CN110257332A - A kind of inducing mesenchymal stem cell differentiation becomes the culture medium and method of dopaminergic neuron - Google Patents
A kind of inducing mesenchymal stem cell differentiation becomes the culture medium and method of dopaminergic neuron Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
Abstract
The present invention relates to stem cells technology field, disclosing a kind of inducing mesenchymal stem cell differentiation becomes the culture medium and method of dopaminergic neuron.Culture medium of the present invention includes preparatory induced medium and dopaminergic neuron induced medium;The preparatory induced medium is basic culture medium with DMEM/F12, contains FBS, bFGF, cAMP and L-AA;The dopaminergic neuron induced medium is basic culture medium with DMEM/F12 and Neurobasal, contains FBS, FGF-8, SHH, cAMP, L-AA, GDNF and BDNF.The present invention carries out induction differentiation to mescenchymal stem cell in two stages by preparatory induced medium and dopaminergic neuron induced medium, the composition of two stage culture mediums of optimum organization simultaneously, dopaminergic neuron can be successfully induced differentiation into, and has higher differentiation rate.
Description
Technical field
The present invention relates to stem cells technology fields, and in particular to a kind of differentiation of inducing mesenchymal stem cell becomes dopaminergic
The culture medium and method of neuron.
Background technique
Parkinson's disease (Parkinson ' s disease, PD) is one of most common neurodegenerative disease, the whole world 65 years old
Above prevalence is 1%~2%, and 85 years old or more population has about 4% people to be perplexed for a long time by it.The master of Parkinson's disease
Wanting clinical symptoms includes static tremor, myotonia, bradykinesia and posture abnormal gait etc..Its characteristic pathology changes
Substantia nigra of midbrain dopaminergic (dopaminergic, DA) neuron is largely denaturalized loss, has Lewy in remaining neuron plasma
Corpusculum is formed.Treatment Parkinson's disease lacks effective treatment means at all at present, and main drug therapy and operative treatment are only
Symptom can be improved and disease progression cannot be prevented, and the problems such as there are side effects.Therefore our poles need to be sought more efficiently
Treatment method.
Cellular transplantation therapy (Cell-replacement therapy) is considered as controlling for one kind most hope and potentiality
Treat the new treatment of Parkinson's disease.Since the pathomechanism of Parkinson's disease is than more visible, damage location Relatively centralized, therefore be well suited for
Make cellular transplantation therapy.Embryonic mesencephalic tissue and embryonic stem cell (embryonic stem are once used in the research of early stage
Cells, ESCs) transplanting method, observation obtains preliminary effect, but ethics problem, donor source be limited and immunological rejection etc.
Problem limits it and further develops;Inductive pluripotent stem cells (induced pluripotent stem cells, iPSCs)
Appearance well solved the immunological rejection and ethics problem that embryonic stem cell faces, however tumorigenic risk is current
Study the biggest obstacle of iPS cell clinical application.Another strategy being widely noticed in recent years is that " small molecule inducing cell is rearranged
Mature somatic induction is divided into other types of functioning cell or progenitor cells by journey " technology.The reprogramming of small molecule inducing cell
Method avoids genetic manipulation, but utilizes the principle of small molecule and cellular endogenous factor interaction, so that autogenous cell regains
The performance of multipotential stem cell.Compared to insertion foreign gene, the advantage of small molecule be it is easy to operate, processing the time easily grasp,
Experimentation cost is reduced, and can be taking human as adjustment concentration and combination.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of differentiation of inducing mesenchymal stem cell to become dopaminergic nerve
The culture medium of member enables the culture medium inducing mesenchymal stem cell to be divided into dopaminergic neuron, and has higher
Differentiation rate, while providing abductive approach based on affiliated culture medium.
To achieve the goals above, the invention provides the following technical scheme:
A kind of inducing mesenchymal stem cell differentiation becomes the culture medium of dopaminergic neuron, including preparatory induced medium
With dopaminergic neuron induced medium;The preparatory induced medium is basic culture medium with DMEM/F12, containing FBS,
BFGF, cAMP and L-AA;The dopaminergic neuron induced medium is using DMEM/F12 and Neurobasal as base
Basal culture medium contains FBS, FGF-8, SHH, cAMP, L-AA, GDNF and BDNF.
Preferably, the preparatory induced medium is basic culture medium with DMEM/F12, contain percent by volume 5%-
10%FBS, 10-100ng/ml bFGF, 0.1-0.5mM cAMP and 0.1-0.2mM L-AA.
In the specific embodiment of the invention, the preparatory induced medium is basic culture medium with DMEM/F12, is contained
Percent by volume 10%FBS, 50ng/ml bFGF, 0.25mM cAMP and 0.2mM L-AA.
Preferably, the dopaminergic neuron induced medium with volume ratio for 1:1 DMEM/F12 and
Neurobasal is basic culture medium, contains percent by volume 0.5%-1%FBS, 10-100ng/ml FGF-8,10-100ng/
Ml SHH, 0.1-0.5mM cAMP, 0.1-0.2mM L-AA, 10-20ng/ml GDNF and 10-20ng/ml BDNF.
In the specific embodiment of the invention, the dopaminergic neuron induced medium is 1:1's with volume ratio
DMEM/F12 and Neurobasal is basic culture medium, contains 1%FBS, 50ng/ml FGF-8,50ng/ml SHH, 0.25mM
CAMP, 0.2mM L-AA, 10ng/ml GDNF and 10ng/ml BDNF.
Meanwhile the present invention carries out induction differentiation, specific method using the culture medium by taking umbilical cord mesenchymal stem cells as an example
It is as follows:
After mescenchymal stem cell is added preparatory induced medium pre-induced 2 days in culture medium described in claim 1, the
Dopaminergic neuron induced medium in culture medium described in 3 days replacement claims 1 continues culture 7 days, changes liquid every other day.
Wherein, the mescenchymal stem cell is P1 for umbilical cord mesenchymal stem cells;The pre-induced and dopaminergic nerve
The environmental condition that first induced medium continues culture is 37 DEG C, 5%CO2, saturated humidity.
The results show that neuronal specificity antibody β III-tubullin (TUJ-1) and dopaminergic neuron specificity are anti-
Body TH expression is positive, and blank control group is feminine gender, shows that culture medium of the invention can be by mesenchyma stem cell differentiation induction
For dopaminergic neuron.
Based on this, the invention proposes the culture mediums to become dopaminergic neuron in inducing mesenchymal stem cell differentiation
In application and/or preparing inducing mesenchymal stem cell differentiation become dopaminergic neuron reagent in application.Wherein,
The mescenchymal stem cell is preferably umbilical cord mesenchymal stem cells.
From the above technical scheme, the present invention passes through preparatory induced medium and dopaminergic neuron induced medium
Induction differentiation, while the composition of two stage culture mediums of optimum organization, Neng Goucheng are carried out to mescenchymal stem cell in two stages
Function induces differentiation into dopaminergic neuron, and has higher differentiation rate.
Detailed description of the invention
Fig. 1, which is shown, uses inductive differentiation medium of the present invention to induce differentiation into as dopamine umbilical cord mesenchymal stem cells
The identified by immunofluorescence of serotonergic neuron;Wherein, A is present invention induction differentiated result;B is blank control group result;TUJ1(βⅢ-
Tubulin): III tubulin of β (neuronal specificity marker);TH: tyrosine hydroxylase (dopamine neuron specificity mark
Will object);DAPI:4', 6-diamidino-2-phenylindole, nuclear targeting;Merge is mixing resultant.
Specific embodiment
The invention discloses culture medium and sides that a kind of differentiation of inducing mesenchymal stem cell becomes dopaminergic neuron
Method, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all
Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This
It invents the culture medium and method to be described by preferred embodiment, related personnel can obviously not depart from the present invention
Hold, culture medium as described herein and abductive approach be modified in spirit and scope or appropriate changes and combinations, realizing and
Using the technology of the present invention.
Just culture of a kind of inducing mesenchymal stem cell differentiation provided by the present invention as dopaminergic neuron below
Base and method are described further.
Embodiment 1: culture medium of the present invention
(1) preparatory induced medium
1. basal medium: DMEM/F12 is purchased from Gibco company (#C11330500BT);
2. active constituent:
1. 5%-10% fetal calf serum (FBS) is purchased from Gibco company (#A3161001);
2. 10-100ng/ml recombination human basic fibroblast growth factor (bFGF) is purchased from peprotech company (#
100-20);
3. 0.1-0.5mM adenosine -3', 5'- cycli phosphate (cAMP) is purchased from peprotech company (#6099240);
4. 0.1-0.2mM vitamin C (L-AA) is purchased from Sigma company (#A4544);
(2) FGF8/SHH dopaminergic neuron induced medium
1. basal medium:
1. 48%DMEM/F12 is purchased from Gibco company (#C11330500BT);
2. 48%Neurobasal culture medium is purchased from Gibco company (#21103-049);
2. active constituent:
1. 0.5%-1% fetal calf serum (FBS) is purchased from Gibco company (#A3161001);
2. 10-100ng/ml fibroblast growth factor-8 (FGF8) is purchased from peprotech company (#100-25);
3. 10-100ng/ml Sonic hedgehog (SHH) is purchased from peprotech company (#100-45);
4. 0.1-0.5mM adenosine -3', 5'- cycli phosphate (cAMP) is purchased from peprotech company (#6099240);
5. 0.1-0.2mM vitamin C (L-AA) is purchased from Sigma company (#A4544);
6. the neurotrophic factor (GDNF) of 10-20ng/ml Deiter's cells source property is purchased from peprotech company
(#450-44);
7. 10-20ng/ml brain-derived neurotrophic factor (BDNF) is purchased from peprotech company (#450-02).
Embodiment 2: the method that inducing mesenchymal stem cell differentiation becomes dopaminergic neuron
The present embodiment is by taking umbilical cord mesenchymal stem cells (be purchased from Sai Laila stem cell company) as an example, using matching in embodiment 1
The inductive differentiation medium of system carries out induction differentiation, and specific abductive approach is as follows: P1 is added for umbilical cord mesenchymal stem cells
The preparatory induced medium pre-induced of bFGF is after 2 days, and replacement FGF8/SHH dopaminergic neuron induced medium continues to train within the 3rd day
It supports 7 days, changes liquid every other day, carry out identified by immunofluorescence after Fiber differentiation 7 days.
As a result, it has been found that: neuronal specificity antibody β III-tubulin (TUJ-1) and dopaminergic neuron specificity are anti-
Body TH expression is positive, and blank control group is negative (see Fig. 1).Therefore, inductive differentiation medium of the invention can be filled by between
Matter stem cell is induced to differentiate into dopaminergic neuron.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the culture medium that a kind of inducing mesenchymal stem cell differentiation becomes dopaminergic neuron, which is characterized in that including preparatory
Induced medium and dopaminergic neuron induced medium;The preparatory induced medium is cultivated based on DMEM/F12
Base contains FBS, bFGF, cAMP and L-AA;The dopaminergic neuron induced medium with DMEM/F12 and
Neurobasal is basic culture medium, contains FBS, FGF-8, SHH, cAMP, L-AA, GDNF and BDNF.
2. culture medium according to claim 1, which is characterized in that the preparatory induced medium is trained based on DMEM/F12
Base is supported, percent by volume 5%-10%FBS, 10-100ng/ml bFGF, 0.1-0.5mM cAMP and 0.1-0.2mM L- are contained
Ascorbic acid.
3. culture medium according to claim 2, which is characterized in that the preparatory induced medium is trained based on DMEM/F12
Base is supported, percent by volume 10%FBS, 50ng/ml bFGF, 0.25mM cAMP and 0.2mM L-AA are contained.
4. culture medium according to claim 1, which is characterized in that the dopaminergic neuron induced medium is with volume ratio
It is basic culture medium for the DMEM/F12 and Neurobasal of 1:1, contains percent by volume 0.5%-1%FBS, 10-100ng/
Ml FGF-8,10-100ng/ml SHH, 0.1-0.5mM cAMP, 0.1-0.2mM L-AA, 10-20ng/ml GDNF
With 10-20ng/ml BDNF.
5. culture medium according to claim 4, which is characterized in that the dopaminergic neuron induced medium is with volume ratio
DMEM/F12 and Neurobasal for 1:1 are basic culture medium, containing 1%FBS, 50ng/ml FGF-8,50ng/ml SHH,
0.25mM cAMP, 0.2mM L-AA, 10ng/ml GDNF and 10ng/ml BDNF.
6. culture medium described in claim 1-5 any one becomes in dopaminergic neuron in inducing mesenchymal stem cell differentiation
Application and/or preparing inducing mesenchymal stem cell differentiation become dopaminergic neuron reagent in application.
7. applying according to claim 6, which is characterized in that the mescenchymal stem cell is umbilical cord mesenchymal stem cells.
8. a kind of method that induction inducing mesenchymal stem cell differentiation becomes dopaminergic neuron, which is characterized in that filled by between
After matter stem cell is added preparatory induced medium pre-induced 2 days in culture medium described in claim 1, replacement right is wanted within the 3rd day
It asks the dopaminergic neuron induced medium in 1 culture medium to continue culture 7 days, changes liquid every other day.
9. method according to claim 8, which is characterized in that the mescenchymal stem cell is P1 dry thin for umbilical cord mesenchyma
Born of the same parents.
10. method according to claim 8, which is characterized in that the pre-induced and dopaminergic neuron induced medium
The environmental condition for continuing culture is 37 DEG C, 5%CO2, saturated humidity.
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Cited By (4)
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CN111690605A (en) * | 2020-07-20 | 2020-09-22 | 青岛赛珥生物医学科技有限公司 | Method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons |
CN113481160A (en) * | 2021-08-20 | 2021-10-08 | 北京太东生物科技有限公司 | Retinal pigment epithelial cell induction culture medium and application thereof |
CN113684181A (en) * | 2021-08-24 | 2021-11-23 | 南通大学 | Method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells |
CN115896024A (en) * | 2022-11-30 | 2023-04-04 | 苏州大学附属第二医院 | Induced differentiation method of dopaminergic neuron and application thereof |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111690605A (en) * | 2020-07-20 | 2020-09-22 | 青岛赛珥生物医学科技有限公司 | Method for inducing adipose-derived stem cells to differentiate into dopaminergic neurons |
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CN113684181A (en) * | 2021-08-24 | 2021-11-23 | 南通大学 | Method for inducing differentiation of human umbilical cord mesenchymal stem cells towards nerve cells |
CN115896024A (en) * | 2022-11-30 | 2023-04-04 | 苏州大学附属第二医院 | Induced differentiation method of dopaminergic neuron and application thereof |
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