CN115125207B - Method for inducing stem cells to differentiate directionally in vitro - Google Patents
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Abstract
The invention belongs to the technical field of stem cell culture, and in particular relates to a culture medium and a method for inducing stem cells to directionally differentiate into nerve cells, wherein the culture medium comprises the following components: EGF; IL-6; PRG4 protein and basal medium. After the umbilical cord mesenchymal stem cells are induced by the culture medium for 10 days, the differentiation proportion of the nerve cells reaches 92.5%, and the nerve specific markers nestin, p-Tubulin III, NSE and GFAP in the prepared nerve cells are positive.
Description
Technical Field
The invention belongs to the technical field of stem cell culture. Relates to a method for inducing stem cells to differentiate directionally in vitro; and more particularly to methods for inducing the directional differentiation of stem cells into neural precursor cells or neural cells in vitro.
Background
For central nervous system diseases such as neuronal damage and loss of function, such as cerebral infarction, alzheimer's Disease (AD), parkinson's Disease (PD), etc., there is no effective method for promoting nerve cell regeneration, so cell replacement therapy is one of the most promising therapeutic strategies for treating neural loss.
Proteoglycans (PRG 4) are glycoproteins having a molecular weight of about 227.5kDa, originally obtained by Swann et al [1] It was found and designated as lubricin (lubricin) encoded by the prg4 gene (consisting of 12 exons) [2] Also known as proteolycan 4 (PRG 4). At present, PRG4 has the possibility of inducing stem cells to differentiate directionally into neural precursor cells or neural cells, which has not been reported so far.
[1]Swann DA，Slayter HS，Silver FH.The molecular structure of lubricat ing glycoprotein-I,the boundary lubricant for articular cartilage[J].J Biol Chem,1981,256(11):5921-5925.
[2]Gleghon JP，Jones AR,Flannery CR,et al.Boundary mode lubrication of articular cartilage by recombinant human lubricin[J].J Orthop Res,2009,27(6):771-777.
Disclosure of Invention
Certain cytokines are known to be closely related to development and neural differentiation of the nervous system, EGF is capable of promoting survival and proliferation of neural precursor cells, but no report has been made before the date of application on EGF in inducing the directed differentiation of stem cells derived from non-neural tissue into neural cells.
Also, it was confirmed by experiments that EGF alone does not have a sufficiently strong ability to induce directional differentiation of non-neural tissue-derived stem cells into a certain type of cells or neural cells, whereas IL-6 does not have a remarkable effect of promoting differentiation of non-neural tissue-derived stem cells into neural precursor cells or neural cells, but unexpectedly, when EGF is combined with IL-6 to induce non-neural tissue-derived stem cells, a strong ability to induce differentiation into neural stem cells can be observed, and thus, it is presumed that EGF is combined with IL-6 to have a synergistic effect of promoting directional differentiation of stem cells into neural cells. And it was unexpectedly observed that the stronger this synergistic effect was as the concentration of IL-6 was reduced, the stronger the synergistic effect was not observed beyond the concentration range defined by the present invention.
The invention is shown in tables 2 and 3 that when EGF and IL-6 are combined to induce stem cells from non-neural tissue, the differentiation proportion of the neural cells is 52.4%, and the nerve specific markers nestin, p-Tubulin III, NSE and GFAP in the prepared neural cells are positive, and the relative expression quantity of the 4 specific markers is obviously higher than that of the EGF group and the IL-6 group which are added singly, so that the two markers have a synergistic effect in the aspect of inducing the directional induction of mesenchymal stem cells.
It is therefore an object of the present invention to provide a medium for inducing directional differentiation of stem cells into neural cells, the medium comprising:
EGF；
IL-6; and
basal medium.
In one embodiment of the invention, the medium comprises:
10～20ng/mL EGF；
IL-6 of 1-3 ng/mL; and
the balance of basal medium.
In one embodiment of the invention, the medium comprises:
15ng/mL EGF；
2ng/mL IL-6; and
the balance of basal medium.
In one embodiment of the invention, the medium comprises:
10ng/mL EGF；
3ng/mL IL-6; and
the balance of basal medium.
In one embodiment of the invention, the medium comprises:
20ng/mL EGF；
IL-6 at 1 ng/mL; and
the balance of basal medium.
In one embodiment of the invention, the medium comprises:
20ng/mL EGF；
3ng/mL IL-6; and
the balance of basal medium.
In one embodiment of the invention, the medium further comprises: PRG4 protein.
It was unexpectedly found that PRG4 protein enhanced this synergy between EGF and IL-6, regulated differentiation and proliferation of stem cells induced by EGF and IL-6 into neural cells, and a higher proportion of neural cell differentiation was observed upon addition of PRG4 protein. It is hypothesized that it may be relevant that the PRG4 protein is capable of modulating IL-6 induced cell differentiation and proliferation.
Table 2 and Table 3 show that the synergistic effect between EGF and IL-6 can be enhanced by adding PRG4 protein, the relative expression quantity of the related specific marker in the prepared nerve cells is highest, the differentiation proportion of the nerve cells is 92.5%, and the nerve cells have a significant difference (P is less than 0.01) compared with the EGF and IL-6 combined group.
The term "differentiated into neural cells" as used herein means that the cells obtained by differentiation of the cells have obvious morphological characteristics of neural cells, and highly express neural cell-specific genes such as nestin, GFAP, p-Tubulin III. The obvious morphological characteristics of the nerve cells mean that the stem cells are changed in a nerve cell shape, such as enlargement of nucleus, bifurcated change of cells, more elongated protrusions extending from the cells, and the like.
In one embodiment of the invention, the concentration of PRG4 protein is generally between 0.1 and 1mg/mL, for example 0.3mg/mL, 0.5mg/mL, and more preferably 0.3mg/mL.
In one embodiment of the invention, the basal medium is dmem, mem or dmem/F12 medium. In representative embodiments of the invention, the basal medium is preferably dmem.
In particular, the culture medium for inducing the directional differentiation of stem cells into nerve cells according to the present invention may be added or not added: serum, vitamins, amino acids, and antibiotics.
In one embodiment of the invention, the stem cells are umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells. In a representative embodiment of the present invention, the stem cells are umbilical cord mesenchymal stem cells.
Another object of the present invention is to provide a method for inducing directional differentiation of stem cells into neural cells, comprising the steps of:
a. taking stem cells with good growth state after identification;
b. culturing in culture medium.
In one embodiment of the invention, in step b, the incubation time is 10 days.
In step a, when the stem cells are umbilical cord mesenchymal stem cells, the stem cells can be isolated from umbilical cord by conventional techniques, and a certain number of cell populations can be obtained through primary culture, passage and expansion.
Wherein the step of isolating umbilical cord mesenchymal stem cells from umbilical cord tissue generally comprises:
cutting cleaned umbilical cord tissue into small segments, removing blood vessel, cutting, placing in culture dish, adding DMEM/f12 culture solution containing 10% FBS, 37deg.C, 5% CO 2 Culturing in an incubator, adding fresh culture solution every 3d, removing tissues and continuing culturing until 4-5 obvious clones appear in a culture dish; when the cells are fully paved in the culture flask by 80-90%, 0.25% trypsin EDTA is digested, and the cells are subjected to passage amplification according to a proportion.
In one embodiment of the invention, the stem cells are umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells. In a representative embodiment of the present invention, the stem cells are umbilical cord mesenchymal stem cells.
It is another object of the invention to provide cells prepared by the method. The cells prepared by the method of the invention are specifically nerve precursor cells or nerve cells, and the cells have important significance in clinic for treating parkinsonism or syndrome, apoplexy and sequelae thereof, alzheimer disease and the like.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
1. Research for inducing directional differentiation of umbilical cord mesenchymal stem cells
1.1 primary culture, passage and expansion of umbilical cord mesenchymal stem cells: taking umbilical cord tissue of newborn from Cesarean section of healthy term, washing with sterile normal saline for several times, cutting into 1cm tissue blocks, removing blood vessel, cutting into pieces of 1mm×1mm, spreading on the bottom of culture dish, adding DMEM/f12 medium containing 10% FBS, 37deg.C, and 5% CO 2 Culturing in an incubator, adding fresh culture solution every 3d, removing tissues and continuing culturing until 4-5 obvious clones appear in a culture dish; when the cells are fully paved in the culture flask by 80-90%, 0.25% trypsin EDTA is digested, and the cells are subjected to passage amplification according to the proportion of 1:2.
1.2 induced differentiation: taking the identified P3 generation mesenchymal stem cells, digesting with 0.05% trypsin EDTA, and adjusting the cell density to 1×10 4 /cm 2 Inoculating to 96-well plate, adding 100 μL/well of the induction culture medium described in Table 1 to induce differentiation for 10 days when the cells reach 80% fusion, observing morphological change of the cells under an inverted microscope, counting the proportion of the neural-like cells (the length of the cell protrusions is 5 times of the cell body diameter), and detecting the expression condition of the specific markers related to the neural cells by adopting a real-time quantitative PCR method.
Table 1: induction medium
1.3 results
1.3.1 cell microscopy: after 10 days of induction, more cells exhibiting neural cell-like changes were observed under both medium 1-2 and 6-8 induced cell microscopes, with the highest proportion of neural-like changed cells induced using medium 1 (Table 2).
1.3.2 expression of neural cell-specific markers: as can be seen from Table 3, although IL-6 does not have a remarkable ability to induce differentiation of umbilical cord mesenchymal stem cells into neural cells, the ratio of the induced differentiation of umbilical cord mesenchymal stem cells into neural cells is 52.4% after 10 days of induction by simultaneous addition of EGF and IL-6, and the neural specific markers nestin, p-Tubulin III, NSE and GFAP in the prepared neural cells are positive, and the relative expression amounts of the 4 specific markers are significantly higher than those of EGF alone and IL-6 alone, showing that both have a synergistic effect in inducing directional induction of mesenchymal stem cells. The synergistic effect between EGF and IL-6 can be enhanced by further adding PRG4 protein, the relative expression amount of the related specific mark in the prepared nerve cells is the highest, and compared with the EGF and IL-6 combined group, the relative expression amount of the related specific mark is obviously different (P is less than 0.01). Although FGF alone induced mesenchymal stem cells more potent than EGF in their directional differentiation, the FGF combined PRG4 and IL-6 induced mesenchymal stem cells more potent than EGF in their combination (table 3).
Table 2: differentiation ratio of neural-like cells
Table 3: relative expression level of neural cell specific marker
Note that: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group 1 of the culture medium, # P＜0.05； ## P＜0.01。
the above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. A culture medium for inducing directional differentiation of umbilical cord mesenchymal stem cells into nerve cells, characterized in that the culture medium consists of the following components:
10～20ng/mL EGF；
1～3ng/mL IL-6；
0.1-1 mg/mLPRG4 protein; and
the balance of basal medium.
2. The culture medium according to claim 1, wherein the basal medium is dmem, mem or dmem/f12 medium.
3. A method for inducing directional differentiation of umbilical cord mesenchymal stem cells into neural cells, comprising the steps of:
a. taking stem cells with good growth state after identification;
b. culturing in a medium according to claim 1 or 2.
4. A method according to claim 3, wherein in step b, the incubation is for a period of 10 days.
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