CN105420193B - Differential medium and its purposes in preparation neural stem cell - Google Patents
Differential medium and its purposes in preparation neural stem cell Download PDFInfo
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Abstract
The invention discloses a kind of differential mediums, it can induce ES cell and/or iPS cell for neural stem cell, the differential medium is the conventional medium of the N2 additive comprising 1%, and the conventional medium is following at least one: RPMI1640 culture medium, DMEM/F12 culture medium, DMEM in high glucose culture medium and α-MEM culture medium.Invention additionally discloses purposes and a kind of method for preparing neural stem cell of the differential medium in preparation neural stem cell.Quick, stable ES cell and/or iPS cell can be induced using the differential medium as neural stem cell.
Description
Technical field
The present invention relates to biological fields, specifically, the present invention relates to culture medium and its application, more specific, the present invention relates to
And a kind of a kind of purposes and side for preparing neural stem cell of differential medium, differential medium in preparation neural stem cell
Method.
Background technique
Stem cell (stem cells) is human body and its various histiocytic primary sources, most significant biological characteristics
Sign is existing self-renewing and the ability that is constantly proliferated, and has the potential of Multidirectional Differentiation.Stem cell is divided into according to different sources
Adult stem cell (somatic stem cells) and embryonic stem cell (embryonic stem cells, ES cell).Adult
Stem cell includes mesenchymal stem cell, pancreatic stem cells, neural stem cell etc., present in adult tissue.
1981, the separation of ES cell and culture succeeded in mouse first, be study so far most extensively, it is most mature
Stem cell system.And the embryonic stem cell of people then starts from 1998, the pungent university of University of Wisconsin-Madison (University of
Wisconsin) scientist's thomson (James A.Thomson) leads research team to extract training from human embryos tissue for the first time
Embryonic stem cell (Embryonic Stem Cell, ES Cell) strain is raised, and confirms that this plant of cell has myeloid-lymphoid stem cell
Feature [Thomson, J.A., et al.Embryonic stem cell lines derived from human
blastocysts.Science,282(1998):1145-1147.].This paper indicates the arrival in an epoch, and Tom
It is gloomy that " father of stem-cell research " is also made by person.
The application prospect of human embryo stem cell (hES) cell research is mainly regenerative medicine field, is led in organizational engineering
Using hES cell as seed cell in domain, a large amount of material can be provided for the transplantation treatment of clinically cell, tissue or organ.
It is lured in vitro by the key molecule gene etc. that control hES cell differentiation culture environment, transfection can promote ES cell directional to break up
Differentiation strategy is led, can get the tissue cell type of specificity.This kind of cell is used for transplantation treatment, will give diabetes, Parkinson
The treatment zone of the diseases such as family name's disease, spinal cord injury, leukaemia, myocardial damage, kidney failure, cirrhosis carrys out new hope.
However, all the time, hES cell research is faced with many problems and dispute, mainly include the following aspects:
(1) source of donor oocyte is difficult, and hES cell establishment efficiency is low.In addition, the immature of SCNT technology will be needed into one
Step expends more human oocytes, so its source is difficult to be guaranteed；(2) immunological rejection, unless using SCNT
Technology, the various cells and tissue that otherwise patient carrys out hES cell differentiation still have immunological rejection；(3) hES cell
With Tumor formation, be transplanted to receptor it is internal after have a possibility that development is tumour, even if using SCNT technology, giving transplanted cells
The counter-measures such as suicide gene are set, this problem also can be not necessarily well solved；(4) hES risk is kept in vitro.Together
Sample, slow-virus transfection technology may there is also similar risks.
For the ethics arguement for avoiding hES cell and therapeutic cloning research, need to find a kind of alternative route, to incite somebody to action
The body cell of the mankind is converted into pluripotent stem cell, provides the autologous stem cells of " personalization " for patient.2003,
The discovery of Gurdon research group, the nucleus of the mouse chest cell broken up completely or adult human peripheral's blood lymphocyte is injected
After xenopus leavis oocytes, the differentiation marker of mammal nuclear is lost, and most characteristic in mammalian stem cell
Marker Oct4 is then in high expression, prompt mammal nuclear can directly by amphibian egg mother cell nuclear vacuole reconstruct to
Express Oct4 [Byrne JA, et al.Nuclei of adult mammalian somatic cells are directly
reprogrammed to oct-4stem cell gene expression by amphibian oocytes.Curr Biol
2003；13:1206-1213.].
2006, Yamanaka research group, Kyoto Univ Japan used outer-gene rotaring dyeing technology, from 24 factors
4 transcription factors such as Oct4, Sox2, c-Myc, Klf4 are filtered out, above-mentioned 4 transcription factors are imported by embryo by retrovirus
Tire l cell or adult mice tail skin fibroblast, obtain under the condition of culture of ES cells
The pluripotent stem cell system of Fbx15+, the cell line is in sides such as cellular morphology, growth characteristics, surface marker, formation teratomas
Face is closely similar with ES cells, and different in terms of gene expression profile, DNA methylation mode and formation chimeric animal
In ES cells, therefore it is named as the multipotent stem cells (iPS cell) of induction.
Yamanaka research group utilize identical technology, by above-mentioned same 4 transcription factors imported into application on human skin at
In fibrocyte, iPS cell also has successfully been obtained.Primary human fibroblast sample synovial cell and be originated from newborn at fiber
The cell line of cell can also equally be reconstructed into as iPS cell.This kind of iPS cell is in cellular morphology, proliferative capacity, surface antigen
Mark, gene expression profile, the epigenetics state of pluripotent stem cell specific gene, telomerase activation etc. and hES
Cell is similar, and when cultivating in vitro and in Mice Body teratoma formed in can be divided into the different cells of 3 germinal layers
Type [Takahashi K et al, Induction of pluripotent stem cells from adult human
fibroblasts by defined factors.Cell 2007；131:861-872.].At the same time, winconsin university
Thomson research group also reports successfully induction fetal fibroblast and is converted into the mankind with hES cell essential characteristic
IPS cell, except that they use slow virus as carrier, and selected in 14 candidate genes Oct4, Sox2,
4 genes such as Nanog, Lin28 are transduceed [Yu J et al, Induced pluripotent stem cell lines
derived from human somatic cells.Science 2007,318:1917-1920].This is known as giving birth to by educational circles
The important breakthrough of object science " milestone " is expected to the ethics helped scientist around clone technology, morals dispute, is medical application
Open gate.
After this, iPS is in terms of the abductive approach of reprogramming, induced efficiency, inducing cell source and biological safety
Important breakthrough is achieved successively.In terms of iPS Cell redifferentiation, certain development has also been obtained.Currently, in iPS cell
Epithelial cell differentiation, hepatocyte differentiation etc., have had been established relatively stable differentiated system [Ahmad Sm, et
al.Differentiation of human embryonic stem cells into corneal epithelial-like
cells by in vitro replication of the corneal epithelial stem cell niche.Stem
Cells,2007,25(5):1145-55.；Metallo CM,et al.Directed differentiation of human
embryonic stem cells to epidermal progenitors.Methods Mol Biol,2010,585:83-
92.；Song ZH,et al.Efficient generation of hepatocyte-like cells from human
induced pluripotent stem cells.Cell Res,2009,19(11),1233-1242.].In neural stem cell
In terms of cell differentiation, iPS cell and ES cell also achieve certain research achievement [Kim DS, et al.Robust
enhancement of neural differentiation from human ES and iPS cells regardless
of their innate difference in differentiation propensity.Stem Cell Rev,2010,6
(2):270-281.]。
Neural stem cell is a kind of mother cell with division potential and self refresh ability, it can pass through not reciprocity point
The mode of splitting generates the various types of cells of nerve fiber.Neural stem cell, which has, to be divided into neural neuron, astroglia and lacks
The ability of prominent spongiocyte, energy self-renewing, so that a large amount of functional cell is provided for nerve fiber, in a variety of nervous systems
There is important application value and research significance in terms of disease.But the source of neural stem cell is extremely limited: from neural group
The middle primary separation of progress is knitted, is largely limited by tissue-derived；From other type adult stem cells
Neural precursor, functionally again there is certain restricted, this is mainly due to adult stem cell differentiation potencies itself
Power is limited, it is difficult to complete regenerating functional neural stem cell.So the source for developing neural stem cell be always Neuscience urgently
Problem to be solved.
The foundation of ES and iPS technology develops new research direction for the regeneration of neural stem cell, both cells self
Updating ability is stronger, and the differentiation capability with height, currently, having multiple seminars establishes ES and iPS cell to nerve
The technical method of stem cell differentiation, such as: EB revulsion, [Bain G, the et al.Embryonic such as PA6 cell co-cultivation method
stem cells express neuronal properties in vitro.Dev Biol.1995,168(2):342-
357.；Okabe S,et al.Kang HC,et al.Behavioral improvement after transplantation
of neural precursors derived from embryonic stem cells into the globally
ischemic brain of adolescent rats.Brain&development.2010,32(8):658-668.].But
It is which kind of method is more suitble to the clinicization of the technology to develop, there is no final conclusion at present.
The new sources for developing neural stem cell are still current Neuscience urgent problem to be solved.
Summary of the invention
The present invention is directed to solve one of above-mentioned technical problem at least to a certain extent or at least provide a kind of selection of business.
One side according to the present invention, the present invention provide a kind of differential medium, and the differential medium can be thin by ES
Born of the same parents and/or the induction of iPS cell are neural stem cell, and the differential medium is the conventional medium of the N2 additive comprising 1%,
The conventional medium is following at least one: RPMI1640 culture medium, DMEM/F12 culture medium, DMEM in high glucose culture medium and α-
MEM culture medium.
This differential medium of the invention, for only added with 1% N2 additive conventional medium, can be thin by ES
Born of the same parents and/or iPS cell rapidly and efficiently, economical and convenient induce as neural stem cell.The neural stem cell of acquisition can be used in mind
The treatment of reparation and the nervous system disease through damaging.
Another aspect according to the present invention, the present invention provide the differential medium of aforementioned present invention one side in preparation nerve
The purposes of stem cell.Using the differential medium culture ES cell and/or iPS cell, can efficiently and stably by ES cell and/
Or the induction of iPS cell is neural stem cell.
Another aspect according to the present invention, the present invention provide a kind of method for preparing neural stem cell, and this method passes through benefit
With the differential medium culture ES cell and/or iPS cell of aforementioned present invention one side, to obtain the neural stem cell.
The method of this preparation neural stem cell of the invention, using the differential medium of one aspect of the present invention i.e. using only
Conventional medium containing 1% N2 additive can rapidly and efficiently, economical and convenient induce ES cell and/or iPS cell
For neural stem cell.The neural stem cell obtained using this method, can be used in the reparation and the nervous system disease of neurotrosis
Treatment.
Detailed description of the invention
Above-mentioned and/or additional aspect and advantage of the invention is from combining in description of the following accompanying drawings to embodiment by change
It obtains obviously and is readily appreciated that, in which:
Fig. 1 shows each stage into neural stem cell induction atomization of the iPS cell in one embodiment of the present of invention
Morphosis.
Fig. 2 shows each stage into neural stem cell induction atomization of the iPS cell in one embodiment of the present of invention
Versatility gene and neural stem cell marker changes in gene expression qPCR result.
Fig. 3 shows that according to one embodiment of present invention, when preparing neural stem cell, iPS cell is to neural stem cell
The immunofluorescence results of neural stem cell marker gene expression in the 7th day are broken up in induction.
Fig. 4 shows iPS cell according to an embodiment of the invention to the 7th day mind of differentiation of neural stem cells
The flow cytomery result expressed through stem cell marker genes.
Specific embodiment
A kind of differential medium provided according to embodiment of the present invention, the differential medium can be thin by ES
Born of the same parents and/or iPS cell directional are induced to differentiate into neural stem cell, and the differential medium is the normal of the N2 additive comprising 1%
Culture medium is advised, the conventional medium is following at least one: RPMI1640 culture medium, DMEM/F12 culture medium, DMEM in high glucose
Culture medium and α-MEM culture medium.
Differential medium in the embodiment, for only added with 1% N2 additive conventional medium, can be by ES
Cell and/or iPS cell rapidly and efficiently, economical and convenient induce as neural stem cell.The neural stem cell of acquisition can be used in
The reparation of neurotrosis and the treatment of the nervous system disease.
So-called N2 additive (N2Supplement), is by transferrins, insulin, flavonoids, putrescine and selenous acid
Sodium composition.N2 additive oneself can be prepared, such as preparing in advance includes transferrins, insulin, flavonoids, putrescine and Asia
Sodium selenate, and make each component concentration be 1mM N2 additive, as mother liquor store it is spare.Commercial product can also be bought,
Referring to the introduction of its product description, is added in the ratio of this embodiment of the present invention, make the differentiation in the embodiment
Culture medium.
A preferred embodiment according to the present invention, the differential medium are the N2 additive comprising 1%
RPMI1640 culture medium.RPMI is Loews dimension Parker memorial institute (Roswell Park Memorial Institute)
Abbreviation.RPMI is a kind of cell culture medium of research institute research and development, and 1640 be culture medium code name, contains 10% fetal calf serum.
RPMI1640 culture medium can be obtained by purchase commercial product or oneself configuration.It only include 1% N2 additive
RPMI1640 culture medium proves that the system is suitable for the induction of neural stem cell through inventor's test of many times.
According to an embodiment of the invention, the differential medium is the conventional medium of the N2 additive comprising 1%, it is described
Conventional medium is following at least one: RPMI1640 culture medium, DMEM/F12 culture medium, DMEM in high glucose culture medium and α-MEM
Culture medium, for example, so-called conventional medium can be also possible to two kinds, three kinds or all four kinds mixed to be one such
It closes.So-called differential medium be only comprising 1% N2 additive conventional medium.
According to another implementation of the invention, the differential medium in any of the above-described embodiment is provided in preparation nerve
The purposes of stem cell.Using the differential medium culture ES cell and/or iPS cell, can efficiently and stably by ES cell and/
Or the induction of iPS cell is neural stem cell.
A kind of method for preparing neural stem cell that another embodiment according to the present invention provides, this method pass through benefit
With the differential medium culture ES cell and/or iPS cell in any of the above-described embodiment, to obtain the neural stem cell.
The method of preparation neural stem cell in the embodiment is using the differential medium in any of the above-described embodiment
Using the conventional medium for the N2 additive for containing only 1%, can by ES cell and/or iPS cell rapidly and efficiently, economical and convenient
Ground induction is neural stem cell.The neural stem cell obtained using this method, can be used in the reparation and nerveous system of neurotrosis
The treatment for disease of uniting.
According to an embodiment of the invention, the ES cell and/or iPS cell in the embodiment come from people, can indicate respectively
For hES and hiPS.
Different from known neural stem cell induced medium and inductive technology, complexity is not added for the method for present embodiment
Signal pathway inhibitor, also do not use known conditioned medium, one kind is only added in conventional medium
People ES cell and/or the induction of iPS cell can be the cell with neural stem cell characteristic, and lured by N2Supplement
It is quick to lead process, method is stablized.
Body cell of the iPS from reprogramming, present embodiment do not make the type of reprogramming body cell and the mode of rearranging into
Limitation.For example, iPS cell from but be not limited to following at least one cell of reprogramming: skin fibroblasts, parodontium are dry
Cell, dental pulp stem cell and urine cell.According to one embodiment of present invention, consider safety and application, iPS cell are
The circles cell of urine cell origin.
According to an embodiment of the invention, using in any of the above-described embodiment differential medium culture ES cell and/or
Before iPS cell, comprising: thin to obtain the ES using following at least one culture medium culture ES cell and/or iPS cell
Born of the same parents and/or iPS cell: mTeSRTM1 culture medium, E8 culture medium and KSR conditioned medium.mTeSRTM1 culture medium, E8 culture medium
It can be obtained by conventional commercial with KSR conditioned medium.
According to an embodiment of the invention, described utilize following at least one culture medium culture ES cell and/or iPS cell,
To obtain the ES cell and/or iPS cell, comprising: cultivated on the culture dish for be coated with matrigel the ES cell and/or
It is thin to digest the ES when the ES cell and/or iPS cell grow to the 60-70% of covering culture dish bottom for iPS cell
It is unicellular to obtain stem cell for born of the same parents and/or iPS cell.Preferred embodiment according to the present invention, the matrigel are type i collagen；
And/or utilize ES cell described in Accutase enzymic digestion and/or iPS cell.Conducive to easily and fast obtain ES cell and/or
IPS cell and its unicellular.
According to an embodiment of the invention, the differential medium culture ES cell using in any of the above-described embodiment and/
Or iPS cell, to obtain the neural stem cell, comprising: utilize the differential medium weight containing 10 μM of ROCK inhibitors
It is unicellular to hang the stem cell, with 0.75-1.0 × 105The density of a cell/mL is seeded in culture plate；Utilize institute within every two days
It states differential medium and liquid once is changed to the cell progress in the culture plate, until obtaining the neural stem cell.The condition is
Inventor attempts by repeatedly adjustment, verification experimental verification determines, is conducive to efficiently by ES cell and/or iPS induction at nerve cord
Cell.
A preferred embodiment according to the present invention, so-called ROCK inhibitor select commercially available Y-27632, are conducive to dry thin
The survival of born of the same parents and launch into neural stem cell induction program.
According to one embodiment of present invention, after being resuspended with the differential medium containing 10 μM of Y-27632, by stem cell
Single cell suspension is with 1.5-2.0 × 105A cells/well, which is seeded in 6 orifice plates, cultivates, to start and realize induction.
The embodiment of the present invention is described below in detail.Embodiment is exemplary, and for explaining only the invention, and cannot be managed
Solution is limitation of the present invention.In the description of the present invention, unless otherwise indicated, the meaning of " plurality " is two or two with
On.
Herein, singular type "one", and " this " includes plural reference, unless the context otherwise clearly statement.
For example, term " (one) cell " includes the cell of plural number, including its mixture.
Herein, unless otherwise indicated, all number marks, such as pH, temperature, time, concentration and molecular weight, packet
Range is included, is all approximation, such as it changes (+) or (-) with 0.1 increment.It is to be understood that although always not describing institute explicitly
Term " about " is all added before some number marks.It will also be understood that although not always specific narration, reagent described herein
Only example, equivalent are known in the art.
Herein, " inducing pluripotent stem cells (the iPS cell) " is such cell, in ES cell culture item
Under part, with ES cell in cellular morphology, growth characteristics, surface marker expression, be transplanted to and can subcutaneously be formed comprising 3 germinal layer groups
It knits cyto-architectural teratoma etc. and people's ES cell is closely similar, and in DNA methylation mode, gene expression profile, dye
Chromaticness state etc. is also closely similar with people's ES cell.
Herein, the term " induction reprogramming " refers to the process of and dedifferentes body cell for multipotent stem cells.
Preferably, body cell is imported by circles plasmid by multipotency sex factor needed for maintaining stem cell versatility, thus
Inducing somatic is dedifferentiated into as multipotent stem cells.The method that the multipotency sex factor imports body cell be can be into this field
Multiple technologies known to technical staff, including virus infection, liposome transfection, electroporation, particle bombardment etc. are various to be transferred to DNA
The method of cell.Preferably, plasmid transfection is carried out using electroporation.
Following embodiments, differential medium component used and its working concentration are preferred are as follows: N2Supplement:1 weight
% is measured, is basic culture medium configuration with RPMI1640.
Experimental method in following embodiments is unless otherwise specified conventional method, such as with reference to " molecular cloning is real
Test guide ", the 3rd edition (2002), Sambrook, Fritsch and Maniatis write；" modern molecular biology experimental method "
(F.M.Ausubel et al. writes (1987))；" Enzymology method " (Academic Press, Inc.)；" PCR2: practical approach ",
M.J.MacPherson, B.D.Hames and G.R.Taylor write, (1995)；" antibody ", Harlow and Lane write,
(1988)；" animal cell culture laboratory manual ", R.I.Freshney writes, (1987)；" stem cell handbook ", volume 2,
W.French Anderson et al. writes.
Test material as used in the following examples is unless otherwise specified from conventional commercial reagent material.
Embodiment one
The routine culture of people ES cell and iPS cell
Material: H1 human embryo stem cell (H1-ES cell), urine cell origin induce multi-potent stem cell that (UC-iPS is thin
Born of the same parents).
Culture medium: mTeSRTM1 culture medium (STEMCELL catalog#05850).
Method and step:
The General Maintenance culture of H1-ES cell and UC-iPS cells pluripotency need to use Metrigel (BD catalog#
354277) it is coated with culture plate, cell is inoculated, uses mTeSRTM1 culture medium is cultivated；Passage in about 4-6 days is primary, can be used
The Dispase enzyme (Gibco catalog#17105-041) of 2mg/ml is digested.
Embodiment two
Material: H1 human embryo stem cell (H1-ES cell), urine cell origin induce multi-potent stem cell that (UC-iPS is thin
Born of the same parents)
Reagent: it is added with the RPMI1640 culture medium (Gibco of N2Supplement (Gibco#17502-048)
Catalog#21870-076), wherein the culture medium contains the N2Supplement of 1 weight %；Y-27632(Sigma
catalog#Y0503)；Accutase(Sigma catalog#A6964)；Ox type i collagen (BD catalog#354231).Y-
27632/ROCK inhibitor。
Method and step:
The H1-ES cell or UC-iPS cell Accutase digestion 3- of covering culture dish bottom 60-70% will be grown to
5min, it is unicellular for blowing and beating, and is seeded in the coated 6 orifice plates of type i collagen, and inoculum density is 1.5-2.0 × 105The hole cells/,
It is cultivated with differential medium, Y-27632 (hereafter changing liquid to be not required to add Y-27632), final concentration of 10 μ is added in culture medium
M, hereafter the next day full dose change liquid, visible typical neural garland spline structure is formed within culture 7 days.
It should be noted that the form for the embryonic stem cell that microscopically observation arrives and its Godwards through stem cell direction
Induce atomization in metamorphosis and differentiated result, induced multi-potent stem cell with people it is essentially identical, here, mainly with people
It is illustrated for inducing multi-potent stem cell.
Before cytomorphology variation in incubation is broken up as shown in Figure 1:, UC-iPS cell is in cloning growth；
Induction differentiation the 1st day, postdigestive unicellular meeting is adherent, and small cloning growth is presented；Induction differentiation the 3rd day, clone is no longer
It is adherent, it is in spherical growth in suspended state；At the 5th day of induction differentiation, the cell of suspension was adherent again, and form is visible obvious
Change；After induction differentiation 7 days, there is typical neural garland spline structure in attached cell, tentatively shows neural stem cell (NSCs)
It is formed, it, can be in spherical suspension growth after Kranz structure is isolated and purified using Mechanical Method (scale is 200 μm in figure).It can be seen that
This method is excessive without EB (embryoid body), it is only necessary to which people's ES or iPS cell differentiation can be nerve cord by simple differentiation culture
Cell.
In atomization, the expression variation of the versatility gene and neural stem cell marker gene in each stage is as schemed
Shown in 2: in atomization, versatility gene expression is gradually lowered, and the expression degree of neural stem cell marker gene gradually rises
Height, most of neural stem cell marker genes reach highest expression on the 7th day after differentiation；But Sox2 is both versatility
Gene is neural stem cell marker gene again, and in entire atomization, variation tendency is little.
To the 7th day neural stem cell of differentiation, We conducted the analysis of the protein level of neural stem cell marker molecule,
Immunofluorescence and flow cytomery result difference are as shown in Figure 3 and Figure 4.
From the immunofluorescence results of Fig. 3: the 7th day neural stem cell formed obviously expresses mind after iPS cell differentiation
Through stem cell marker genes (Nestin, Pax6 and Sox1).One column of left side is the fluorescence dye observed under fluorescence microscope in figure
For color as a result, an intermediate column is the nucleus DAPI coloration result in the corresponding visual field, one column of right side is the composite diagram of the former two, figure acceptance of the bid
Ruler is 50 μm.
From Fig. 4 flow cytomery result as it can be seen that after iPS cell differentiation the 7th day formed neural stem cell Nestin
Expression efficiency 90% or more, Pax6 expression efficiency close to 80%, thus illustrate that the differentiation efficiency of this method is higher.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (5)
1. a kind of method for preparing neural stem cell, which is characterized in that thin using differential medium culture ES cell and/or iPS
Born of the same parents, to obtain the neural stem cell,
Before using differential medium culture ES cell and/or iPS cell, comprising:
It is thin to obtain the ES cell and/or iPS using following at least one culture medium culture ES cell and/or iPS cell
Born of the same parents: mTeSRTM1 culture medium, E8 culture medium and KSR conditioned medium, it is described to utilize following at least one culture medium culture ES cell
And/or iPS cell, to obtain the ES cell and/or iPS cell, comprising:
The ES cell and/or iPS cell are cultivated on the culture dish for be coated with matrigel, when the ES cell and/or iPS are thin
When the intracellular growth extremely 60-70% of covering culture dish bottom, the ES cell and/or iPS cell are digested, it is slender to obtain stem cell
Born of the same parents,
Wherein, the differential medium be only added with 1% N2 additive conventional medium, the conventional medium be with
Lower at least one: RPMI1640 culture medium, DMEM/F12 culture medium, DMEM in high glucose culture medium and α-MEM culture medium,
Wherein, the matrigel is type i collagen,
Wherein, the ES cell is H1 human embryo stem cell.
2. method of claim 1, which is characterized in that the ES cell and/or the iPS cell come from people.
3. method of claim 1, which is characterized in that following at least one cell of the iPS cell from reprogramming: skin
Fibroblast, periodontal ligament stem cell, dental pulp stem cell and urine cell.
4. method of claim 1, which is characterized in that utilize ES cell and/or iPS cell described in Accutase enzymic digestion.
5. method of claim 1, which is characterized in that described thin using the differential medium culture ES cell and/or iPS
Born of the same parents, to obtain the neural stem cell, comprising:
It is unicellular to be resuspended the stem cell using the differential medium containing 10 μM of ROCK inhibitors, with 0.75-1.0 ×
105The density of a cell/mL is seeded in culture plate；
Liquid once is changed to the cell progress in the culture plate using the differential medium within every two days, until obtaining the nerve
Stem cell.
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| CN111793608B (en) * | 2017-07-28 | 2022-05-17 | 杨涛 | HS5 conditioned medium for directionally inducing differentiation of hipscs into neural cell system |
| CN108315301B (en) * | 2018-02-23 | 2019-02-12 | 武汉睿健医药科技有限公司 | A kind of serum free medium of induced nerve stem cells and its application |
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