CN103131671B - A kind of method of new inducing somatic reprogramming, kit and purposes - Google Patents
A kind of method of new inducing somatic reprogramming, kit and purposes Download PDFInfo
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Abstract
The invention provides a kind of method of new inducing somatic reprogramming, kit and purposes.It is used for the purposes that induced pluripotent stem cells are produced as inducible factor the present invention relates to mesendoderm differentiation and development correlation factor.The present invention provides inducible factor so as to induce generation induced pluripotent stem cells (iPS cells) by the cell of differentiation, and the inducible factor includes mesendoderm differentiation and development correlation factor, Sox2, Klf4 and optionally c Myc.In the present invention, the iPS cells for substituting Oct4 generations by mesendoderm differentiation and development correlation factor have the characteristic similar with mouse embryonic stem cell, i.e., with the ability for maintaining self-renewing and the totipotency of differentiation.
Description
Technical field
The present invention relates to a kind of method of inducing somatic reprogramming, kit and purposes.
Background technology
(1) embryonic stem cell brief introduction
Embryonic stem cell (embryonic stem cell, ESCs, abbreviation ES cells.) embryonic stem cell is body early embryo
The class cell that (before gastrula stage) or original sexual gland kind are separated, it has in vitro culture infinite multiplication, self-renewing
With the characteristic of Multidirectional Differentiation.
(2) the characteristics of stem cell versatility (pluripotency)：
Embryonic stem cell is referred to as " pluripotency " stem cell because having to three differentiation capabilities of endoderm cell, this
Outward, embryoma (EC) cell is respectively provided with the versatility similar with embryonic stem cell with embryonic germ (EG) cell.Pluripotency is dry thin
The characteristics of born of the same parents are common be：ES cells have the morphosis similar to body early embryo cell, and nucleus is big, there is one or several cores
Benevolence, is generally euchromatin in karyon, and kytoplasm endochylema is few, simple structure.During in vitro culture, cell arrangement closely, is given birth in colony shape
It is long.There is obvious boundary in cell clone and surrounding, boundary is unclear each other for the clone cell of formation, and cell surface has refractive power stronger
Smectic droplet.Cell clone form of diverse, (human embryo stem cell is more flat, and mouse embryonic stem is thin in island or nido for majority
Born of the same parents are more swelled, and cell boundary is not obvious).With AP enzymatic activitys, the surfaces such as SSEA4, TRA1-60, TRA1-81 are specifically expressed
Mark (mouse embryo stem cell also specifically expresses SSEA1, and human embryo stem cell does not express SSEA1), and Oct4,
The significant genes such as Nanog, Sox2, rex1 (ZFP42), GDF3, lin28, mbd2, TEGF1.Further, it is also possible to be trained suspending
Under the conditions of supporting, embryoid body (Embryonid bodies) structure is formed, concurrently give birth to Spontaneous Differentiation, EB is adherent after being formed 6-9 days
Culture, can detect inwardly, in, the cell of outer three differentiation of germinal layers.
(3) research of embryonic stem cell
The thing that mammal never occurs under normal circumstances is cell " dedifferenting " --- cell reverses hair
The process educated, it is a kind of more original type to return.In fact it is tumour cell for the sole exception of this rule, compares
In the histocyte that it is originated, their differentiation degree is than relatively low.Unfortunately, some tumour cells can endless division,
The characteristics of showing immortalization, and multipotential cell also have it is similar the characteristics of.
Up to date, it is by a kind of fine behaviour by unique method that an intracellular biological clock of Normal adult is reversed
Make, allow cell to return the state as embryo's like cell, this process is referred to as the reprogramming of cell.Realize the most ancient of reprogramming
Old method is the nuclear transfer technology of body cell, or referred to as " is cloned ", and its concrete operations is that a Normal adult is intracellular
Inhereditary material entered in egg cell by microinjection, and be removed before egg cell DNA in itself.This DNA and ovum
Plastidogenetic heterozygote can bud into body early embryo, and the stem cell of versatility can be extracted from this body early embryo.
Since the birth of sheep Dolly in 1997 and 1998 first isolated hESC, nuclear transfer skill
Art with the approach of customized acquisition multipotential cell, and then can transplant replacement disease damaged tissues and wound as a kind of
Tissue, it is of great interest.Some factors (we solve also little) in egg cell are really by adult donor cells
Interior inhereditary material youthens, or even including telomere.Telomere protects the end of chromosome just as a cap, and
Gradually shorten with advancing age, but reprogram the state for remaining to allow telomere to keep rejuvenation.Although clone technology is in animal
Considerable progress is achieved in vivo, but is still unsuccessful by its effort for being used to generate hESC.
(4) research of inducing pluripotent stem cells (IPS cells)
In August, 2006, stretches more (Yamanaka) laboratory first in Kyoto Univ Japan regeneration doctor Science Institute professor mountain
First announce that 4 genes (Oct4, Sox2, c-Myc and KLF4) are imported in the fibroblast of mouse is successfully induced weight
Stem cell of the programming as totipotency, its property is similar with embryonic stem cell.(Takahashi and Yamanaka, 2006) from
And disclosing can set up totipotency first by transgenosis in the cell of differentiation.
In this experiment, Yamanaka et al. have chosen and mouse embryo stem cell self-renewing and maintenance versatility first
They are cloned into retroviral vector by 24 related genes respectively, and cotransfection is carried out to embryo fibroblast, are being entered
After screening of the row based on Fbx15 reporters, they observed the formation of the cell colony of ES-like.By to this 24
The further analysis of individual gene, they have found that only transduce Oct4, Sox2, c-Myc, and KLF4 can just make embryo fibroblast
Be completely converted into for induction pluripotent stem cell (iPS cell-Induced Pluripotent Stem Cells, are commonly called as " luring
The pluripotent stem cell led ").The IPS cells possess normal caryogram, the molecular marker of the similar embryonic stem cell of expression, can be with
Be induced to differentiate into vitro it is interior, in, the cell of the terminal differentiation of outer three germinal layers, teratoma can be formed in nude mouse,
In the teratoma containing in, in, the noble cells of outer three germinal layers.Additionally, as embryonic stem cell, IPS cells are in Nanog
With the hypomethylation and acetylation high that H3 is detected on Oct4 promoter.However, using Fbx15 genes as reporter
The cell that screens and without complete totipotency, such as：The dryness such as endogenous Oct4 gene is still no can normally to be opened
Dynamic expression, it is impossible to which the method injected by blastaea is made gomphosis mouse etc..
Meanwhile, research find only use Oct4, Sox2 and KLF4 as can obtain IPS cells, cMyc is not body cell
Transcription factor necessary to reprogramming.
Hereafter, using the strategy of Yamanaka, some new factors for producing induced multi-potent stem cell are screened out
Come.The Huck-Hui Ng seminar of Singapore finds that Nr5a2 and Klf4, Sox2, cMyc complete to reprogram (Jian- together
Chien Dominic Heng et al., 2009), and Oct4, Esrrb, Klf4, and cMyc can also complete to rearrange together
Journey.Meanwhile, research is found only with Oct4 and in special cell type, such as NSC (Kim et al., 2009), is grown
Supporting ectoderm cell (Tong Wu et al., 2011) etc. can just realize that by reprogramming of somatic cells be induced multi-potent stem cell.
Also, can also realize for l cell being changed into induced multi-potent stem cell (Yanqin in the case of plus small molecule
Li et al., 2010) and people keratinocyte be changed into induced multi-potent stem cell (Saiyong Zhu et al.,
2010)。
So far, different seminar have attempted reprogramming technology on the different types of cell of many kinds, and take
Obtained successfully.The improvement of many is there has also been in the methodology of induction.2008, Hochedlinger seminars utilized unconformity
Genome adenovirus (Adenovirus) carrier success induction of IPS cells (Matthias Stadtfeld et al.,
2008)；The same year, Yamanaka using plasmid vector also succeed induction of IPS cells (Keisuke Okita et al.,
2008)；PiggyBac transposon system also confirms that the method that can cut away external source insertion gene completes reprogramming later
(Woltjen K et al., 2009)；Later Ding Sheng seminars also successfully induce IPS cells using the albumen of four factors
(Zhou H et al., 2009)；Recently, with RNA (Luigi Warren et al., 2010) and tiny RNA (micro RNA)
Successfully reprogramming (Frederick Anokye-Danso et al., 2011and Alessandro Rosa et can be realized
Al., 2011).
Gata3, Gata6, Gata4, Pax1, Six1, Gata2, Foxa2 are extensive as mesendoderm differentiation gene
Report, but its effect during reprogramming is there is presently no report.Gata3 has important regulating and controlling for the specialization of T cell
Effect (C.N.Ting et al., 1996)；Gata6 and Gata4 are the typical marker of entoderm, for cardiac muscle cell's
Specialization plays an important role (J.K.Takeuchi et al., 2009)；Pax1 plays a crucial role for the maturation of T cell
(J.Wallin et al., 1996)；Six1 plays an important role (X.Li et al., 2003) for the kidney development of mouse；
Gata2 is required (F.Y.Tsai et al., 1997) for the amplification of hemopoietic forebody cell；Foxa2 is the important of liver cell
Marker, and the transdifferentiation (S.Sekiya et al., 2011) from body cell to liver cell can be together completed with HNF4 α.
(5) application prospect
The foundation of induced multi-potent stem cell solves the ethics problem of embryo cleavage well, for life science
And the healthy important in inhibiting of the mankind.The research of induced multi-potent stem cell can help disclose the developmental mechanism of people and animal
And influence factor；Transgenic animals or gene-targeted animals are set up, human disease model is prepared；Differentiation Induction in vitro provides various
The human body cell of type, carries out study of pharmacy；By forming chimera, make under tight control some organ origins of animal in
Human body cell, so as to be used for clinical transplantation；Most tempting prospect is exactly the target cell for cell therapy and gene therapy, is thin
Born of the same parents are transplanted in the material for providing non-immunogenicity and the human body cell transplanting ex vivo for passing through genetic modification transformation, reach healing disease
Purpose of disease etc..
The content of the invention
It is this kind of the invention provides a kind of by importing the method that mesendoderm differentiation associated gene inducing cell is reprogrammed
Gene can be with other reprogramming genes such as Sox2 together, and induction produces induced pluripotent stem cells (iPS cells).The method passes through
To providing mesendoderm differentiation and development related gene (such as gata3) in the cell of differentiation, in other genes such as external source Sox2
Under collective effect, induction produce IPS cells-induction pluripotent stem cell (Induced Pluripotent Stem Cells,
It is commonly called as " omnipotent cell ").
Therefore, one aspect of the present invention is related to mesendoderm differentiation and development correlation factor to be dived for inducing as inducible factor more
The purposes that energy stem cell produces.
Further aspect of the present invention is related to a kind of method for preparing induced pluripotent stem cells, including is provided to the cell of differentiation
The step of inducible factor.In some embodiments of the present invention, preparation method of the invention include by the cell of differentiation with lure
The step of inducement son contact.In some embodiments of the present invention, the inducible factor includes mesendoderm differentiation and development phase
Close the factor.In some embodiments of the present invention, the inducible factor includes mesendoderm differentiation and development correlation factor, Sox2
With the combination of Klf4.In some embodiments of the present invention, the inducible factor further includes c-Myc.Of the invention
In some embodiments, the inducible factor includes mesendoderm differentiation and development correlation factor, the group of Sox2, Klf4 and c-Myc
Close.In some embodiments of the present invention, the method for the present invention includes providing mesendoderm differentiation and development to the cell of differentiation
The combination of correlation factor, Sox2 and Klf4.In some embodiments of the present invention, the method for the present invention is included to the thin of differentiation
Born of the same parents provide mesendoderm differentiation and development correlation factor, the combination of Sox2, Klf4 and c-Myc.In the method for the invention, interior embryo in
Layer gene can substitute Oct4 and/or c-Myc, therefore be required for Oct4 and/or c-Myc.
In the present invention, the cell of the differentiation for being used can be the cell of any differentiation known in the art, for example, skin
Skin cell, liver cell, gastric cells, horn cell or blood cell, it is preferable that the cell come from embryonic period, embryonic phase, just birth after or
Adult stage.
In the present invention, the cell of the differentiation for being used can derive from mammal or nonmammalian.In the present invention
Some embodiments in, the cell derived of differentiation that the present invention is used is in people.In some embodiments of the present invention, this hair
The cell derived of the bright differentiation for using is in non-human mammal.In some embodiments of the present invention, the present invention is used
The cell derived of differentiation is in mouse such as mouse or rat or primate.
Another aspect of the invention is related to a kind of kit for preparing induced pluripotent stem cells, including inducible factor.At this
In some embodiments of invention, the kit also includes the reagent and carrier for transformed cells.The carrier can be
Such as carrier of slow virus system.Culture medium and specification can also be included in kit of the invention.More of the invention
In embodiment, the inducible factor for being used includes mesendoderm differentiation and development correlation factor, Sox2, Klf4 and optionally c-
Myc.In some embodiments of the present invention, kit of the invention includes mesendoderm differentiation and development correlation factor, Sox2
With the combination of Klf4.In some embodiments of the present invention, the method for the present invention include mesendoderm differentiation and development it is related because
The combination of son, Sox2, Klf4 and c-Myc.
Herein, inducible factor is used with its largest sense, including produces gene (its of induced pluripotent stem cells
Coded polynucleotide or its protein product) or gene combination, inducible factor can be nucleic acid such as DNA, mRNA or protein form.
Herein, mesendoderm differentiation and development correlation factor is used in its broadest sense, including known in the art
Any mesendoderm differentiation and development related gene, the albumen that produces of the polynucleotides that encode the gene, the gene expression
Matter.For example, in some embodiments of the present invention, the mesendoderm differentiation and development correlation factor includes GATA3, GATA6,
At least one of GATA4, PAX1, GATA2, SIX1 or FOXA2.In some embodiments of the present invention, interior embryo in described
Layer differentiation and development correlation factor is selected from least one of GATA3, GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2.
In some embodiments of the invention, the mesendoderm differentiation and development correlation factor is selected from GATA3, GATA6, GATA4,
PAX1, GATA2, SIX1 or FOXA2.In some embodiments of the present invention, using only selected from GATA3, GATA6, GATA4,
A mesendoderm differentiation and development correlation factor in PAX1, GATA2, SIX1 or FOXA2.In some embodiments of the invention
In, using selected from GATA3, two or more mesendoderm differentiation in GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2
Develop the combination of correlation factor.In some embodiments of the present invention, the mesendoderm differentiation and development correlation factor for using is
GATA3。
Another aspect of the invention is directed to use with induced pluripotent stem cells prepared by the method for the present invention or kit.
Further aspect of the present invention is related to the purposes of prepared induced pluripotent stem cells.For example, for known in the art
Various uses.
As demonstrated in the present invention, it is dry thin that mesendoderm differentiation and development correlation factor can be used for preparation induction pluripotency
Born of the same parents.In this regard, mesendoderm differentiation and development correlation factor can be with Sox2, Klf4 and optionally c-Myc is combined for making
Standby induced pluripotent stem cells.
As demonstrated in the present invention, mesendoderm differentiation and development correlation factor can be also used for improving reprogramming efficiency.
In this regard, mesendoderm differentiation and development correlation factor can be combined with Sox2, Klf4 and c-Myc for improving reprogramming effect
Rate.
Brief description of the drawings
Fig. 1：A, b show mesendoderm gene can substitute OCT4 and ectoderm gene can not, in a MEF be mice embryonic into
Fibrocyte；MDF is mouse dermal fibroblast in b；GFP is green fluorescent protein.
Fig. 2：A, b, c show that mesendoderm gene GATA3 can substitute OCT4 and c-MYC, and can improve reprogramming
Efficiency.
Fig. 3 induces the timetable schematic diagram of iPS cells with GATA3 and SOX2, KLF4, c-MYC (SKM).
Fig. 4 GATA3 effects that each different time stage plays in iPS cells are induced.
The iPS primary cell colonies that Fig. 5 is induced with GATA3 and SOX2, KLF4, c-MYC (SKM).Engineer's scale is 100 μm.
Fig. 6 keeps AP (alkaline phosphatases with the iPS cell line that GATA3 and SOX2, KLF4, c-MYC (SKM) are induced through passage
Enzyme) stained positive.Engineer's scale is 100 μm.
Fig. 7 expresses multipotency marker protein with the iPS cell line that GATA3 and SOX2, KLF4, c-MYC (SKM) are induced,
Such as Nanog, Utf1 and SSEA1.Engineer's scale is 10 μm.
There is no OCT4 external sources to reprogram base in the iPS cell line that Fig. 8 GATA3 and SOX2, KLF4, c-MYC (SKM) are induced
The integration of cause.
Fig. 9 expresses dryness marker gene with the iPS cell line that GATA3 and SOX2, KLF4, c-MYC (SKM) are induced.
The external source of the iPS cell line that Figure 10 is induced with GATA3 and SOX2, KLF4, c-MYC (SKM) has reprogrammed gene
Basic silence.
The Oct4 of the iPS cell line that Figure 11 is induced with GATA3 and SOX2, KLF4, c-MYC (SKM), Nanog promoter DNAs
Methylation level is very low, similar with embryonic stem cell.
The iPS cell line gene expression profile and embryo that Figure 12 is induced with GATA3 and SOX2, KLF4, c-MYC (SKM) are dry thin
Born of the same parents are close to
Figure 13 has three embryonic tissue cells with the iPS cell line that GATA3 and SOX2, KLF4, c-MYC (SKM) are induced
The differentiation capability of type.Engineer's scale is 100 μm.
Figure 14 GATA3, GATA4, GATA6 and SOX2, the iPS cell line of KLF4, c-MYC (SKM) induction have blastaea
Injection produces the ability of gomphosis mouse.
There is Figure 15 iPS cell lines that GATA3 and SOX2, KLF4, c-MYC (SKM) are induced blastaea to inject generation reproduction
Chimeric ability.
The iPS cell line that Figure 16 GATA3 and SOX2, KLF4, c-MYC (SKM) are induced injects the chimeric of generation through blastaea
Each histoorgan of mouse can detect the chimeric of donor iPS cells.
Specific embodiment
The present invention is further illustrated with by embodiment.It will be understood by those skilled in the art that the present invention is simultaneously
It is not limited to the embodiment, and one of ordinary skill in the art can be based on the teaching of specification and embodiment is repaiied
Change.These modifications are also contained in the scope of the present invention as defined in appended claims of the invention.
Experimental technique in following embodiments, unless otherwise specified, is conventional method.
1. mouse species
Trangenic mice strain：TgN (GOFGFP) 11Imeg (OG) is purchased from RIKEN BioResource Center.Mouse product
It is ICR magnificent purchased from dimension tonneau.
2. cell culture
Primary mouse embryonic fibroblast (MEF) is all from ICR × OG transgenosis with newborn mice Skin Cell (MDF)
Mouse.MEF and 293T cell culture mediums are DMEM (Hyclone) cultures containing 10% hyclone (FBS, Invitrogen)
Base.Mice embryonic stem cell system R1 and iPS cell culture medium is：80%DMEM/F12 (Invitrogen), N2, B27,1mM L-
Glutamine, 1% nonessential amino acid, 0.055mM beta- mercaptoethanols (being purchased from Invitrogen) and 1 μM
PD0325901,3 μM of CHIR99021 (Stemgent).Mice embryonic stem cell system R1 and iPS cell culture is in mitomycin C
On MEF feeder cells after treatment, culture medium changes liquid daily.Use pancreatin had digestive transfer culture.
The generation of 3.iPS
Tet-on slow virus systems refer to (N.Maherali et al., 2008).The generation of slow virus, collects and infection
Refer to (Y.Zhao et al., 2008).Culture medium of the cell infection after 24 hours is 80%knockout DMEM, 10% tire ox
Serum, 10% serum substitute (KSR) for knocking out, nonessential amino acid, 100units/ml is dual anti-, 1mM Glus, 55
μM beta -mercaptoethanol, 1 μ g/ml Dox.Once changed liquid within every 3 days.After infection 8 days, the culture medium of cell is changed into mice embryonic
Stem cell media.After infection 12 days, GFP is positive and form is come and to pass on mouse embryonic stem thin as the clone of ips is picked
In born of the same parents' culture medium.
The identification of 4.ips
RT-PCR is analyzed, and alkaline phosphatase detection, DNA chip, teratoma refers to (Y.Li et with chimera authentication method
Al .2010).SABC identifies that primary antibody used is SSEA-1 (1: 100；Chemicon), UTF1 (1: 200；Abcam), and
Nanog(1∶100；R&D).Secondary antibody is the donkey anti-rabbit IgG of rhodamine mark and the sheep anti mouse IgM (Santa of rhodamine mark
Cruz Biotechnology, 1: 200).DAPI is used for carrying out core dye.Genomic PCR primer is shown in Table lattice 1, and RT-PCR primer is shown in
Form 2.The kit of methylation analysis is the CpGenomeTM Fast DNA modification kit
(Millipore).Detection primer used by methylation analysis is referred to (K.Takahashi, S.Yamanaka, 2006).
The Genomic PCR primer of form 1
The RT-PCR primer of form 2
Mesendoderm differentiation gene substitutes the exploration of OCT4 during reprogramming.
We have found that GATA3 can substitute Oct4 (Fig. 2 a-c) during OSKM, OSK reprogrammings first.And GATA3
Also substitute cMyc and improve the effect (Fig. 2 a) of OSKM reprogramming efficiencies.GATA3 substitutes the ips induction times and OSKM of Oct4
Basically identical (Fig. 3).The experiment for testing DOX induction times shows that the effect of the replacement Oct4 of GATA3 is mainly played in later stage (figure
4).Induce iPS clones (Fig. 5) that produces can Long Term Passages in vitro, while after testing, embryo is also expressed except Oct4 dry thin
The key gene of born of the same parents such as alkaline phosphatase (Fig. 6), Nanog, Utf1 and SSEA1 (Fig. 7).Genomic PCR identification is dirty without Oct4
Dye (Fig. 8).RT-PCR detections show that these iPS cell lines express the mark of some multipotencies as embryonic stem cell line
Gene, and induce the body cell of iPS cell line not express (Fig. 9) then.
Additionally, the external source of the iPS cell line induced with GATA3 and Sox2, Klf4, c-Myc (SKM) has reprogrammed gene
Basic silence (Figure 10).Oct4 the and Nanog promoter demethylation degree of this kind of iPS cells of DNA methylation analytical proof with
And (Figure 11) similar with mouse embryonic stem cell.Gene microarray analysis result also shows that GATA3 substitutes the iPS cells that Oct4 is produced
Its gene expression profile and the iPS cells of four factors induction are similar with mouse embryonic stem cell (Figure 12).Additionally, GATA3 substitutes Oct4
The iPS cells of generation are transplanted in immunodeficient mouse body can produce teratoma (Figure 13).When GATA3 substitutes what Oct4 was produced
IPS cells and eight cell stages are merged and are implanted into Mouse Blastocysts, then the cell in this group of iPSC sources can be in Mice Body
It is interior to continue to develop, generation Chi-meric mice (Figure 14).Also, the positive cells of GFP are able to observe that in the embryo of 17 days, it was demonstrated that
It is reproduction cell (Figure 15) that the cell in iPSC sources can develop.The histoorgan for separating Chi-meric mice carries out Genomic PCR detection
Can find, the special Oct4-GFP genetic fragments (Figure 16) of donorcells are can detect that in each histoorgan of Chi-meric mice,
Show that donor iPS cell differentiation products have been incorporated into each histoorgan of Recipient mice.Therefore, as a whole, GATA3
Substitute Oct4 produce iPS cells have the characteristic similar with mouse embryonic stem cell, i.e., with maintenance self-renewing ability with
And the totipotency of differentiation.
Because GATA3 belongs to mesendoderm differentiation gene, it is presumed that other mesendoderm differentiation genes are possible to also
Substitute Oct4.Experimental result confirms our supposition, other mesendoderm differentiation genes GATA 2,3,4,6 and FOXA2,
SIX1, PAX1 also have the ability (Fig. 1) that Oct4 is substituted during reprogramming.
Bibliography：
The of 1.N.Maherali et al., Cell Stem Cell 3,340 (2008)
The of 2.Y.Zhao et al., Cell Stem Cell 3,475 (2008)
3.Y.Li et al., Cell Research 21,196.
The of 4.K.Takahashi, S.Yamanaka, Cell 126,663 (2006)
Claims (20)
1. mesendoderm differentiation and development correlation factor is used for during OSKM or OSK is reprogrammed by substituting as inducible factor
Oct4 carrys out the purposes of induced pluripotent stem cells generation, wherein the mesendoderm differentiation and development correlation factor is selected from GATA3,
At least one of GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2.
2. a kind of method for preparing induced pluripotent stem cells, including the step of provide inducible factor to the cell of differentiation, it is described
Inducible factor is included in Sox2, the Klf4 during OSK reprogrammings and substitutes the mesendoderm differentiation and development correlation factor of Oct4,
The cell derived of wherein described differentiation in non-human mammal, wherein the mesendoderm differentiation and development correlation factor be selected from
At least one of GATA3, GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2.
3. the method for claim 2, wherein the inducible factor is also included in the c-Myc during OSKM reprogrammings.
4. the method for Claims 2 or 3, wherein the cell of the differentiation is Skin Cell, liver cell, gastric cells, horn cell
Or blood cell.
5. the method for Claims 2 or 3, wherein the cell comes from embryonic period, embryonic phase, just after birth or the adult stage.
6. the method for Claims 2 or 3, wherein the cell derived of the differentiation is in mouse or primate.
7. a kind of method for preparing induced pluripotent stem cells, including the step of provide inducible factor to the cell of differentiation, it is described
Inducible factor is included in Sox2, the Klf4 during OSK reprogrammings and substitutes the mesendoderm differentiation and development correlation factor of Oct4,
The cell derived of wherein described differentiation is in people, wherein the cell is come from after being just born or the adult stage, wherein the mesendoderm
Differentiation and development correlation factor is at least one of GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2 selected from GATA3.
8. the method for claim 7, wherein the inducible factor is also included in the c-Myc during OSKM reprogrammings.
9. the method for claim 7 or 8, wherein the cell of the differentiation is Skin Cell, liver cell, gastric cells, horn cell
Or blood cell.
10. a kind of kit for preparing induced pluripotent stem cells, including inducible factor, the inducible factor is included in OSK weights
The mesendoderm differentiation and development correlation factor of Sox2, Klf4 and replacement Oct4 in programming process, wherein the mesendoderm point
It is at least one of GATA6, GATA4, PAX1, GATA2, SIX1 or FOXA2 selected from GATA3 to change development correlation factor.
The kit of 11. claims 10, wherein the inducible factor is also included in the c-Myc during OSKM reprogrammings.
The purposes of 12. claims 1, wherein the mesendoderm differentiation and development correlation factor is GATA3.
The method of 13. Claims 2 or 3, wherein the mesendoderm differentiation and development correlation factor is GATA3.
The method of 14. claims 7 or 8, wherein the mesendoderm differentiation and development correlation factor is GATA3.
The kit of 15. claims 10 or 11, wherein the mesendoderm differentiation and development correlation factor is GATA3.
The purposes of 16. claims 1, wherein the inducible factor is DNA, mRNA or protein form.
The method of 17. Claims 2 or 3, wherein the inducible factor is DNA, mRNA or protein form.
The method of 18. claims 7 or 8, wherein the inducible factor is DNA, mRNA or protein form.
The kit of 19. claims 10 or 11, wherein the inducible factor is DNA, mRNA or protein form.
Induction pluripotency prepared by the method for any one of 20. claim 2-9 or the kit of claim 10 or 11 is dry thin
Born of the same parents.
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| CN101014702A (en) * | 2004-07-09 | 2007-08-08 | 赛瑟拉公司 | Preprimitive streak and mesendoderm cells |
| CN101835890A (en) * | 2008-06-27 | 2010-09-15 | 国立大学法人京都大学 | Method of efficiently establishing induced pluripotent stem cells |
| WO2010117879A1 (en) * | 2009-04-08 | 2010-10-14 | Ld Biopharma, Inc. | Generating ips cells by protein transduction of recombinant potency-determining factors |
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| CN101014702A (en) * | 2004-07-09 | 2007-08-08 | 赛瑟拉公司 | Preprimitive streak and mesendoderm cells |
| CN101835890A (en) * | 2008-06-27 | 2010-09-15 | 国立大学法人京都大学 | Method of efficiently establishing induced pluripotent stem cells |
| WO2010117879A1 (en) * | 2009-04-08 | 2010-10-14 | Ld Biopharma, Inc. | Generating ips cells by protein transduction of recombinant potency-determining factors |
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