CN108148808A - Contribute to the inducing culture of inductive formation neural precursor - Google Patents
Contribute to the inducing culture of inductive formation neural precursor Download PDFInfo
- Publication number
- CN108148808A CN108148808A CN201611101061.2A CN201611101061A CN108148808A CN 108148808 A CN108148808 A CN 108148808A CN 201611101061 A CN201611101061 A CN 201611101061A CN 108148808 A CN108148808 A CN 108148808A
- Authority
- CN
- China
- Prior art keywords
- cell
- neural precursor
- culture
- gibco
- growth factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of inducing cultures for contributing to inductive formation neural precursor.Its composition and content are：87%DMEM/F12 culture mediums (Gibco), 1%GlutaMAX (Gibco), 2%B27 minus vitamin A (Gibco), 1~10 μ g/mL heparin (Stem Cell Technologies), 1000units/mL LIF (mLIF or hLIF) (Merck Millipore), 10~20ng/mL basic fibroblast growth factor (bFGF；R＆D Systems), 10~40ng/mL epidermal growth factor (EGF；Peprotech).Comprising the lineage-specific cellular factor in the culture medium, it is combined with a certain concentration.It, can be in vitro under environment, by the functional neural precursor of mammalian somatic cell induced synthesis by the inducing culture.
Description
Technical field
The present invention relates to regenerative medicine field, particularly a kind of Fiber differentiation for contributing to inductive formation neural precursor
Base.
Background technology
Neurodegenerative disease is nervous system disease caused by the structure and function of the nerve cell of brain or spinal cord is lost
Disease.Such disease, including the course of disease longer (20 years or longer) as Alzheimer's disease, parkinsonism and a small number of disease progressions are very fast
The amyotrophic lateral sclerosis of (2-3) etc. constitutes huge threat to the health of the mankind particularly the elderly.It is existing
Although some chemotherapy means can also alleviate these illnesss, all without obtaining fairly obvious radical cure effect.Cause
This, rebuilding the nerve fiber of lesion and the therapeutic strategy of organ by the means of regenerative medicine is particularly important.
The final goal of regenerative medicine is to replace the tissue of aging, lesion or damage, at present can be by two big approach come real
Existing, i.e., cell reprograms technology and cells transdifferentiate technology.The innovation of cell reprogramming technology brings hope for cell therapy,
It is existing research shows that the special multipotential cell system of established a variety of diseases, it is thin can be divided into a plurality of types of bodies in vitro
Born of the same parents provide sufficient seed source for cell therapy.At the same time, the appearance of cells transdifferentiate technology also results in the pole of people
Big concern.By cells transdifferentiate, the fibroblast of patient can be translates directly into other kinds of body cell, without passing through
The stage of multipotential stem cell.Therefore, this method is more direct and efficient, this technology is similarly cell therapy and has lighted new wish
It hopes.At present, by cell reprogramming and cells transdifferentiate both technologies can obtain neurodegenerative disease patient lesion or
The cell type of damaged nerve tissue, the function of organization to repair impaired provide strong means and completely new treatment plan
Slightly, there is potential application value in terms of cell therapy.In following application, in some instances it may even be possible to by thin near in situ change
The gene expression of born of the same parents or microenvironment supplement the cell type of damaged tissues.
Although the reprogramming of traditional transcription factor mediation and transdifferentiation technology bring unprecedented for cell therapy
Dawn, but also have the problems urgently to be resolved hurrily from the application for acquiring clinical treatment of new type cell.Due to transcription factor
Foreign gene is often imported into cell by means of modes such as retrovirus, slow virus in the Induction Process of mediation, although this kind of side
Formula has higher gene transduction efficiency, but since virus sequence is integrated into the genome of cell, gene can be caused to be inserted into
Mutation occurs or even with oncogenicity, so the method for gene introduction of this potentially dangerous property is obviously unfavorable for it again
The application of raw medical domain, thus how different research groups will realize that transdifferentiation is used as using circles abductive approach and grind
Study carefully hot spot.
The foreign gene of oncogenicity and traditional transdifferentiation different from induced multi-potent stem cell is inserted into, compound inductive formation
Neural precursor has successfully evaded many applications limitation of conventional method, is more suitable for being directly used in clinical treatment.2014, together
Help that university professor Pei Gang organizes it was discovered by researchers that the method induced by pure chemical small molecule, can turn body cell
It is divided into neural precursor.They pass through the combination of specified chemical composition, the i.e. VCR (inhibitor of VPA, HDAC；
The inhibitor of CHIR99021, GSK3；The inhibitor of Repsox, TGF β accesses), it, can be by the embryo of mouse along with low-oxygen environment
Fibroblast is induced into neural precursor.In the recent period, Chinese's science retainer of a big family wins professor and its colleague has found and reflects from mechanism
Determine multiple completely new micromolecular compounds, they are combined into a kind of culture medium of entitled M9, including CHIR99021, A83-
01, RG108, Parnate, SMER28, Hh-Ag1.5, retinoic acid, LDN193189 and bFGF can promote lactation to move
Induction of the object fibroblast to neural precursor is broken up.The neural precursor of this kind of compound method induction and lactation are moved
The neural precursor in object brain source is similar at many aspects, such as increment and self-renewal capacity, gene expression profile and body
Inside and outside differentiation potential etc..
The studies above result is all confirmed without the importing by exogenous virus, it is possible to be moved lactation by certain way
The somatic induction of object is changed into neural precursor, and the main effect model of small molecule, before being active cell endogenous neural
The expression of body gene.Therefore, it is proposed that by the combination of the specific cells factor, to activate neural precursor mark in noble cells
Gene is expressed again, can also realize the Induction Transformation to neural precursor by mammalian fibroblasts.Further, since
Cell factor relative low price, and it is easy to operate, convenient for being added in cell culture medium.Therefore, this addition is special
After determining cell factor, the culture medium for contributing to inductive formation neural precursor of acquisition will be a kind of safe efficient and grasp
Make easy culture systems, the adult cell of differentiation can not only be promoted to be changed into neural precursor, be more suitably applied to face
The application of bed translational medicine.
Invention content
The purpose of the present invention is to provide a kind of inducing cultures for contributing to inductive formation neural precursor, main to solve
Comprising the lineage-specific cellular factor in the culture medium, group is carried out with a certain concentration for the problems of certainly above-mentioned prior art
It closes.It, can be in vitro under environment, by the functional neural precursor of mammalian somatic cell induced synthesis by the inducing culture
Cell.
To achieve the above object, the technical scheme is that:
A kind of inducing culture for contributing to inductive formation neural precursor, it is characterised in that：Its composition and content are：
87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco),
2%B27minus vitamin A (Gibco),
1~10 μ g/mL heparin (Stem Cell Technologies),
1000units/mL LIF (mLIF or hLIF) (Merck Millipore),
10~20ng/mL basic fibroblast growth factor (bFGF；R＆D Systems),
10~40ng/mL epidermal growth factor (EGF；Peprotech).
In this formula, the specific combination of more than growth factor, can play makes somatic conversion be neural precursor shape
The effect of state.
The effect that mammalian somatic cell transdifferentiation is neural precursor is carried out using above-mentioned inducing culture to realize,
Concrete operation step is as follows：
1. it is conducive to mammalian fibroblasts culture systems culture mice embryonic or adult fibroblast or people into fibre
Tie up cell.
2. fibroblast is inoculated with certain proportion, and the above-mentioned induction by being added to the special cells factor is trained
Foster base is cultivated, and is completed the process that mammalian somatic cell transdifferentiation is neural precursor state, which needs about
The time of 2-4 weeks.
3. culture systems are hereafter stablized using neural precursor, it will including N2, B27 (Illuminex), FGF2, EGF etc.
The neural precursor that this transdifferentiation obtains is stablized in functional neural precursor state.The process needs about 1-2 weeks
Time.
4. finally this kind of neural precursor can be expanded or be preserved.
The neural precursor obtained by inducing culture, with the neural precursor with being obtained in conventional bulk very
Similar characterization of molecules shows as that there is certain self-renewal capacity and the transcript profile of specificity to be divided into nervous system
The potential of cell.
The cell that the inducing culture of the present invention is suitble to is preferably mammalian cell, more preferable mouse embryo fibroblast
Cell and mouse adult fibroblast.
The beneficial effects of the invention are as follows regulating and controlling the signal path of upstream and downstream by adjusting the growth factor in culture medium, from
And realize the external evoked generation neural precursor of circles.It is this culture medium realize cells transdifferentiate by way of,
Not only operating aspect, it is more safe and efficient, and also stability is high, is suitble to mammalian cell answering in terms of translational medicine
With, can promote differentiation adult cell be changed into neural precursor.
Description of the drawings
Fig. 1 shows the inducing culture by adding particular growth factor, carries out mouse embryonic fibroblasts transformation
For the flow of neural precursor, selection and time point including culture medium.
Fig. 2 shows the inducing culture of addition particular growth factor, inducing mouse embryonic fibroblast generation nerve
The form photo in precursor each period.
Fig. 3 shows the inducing culture of addition particular growth factor, and induction Nestin-GFP mouse embryo fibroblasts are thin
Born of the same parents generate the fluorescence micrograph of neural precursor several time points.
Fig. 4 shows the inducing culture of addition particular growth factor, and induction Nestin-GFP mouse embryo fibroblasts are thin
Born of the same parents generate the flow cytometer showed Efficiency Statistics figure of neural precursor.
Fig. 5 shows RNA-Seq bis- generations sequencing results, show the inducing culture can promote in induction into fiber finer
Cellular expression neural precursor specific gene promotes its Godwards premenstrual somatic conversion.
Fig. 6 shows RNA-Seq bis- generations sequencing results, show the inducing culture can inhibit in induction into fiber finer
Originally some into fiber-specific gene, promotes it to break intrinsic somatic cell gene express spectra, so as to turn to neural precursor to born of the same parents
Become.
Fig. 7 shows real-time quantitative PCR as a result, showing the neural precursor expression nerve cord of inducing culture generation
Cell-specific genes.
It is that Fig. 8 shows immunofluorescence as a result, showing the neural precursor expression god of the inducing culture inductive formation
Through stem cell specific gene Nestin, Sox1 and Sox2.
Fig. 9 shows the inducing culture by adding particular growth factor, carries out mouse adult fibroblast transformation
Flow for neural precursor.
Figure 10 shows the inducing culture of addition particular growth factor, induces Nestin-GFP mouse adult into fiber finer
Born of the same parents generate the fluorescence micrograph of neural precursor several time points.
Figure 11 shows real-time quantitative PCR as a result, showing that the inducing culture can promote the adult in induction into fiber
Cell expresses neural precursor specific gene, promotes its Godwards premenstrual somatic conversion.
Figure 12 shows real-time quantitative PCR as a result, showing that the inducing culture can inhibit the fibroblast in induction
Originally some promotes it to break intrinsic adult cell gene expression profile, so as to turn to neural precursor into fiber-specific gene
Become.
It is that Figure 13 shows immunofluorescence as a result, showing the neural precursor expression god of the inducing culture inductive formation
Through stem cell specific gene Nestin, Sox1 and Sox2.
Figure 14 shows the inducing culture by adding particular growth factor, carries out human fibroblasts and is changed into nerve
The flow of precursor.
Figure 15 shows the inducing culture of addition particular growth factor, and induction human fibroblasts are changed into neural precursor
The aspect graph of cell.
Specific embodiment
Detailed description for the present invention and technology contents, cooperation schema is described as follows, however institute's accompanying drawings only provide ginseng
It examines and illustrates to use, it is non-limiting the present invention.
1st, culture medium
(1) fibroblastic culture medium composition is：
89% basal medium DMEM (Gibco),
10% fetal calf serum (FBS；Gibco),
1%L- glutamine (Glutamax；Gibco).
(2) contribute to the inducing culture of inductive formation neural precursor described in invention, suitable for mice embryonic into fibre
Tie up cell Godwards premenstrual somatic conversion, Media Components and content：
87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco),
2%B27minus vitamin A (Gibco),
1-2.5 μ g/mL heparin (Stem Cell Technologies),
1000units/mL mLIF (Merck Millipore),
10-15ng/mL basic fibroblast growth factor(bFGF；R＆D Systems),
10-15ng/mL epidermal growth factor(EGF；Peprotech).
(3) contribute to the inducing culture of inductive formation neural precursor described in invention, suitable for mouse adult into fibre
Tie up cell Godwards premenstrual somatic conversion, Media Components and content：
87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco),
2%B27minus vitamin A (Gibco),
2.5-5 μ g/mL heparin (Stem Cell Technologies),
1000uni ts/mL mLIF (Merck Millipore),
15-20ng/mL basic fibroblast growth factor(bFGF；R＆D Systems),
15-20ng/mL epidermal growth factor(EGF；Peprotech).
(4) contribute to the inducing culture of inductive formation neural precursor described in invention, suitable for human fibroblasts
Godwards premenstrual somatic conversion, Media Components and content：
87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco),
2%B27minus vitamin A (Gibco),
10 μ g/mL heparin (Stem Cell Technologies),
1000units/mL hLIF (Merck Millipore),
20ng/mL basic fibroblast growth factor(bFGF；R＆D Systems),
40ng/mL epidermal growth factor(EGF；Peprotech).
(5) mouse Nerve precursor stabilization culture medium composition is：DMEM/F12 culture mediums (Gibco), additional 1%N2
(Invitrogen), 2%B27 (Invitrogen), 20ng/mL basic fibroblast growth factor (bFGF；
R＆D Systems) and 10ng/mL epidermal growth factor (EGF；Peprotech).
(6) people's derived neural stem cell non-serum culture medium：Using AngCell people's derived neural stem cell of Angecon companies
Serum free medium.
2nd, for the cell of transdifferentiation induction
The cell somatic types that transdifferentiation induction uses are respectively mouse embryonic fibroblasts, and mouse adult is into fiber finer
Born of the same parents and human fibroblasts, this three classes cell are that inventor voluntarily prepares.In addition, inventor further preferably goes out one kind
The fibroblast of Nestin-GFP transgenic mices, the cell can express green fluorescence when being changed into neural precursor
Green is presented in albumen (GFP) under fluorescence microscope, and the green fluorescent protein is not expressed in fibroblast.Pass through stream
The ratio of formula Cytometric Analysis green cells, it can be verified that the induced efficiency of the inducing culture.
Embodiment 1
Prepare common mouse embryonic fibroblasts, cultivated using fibroblast culture medium, culture medium composition
For：89% basal medium DMEM (Gibco), 10% fetal calf serum (FBS；) and 1%L- glutamine (Glutamax Gibco；
Gibco)。
(1) it is applicable in the density repopulating cell of 150000 cells/wells in 24 porocyte culture plates using invention is described
In the inducing culture of mouse embryonic fibroblasts Godwards premenstrual somatic conversion：87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco), 2%B27minus vitamin A (Gibco), 1 μ g/mL heparin (Stem Cell
Technologies), 1000units/mL mLIF (Merck Millipore), 10ng/mL basic fibroblast
growth factor(bFGF；R＆D Systems), 10ng/mL epidermal growth factor (EGF；
Peprotech).In 7 days of induction starting, cell is gently blown and beaten once a day, it is therefore an objective to which the cell for making into agglomerate aggregation suspends
Get up, form preferable cell spherical shape, it is primary to replace fresh inducing culture within every two days.After 7 days, cell, cell are no longer blown and beaten
Start adherent, be continuing with mouse embryonic fibroblasts inducing culture, it is thin to replace fresh mouse embryo fibroblast within every two days
Born of the same parents' inducing culture is primary.At this point, cell from adherent agglomerate get up rapidly by amplification, cell can be observed and increase in regional
It grows.
(2) after induction starting 14 days, with trypsin/EDTA (Invitrogen) by each hole in 24 well culture plates
Cell digests respectively, is taped against in a corresponding 35mm Tissue Culture Dish, it is steady to be changed to mouse Nerve precursor at this time
Determine culture medium：DMEM/F12 culture mediums (Gibco), additional 1%N2 (Invitrogen), 2%B27 (Invitrogen), 20ng/
mL basic fibroblast growth factor(bFGF；R＆D Systems) and 10ng/mL epidermal growth
factor(EGF；Peprotech).After continuing culture 7 days, the edge that attached cell can be observed is climbed out of on many similar nerves
Skin like cell forms neural network shape structure, these cells are expanded after digestion, can finally form ripe neural precursor
Cell has typical neural precursor form.
(3) Media Components and usage time are as shown in Figure 1.Cellular change form is as shown in Figure 2.
Embodiment 2
Prepare Nestin-GFP mouse embryonic fibroblasts, cultivated using fibroblast culture medium, the culture
Base forms：89% basal medium DMEM (Gibco), 10% fetal calf serum (FBS；) and 1%L- glutamine Gibco
(Glutamax；Gibco).
(1) with the density of 150000 cells/wells, it is thin that Nestin-GFP mouse embryonic fibroblasts are inoculated in 24 holes
In born of the same parents' culture plate, using the invention Fiber differentiation suitable for mouse embryonic fibroblasts Godwards premenstrual somatic conversion
Base：87%DMEM/F12 culture mediums (Gibco), 1%GlutaMAX (Gibco), 2%B27minus vitamin A (Gibco),
2.5 μ g/mL heparin (Stem Cell Technologies), 1000units/mL mLIF (Merck Millipore),
15ng/mL basic fibroblast growth factor(bFGF；R＆D Systems), 15ng/mL epidermal
growth factor(EGF；Peprotech).In 7 days of induction starting, cell is gently blown and beaten once a day, it is therefore an objective to make into
The cell of agglomerate aggregation suspends, and it is spherical to form preferable cell, and it is primary to replace within every two days the fresh inducing culture, at this time
Visible cell gradually appears green fluorescence.After 7 days, cell is no longer blown and beaten, cell starts adherent, is continuing with the Fiber differentiation
It is primary to replace fresh culture in every two days for base.At this point, cell from adherent agglomerate get up rapidly by amplification, cell can be observed
It is proliferated in regionality, and green cells become more, green florescent signal becomes strong.
(2) after induction starting 14 days, with trypsin/EDTA (Invitrogen) by each hole in 24 well culture plates
Cell digests respectively, is taped against in a corresponding 35mm Tissue Culture Dish, it is steady to be changed to mouse Nerve precursor at this time
Determine culture medium：DMEM/F12 culture mediums (Gibco), additional 1%N2 (Invitrogen), 2%B27 (Invitrogen), 20ng/
mL basic fibroblast growth factor(bFGF；R＆D Systems) and 10ng/mL epidermal growth
factor(EGF；Peprotech).After continuing culture 7 days, the edge that attached cell can be observed is climbed out of on many similar nerves
Skin like cell forms neural network shape structure, strong green florescent signal is presented.These cells are expanded after digestion, finally
Ripe neural precursor can be formed, there is typical neural precursor form.
(3) Media Components, usage time and operating process are as shown in Figure 1.Cellular change form is as shown in Figure 3.
(4) due to the initiator cell that Nestin-GFP mouse embryonic fibroblasts is selected to be induced as transdifferentiation, glimmering
Under light microscope, it is seen that successfully realize that the cell of transdifferentiation forms the form of nerve ball sample and the expression of green fluorescence is presented,
As shown in Figure 3.Different time points during whole operation, using Fluorescein activated cell sorter to expressing green cells
Ratio analyzed, it is seen that operate 14 days when, green cells ratio be more than 80%, and at this time using invention institute
The inducing culture stated highly proves that the culture medium can promote transformation of the fibroblast to neural precursor.Such as Fig. 4 institutes
Show.
(5) during cell is changed, the RNA of cell is extracted, passes through two generation sequencing technologies -- RNA-Seq is to turning
Cell during change carries out gene expression spectrum analysis, it was demonstrated that these have used the induction of the specific cells factor containing invention
The cell that culture medium is cultivated, gradually start express neural precursor marker gene, and expression quantity can maintain compared with
High level, it was demonstrated that the inducing culture can promote transformation of the fibroblast to neural precursor, activate endogenous nerve
The gene regulation access of precursor.As shown in Figure 5.
(6) in addition, further passing through two generation sequencing technologies -- RNA-Seq carries out gene expression to the cell in transition process
Spectrum analysis finds these cells that the inducing culture of the specific cells factor containing invention has been used to be cultivated, former
This distinctive express spectra of fibroblast is gradually erased, and fiber finer can be inhibited by further demonstrating the inducing culture
The body cell express spectra that born of the same parents originally have, and the express spectra of neural precursor is activated, so as to promote fibroblast to neural precursor
The transformation of cell.As shown in Figure 6.
(7) it is detected by real-time quantitative PCR, it was demonstrated that after employing inducing culture, the mark of fibroblast-like cell specific
The expression quantity of gene Thy1 continuously decreases, and the expression quantity of the marker gene Nestin of neural precursor gradually rises, it was demonstrated that
The inducing culture of the invention has the function of to promote fibroblast Godwards premenstrual somatic conversion.As shown in Figure 7.
(8) protein markers staining analysis is carried out to the neural precursor that the inducing culture obtains, it was demonstrated that it is from albumen
Level is high to express neural precursor marker protein Nestin, Sox1 and Sox2.As shown in Figure 8.
Embodiment 3
Prepare Nestin-GFP mouse adult fibroblasts, cultivated using fibroblast culture medium, the culture
Base forms：89% basal medium DMEM (Gibco), 10% fetal calf serum (FBS；) and 1%L- glutamine Gibco
(Glutamax；Gibco).
(1) cultivating system is changed to applicable in 24 well culture plates with the density repopulating cell of 200000 cells/wells
In the inducing culture of mouse adult fibroblast Godwards premenstrual somatic conversion：87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco), 2%B27minus vitamin A (Gibco), 5 μ g/mL heparin (Stem Cell
Technologies), 1000units/mL mLIF (Merck Millipore), 20ng/mL basic fibroblast
growth factor(bFGF；R＆D Systems), 20ng/mL epidermal growth factor (EGF；
Peprotech).In 7 days of induction starting, cell is gently blown and beaten once a day, it is therefore an objective to which the cell for making into agglomerate aggregation suspends
Get up, it is spherical to form preferable cell, replaces within every two days that fresh inducing culture is primary, and visible cell gradually appears green at this time
Fluorescence.Hereafter, cell is no longer blown and beaten, cell starts adherent, is continuing with inducing culture, replaces the inducing culture within every two days
Once.At this point, cell from adherent agglomerate get up rapidly by amplification, cell can be observed and be proliferated in regional, and green is glimmering
Photo-cell becomes more, and green florescent signal becomes strong.After induction starting 21 days, 24 holes are trained with trypsin/EDTA (Invitrogen)
The cell for supporting each hole in plate digests respectively, is taped against in a corresponding 35mm Tissue Culture Dish, is changed at this time small
Mouse neural precursor stablizes culture medium：DMEM/F12 culture mediums (Gibco), additional 1%N2 (Invitrogen), 2%B27
(Invitrogen), 20ng/mL basic fibroblast growth factor (bFGF；R＆D Systems) and 10ng/
mL epidermal growth factor(EGF；Peprotech).After continuing culture 7 days, strong green fluorescence letter is presented in cell
Number.These cells are expanded after digestion, can finally form ripe neural precursor, have typical neural precursor
Form.
(2) Media Components, usage time and operating process are as shown in Figure 9.Cellular change form is as shown in Figure 10.
(3) during adult fibroblast is changed to neural precursor, the RNA of cell is extracted, is passed through
Real-time quantitative PCR detects, it was demonstrated that after employing inducing culture, the gradual high level expression neural precursor of adult fibroblast is thin
The marker gene of born of the same parents such as Pax6, Sox2 and Nestin, it was demonstrated that the inducing culture of the invention have promote fibroblast to
The function of neural precursor transformation.As shown in figure 11.
(4) it is detected by real-time quantitative PCR, it was demonstrated that after employing inducing culture, adult fibroblast specificity
The expression quantity of marker gene Thy1 and Col1a1 continuously decrease, and fiber finer can be inhibited by further demonstrating the inducing culture
The body cell express spectra that born of the same parents originally have, and the express spectra of neural precursor is activated, so as to promote fibroblast to neural precursor
The transformation of cell.As shown in figure 12.
(5) protein markers staining analysis is carried out to the neural precursor that the inducing culture obtains, it was demonstrated that it is from albumen
Level, high level expression neural precursor marker protein Nestin, Sox1 and Sox2.As shown in figure 13.
Embodiment 4
Prepare human fibroblasts, cultivated using fibroblast culture medium, culture medium composition is：89% basis
Culture medium DMEM (Gibco), 10% fetal calf serum (FBS；) and 1%L- glutamine (Glutamax Gibco；Gibco).
(1) with the density repopulating cell of 350000 cells/wells in 24 well culture plates, by cultivating system replace for people into
Fibrocyte inducing culture：87%DMEM/F12 culture mediums (Gibco), 1%GlutaMAX (Gibco), 2%B27minus
Vitamin A (Gibco), 10 μ g/mL heparin (Stem Cell Technologies), 1000units/mL hLIF
(Merck Millipore), 20ng/mL basic fibroblast growth factor (bFGF；R＆D Systems),
40ng/mL epidermal growth factor(EGF；Peprotech).In 14 days of induction starting, once a day gently
Blow and beat cell, it is therefore an objective to which the cell for making into agglomerate aggregation suspends, and forms preferable cell spherical shape, replaces Freshman within every two days
Fibroblast inducing culture is primary.Hereafter, cell is no longer blown and beaten, cell starts adherent, is continuing with human fibroblasts
It is primary to replace the inducing culture in every two days for inducing culture.At this point, cell from adherent agglomerate slowly get up by amplification, it can
Observe that part cell is proliferated in regional.After induction starting 28 days, 24 holes are trained with trypsin/EDTA (Invitrogen)
The cell for supporting each hole in plate digests respectively, is taped against in a corresponding 35mm Tissue Culture Dish, is changed at this time
AngCell people's derived neural stem cell non-serum culture medium of Angecon companies.After continuing culture 14 days, maturation can be finally formed
Neural precursor.These cells are expanded after digestion, have typical neural precursor form.
(2) Media Components, usage time and operating process are as shown in figure 14.Induce the people's neural precursor shape obtained
State is as shown in figure 15.
Therefore, the present invention obtains, with the inducing culture of the factor of the present invention containing specific cells, can will include
Mouse embryonic fibroblasts, mouse adult fibroblast and the lactation mammal including human fibroblasts are into fiber finer
Born of the same parents are changed into neural precursor state, and are stably maintained at this state by corresponding neural precursor culture systems.This
The advantage of the invention culture medium progress cells transdifferentiate for contributing to inductive formation neural precursor is, without disease
The integration of malicious foreign gene, use is safer, while easy to operate, efficient to greatly enhance the latent of clinical practice
Energy.
As described above, preferable possible embodiments only of the invention, therefore, it does not limit the patent models of the present invention
It encloses, changes such as with the equivalent structure carried out by description of the invention and schema content, reason is the same as the right for being contained in the present invention
In the range of, Chen Ming is given in conjunction.
Claims (1)
1. a kind of inducing culture for contributing to inductive formation neural precursor, it is characterised in that：Its composition and content are：：
87%DMEM/F12 culture mediums (Gibco),
1%GlutaMAX (Gibco),
2%B27minus vitamin A (Gibco),
1~10 μ g/mL heparin (Stem Cell Technologies),
1000units/mL LIF (mLIF or hLIF) (Merck Millipore),
10~20ng/mL basic fibroblast growth factor (bFGF；R＆D Systems),
10~40ng/mL epidermal growth factor (EGF；Peprotech).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611101061.2A CN108148808B (en) | 2016-12-05 | 2016-12-05 | Induction medium for inducing generation of neural precursor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611101061.2A CN108148808B (en) | 2016-12-05 | 2016-12-05 | Induction medium for inducing generation of neural precursor cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108148808A true CN108148808A (en) | 2018-06-12 |
| CN108148808B CN108148808B (en) | 2020-12-11 |
Family
ID=62469900
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611101061.2A Active CN108148808B (en) | 2016-12-05 | 2016-12-05 | Induction medium for inducing generation of neural precursor cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148808B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574441A (en) * | 2022-03-16 | 2022-06-03 | 中山大学·深圳 | Novel biological application of black phosphorus nanosheet |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010057039A2 (en) * | 2008-11-14 | 2010-05-20 | Cythera, Inc. | Encapsulation of pancreatic cells derived from human pluripotent stem cells |
| WO2012018933A2 (en) * | 2010-08-04 | 2012-02-09 | Cellular Dynamics International, Inc. | Reprogramming immortalized b cells |
| CN103740757A (en) * | 2014-01-21 | 2014-04-23 | 中国科学院生物物理研究所 | Method for preparing pig neural stem cells by reprogramming |
| CN104894060A (en) * | 2014-03-03 | 2015-09-09 | 中国科学院上海生命科学研究院 | Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof |
-
2016
- 2016-12-05 CN CN201611101061.2A patent/CN108148808B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010057039A2 (en) * | 2008-11-14 | 2010-05-20 | Cythera, Inc. | Encapsulation of pancreatic cells derived from human pluripotent stem cells |
| WO2012018933A2 (en) * | 2010-08-04 | 2012-02-09 | Cellular Dynamics International, Inc. | Reprogramming immortalized b cells |
| CN103740757A (en) * | 2014-01-21 | 2014-04-23 | 中国科学院生物物理研究所 | Method for preparing pig neural stem cells by reprogramming |
| CN104894060A (en) * | 2014-03-03 | 2015-09-09 | 中国科学院上海生命科学研究院 | Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| BEHNAM EBRAHIMI: "Engineering cell fate: Spotlight on cell-activation and signaling-directed lineage conversion", 《TISSUE AND CELL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574441A (en) * | 2022-03-16 | 2022-06-03 | 中山大学·深圳 | Novel biological application of black phosphorus nanosheet |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108148808B (en) | 2020-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kaltschmidt et al. | Adult craniofacial stem cells: sources and relation to the neural crest | |
| Wang et al. | Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells | |
| Conti et al. | Neural stem cell systems: physiological players or in vitro entities? | |
| CN103396993B (en) | The oligodendroglia of myelinization again and the treatment for spinal cord injury of derived from primate embryonic stem cells | |
| CN104419661B (en) | The preparation method of NSC | |
| CN110396499A (en) | A kind of method and its application of induced nerve stem cells | |
| CN107326013A (en) | Nerve cell system, abductive approach and application after directional induction hiPSC differentiation | |
| Xiong et al. | Optimal time for passaging neurospheres based on primary neural stem cell cultures | |
| CN105331575B (en) | A kind of efficient amplification cultivating system of human vascular endothelial progenitor cells | |
| WO2019182044A1 (en) | Differentiation promoter for pluripotent stem cells | |
| CN103484432A (en) | Induction medium for inducing neural stem/progenitor cells to be differentiated into oligodendrocyte precursor cells and induction method and application thereof | |
| US20210355439A1 (en) | Differentiation medium and method for preparing oligodendrocyte precursor | |
| CN104774808B (en) | The method that umbilical cord mesenchymal stem cells are induced differentiation into GABAergic neuron | |
| Pandamooz et al. | The influence of cerebrospinal fluid on epidermal neural crest stem cells may pave the path for cell-based therapy | |
| WO2023011251A1 (en) | Rapid and efficient clinical grade pigment epithelium induction method, kit, and application | |
| US20120003186A1 (en) | Method for dedifferentiating adipose tissue stromal cells | |
| Dai et al. | The Human Skin‐Derived Precursors for Regenerative Medicine: Current State, Challenges, and Perspectives | |
| CN101294146B (en) | System for inducing nerve stem cell differentiation and inducing method thereof | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN104178456A (en) | Passage method and application of human induced pluripotent stem cells | |
| Zhang et al. | Schwann cells originating from skin-derived precursors promote peripheral nerve regeneration in rats | |
| CN108795842B (en) | Method for generating autologous melanocytes by inducing iPS cells through 3D suspension and application | |
| CN105087475B (en) | A kind of method that cell culture fluid and its application and induction dental pulp stem cell break up to neural-like cells | |
| CN108148808A (en) | Contribute to the inducing culture of inductive formation neural precursor | |
| CN103305458B (en) | Method for inducing cholinergic neurons from pluripotent stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |