CN109266610B - Method for promoting mesenchymal stem cells to differentiate into neurons - Google Patents

Method for promoting mesenchymal stem cells to differentiate into neurons Download PDF

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CN109266610B
CN109266610B CN201811095661.1A CN201811095661A CN109266610B CN 109266610 B CN109266610 B CN 109266610B CN 201811095661 A CN201811095661 A CN 201811095661A CN 109266610 B CN109266610 B CN 109266610B
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mesenchymal stem
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陈金虎
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Ningbo Jinwei Biotechnology Co ltd
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Abstract

The invention discloses a method for promoting differentiation of mesenchymal stem cells into neurons, which comprises the following steps: 1) preparing human mesenchymal stem cells and subculturing; 2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium; 3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium. The differentiation into neurons was promoted by the addition of neurotrophic factors to the induction medium, and the results showed that the cells expressing the mature neural marker (MAP2ab) and the immature neural marker (β -tubulin III) were significantly increased relative to the blank control. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.

Description

Method for promoting mesenchymal stem cells to differentiate into neurons
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for promoting mesenchymal stem cells to be differentiated into neurons.
Background
Spinal Cord Injury (SCI) is a serious central system disorder, the most serious consequence of which is severe dysfunction of the limbs below the injured segment, and the resulting paraplegia can cause patients to lose their ability to self-care, which not only causes serious physical and psychological injuries to the patients, but also causes heavy burden to the family.
In the treatment of spinal cord injured patients today, it is common to reduce the extent of paralysis by means of the use of some kind of medication in combination with physical therapy. However, the quality of life of the patient cannot be improved well by such treatment.
How to repair and restore the function of central nerves after spinal cord injury is still a problem. Although the discovery of nerve growth factor brings hope to clinical drug treatment of central nerve injury and achieves certain effect, the molecular weight of the neurotrophic substances is too large to penetrate the blood brain barrier and enter central nerve tissues to exert the activity of the neurotrophic substances.
In spinal cord injury, transplantation may be an operable method for central nervous system repair. However, in order to realize transplantation therapy of cells, it is first necessary to study and evaluate the ability of cells to differentiate into neurons and to develop a method for promoting cell differentiation.
Accordingly, those skilled in the art have been devoted to developing a method of promoting differentiation of stem cells into neurons.
Disclosure of Invention
To achieve the above objects, the present invention provides a method of promoting differentiation of mesenchymal stem cells into neurons.
In a preferred embodiment of the present invention, the method comprises the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium.
Preferably, the step 3) is to break the human mesenchymal stem cell-neurosphere in the step 2), inoculate the broken human mesenchymal stem cell-neurosphere into a culture container which is double-coated by poly-L-lysine and laminin and contains the induction medium, and perform culture at 37 ℃ and 5% CO2Culturing and differentiating for 7-10 days.
Further, the induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF.
In another preferred embodiment, the step 3) is to break up the human mesenchymal stem cell-neurosphere in the step 2), inoculate the broken human mesenchymal stem cell-neurosphere into a culture container which is double-coated by poly-L-lysine and laminin, firstly use a first induction culture medium containing BDNF to perform induced differentiation for 5-6 days,then using a second induction medium containing TGF-beta 1 to perform induced differentiation for 4-5 days, and the cells are cultured at 37 ℃ and 5% CO2And (5) culturing.
Further, after adding a second induction medium containing TGF-. beta.1, the cells were first incubated at 40 ℃ with 5% CO2Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
Further, the first induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF; the second induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 1-5ng/L TGF-. beta.1.
Further, the step 1) is specifically to disinfect the umbilical cord with alcohol, remove blood vessels in balanced salt solution, and cut the mesenchymal tissue in the Wharton's jelly to 0.4-0.6cm3Size, centrifuging to obtain mesenchymal tissue part, and washing by using serum-free DMEM/F12 culture medium; washing the precipitate obtained after centrifugation for enzymatic digestion; counting cells of the suspension liquid after enzyme digestion, and then carrying out inoculation culture to obtain the human mesenchymal stem cells; passages were performed when cells grew to 70% -80% confluency.
Preferably, step 2) is performed using the human mesenchymal stem cells of the fourth to sixth generations of step 1).
Further, step 2) comprises: separating the human mesenchymal stem cells cultured in the step 1) by an enzyme method, and culturing the human mesenchymal stem cells in a tissue culture low-adhesion plastic flask containing a differentiation culture medium; wherein the differentiation culture medium is NB culture medium containing 20ng/mL epidermal growth factor, 20ng/mL basic cell-forming growth factor and 1:50 diluted B27; cells were incubated at 37 ℃ with 5% CO2And (4) culturing, adding fresh epidermal growth factor, basic cell growth factor and B27 every 3-4 days, and replacing the differentiation culture medium once a week.
Another aspect of the present invention also provides an application of the method for promoting differentiation of mesenchymal stem cells into neurons in studying nerve repair after spinal cord injury.
By using the method disclosed by the invention, the proportion of mesenchymal stem cells differentiated into neurons can be effectively improved. Compared with a blank control group, the proportion of cells expressing a mature nerve marker (MAP2ab) and an immature nerve marker (beta-tubulin III) in the cells subjected to induced differentiation by using an induction medium containing BDNF is remarkably improved and can respectively reach 8 +/-1.9% and 38.6 +/-2.9%. In the cells induced and differentiated by using the first induction medium containing BDNF and the second induction medium containing TGF-beta 1, the proportion of the cells expressing the mature nerve marker (MAP2ab) and the immature nerve marker (beta-tubulin III) is also obviously improved compared with that of the blank control group, and the proportion of the two positive cells is further improved compared with that of the cells induced and differentiated by using only the induction medium containing BDNF. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.
The conception, the specific steps, and the technical effects produced by the present invention will be further described in conjunction with the accompanying drawings to fully understand the objects, the features, and the effects of the present invention.
Drawings
Fig. 1 shows two cell morphologies in the primary culture of human mesenchymal stem cells according to a preferred embodiment of the present invention, wherein fig. 1a is a long bar shape and fig. 1b is a circular shape.
FIG. 2 is the cell morphology of the third generation passage cell of the human mesenchymal stem cell cultured to 80% fusion rate according to a preferred embodiment of the present invention;
FIG. 3 is a diagram illustrating the initial aggregation of human mesenchymal stem cells 1-2 days after re-seeding of the human mesenchymal stem cells into a neurosphere differentiation medium according to a preferred embodiment of the present invention.
Fig. 4 is a diagram showing that the human mesenchymal stem cell according to a preferred embodiment of the present invention shows a plurality of spheroids of floating cells 3 to 4 days after transformation.
Fig. 5 is the result of the 3 rd generation CD44 staining of human mesenchymal stem cells according to a preferred embodiment of the present invention.
FIG. 6 is a nesting positive marker map of neurospheres derived from human mesenchymal stem cells according to a preferred embodiment of the present invention.
Fig. 7 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into β -tubulin III (immature neural marker) -positive cells according to a preferred embodiment of the present invention.
Fig. 8 is a graph showing the results of immunofluorescence histochemistry method of differentiation of human mesenchymal stem cells into GFAP (astrocyte marker) -positive cells according to a preferred embodiment of the present invention.
Fig. 9 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into CalC (oligodendrocyte marker) positive cells according to a preferred embodiment of the present invention.
Fig. 10 is a graph showing the results of immunofluorescence histochemistry assay of human mesenchymal stem cells differentiated into MAP2ab (mature neural marker) -positive cells according to a preferred embodiment of the present invention.
FIG. 11 is a graph showing the results of protein-blot analysis of the protein levels associated with the differentiation of human mesenchymal stem cells into different neural marker-positive cells according to a preferred embodiment of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
The experimental methods used in the embodiments of the present invention are conventional in the art unless otherwise specified. The reagents used in the embodiments of the present invention can be obtained by purchase, unless otherwise specified.
Human mesenchymal stem cells have many advantages of both embryonic stem cells and adult stem cells, and have advantages over other stem cells in that: the plasticity is strong; is readily obtainable by a less invasive means and is capable of rapid proliferation; immune compatibility, the patient's own human mesenchymal stem cells can be used for autologous transplantation. Therefore, the present invention uses human mesenchymal stem cells as starting materials for differentiation studies.
In neurogenesis, micro-environmental factors have a very important influence on proliferation and differentiation. Among them, BDNF is widely distributed in the developing and mature nervous system and plays an important role in the development, survival and repair of nerve cells.
The effect of differentiation of human mesenchymal stem cells into neurons was observed by the present study through the combination of different trophic factors with different conditions.
Example 1 preparation of human mesenchymal Stem cells
The umbilical cord of the newborn was obtained, placed in hanks' balanced salt solution (HBSS, Gibco, USA), and stored at 4 ℃. The umbilical cord was sterilized with 75% alcohol for 30 seconds. In HBSS, umbilical vessels are cleared. The mesenchymal tissue present in Wharton's jelly was cut to about 0.5cm3And centrifuged at 1,200rpm for 5 minutes. After centrifugation, the supernatant was removed, and the precipitated portion of the mesenchymal tissue was washed with serum-free DMEM/F12 medium (Gibco, USA), followed by centrifugation at 1,200rpm for 5 minutes. The pellet fraction was enzymatically separated using 0.075% collagenase type II (Sigma) at 37 ℃ for 18 hours, and then further digested using 0.125% trypsin/EDTA (Gibco) at 37 ℃ for 30 minutes. The suspension was neutralized using DMEM/F12 containing 10% (v/v) fetal bovine serum, and the cells in the suspension were counted under a microscope. Adjusting to 5-7 × 103Per cm2Inoculating into tissue culture flask, and culturing at 37 deg.C with 5% CO2The culture was performed while maintaining a sub-confluent state. When cells were grown to 70% confluence, the cells were incubated at 1: 3, subculture. Subsequent studies were performed using cells from fourth to sixth passage.
As shown in fig. 1a and 1b, in the primary culture of human mesenchymal stem cells, two cell morphologies, one being a long strip and one being a round one, appeared. After three to five passages, the human mesenchymal stem cells exhibit a flat cell morphology of a monolayer, and the morphology of all cells tends to be similar. FIG. 2 shows the cell morphology in the third generation subculture to 80% confluency.
Example 2 formation of human mesenchymal Stem cell-neurosphere from human mesenchymal Stem cell
Separating the fourth generation of human mesenchymal stem cells with 0.125% trypsin/0.02% EDTA, wherein the separated human mesenchymal stem cells are 2 × 105Per cm2The density of (a) was inoculated into a tissue culture low-adhesion plastic flask containing a differentiation medium. The differentiation medium was NB medium (Invitrogen) containing 20ng/mL Epidermal Growth Factor (EGF), 20ng/mL basic fibroblast growth factor (bFGF) and 1:50 dilution of B27 (Gibco).
Cells were incubated at 37 ℃ with 5% CO2And (5) culturing. Fresh growth factors were added every 3-4 days and the medium was changed once a week. Sphere formation was observed 4-5 days after initial differentiation. Cells were cultured in differentiation medium until passage.
FIG. 3 shows the cell morphology of human mesenchymal stem cells re-seeded into neurosphere differentiation medium for 1-2 days, and the cells begin to aggregate to form human mesenchymal stem cells-neurospheres. Spheres of many floating cells appeared 3-4 days after transformation (i.e., around 7 days after reseeding), as shown in fig. 4.
The passaging is performed enzymatically every 10-12 days, i.e., with 0.025% trypsin plus 0.6% glucose. These neurosphere-like structures continue to expand for 4-6 weeks (approximately 3-4 generations) before the neurons terminally differentiate.
Almost all passage 3 human mesenchymal stem cells showed strong CD44 staining (fig. 5). Neurospheres derived from human mesenchymal stem cells were confirmed by nestin antibody (fig. 6).
Example 3 further differentiation of human mesenchymal Stem cell-neurosphere (BDNF Induction)
Human mesenchymal stem cell-neurospheres cultured in example 2 were collected, broken up, and re-seeded into poly-L-lysine and laminin double-coated six-well chamber slides (numcs) containing induction medium. Cells were incubated at 37 ℃ with 5% CO2Culturing and differentiating for 7-10 days.
Wherein the induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco) and 10ng/mL recombinant human BDNF (rhBDNF, R & D Systems).
In the control group, the experimental conditions were the same as described above except that the induction medium was replaced with a control induction medium without BDNF, i.e., NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, and 1% penicillin/streptomycin (all purchased from Gibco).
The differentiated cells were collected for analysis:
human mesenchymal stem cells differentiated into beta-tubulin III (immature neural marker), GFAP (astrocytic marker), CalC (oligodendrocyte marker) and MAP2ab (mature neural marker) positive cells, and the results of immunofluorescence histochemistry for positive cells of different markers were shown in FIGS. 7-10. By Hoechst33342+And (3) dyeing, quantifying various differentiated positive cells, and calculating a quantitative result by the following method: corresponding number of positive cells/Hoechst 33342+The number of stained cells was 100%, and the results are shown in Table 1.
Table 1: quantitative percentage of positive cells
Induction medium Beta-tubulin III GFAP CalC MAP2ab
Control 19.2±3.0％ 42.8±3.8％ 27±2％ 4.4±1.8％
BDNF Induction 38.6±2.9％ 15.8±4.5％ 20.6±4.6％ 8±1.9％
The results show that beta-tubulin III positive cells and MAP2ab positive cells were significantly higher produced using the neural specific induction method than using the conventional induction method.
Protein imprinting further verified:
protein imprinting is a routine procedure in the art, and in this study was treated with a primary antibody against β -tubulin III (1:500), MAP2ab (1:500), followed by a secondary antibody linked to peroxidase. Development is performed by an enhanced chemiluminescence method. Beta-agonist protein (beta-Actin) was used as an internal control. Antibodies were all from Chemicon.
The results of the protein blot verification are shown in fig. 11, and the results match the immunohistochemical results.
The results show that after BDNF is added, the proportion of cells expressing a mature nerve marker (MAP2ab) and an immature nerve marker (beta-tubulin III) is obviously improved compared with that of a blank control group, namely, the proportion of umbilical cord derived neural stem cells which are transformed into neurons is obviously improved by adding a brain derived nerve growth factor group.
Example 4 further differentiation of human mesenchymal Stem cell-neurosphere (BDNF + TGF-. beta.1 Combined Induction)
Human mesenchymal stem cell-neurospheres cultured in example 2 were collected, ground, and re-seeded into poly-L-lysine and laminin double-coated six-well chamber slides (chamber slides, NUNC) containing BDNF induction medium. Cells were incubated at 37 ℃ with 5% CO2Culturing and differentiating for 5-6 days. After that time, the user can use the device,replacing BDNF induction culture medium with TGF-beta 1 induction culture medium, firstly, at 40 deg.C and 5% CO2Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
Wherein the BDNF induction medium is NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco) and 10ng/mL recombinant human BDNF (rhBDNF, R & D Systems);
TGF-. beta.1 Induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin (all purchased from Gibco), and 1-5ng/L TGF-. beta.1 (Sigma).
In the control group, the experimental conditions were the same as described above except that both BDNF-inducing medium and TGF-. beta.1-inducing medium were replaced with BDNF-free control inducing medium, i.e., NB medium supplemented with 0.5umol/L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, and 1% penicillin/streptomycin (all purchased from Gibco).
The differentiated cells were collected for analysis:
the positive cells of different markers were observed by immunofluorescence histochemistry and were examined by Hoechst33342+And (3) dyeing, quantifying various differentiated positive cells, and calculating a quantitative result by the following method: corresponding number of positive cells/Hoechst 33342+The number of stained cells was 100%, and the results are shown in Table 2.
Table 2: quantitative percentage of positive cells
Figure BDA0001805473380000061
The results show that beta-tubulin III positive cells and MAP2ab positive cells were significantly higher produced using the neural specific induction method than using the conventional induction method. Also, the proportion of cells expressing the mature neural marker (MAP2ab) and the immature neural marker (β -tubulin III) was increased to some extent, compared to the BDNF alone induction method in example 3.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (7)

1. A method of promoting differentiation of mesenchymal stem cells into neurons, comprising the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium; specifically, the human mesenchymal stem cells-neurospheres in the step 2) are smashed and inoculated into a culture container which is double-coated by poly-L-lysine and laminin and contains the induction culture medium, and the culture container is heated at 37 ℃ and 5% CO2Culturing and differentiating for 7-10 days;
wherein the induction medium is NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF.
2. A method of promoting differentiation of mesenchymal stem cells into neurons, comprising the steps of:
1) preparing human mesenchymal stem cells and subculturing;
2) allowing the human mesenchymal stem cells after subculture to differentiate into human mesenchymal stem cell-neurospheres in a differentiation medium;
3) differentiating the human mesenchymal stem cell-neurospheres into neurons in an induction medium; specifically, the human mesenchymal stem cells-neurospheres in the step 2) are smashed and connectedPlanting into culture container with double coatings of poly-L-lysine and laminin, inducing differentiation for 5-6 days with first inducing culture medium containing BDNF, and inducing differentiation for 4-5 days with second inducing culture medium containing TGF-beta 1, and culturing at 37 deg.C with 5% CO2Culturing;
wherein the first induction medium is an NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 10ng/mL recombinant human BDNF; the second induction medium was NB medium supplemented with 0.5umol/L all-trans retinoic acid, 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin/streptomycin, and 1-5ng/L TGF-. beta.1.
3. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 2, wherein the cells are first cultured at 40 ℃ and 5% CO after the addition of the second induction medium containing TGF- β 12Culturing for 3-4 hr, transferring to 37 deg.C, and culturing with 5% CO2And continuously culturing and differentiating for 4-5 days.
4. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2, wherein the step 1) is specifically to sterilize umbilical cord with alcohol, remove blood vessels in balanced salt solution, and cut the mesenchymal tissue in Wharton's jelly to 0.4-0.6cm3Size, centrifuging to obtain mesenchymal tissue part, and washing by using serum-free DMEM/F12 culture medium; washing the precipitate obtained after centrifugation for enzymatic digestion; counting cells of the suspension liquid after enzyme digestion, and then carrying out inoculation culture to obtain the human mesenchymal stem cells; passages were performed when cells grew to 70% -80% confluency.
5. The method for promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2, wherein step 2) is performed using the human mesenchymal stem cells of the fourth to sixth generations of step 1).
6. The method of claim 5A method for promoting differentiation of mesenchymal stem cells into neurons, wherein the step 2) comprises: separating the human mesenchymal stem cells cultured in the step 1) by an enzyme method, and culturing the human mesenchymal stem cells in a tissue culture low-adhesion plastic flask containing a differentiation culture medium; wherein the differentiation culture medium is NB culture medium containing 20ng/mL epidermal growth factor, 20ng/mL basic cell-forming growth factor and 1:50 diluted B27; cells were incubated at 37 ℃ with 5% CO2And (4) culturing, adding fresh epidermal growth factor, basic cell growth factor and B27 every 3-4 days, and replacing the differentiation culture medium once a week.
7. Use of the method of promoting differentiation of mesenchymal stem cells into neurons according to claim 1 or 2 for studying nerve repair after spinal cord injury.
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