CN108823164A - Induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell - Google Patents
Induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell Download PDFInfo
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Abstract
It the invention belongs to stem cell biology and technical field of cell culture, proposes and promotes to induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell, specifically include serum-free differentiation minimal medium and promote the additive of nerve group cell differentiation.Wherein, serum-free minimal medium neccessary composition includes：DMEM/F12 culture medium, human serum albumins, L-Glutamine, D-Glucose, vitamin C, N2/B27 cell culture additive, nonessential amino acid.The additive of neural progenitor cell differentiation is promoted to specifically include：SB431542, dermorphin, Fibroblast growth factors 2, vitamin A acid, brain-derived neurotrophic factor.The differentiation of the neural progenitor cell in mixing with cells system can be helped to improve with this culture medium, the differentiation to other types cell is reduced, to finally obtain the neural progenitor cell of high-purity.
Description
Technical field
The invention belongs to stem cell biologies and technical field of cell culture, relate in particular to a kind of for promoting induction
The serum free culture system that multipotential stem cell breaks up to neural progenitor cell.
Background technique
Neurodegenerative disease is a kind of slowly onset, the course of disease in the disease for carrying out sexual development, prognosis mala, is still lacked so far
Effective radical cure method.Clinical common neurodegenerative disease has Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy
With Alzheimer disease etc..Though these disease causes of disease are different but pathologically having nervous centralis different parts and different degrees of
N euron loss and dysfunction.The neuron lost is substituted, functional rehabilitation is allowed to, is the new approaches for treating this kind of disease.Closely
The stem cell of research discovery in vitro culture over year can not only be proliferated, break up, but also " transdifferentiation ", Ye Jiyi can occur
The tissue-derived cell of kind can be divided into other incoherent tissue cell types.For example, candidate stem cell, mesenchyma are dry thin
Born of the same parents etc. can be to mature neural cellular differentiation under certain conditions in vitro.Experiment in vivo also confirms that neural stem cell or non-nerve
Tissue-derived stem cell transplantation, which enters, can be divided into mature nerve cell, newborn nerve cell energy in impaired brain tissue
The cells play function of substituting missing, provides new treatment method for this kind of nervous system refractory disease.
In histology engineering and clinical treatment, inductive pluripotent stem cells can theoretically induce differentiation into all kinds of nerves
Cell, these cells can be widely applied to the reparation of tissue nervous system and reconstruction atrophy, necrosis, the neuron and nerve of defect
Colloid.And by inductivity stem cell induce the functional nerve member hypotype with specific neurotransmitter phenotype be cell transplantation and
The carrier of gene therapy provides well using means, has a wide range of applications for the treatment of central nervous diseases.
Contain fetal calf serum ingredient in the culture medium of multipotential stem cell culture and differentiating into nerve cells mostly, serum
Ingredient is unknown and complicated, since the serum product of different manufacturers is batch widely different, causes the repeatability of experiment extremely low.This
Outside, serum is also possible to carry pathogen and other virulence factors, and the nerve cell for greatly reducing differentiation is detected in drug screening
Or the application value and safety of cell therapy etc..
Summary of the invention
The purpose of the present invention is on the basis of serum free medium, add a series of substances to promote induced multi-potent dry thin
Born of the same parents are applied to drug screening, the foundation of disease model to neural progenitor cell directed differentiation, available a large amount of neural progenitor cell
And stem cell regenerating medical domain.
Culture medium of the invention, can will induce multi-potent stem cell to neural progenitor cell break up, inhibit neoblast and
Growth to other types cell obtains the neural progenitor cell of high-purity in the later period of differential period, for grinding for scientific research field
Study carefully and business application.
The present invention provides a kind of promotions to induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell, tool
Body includes serum-free differentiation minimal medium and the additive for promoting neural progenitor cell differentiation.Wherein, serum-free minimal medium,
Neccessary composition includes：Basal medium, seralbumin, L-Glutamine, D-Glucose, vitamin C, N2 cell culture addition
Agent, B27 cell culture additive and nonessential amino acid.Promote the additive of neural progenitor cell differentiation, specific ingredient includes：
SB431542, dermorphin, Fibroblast growth factors 2, vitamin A acid, brain-derived neurotrophic factor.
In one or more embodiments, the basal medium in the minimal medium is selected from DMEM/F12,
One or more of Neurobasal, RPMI 1640, DMEM and IMDM culture medium.
In one or more embodiments, seralbumin in the serum-free minimal medium ingredient, L- glutamy
The concentration of amine, D-Glucose, vitamin C, N2 cell culture additive, B27 cell culture additive and nonessential amino acid point
It is not：Seralbumin is 0.25 g/L-25 g/L, L-Glutamine 1-10nM, D-Glucose 1-10g/L, vitamin
C is the N2 cell culture additive of 100-1000 μ g/L, 0.5-5%, the B27 cell culture additive and nonessential ammonia of 0.5-5%
Base acid is 10-100 μM.
In one or more embodiments, SB431542 in the additive for promoting neural progenitor cell differentiation, skin coffee
The concentration of peptide, Fibroblast growth factors 2, vitamin A acid, brain-derived neurotrophic factor is respectively：SB431542 is 5-30 μM, skin
Deltorphin delta is 1-10 μM, and Fibroblast growth factors 2 is 5-30 μ g/L, and vitamin A acid is 1-10 μM, brain-derived neurotrophic factor 1-
10μg/L。
It is proposed using the present invention for promoting to induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell,
Its advantage is that：Neural progenitor cell differential medium eliminates animal sources serum composition, reduces the batch sex differernce of differentiation, improves
The safety and application value of the neural progenitor cell of source of human stem cell；Neural progenitor cell can be induced from inductivity stem cell to be divided
Change, and inducing differentiated system is the mixed system of different cell types, can be helped to improve mixing with cells with this culture medium
Neural progenitor cell differentiation in system, reduces the differentiation to other types cell, so that the neural ancestral for finally obtaining high-purity is thin
Born of the same parents；The neural progenitor cell that the system prepares can be applied to drug screening, the foundation of disease model and stem cell regenerating
Medical domain.
Detailed description of the invention
Fig. 1 is used to detect the marker gene of neural progenitor cell differentiation and maturation（Pax6, Nestin, Oct4）Expression
Situation.
Specific embodiment
With reference to the accompanying drawings and examples, more specifically the present invention is illustrated, but should not be considered limited to
This embodiment illustrated.
It is basic culture medium with DMEM/F12, adds following component thereto, preparation obtains serum-free minimal medium：
The seralbumin of 2.5g/L, the L-Glutamine of 5nM, the D-Glucose of 2g/L, the vitamin C of 200 μ g/L, 1% N2 cell
Cultivate additive, 2% B27 cell culture additive, 100 μM of nonessential amino acid.
Promote the additive of neural progenitor cell differentiation, specific constituent concentration is：10 μM of SB431542,1 μM of dermorphin,
The Fibroblast growth factors 2 of 10 μ g/L, 1 μM of vitamin A acid, the brain-derived neurotrophic factor of 1 μ g/L.
People induces multi-potent stem cell to be cultivated in serum-free minimal medium system, forms embryoid volume morphing.In base
10 μM of SB431542,1 μM of dermorphin, the Fibroblast growth factors 2 of 10 μ g/L, lasting training are added in basal culture medium system
It supports 1 week.Embryoid body cell is subjected to enzymatic hydrolysis separation later, be added in minimal medium system 20 μ g/L at fiber growth because
Son -2 carries out adhere-wall culture.
The expression of marker gene relevant to neural progenitor cell differentiation and maturation is detected using real time quantitative PCR method.
As a result as shown in Figure 1, in serum free culture system of the invention, induction versatile stem cell differentiation becomes nerve
Progenitor cells.
Above-described embodiment is one embodiment of the present invention, but embodiments of the present invention are not by present embodiment
Limitation, it is other it is any without departing from the change made in the case of essence of the invention and principle, substitution, combine, simplification and modification,
It should be equivalence replacement mode, be included within the scope of the present invention.
Claims (6)
1. inducing multi-potent stem cell the serum free culture system broken up to neural progenitor cell, which is characterized in that the wherein nothing
Serum free culture system system includes serum-free differentiation minimal medium and the additive for promoting neural progenitor cell differentiation.
2. according to claim 1 induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell, spy
Sign is that the serum-free differentiation minimal medium includes basal medium and culture medium additive：Basal medium includes
One or more of DMEM/F12, Neurobasal, RPMI 1640, DMEM and IMDM culture medium；Culture medium additive includes
The seralbumin of 0.25 g/L-25 g/L, the L-Glutamine of 1-10nM, the D-Glucose of 1-10g/L, 100-1000 μ g/L
Vitamin C, the N2 cell culture additive of 0.5-5%, the B27 cell culture additive of 0.5-5%, 10-100 μM nonessential
Amino acid.
3. according to claim 1 induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell, spy
Sign is that the additive for promoting neural progenitor cell differentiation specifically includes：5-30 μM of SB431542,1-10 μM of skin
Deltorphin delta, the Fibroblast growth factors 2 of 5-30 μ g/L, 1-10 μM of vitamin A acid, the brain-derived neurotrophic factor of 1-10 μ g/L.
4. serum-free differentiation minimal medium according to claim 2, which is characterized in that the nonessential amino acid choosing
From l-Alanine, L-arginine, L-Aspartic acid, altheine, L-cysteine, L-Glutamine, Pidolidone, sweet ammonia
One or more of acid, L-Histidine, L-PROLINE, Serine, L- tyrosine.
5. according to claim 1 induce multi-potent stem cell the serum free culture system broken up to neural progenitor cell, spy
Sign is, induces multi-potent stem cell in different incubation time points, be added in cultivating system serum-free minimal medium and
One or more promote the additive of neural progenitor cell differentiation, persistently cultivate 2 weeks, carry out the analysis of neural progenitor cell maturity.
6. neural progenitor cell maturity analysis according to claim 5, which is characterized in that detection technique includes real-time quantitative
PCR and immunostaining.
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Cited By (2)
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| CN115125209A (en) * | 2022-08-31 | 2022-09-30 | 华科星河(北京)生物科技有限公司 | Method for inducing cervical spinal cord neural stem cells from induced pluripotent stem cells |
| CN115125210A (en) * | 2022-08-31 | 2022-09-30 | 华科星河(北京)生物科技有限公司 | Culture medium and method for lumbosacral segment spinal cord neural stem cells induced from iPSC |
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Cited By (4)
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