speed of the agitator to a constant speed of 40-45 rpm / min on the second day, and setting the speed of the agitator to a constant speed of 50-55 rpm / min from the third day to the fourteenth day, and culturing the cells in an incubator with 5 volume % CO2-95 volume % air at 36.5-37°C; In step (2), the amount of neural basal culture medium added is 50-200 ml; the amount of microcarrier added is 5-10 pieces, 20 mg / piece, and the cell seeding density is 4.5×10 5 ~5×10 5 Cells / microcarrier sheet; In step (2), the neural basal medium contains 1% by volume GlutaMAX™, 2% by volume 50×B-27, and 10 ng / mL bFGF.

2. The method according to claim 1, characterized in that: In step (1), the method of using human nasal polyps as a tissue source to obtain human nasal mucosal mesenchymal stem cells includes: washing the nasal polyps, removing mucus and blood, cutting the nasal polyps into multiple 1-2 mm tissue blocks, placing them in a culture dish, and infiltrating each tissue block with a small amount of complete culture medium; after 2 hours, completely immersing the tissue blocks in the culture dish in complete culture medium, placing them in an incubator at 36-37°C for 10-12 hours, and then replacing the culture medium every two days; after another 6-7 days, cells begin to grow out of the tissue blocks and invade the culture dish, and when the confluence reaches 80-90%, the cells are digested with trypsin-EDTA digestion solution and centrifuged to collect the cells, thereby obtaining P0 generation human nasal mucosal mesenchymal stem cells.

3. The method according to claim 2, characterized in that In step (1), the preparation method of the complete culture medium is as follows: penicillin, streptomycin, amphotericin B and fetal bovine serum are added to DMEM / F12 to prepare the complete culture medium, wherein the fetal bovine serum concentration is 9-10% by volume, the penicillin concentration is 90 IU / ml-100 IU / ml, the streptomycin concentration is 90 IU / ml-100 IU / ml, and the amphotericin B concentration is 0.23-0.25 μg / ml.

4. The method according to claim 1, characterized in that The method further comprises step (3), wherein the neural stem cells obtained in step (2) are subjected to multidirectional differentiation identification.

5. The method according to claim 4, characterized in that The step (3) comprises: The neural stem cells obtained in step (2) are cultured in a neuronal differentiation medium for 21 to 23 days to differentiate into neurons and glial cells, and then the glial cell marker GFAP and the neuronal marker TH are identified.

6. The method according to claim 5, characterized in that In the step (3), the neuronal differentiation medium is prepared as follows: DMEM / F12 medium and neurobasal medium are mixed in a volume ratio of 1:1 to obtain a mixture, and fetal bovine serum, N2 supplement, B27 supplement and 100× penicillin / streptomycin / glutamine are added to the mixture.

7. The method according to claim 6, characterized in that In the step (3), in the neuronal differentiation medium, the concentration of fetal bovine serum is 9-10% by volume, the concentration of N2 supplement is 1-1.5% by volume, the concentration of B27 supplement is 2-2.5% by volume, the concentration of penicillin is 90 IU / ml-100 IU / ml, and the concentration of streptomycin is 90 IU / ml-100 IU / ml.

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