CN108384755A - A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating - Google Patents
A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Abstract
The invention discloses it is a kind of efficiently, efficiently inductive pluripotent stem cells belong to Neuscience stem cells technology field to neural stem cell differentiating method.Step 1：Activate inductive pluripotent stem cells monolayer cultivation；Step 2：Inductive pluripotent stem cells Fiber differentiation；Step 3：Inductive pluripotent stem cells induction differentiation neural stem cell.The present invention has many advantages, such as that simple operation, at low cost, yield is big, differentiation purity is high, repeatability is strong.The entire non-animal derived property cell of link and other ingredients, not only greatly shorten the time needed for induced nerve stem cells, the safety for also substantially increasing iPS Derived Nerve stem cells, the neural stem cell further to mass produce clinical application rank using the iPS of autologous patient cell induction provide a kind of completely new method.
Description
Technical field
The present invention relates to it is a kind of efficiently, efficiently inductive pluripotent stem cells belong to neural stem cell differentiating method
Neuscience stem cells technology field.
Background technology
For a long time, it is believed that mammalian nervous system can not regenerate after injury.Traditional treatment means for
The therapeutic effect of nervous system injury is extremely limited.But with the development of science and technology stem cell biology and Neuscience into
Exhibition provides possibility for the treatment of the neurodegenerative disease and neurotrosis that are difficult to cure.Since stem cell has self more
Potential that is new and being divided into different cells, stem cell therapy pass through again by the god of neuron and other nerve cell implant damages
Through tissue, for the maincenter for the treatment of and peripheral nervous disease and damage.
What stem cell was relatively conventional at present is embryonic stem cell, has and treats many disease, especially nervous system diseases
The great potential of disease.But there is inevitable ethics problems for the application of embryonic stem cell, and will also after heteroplastic transplantation
Immunological rejection is generated, is above subject to many limitations in application.
It 2006, stretches in Kyoto Univ Japan mountain and research group is more led to find adult cell " reprogramming ", then pass through
4 genes (OCT3/4, SOX2, KLF4 and MYC, they are collectively referred to as OSKM) are inserted into the DNA of adult cell, are just generated
These " inductive pluripotent stem cells " (induced Pluripotent Stem Cells, iPSC).Using iPSC can with gram
The disadvantages mentioned above of embryonic stem cell is taken, and iPSC equally has the potential for being induced to differentiate into various kinds of cell, especially in nerve cord
In terms of cell differentiation, the remote super other cell types of cell efficiency and success rate after differentiation.
Neural stem cell made of induction is the precursor for having self-renewing and generating differentiation, has and generates different spectrums
Neuron, the spongiocyte of system, such as the function of oligodendroglia or astroglia, for treating the nervous system disease,
Especially degenerative neural lesion and neurotrosis possess the meaning across the epoch.
Currently, the shortcomings that method for induced nerve stem cells of the prior art, is as follows：To originating iPSC conditions dictates
Height depends on feeder layer co-cultured cell, and there are uncertain medium exchange (such as serum, conditioned medium), induction durations
It is long, process is complicated and has that uncertain (such as using embryoid body), differentiation efficiency is poor, purity is low.Therefore, it is a kind of that there is an urgent need for research and development
Safer, quick, repeatable strong abductive approach, with overcome the deficiencies in the prior art.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide it is a kind of efficiently, efficiently inductive pluripotent stem cells
To neural stem cell differentiating method.The present invention has that simple operation, at low cost, yield are big, differentiation purity is high, repeatability is strong
The advantages that.The entire non-animal derived property cell of link and other ingredients, not only greatly shorten the time needed for induced nerve stem cells,
The safety of iPS Derived Nerve stem cells is also substantially increased, further to utilize the iPS of autologous patient cell induction extensive
The neural stem cell of production clinical application rank provides a kind of completely new method.
The technical solution that the present invention solves above-mentioned technical problem is as follows：It is a kind of efficiently, efficiently inductive pluripotent stem cells
To neural stem cell differentiating method, include the following steps：
Step 1：By the inductive pluripotent stem cells of activation, by ten thousand/cm of 2-82Density is inoculated in Matrigel matrix bed boards
In culture dish or culture bottle afterwards, E8 complete mediums are added, is placed in cell incubator and cultivates；
Step 2：Replace the culture dish of inoculation inductive pluripotent stem cells or the E8 complete mediums in culture bottle in step 1
For nerve-inducing complete medium, after concussion, it is placed in cell incubator and cultivates；
Step 3：The nerve-inducing complete medium that more renews simultaneously observes cellular morphology daily, culture to the 7-8 days to get
To the neural stem cell of differentiation.
The shortcomings that the present invention overcomes traditional abductive approach, be it is a kind of it is novel, safely, quickly, the strong induction of repeatability
Method, scientific research and clinical application wide market, can benefit many patients.This abductive approach does not need feeder cells,
And non-animal derived property cell and other ingredients, the time required to greatly shortening induced nerve stem cells.
In the step 1 of the present invention, inductive pluripotent stem cells used are used using induction agent box inducing somatic
Induction agent box Epi5TMEpisomal iPSC Reprogramming Kit match silent winged generation that science and technology, MAN0008352.
In step 1, inoculum density is ten thousand/cm of 2-82.This inoculum density, it is ensured that induced in 6-8 days neural stem cell
Cell reaches complete fusion when success.If the excessively dilute (20,000/cm of ＜ of inoculum density2), the possible mortality of induction incipient cell,
It is difficult to reach the required millions cell quantity for the treatment of after 6-8 days.If overstocked (80,000/the cm of ＞ of inoculum density2), induction is just
Phase cell almost merges, and growing space is limited, has a certain impact to cell state.
In step 2, culture was to the 7-8 days, and cell breakthrough monolayer cultivation is to get to the neural stem cell of differentiation.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step 1, the preparation method of the inductive pluripotent stem cells of the activation is：
Step 1.1：By Matrigel matrix, bed board, addition E8 complete mediums are placed in cell training on tissue culture plate
It supports in case and stands one day, obtain the tissue culture plate after bed board；
Step 1.2：Cell fusion is reached to the inductive pluripotent stem cells of 70-90% density, by per 10cm2Cell training
The ratio that board bottom area liquid volume added is 1mL is supported, cell dissociation buffer is added and is placed in cell incubator, digests 3-5min；
Step 1.3：By every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added DMEM/F12 culture
Base, dilution enzymolysis, obtains cell suspension；
Step 1.4：Cell suspension is transferred in centrifuge tube, DMEM/F12 basal mediums are added, is centrifuged, room
Temperature, 200g centrifuge 5min, remove supernatant, are resuspended with E8 complete mediums, obtain cell suspension and count；
Step 1.5：The cell suspension that step 1.4 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000
The ratio of a cell is added in the tissue culture plate after the bed board that step 1.1 obtains, adds E8 complete mediums, 10mM
ROCK inhibitor is to get to the inductive pluripotent stem cells of the activation.
It is using above-mentioned further advantageous effect：The survival rate of inducing cell can be increased.
Further, in step 1.5, the ROCK inhibitor be Y-27632, Thiazovivin, Fasudil,
The combination of one or more of GSK429286A.
Further, the ROCK inhibitor is Y-27632.
It is using above-mentioned further advantageous effect：Y-27632 is a kind of permeable cell membrane, high-effect and pass through
Reverse transcriptase ATP is attached to catalytic site, the compound of selective depression ROCK1 and ROCK2.
Further, the model 72304 of the Y-27632 is purchased from StemCell Technologies companies.
Further, in step 1, the E8 complete mediums are TeSRTM-E6、mTeSRTM1、TeSRTM-E8TMIn one kind
Or two or more combination.
Further, the E8 complete mediums are TeSRTM-E8TM。
It is using above-mentioned further advantageous effect：Without feeder layer, non-animal derived property culture medium, it is conducive to Transformation Application.
Further, the TeSRTM-E8TMModel #05940, be purchased from STEMCELL Technologies companies.
Further, in step 2, in step 2, the nerve-inducing complete medium is in half complete medium of nerve-inducing
Middle 70-120 μM of retinoic acid of addition.
It is using above-mentioned further advantageous effect：It can promote the induced efficiency of neural stem cell.
Wherein, retinoic acid, also known as vitamin A acid, Retinoic Acid, abbreviation RA are the biologically active metabolite institutes of vitamin A
A kind of fat-soluble small-molecule substance generated, is mainly made of cyclic ethylene, side chain and polar group.It is of the present invention to regard
The model R2625 of yellow acid is purchased from Sigma-Aldrich companies.Retinoic acid, which should be noted that, to be protected from light.Half complete medium of nerve-inducing
It needs now to add retinoic acid daily, nerve-inducing complete medium can just be prepared.
Further, half complete medium of the nerve-inducing is that 10 μM of selectivity ALK5 suppressions are added in basal medium
Preparation, 250nM selectivity BMP signal pathway inhibitors and 25 μ g/mL insulin.
It is using above-mentioned further advantageous effect：Above-mentioned selectivity ALK5 inhibitor, signal pathway inhibitor and pancreas
Island element is small molecule, and the induced efficiency that double Smad inhibit is much larger than a kind of single induced efficiency of the factor.
Further, the basal medium be DMEM/F12 culture mediums in be added 1wt% nonessential amino acid,
The Pen .- Strep of 1wt% is dual anti-, the 2- sulfydryl second of the GlutaMAX-1 and 0.1wt% of 16wt% glucose, 1wt%
Alcohol.
Advantageous effect using above-mentioned additive is：Using DMEM/F12 culture mediums in the prior art instead of existing skill
Advanced DMEM/F12 culture mediums in art, ingredient is more succinct, and inducing effect more preferably, need not additionally add animal derived blood
Clearly, thus derived cost is lower.
DMEM/F12 culture mediums of the present invention are purchased from Life Technologies companies, model 11039021.
Advanced DMEM/F12 culture mediums are purchased from Life Technologies companies, model 12634028.
DMEM/F12 culture mediums lack ascorbic acid phosphoric acid esters, rich fat ox blood compared with advanced DMEM/F12 culture mediums
Pure albumen, people's Holo-transferrin, insulin recombinate full chain, glutathione, ammonium metavanadate, manganous chloride and sodium selenite etc.
Ingredient, but have l-GLUTAMINE and HEPES.It can be seen that DMEM/F12 culture mediums are instead of in the prior art advanced
DMEM/F12 culture mediums, ingredient are more succinct.
Nonessential amino acid refers to synthesizing in animal body, need not be from the amino acid of external complement as nutrient source.
It is generally synthesized by itself in plant, microorganism essential amino acid, these are referred to as nonessential amino acid.It is non-for people must
It is glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, glutamine, asparagus fern to need amino acid
Propylhomoserin, glutamic acid.These amino acid synthesize carbochain by the metabolin of carbohydrate or by essential amino acid, further by amino
Transfer reaction introduces amino and generates amino acid.Nonessential amino acid of the present invention is public purchased from Life Technologies
Department, model 11140-050.
Pen .- Strep of the present invention is dual anti-to be purchased from Life Technologies companies, model
15070063。
Glucose of the present invention is purchased from Sigma-Aldrich companies, model G5767.
GlutaMAX-I is a kind of advanced cell culture additive, can directly substitute the L- glutamy in cell culture medium
Amine.L-Glutamine is that cell carries out a kind of nutrition necessary to energy generation and protein and nucleic acid synthesis in cell culture
Element.L-Glutamine in cell culture medium can spontaneous degradation, the by-product of generation is ammonia and pyroglutamic acid.GlutaMAX-I has
There are high ease of solubility, thermal stability, helps to improve adherent and suspension mammalian cell culture efficiency, it avoids incubation
In with the relevant problem of L-Glutamine spontaneous degradation, so that incubation time is extended.Meanwhile it is also used as and configures many type cultures
The supplement of base.GlutaMAX-I of the present invention is purchased from Life Technologies companies, model 35050079.
2 mercapto ethanol is purchased from Life Technologies companies, model 21985023.
Further, the selectivity ALK5 inhibitor is SB431542.
It is using above-mentioned further advantageous effect：P38, MAPK are compared to the effect of ALK5 and other kinases are strong by 100
Times.
Further, the model 04-0010 of the SB431542 is purchased from Stemgent companies.
Further, the BMP signal pathway inhibitors are LDN193189.
It is using above-mentioned further advantageous effect：The main transcriptional activity for inhibiting BMPI receptors ALK2 and ALK3,
BMP is acted on to be compared to be used for 200 times of TGF-β high selectivity.
Further, the model 04-0074 of the LDN193189 is purchased from Stemgent companies.
Further, the cell incubator condition of culture is 37 DEG C, 5%v/v CO2。
The beneficial effects of the invention are as follows：
1, the present invention has many advantages, such as that simple operation, at low cost, yield is big, differentiation purity is high, repeatability is strong.Entire ring
Non-animal derived property cell and other ingredients are saved, the time needed for induced nerve stem cells is not only greatly shortened, also substantially increases
The safety of iPS Derived Nerve stem cells, further to utilize the iPS of autologous patient cell induction to mass produce clinical application
The neural stem cell of rank provides a kind of completely new method.
2, present invention only requires add to commonly use chemical substance and specific small in the DMEM/F12 culture mediums of the prior art
Molecular compound, such as LDN193189, SB431542 and RA, you can (7 days most fast) in a short time, induce purity 99% with
On neural stem cell, and in terms of function prove these cells can downstream break up, an one-step inducing of going forward side by side be neuron,
Oligodendroglia and astroglia etc..
3, the shortcomings that the present invention overcomes traditional abductive approach, be it is a kind of it is novel, safely, quickly, repeatability is strong lures
Guiding method, wide market can benefit many patients.
Description of the drawings
Fig. 1 is the density schematic diagram (amplification factor 4X fields of microscope) of the unicellular inoculation in the 0th day of the present invention.
Fig. 2 is the density schematic diagram (amplification factor 10X fields of microscope) of the unicellular inoculation in the 0th day of the present invention.
Fig. 3 is that the 1st day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Fig. 4 is that the 2nd day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Fig. 5 is that the 3rd day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).Margin of colonies cell
Form continues to significantly change.
Fig. 6 is that the 4th day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Fig. 7 is that the 5th day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Fig. 8 is that the 6th day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Fig. 9 is that the 7th day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Figure 10 is that the 8th day cell of the present invention induces schematic diagram (amplification factor 10X fields of microscope).
Figure 11 is induction the 0th day Oct4 and Nestin the expression figure (amplification factor 10X fields of microscope) of the present invention.
Figure 12 is induction the 2nd day Oct4 and Nestin the expression figure (amplification factor 10X fields of microscope) of the present invention.
Figure 13 is induction the 4th day Oct4 and Nestin the expression figure (amplification factor 10X fields of microscope) of the present invention.
Figure 14 is induction the 5th day Oct4 and Nestin the expression figure (amplification factor 10X fields of microscope) of the present invention.
Figure 15 is induction the 7th day Oct4 and Nestin the expression figure (amplification factor 10X fields of microscope) of the present invention.
Figure 16 is that the 8th day Sox2 and Nestin of induction of the present invention expresses Fig. 1 (amplification factor 10X fields of microscope).
Figure 17 is that the 8th day Sox2 and Nestin of induction of the present invention expresses Fig. 2 (amplification factor 10X fields of microscope).
Figure 18 is that the 8th day Sox2 and Nestin of induction of the present invention expresses Fig. 3 (amplification factor 10X fields of microscope).
Figure 19 is that the 11st day Sox1 and Nestin of induction of the present invention expresses Fig. 1 (amplification factor 10X fields of microscope).
Figure 20 is that the 11st day Sox1 and Nestin of induction of the present invention expresses Fig. 2 (amplification factor 10X fields of microscope).
Figure 21 is that the 11st day Sox1 and Nestin of induction of the present invention expresses Fig. 3 (amplification factor 10X fields of microscope).
Specific implementation mode
Principles and features of the present invention are described below in conjunction with specific embodiment, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
Embodiment 1
The present embodiment it is efficient, efficiently inductive pluripotent stem cells are to neural stem cell differentiating method, including as follows
Step：
Step 1：Unicellular inoculation in 0th day
First prepare the inductive pluripotent stem cells of activation：
Step 1.1：By Matrigel matrix on tissue culture plate bed board, be added E8 complete mediums, be placed in 37 DEG C,
5%v/v CO2One day is stood in cell incubator, obtains the tissue culture plate after bed board.Wherein, E8 complete mediums are
TeSRTM-E8TM, model #05940, purchased from STEMCELL Technologies companies.
Step 1.2：Cell fusion is reached to the inductive pluripotent stem cells of 80% density, by per 10cm2Cell culture
Board bottom area liquid volume added is the ratio of 1mL, and cell dissociation buffer is added and is placed in cell incubator, digests 4min.Wherein, used to lure
The property led multipotential stem cell is using induction agent box inducing somatic, induction agent box Epi5 usedTM Episomal iPSC
Reprogramming Kit match silent winged generation that science and technology, MAN0008352.
Step 1.3：By every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added DMEM/F12 culture
Base, dilution enzymolysis, obtains cell suspension.Wherein, DMEM/F12 culture mediums are purchased from Life Technologies companies, model
It is 11039021.
Step 1.4：Cell suspension is transferred in centrifuge tube, DMEM/F12 basal mediums are added, is centrifuged, room
Temperature, 200g centrifuge 5min, remove supernatant, are resuspended with E8 complete mediums, obtain cell suspension and count.
Step 1.5：The cell suspension that step 1.4 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000
The ratio of a cell is added in the tissue culture plate after the bed board that step 1.1 obtains, adds E8 complete mediums, 10mM
ROCK inhibitor is to get to the inductive pluripotent stem cells of the activation.Wherein, ROCK inhibitor Y-27632, model
72304, it is purchased from StemCell Technologies companies.
Step 2：Replace the culture dish of inoculation inductive pluripotent stem cells or the E8 complete mediums in culture bottle in step 1
For nerve-inducing complete medium, after concussion, it is placed in cell incubator and cultivates.
Wherein, the nerve-inducing complete medium is 100 μM of retinoic acids of addition in half complete medium of nerve-inducing.
Half complete medium of the nerve-inducing be in basal medium be added 10 μM of selectivity ALK5 inhibitor,
250nM selectivity BMP signal pathway inhibitors and 25 μ g/mL insulin.
The basal medium is the nonessential amino acid that 1wt% is added in DMEM/F12 culture mediums, the mould of 1wt%
Element-streptomysin is dual anti-, the 2 mercapto ethanol of the GlutaMAX-1 and 0.1wt% of 16wt% glucose, 1wt%.
The selectivity ALK5 inhibitor is SB431542, model 04-0010, is purchased from Stemgent companies.
The BMP signal pathway inhibitors are LDN193189, model 04-0074, are purchased from Stemgent companies.
Step 3：The nerve-inducing complete medium that more renews simultaneously observes cellular morphology daily, culture to the 7-8 days to get
To the neural stem cell of differentiation.
Conclusion
From Fig. 1-2 as can be seen that when collecting within the 0th day cell resuspension, certain form can be formed after 3-9 cell mass inoculation
Clone colony.
As seen from Figure 3, it induces the 1st day, cellular morphology has significantly changed, and especially margin of colonies is thin
Born of the same parents.It is induced the 2-4 days it can be seen from Fig. 4-6, margin of colonies cellular morphology continues to significantly change.It can be seen by Fig. 7
Go out, induces the 5th day, cell aggregation is may be seen indistinctly " garland shape " at definite shape.As seen from Figure 8, it induces the 6th day, cell
Aggregation becomes close.It is induced the 7-8 days it can be seen from Fig. 9-10, cell breaks through monolayer cultivation, or even forms 3D Constituent cells group.
Passage amplification at this time directly freezes, while the Quality Control marker (Nestin, Oct4) of reserved a small amount of cell detection induction NSC.
NSC immunofluorescences are observed and imaging
The cell for taking the 8th day after inducing successfully, by immunofluorescence dyeing conventional steps be incubated respectively it is red (Pax6,
) and green (Sox1, Sox2) cell marker Nestin.It is first under 10X fields of microscope with inversion or upright fluorescence microscope
Cell mass is found with DAPI, then red (Pax6, Nestin) and green are observed with 20X fields of microscope or 40X fields of microscope
(Sox1, Sox2) fluorescent marker is imaged after selection and preserves.
Nestin marks each NSC cell body, Sox1 to mark overwhelming majority NSC nucleus.Show to induce NSC successes.
In actually detected, it is only necessary to detect the 6-8 days a large amount of Nestin positive expressions, and Oct4 feminine genders are expressed.
From Figure 11-15 as can be seen that inducing the 0th day, it is seen that a large amount of Oct4 expression are expressed without Nestin.With luring
Progress is led, Oct4 is completely disappeared within the 2nd day, the 4th day, the 5th day and the 7th day, and Nestin replaces, and gradually expression increases.
From Figure 16-18 as can be seen that inducing the 8th day, Sox1, Sox2 and Nestin have great expression.
From Figure 19-21 as can be seen that inducing the 11st day, Sox1, Sox2 and Nestin have great expression.
It can be seen that the induced efficiency of this method is very high, 99% or more.The present invention have simple operation, it is at low cost,
The advantages that yield is big, differentiation purity is high, repeatability is strong.The entire non-animal derived property cell of link and other ingredients, not only significantly
Shorten the time needed for induced nerve stem cells, also substantially increase the safety of iPS Derived Nerve stem cells, for further profit
The neural stem cell that the iPS induced with autologous patient cell mass produces clinical application rank provides a kind of completely new side
Method.
Embodiment 2
The present embodiment it is efficient, efficiently inductive pluripotent stem cells are to neural stem cell differentiating method, including as follows
Step：
Step 1：Unicellular inoculation in 0th day
First prepare the inductive pluripotent stem cells of activation：
Step 1.1：By Matrigel matrix on tissue culture plate bed board, be added E8 complete mediums, be placed in 37 DEG C,
5%v/v CO2One day is stood in cell incubator, obtains the tissue culture plate after bed board.Wherein, E8 complete mediums are
TeSRTM-E6。
Step 1.2：Cell fusion is reached to the inductive pluripotent stem cells of 70% density, by per 10cm2Cell culture
Board bottom area liquid volume added is the ratio of 1mL, and cell dissociation buffer is added and is placed in cell incubator, digests 3min.Wherein, used to lure
The property led multipotential stem cell is using induction agent box inducing somatic, induction agent box Epi5 usedTM Episomal iPSC
Reprogramming Kit match silent winged generation that science and technology, MAN0008352.
Step 1.3：By every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added DMEM/F12 culture
Base, dilution enzymolysis, obtains cell suspension.Wherein, DMEM/F12 culture mediums are purchased from Life Technologies companies, model
It is 11039021.
Step 1.4：Cell suspension is transferred in centrifuge tube, DMEM/F12 basal mediums are added, is centrifuged, room
Temperature, 200g centrifuge 5min, remove supernatant, are resuspended with E8 complete mediums, obtain cell suspension and count.
Step 1.5：The cell suspension that step 1.4 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000
The ratio of a cell is added in the tissue culture plate after the bed board that step 1.1 obtains, adds E8 complete mediums, 10mM
ROCK inhibitor is to get to the inductive pluripotent stem cells of the activation.Wherein, ROCK inhibitor Thiazovivin.
Step 2：Replace the culture dish of inoculation inductive pluripotent stem cells or the E8 complete mediums in culture bottle in step 1
For nerve-inducing complete medium, after concussion, it is placed in cell incubator and cultivates.
Wherein, the nerve-inducing complete medium is 100 μM of retinoic acids of addition in half complete medium of nerve-inducing.
Half complete medium of the nerve-inducing be in basal medium be added 10 μM of selectivity ALK5 inhibitor,
250nM selectivity BMP signal pathway inhibitors and 25 μ g/mL insulin.
The basal medium is the nonessential amino acid that 1wt% is added in DMEM/F12 culture mediums, the mould of 1wt%
Element-streptomysin is dual anti-, the 2 mercapto ethanol of the GlutaMAX-1 and 0.1wt% of 16wt% glucose, 1wt%.
The selectivity ALK5 inhibitor is SB431542, model 04-0010, is purchased from Stemgent companies.
The BMP signal pathway inhibitors are LDN193189, model 04-0074, are purchased from Stemgent companies.
Step 3：The nerve-inducing complete medium that more renews simultaneously observes cellular morphology daily, culture to the 7th day to get to
The neural stem cell of differentiation.
Embodiment 3
The present embodiment it is efficient, efficiently inductive pluripotent stem cells are to neural stem cell differentiating method, including as follows
Step：
Step 1：Unicellular inoculation in 0th day
First prepare the inductive pluripotent stem cells of activation：
Step 1.1：By Matrigel matrix on tissue culture plate bed board, be added E8 complete mediums, be placed in 37 DEG C,
5%v/v CO2One day is stood in cell incubator, obtains the tissue culture plate after bed board.Wherein, E8 complete mediums are
mTeSRTM1。
Step 1.2：Cell fusion is reached to the inductive pluripotent stem cells of 90% density, by per 10cm2Cell culture
Board bottom area liquid volume added is the ratio of 1mL, and cell dissociation buffer is added and is placed in cell incubator, digests 5min.Wherein, used to lure
The property led multipotential stem cell is using induction agent box inducing somatic, induction agent box Epi5 usedTM Episomal iPSC
Reprogramming Kit match silent winged generation that science and technology, MAN0008352.
Step 1.3：By every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added DMEM/F12 culture
Base, dilution enzymolysis, obtains cell suspension.Wherein, DMEM/F12 culture mediums are purchased from Life Technologies companies, model
It is 11039021.
Step 1.4：Cell suspension is transferred in centrifuge tube, DMEM/F12 basal mediums are added, is centrifuged, room
Temperature, 200g centrifuge 5min, remove supernatant, are resuspended with E8 complete mediums, obtain cell suspension and count.
Step 1.5：The cell suspension that step 1.4 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000
The ratio of a cell is added in the tissue culture plate after the bed board that step 1.1 obtains, adds E8 complete mediums, 10mM
ROCK inhibitor is to get to the inductive pluripotent stem cells of the activation.Wherein, ROCK inhibitor Fasudil.
Step 2：Replace the culture dish of inoculation inductive pluripotent stem cells or the E8 complete mediums in culture bottle in step 1
For nerve-inducing complete medium, after concussion, it is placed in cell incubator and cultivates.
Wherein, the nerve-inducing complete medium is 100 μM of retinoic acids of addition in half complete medium of nerve-inducing.
Half complete medium of the nerve-inducing be in basal medium be added 10 μM of selectivity ALK5 inhibitor,
250nM selectivity BMP signal pathway inhibitors and 25 μ g/mL insulin.
The basal medium is the nonessential amino acid that 1wt% is added in DMEM/F12 culture mediums, the mould of 1wt%
Element-streptomysin is dual anti-, the 2 mercapto ethanol of the GlutaMAX-1 and 0.1wt% of 16wt% glucose, 1wt%.
The selectivity ALK5 inhibitor is SB431542, model 04-0010, is purchased from Stemgent companies.
The BMP signal pathway inhibitors are LDN193189, model 04-0074, are purchased from Stemgent companies.
Step 3：The nerve-inducing complete medium that more renews simultaneously observes cellular morphology daily, culture to the 8th day to get to
The neural stem cell of differentiation.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of, efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating method, which is characterized in that including such as
Lower step：
Step 1：By the inductive pluripotent stem cells of activation, by ten thousand/cm of 2-82Density is inoculated in the training after Matrigel matrix bed boards
It supports in ware or culture bottle, E8 complete mediums is added, is placed in cell incubator and cultivates；
Step 2：It is god to replace the culture dish of inoculation inductive pluripotent stem cells or the E8 complete mediums in culture bottle in step 1
Through inducing complete medium, after concussion, it is placed in cell incubator and cultivates；
Step 3：The nerve-inducing complete medium that more renews simultaneously observes cellular morphology daily, and culture is to the 7-8 days to get to dividing
The neural stem cell of change.
2. according to claim 1, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that in step 1, the preparation method of the inductive pluripotent stem cells of the activation is：
Step 1.1：By Matrigel matrix, bed board, addition E8 complete mediums are placed in cell incubator on tissue culture plate
It is middle to stand one day, obtain the tissue culture plate after bed board；
Step 1.2：Cell fusion is reached to the inductive pluripotent stem cells of 70-90% density, by per 10cm2Tissue culture plate
Floor space liquid volume added is the ratio of 1mL, and cell dissociation buffer is added and is placed in cell incubator, digests 3-5min；
Step 1.3：By every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added DMEM/F12 culture mediums, it is dilute
Enzymolysis is released, cell suspension is obtained；
Step 1.4：Cell suspension is transferred in centrifuge tube, DMEM/F12 basal mediums are added, is centrifuged, room temperature,
200g centrifuges 5min, removes supernatant, is resuspended with E8 complete mediums, obtains cell suspension and count；
Step 1.5：The cell suspension that step 1.4 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000 it is thin
The ratio of born of the same parents is added in the tissue culture plate after the bed board that step 1.1 obtains, and adds E8 complete mediums, 10mM ROCK
Inhibitor is to get to the inductive pluripotent stem cells of the activation.
3. according to claim 2, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that in step 1.5, the ROCK inhibitor be Y-27632, Thiazovivin, Fasudil,
The combination of one or more of GSK429286A.
4. according to claim 1, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that in step 1, the E8 complete mediums are TeSRTM-E6、mTeSRTM1、TeSRTM-E8TMIn one kind or
Two or more combinations.
5. according to any one of claims 1 to 4, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell
The method of differentiation, which is characterized in that in step 2, the nerve-inducing complete medium is in half complete medium of nerve-inducing
Middle 70-120 μM of retinoic acid of addition.
6. according to claim 5, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that half complete medium of the nerve-inducing is that 7-12 μM of selectivity ALK5 suppression is added in basal medium
Preparation, 200-290nM selectivity BMP signal pathway inhibitors and 20-30 μ g/mL insulin.
7. according to claim 6, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that the basal medium be DMEM/F12 culture mediums in be added 0.1-2wt% nonessential amino acid,
The Pen .- Strep of 0.1-2wt% is dual anti-, the GlutaMAX-1 and 0.1- of 11-206wt% glucose, 0.1-2wt%
The 2 mercapto ethanol of 1wt%.
8. according to claim 6, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that the selectivity ALK5 inhibitor is SB431542.
9. according to claim 6, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that the BMP signal pathway inhibitors are LDN193189.
10. according to claim 1, a kind of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating side
Method, which is characterized in that the cell incubator condition of culture is 37 DEG C, 5%v/vCO2。
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