CN103667349B - Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) - Google Patents
Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) Download PDFInfo
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Abstract
The invention discloses a method for efficiently acquiring inductive pluripotent stem cells (iPSCs). Adipose-derived stem cells (ADSCs) are induced and reprogrammed to form the iPSCs by using the method. The method concretely comprises the following steps: (1) inducing cDNA (Complementary Desoxvribose Nucleic Acid) of a multifunctional factor to the ADSCs; (2) culturing the ADSCs by using a serum-free culture medium in a feed-layer-free system, wherein the ADSCs is obtained in the step (1); (3) after cloning sample cells of embryonic stem cells (ESCs), further culturing by using a culture medium with an MEK (Methyl Ethyl Ketone) and GSK3 (Glaxo Smith Klein) signal channel inhibitor, selecting cells to clone, and enlarging culture; and (4) authenticating the cell cloning pluripotency. In the method, the ADSCs are low in cost and easily available on a large scale, and the reprogramming speed and efficiency are very high; the feed-layer-free system can be used for increasing the cloning purity of positive iPSCs; the serum-free culture medium can be used for avoiding unstable factors brought by undefined components in serum and batch difference; the signal channel inhibitor can be used for promoting cells to be up to the sufficient reprogramming state and improving the cloning quality of the positive iPSCs.
Description
Technical field
The present invention relates to cell field, particularly to a kind of method of effective acquisition pig inductive pluripotent stem cells.
Background technology
Stem cell is human body and its various histiocytic primary source, its most significant biological property be existing self
Update and the continuous ability bred, have the potential of Multidirectional Differentiation again.Stem cell is divided into adult stem cell according to different sources
（Adult stem cells）And embryonic stem cell（Embryonic stem cells,ESCs）.Adult stem cell includes bone marrow
Mescenchymal stem cell, pancreatic stem cells, neural stem cell, fat stem cell etc., present in adult tissue.
1981, the separation of ESCs and culture succeeded first in mice, be study so far the most extensively, the most ripe
Stem cell system.And the generation of the stem cell of people starts from 1998, American scientist James A.Thomson leads research team first
Extract the secondary tissue from human embryos and turn out ESCs strain, and confirm that this plant of cell has myeloid-lymphoid stem cell feature, this grinds
Study carefully paper publishing in top academic journal " Science " above.The application prospect of people's ESCs cell research is mainly regenerative medicine
Field, using people ESCs as seed cell in tissue engineering field, can control for the transplanting of clinically cell, tissue or organ
Treat and substantial amounts of material is provided.The key of people's ESCs directed differentiation can be promoted by controlling people's ESCs differentiation culture environment, transfection
The Differentiation Induction in vitro strategy such as molecular gene, can obtain specific tissue cell type.This kind of cell is used for transplantation treatment, will
Bring newly to the treatment of the diseases such as diabetes, parkinson, spinal cord injury, leukemia, myocardial damage, renal failure, liver cirrhosis
Hope.
However, all the time, people ESCs research is faced with many difficult problems and dispute, mainly includes the following aspects：（1）
The source of donor oocyte is difficult, and people's ESCs establishment efficiency is low.Additionally, body-cell neucleus transplanting（Somatic cell
nuclear transfer,SCNT）The immature of technology will need to expend more human oocytes further, so its
Source is difficult to be guaranteed；（2）Immunological rejection, unless adopted SCNT technology, otherwise patient differentiates to people ESCs
Various cells and tissue yet suffer from immunological rejection；（3）People ESCs has Tumor formation, be transplanted to receptor internal after have send out
Open up the probability for tumor, even if using SCNT technology, to counter-measures such as transplanted cells setting suicide genes, also differing surely
Enough solve this problem well.
For avoiding the ethics arguement of people ESCs and therapeutic cloning research, need to find a kind of alternative route, so that will
The somatic cell of the mankind is converted into pluripotent stem cell, provides the autologous stem cells of " personalized " for patient.2003, Britain
Cambridge University Gurdon research group finds, the mouse chest cell of differentiation or adult human peripheral's blood lymphocyte completely is thin
After karyon injection Xenopus Oocytes, the differentiation mark of mammal nuclear is lost, and in mammalian stem cell
Most distinctive mark Oct4 gene is in then high expression, points out mammal nuclear directly female thin by Amphibian ovum
Karyon bubble is reconstructed thus expressing Oct4 gene.
2006, Kyoto Univ Japan's Yamanaka research group adopted outer-gene rotaring dyeing technology, from 24 factors
Filter out 4 multipotency sex factors such as Oct4, Sox2, Klf4, c-Myc gene, by retrovirus by above-mentioned 4 versatilities because
Son imports mouse fetal fibroblast or adult mice tail skin fibroblast, obtains under the condition of culture of mice ESCs
Obtained pluripotent stem cell system, this cell line in cellular morphology, growth characteristics, gene expression profile, surface antigen markers thing, dive more
The aspects such as the epigenetics state of the dry cell-specific genes of energy, telomerase activation, formation teratoma and mice ESCs are very
Similar, therefore it is named as inductive pluripotent stem cells（Induced pluripotent stem cells,iPSCs）.
Then, Yamanaka research group utilizes identical technology, and above-mentioned 4 same multipotency sex factors are imported to people
In skin flbroblast, have successfully been obtained people iPSCs.Primary human fibroblast sample synovial cell becomes with from neonate
Fibrocellular cell line equally also can be reconstructed into as people iPSCs.The each side characteristic of this kind of iPSCs and people ESCs very phase
Seemingly, and in vitro cultivate formation embryoid body when and in mice body formed teratoma in all can be divided into three germinal layers
Different cell types.Meanwhile, winconsin university Thomson research group also reports and successfully induces fetus to become fiber finer
Born of the same parents reprogram the iPSCs for having people's ESCs basic feature, except that they are used slow viruss as carrier, and at 14
Have selected 4 genes such as Oct4, Sox2, Nanog, Lin28 in candidate gene to be transfected.This is referred to as bioscience by educational circles
The important breakthrough of " milestone " is expected to ethics, the morals dispute helping scientist to bypass clone technology, is that medical application is opened greatly
Door.
Although scientists are made that significant achievement in terms of people ESCs, from ESCs, iPSCs is to can move in other words
The tissue planted and organ, road is still hard and remote.For another angle, part scientist has begun to searching can
Organ with directly transplanting.But the limitation due to human organ source, other species, the especially device of ungulate mammal
Official initially enters in the visual field of researcher, and pig is exactly one of typical example.Because pig is in organ morphology, volume and life
The similarity of reason function aspects and people, and lasting a long time, and is compared using extensive mice at present, and more conducively the mankind are carried out thoroughly
Go deep into and targetedly scientific experimentss.Mice ESCs widely teaches that with example, on ESCs, can very efficiently
Realize genetic modification chimeric with reproduction, and then produce with specific trait animal offspring.Equally, with genetic engineering means pair
The ESCs of pig carries out genetic manipulation, can cultivate the colony of for example anti-Hyperacute immunological rejection of pig of medical prospect, have
Hope the organ transplantation treatment realizing the mankind.In the past few decades, the scientific research for pig has been achieved for welcome advance.
Although however, many scientists once attempted separating the ESCs of pig, all ending in failure, including cattle, sheep etc., obtaining
Also it is only class ESCs, these cells cannot maintain the state of versatility and self renewal in vitro for a long time.Therefore, at present for
The genetic manipulation of pig cell is mostly based on somatic cell nuclear transfer technique.But, because oocyte is rearranged for somatic cell nuclear
Journey is not thorough, and the offspring of generation often has deformity, and nuclear transplantation long the production cycle, thus low in efficiency, waste time and energy.Thus,
Pig iPSCs becomes the selection of great application prospect instantly.
Additionally, before people iPSCs is applied to clinic, the medical effect that it is played awaits animal experiment assessment, peace
Full problem tumor sex chromosome mosaicism be also required to strictly be detected.Because the life-span of mice is shorter, and the raising of primate monkey
More difficult, cost is very high, further relates to ethics morals problem.Thus, except for organ transplantation, pig can candidate as another
Living model, also begin to be praised highly by a lot of scientists and clinician, such as by setting up the multiple hereditary of the mankind
The pig model of character analyzes clinical efficacy in tissue regeneration medical research for the iPSCs, safety to monitor, for accelerating the mankind
The clinical practice of iPSCs provides science data.In addition, in pharmaceutical field, being that pharmacology, medicine are carried out for laboratory animal with mice for a long time
Effect research, produces, based on pig iPSCs, the popularization of disease model obtaining, undoubtedly also can by greatly reinforce test before clinical drug
By property, improve the quality of medical experiment.
Currently, had multiple country research groups obtain pig iPSCs, although verified transfection a series of external sources because
Son is so that the fibroblastic epigenetic modification of pig and genetic transcription are reset to the state close to ESCs, but pig iPSCs
Induced efficiency still very low, the cycle that efficiency value is about needed for 0.1-0.2%, and positive iPSCs Clone formation is longer,
It is typically more than 2 weeks.Meanwhile, it is limited by and enough understanding and experience, reprogramming of somatic cells condition are lacked to pig ESCs culture
Imperfection in setting, the versatility level of set up cell line is also and insufficient, and these all will be an impediment to it industrially
Subsequent applications.Different cell types is different on reprogramming efficiency, identical cell type weight under different inductive conditions
Programming efficiency is also different, thus finds highly efficient pig iPSCs reprogramming technical system, is to need badly gram in research from now on
The problem of clothes.
Content of the invention
In order to overcome, above-mentioned induction pig iPSCs efficiency is low, cycle length problem, it is an object of the invention to provide a kind of efficiently
Obtain the technical method of pig iPSCs, the method is under no feeder layer, serum-free culturing conditions, ADSCs induction reprogramming is
Pluripotent stem cell, and the moment during reprogramming, using MEK signal pathway inhibitor PD0325901 and GSK3
Signal pathway inhibitor CHIR99021 processes cell.
A kind of method of effective acquisition pig inductive pluripotent stem cells of the present invention, used by the fat from pig
Fat stem cell（ADSCs）Induction reprogramming is for inductive pluripotent stem cells it is characterised in that comprising the steps：
（1）The cDNA of multipotency sex factor is imported ADSCs, wherein said multipotency sex factor is Oct4, Sox2, Klf4,
C-Myc gene；
（2）No raising in coating systems, using serum-free medium or add the culture medium culturing for serum additive
Step（1）The ADSCs obtaining；
（3）After embryonic stem cell-like cell clone occurs, described culture medium is added MEK signal pathway inhibitor
PD0325901 and GSK3 signal pathway inhibitor CHIR99021, continues culture 3 days, then picking cell clone amplification culture；
（4）The versatility of identification of cell clone, includes detecting the alkaline phosphatase activitieses of cell, endogenous versatility gene
Expression, embryonic stem cell（Embryonic stem cells,ESCs）The expression of label, differentiation in vivo form teratomatous energy
Power.
According to the further feature of method of the present invention, described step（1）The cDNA of middle multipotency sex factor passes through disease
Poisonous carrier imports described ADSCs.Preferably, described viral vector is slow virus carrier.It is highly preferred that described slow virus carrier is
Medicine derivable RevTet-On type expression vector.RevTet-On type Lentiviral makes to reprogram acquisition
In iPSCs, the expression of the external source versatility factor has Modulatory character at any time, such as iPSCs is being carried out with the heredity behaviour such as genetic modification
After work, can select to keep original state, normal somatic cell can be divided into again, be easy to be transported accordingly as needed
With processing.
According to method of the present invention, in step（2）In, using no raising coating systems, positive iPSCs gram can be improved
Grand purity.Preferably for described no raising coating systems, need matrigel is coated in advance on culture dish.Matrigel can
Automatically assemble and form the three dimensional matrix with biologic activity, the structure of simulation cells in vivo basement membrane, composition, physical characteristics and
Function, is conducive to the culture of cell in vitro.
According to method of the present invention, in step（2）In, it is preferred to use the culture medium of serum-free, it is by for chemistry
Determinate each group assignment system forms, and composition is clear, clear and definite, is convenient for optimizing improvement, transformation further.Preferably,
According to the further feature of method of the present invention, described step（2）In serum-free medium can add generation
Serum additive, preferably 15%（Percent by volume）Serum substitute（Such as KnockOutTMSR）Or 5mg/mL high fat Ox blood serum
Albumin（For exampleⅡ）.For serum additive can avoid not clear and definite component in serum and batch wise differences to
IPSCs cultivation conditions bring unstable factor.
According to the further feature of method of the present invention, described serum-free medium also comprises：40%DMEM/F-12
Culture medium, 40%Culture medium, 1%N-2 additive, 1%B-27 additive, 1%Additive,
0.1mM beta -mercaptoethanol, 1000U/mL leukaemia inhibitory factor, 2 μ g/mL doxycyclines.
According to the further feature of method of the present invention, described leukaemia inhibitory factor is originated for mice.This
The bright leukaemia inhibitory factor that other source of species may also be employed, but the leukaemia inhibitory factor in mice source is relative on cost
Ground is more cheap.
According to method of the present invention, described step（2）The inoculum density of the ADSCs of middle culture is the key of the present invention
One of factor.If cell-seeding-density is too low, the cell radix very little of reprogramming, the yield of positive iPSCs clone can be reduced,
It is unfavorable for the secretion interaction behavior of iuntercellular beneficial agents simultaneously；Whereas if cell-seeding-density is too high and spacing is too small, then
Different unicellular propagation during reprogramming and the cell clone come can come in contact so that convergeing to together, thus causing not
Produce cross-contamination with the iuntercellular of reprogramming level it is impossible to obtain the positive iPSCs clone of reprogramming completely.Preferably, institute
State step（2）The ADSCs of middle culture is with 2,500 cell/cm2Density be inoculated on culture dish.Experiment shows, this inoculation
ADSCs under density then ensure that and favorably accomplishes reprogramming process, obtains simple positive iPSCs clone.
According to the further feature of method of the present invention, described step（3）Middle added MEK signal path suppression
Agent can be the PD0325901 product of Selleckchem company（Article number is S1036）；The GSK3 signal path suppression added
Preparation can be the CHIR99021 product of Selleckchem company（Article number is S2924）.Experiment shows, 0.5 μM of MEK with
3 μM of GSK3 signal pathway inhibitor, not only can promote the generation of cell reprogramming, in the later stage of induction reprogramming,
That mek inhibitor can also promote not to be reprogrammed and part reprogramming then move towards the apoptosis of differentiation again.
Compared with prior art, the present invention has the advantages that：
Strategies for obtain pig iPSCs other from disclosed in prior art are different, method of the present invention than with
Toward having carried out comprehensive optimization, the adult stem cell ADSCs being easy to efficiently reprogram be have chosen on the cell material of source, in weight
Employ safer no feeder layer, serum-free system in programmed environment, apply screen in the later stage of reprogramming process
Cover the pathway inhibitor of cell differentiation signal.Therefore, ADSCs used not only can induced synthesis pig iPSCs, and induce weight
The efficiency of programming is noticeably greater than conventional fibroblast, after pig ADSCs transcription multipotency sex factor, in no feeder layer, serum-free
Under the conditions of, can induce as iPSCs, efficiency is about 1.53% about, and compared with fibroblast, induction is rearranged efficient, high-purityly
The efficiency of journey improves more than 6 times, and cell completes the cycle time at least more than 4 days needed for reprogramming process；Produce these
The expression of iPSCs pluripotency marker's thing and ESCs（With reference to mice, people）Close, the expression of exogenous factor simultaneously can be complete
Full silence；The moment rearranged in ADSCs is processed hence it is evident that being improve pig using specific signal pathway inhibitor
The quality of iPSCs, shows cell with some genes of reprogramming horizontal relevance, the expression in epigenetic modification site
Reach abundant reprogramming state, the research for this pig pluripotent stem cell field provides good platform.Additionally, with respect to current difficulty
Directly to extract required higher human and material resources, time cost, the method in ESCs, and its extraction process from the embryo of pig
By the iPSCs by effective acquisition pig, it is that genetic manipulation provides great convenience, promote the organ set up based on pig iPSCs
Transplanting pig, the production of human genetic disease's swine model and popularization, serve the clinical practice of physianthropy.
Brief description
Fig. 1 is the pig iPSCs aspect graph that ADSCs and its induction reprogramming obtain.
Fig. 2 is the schematic diagram that pig iPSCs induces reprogramming process.
Fig. 3 is alkaline phosphatase staining result and its reprogramming efficiency analysis chart of pig iPSCs.
Fig. 4 is versatility gene and the ESCs marker representation qualification figure of pig iPSCs.
Fig. 5 is the teratoma differentiation qualification figure of pig iPSCs.
Specific embodiment
1. definition and technology：
Except as otherwise noted, by the conventional art using molecular biology, cytobiology, it belongs to for the practice of the present invention
Art technology scope.Referring to《Molecular Cloning:A Laboratory guide》, J.Sambrook et al. writes（2008）；《Fine works molecular biosciences
Learn experiment guide》, F.M.Ausubel et al. writes（2008）；《Zooblast culture medium this technology guide》,
R.I.Freshney et al. writes（2008）；《Stem cell handbook》, R.Lanza et al. writes（2013）.
Used in the present invention, some terms have the implication of following definition：All of Digital ID, such as pH, temperature,
Time, concentration and molecular weight, including scope, are all approximations.It is to be understood that although always clearly not describing, all of numeral
Term " about " is all added before mark.It will also be understood that although always clearly not describing, the reagent described in the present invention is only
Example, its equivalent is known in the art.
" inductive pluripotent stem cells of the present invention（iPSCs）" it is such cell, it is in embryonic stem cell
（ESCs）Under condition of culture, bag can be differentiated to form with ESCs in cellular morphology, growth characteristics, surface marker expression, inside and outside
The aspects such as the organizational structure containing three endoderm cell are closely similar, and genomic DNA methylation level mode, gene expression profile,
The aspects such as chromatin state are also quite similar.
Fat stem cell of the present invention（ADSCs）It is the ADSCs from mammal, it is from the back of the body, subcutaneous abdomen
In fatty tissue, separation and Extraction obtains, and has plasticity.Fatty tissue is located at below skin, belongs to a kind of loose connective tissue
Structure, is distributed in animal body in a large number, according to the difference of adipose cell 26S Proteasome Structure and Function, fatty tissue is divided into white（Yellow）
Fatty tissue, brown adipose tissue, experiment is drawn materials and used is belonged to the former.Fatty tissue is except the adipose cell by a large amount of clusters
Constitute, be also rich in the ADSCs with self-renewal capacity, the injury repairing of the tissues such as skin can be participated in.
The ADSCs of culture is in that fibroblast-like short fusiformis is adherent, vortex shape growth, form is full and refractivity very
By force；Secrete some adhesion molecules, extracellular matrix proteins, stem cell factor and somatomedin, expression mescenchymal stem cell is special
Specific labels CD44, CD90, CD29；It is capable of plasticity under certain inductive condition, show as laterally or longitudinally
Differentiation capability, such as to adipose cell, osteocyte, Chondrocyte Differentiation.
The ADSCs of various animal origins all can be easily separated from animal, be easy to process, it studied and also will not relate to
And ethics morals problem.For example separate from physianthropy improves looks the postoperative fatty tissue garbage of related surgical and obtain, or
Extract from the fatty tissue fattening domestic animal.
Term " induction reprogramming " of the present invention refers to the process of dedifferente somatic cell for multipotent stem cells.Excellent
Selection of land, by the multipotency sex factor cDNA needed for stem cell versatility being maintained to import the i.e. described ADSCs of somatic cell, can lure
Conductor cell de-differentiation becomes multipotent stem cells.Wherein it is preferred to, described multipotency sex factor include Oct4, Sox2,
Klf4, and c-Myc gene.Most preferably, described multipotency sex factor behaviour source Oct4, Sox2, Klf4, and c-Myc gene.
Specifically, described multipotency sex factor is Oct4, and NCBI accession number is NM_002701；Sox2, NCBI accession number is NP_003097；
Klf4, NCBI accession number is NP_004226；C-Myc, NCBI accession number is NP_002458.
The described multipotency sex factor cDNA somatic method of importing can be multiple skill well known to those skilled in the art
Art, including various methods that DNA is proceeded to cell such as viral infection, liposome transfection, electroporations.Preferably, using comprising
The viral vector of cDNA is transfected, and described viral vector includes multiple virus such as slow virus carrier, retroviral vector and carries
Body.Preferably slow virus carrier（The RevTet-On type carrier of certain drug regulating and expressing at any time for example can be subject to）, such as implement
Described in example.
" no feeder layer, serum-free culturing conditions " of the present invention are on this area convenient stem cells condition of culture basis
On optimization processing, and include some each concrete cell lines suitable, but do not affect the modification of cell fundamental property.Culture
Method and condition of culture referring to《Stem cell handbook》, R.Lanza et al. writes（2013）.
The process of the technical method preferred embodiment of effective acquisition pig inductive pluripotent stem cells of the present invention is as follows, Fig. 1
It is the pig iPSCs aspect graph that ADSCs and its induction reprogramming obtain；Fig. 2 is the schematic diagram that pig iPSCs induces reprogramming process；
Fig. 3 is alkaline phosphatase staining result and its reprogramming efficiency analysis chart of pig iPSCs；Fig. 4 is the versatility gene of pig iPSCs
With ESCs marker representation qualification figure；Fig. 5 is the teratoma differentiation qualification figure of pig iPSCs.
2. embodiment
For making the present invention easier to understand, the specific embodiment of the present invention is further illustrated below.
Following row of implementing schematically illustrate the standard laboratory practices of inventor, for the pattern of the example present invention, and should be by
The present invention is interpreted as being defined in the scope of these embodiments.These embodiments, according to present invention disclosure and those skilled in the art
General level, technical staff will be understood that following be for illustration only, various changes can be carried out in less than the scope of the present invention
Move, modify and transform.Wherein involved technology, unless stated otherwise, is all molecular biosciences well known to those skilled in the art
The routine techniquess of the aspects such as, cytobiology.
The preparation of embodiment 1. cell and culture
1.1 pork fat stem cell（ADSCs）Culture
Isolate fatty tissue from the pig back of the body, subcutaneous abdomen, remove blood vessel, muscle residue, DPBS buffer（Gibco is public
Department）Washing is fully washed, and then surgically cuts and fully shreds fatty tissue to almost without shearing resistance, is transferred to centrifugation
In pipe, add be equivalent to 2～3 times of fatty tissue cumulative volume 0.09% type i collagen enzymic digestion liquid（Sigma company）, it is placed in 37
Vibration, digestion process in DEG C water-bath；After tissue suspension gelatinizing, repeatedly blow and beat with dispersion tissue's chip with pasteur pipet, 1,
200rpm room temperature is centrifuged 5min, discards ripe fatty tissue, the Digestive system in middle level on centrifuge tube upper strata, then with containing 10% tire Sanguis Bovis seu Bubali
Clear DMEM/F-12 basal medium（HyClone company）The fully cell of resuspended centrifuge tube bottom；With aperture it is successively
250 μm, 80 μm, 25 μm of nylon screen filtration cell suspension, discard basal medium, with fresh basal medium after centrifugation
The cell pellet of cyclic washing centrifuge tube bottom；Recentrifuge after discarding basal medium, with pig ADSCs complete medium
Fully re-suspended cell precipitate, and be inoculated in Tissue Culture Flask, put into 37 DEG C, 5%CO2Cell culture incubator in culture.Institute
State in pig ADSCs complete medium and comprise DMEM/F-12 culture medium（HyClone company）, 10% hyclone（Gibco is public
Department）, 10ng/mL basic fibroblast growth factor（Peprotech company）, 50 μ g/mL L-AAs（Sigma is public
Department）, 2mM L-Glutamine（Gibco company）.
1.2 pig inductive pluripotent stem cells（iPSCs）Culture
40%DMEM/F-12 culture medium is comprised in pig iPSCs Serum-free complete medium（Gibco company）、40%Culture medium（Gibco company）, 15% serum substitute（KnockOutTMSR）Or 5mg/mL high fat Sanguis Bovis seu Bubali is pure
Albumen（Ⅱ）（Gibco company）, 1%N-2 additive（Gibco company）, 1%B-27 additive（Gibco is public
Department）、1%Additive（Gibco company）, 0.1mM beta -mercaptoethanol（Gibco company）, 1000U/mL leukemia
Inhibitive factor（Millipore company）, 2 μ g/mL doxycyclines（Clontech company）.Additionally, processing the little of cell clone
The working concentration of molecule inhibitor PD0325901 is 0.5 μM, and the working concentration of CHIR99021 is 3 μM.
The culture of 1.3 other cells
293T cell be used as slow viruss package cell line, using with fibroblast identical culture medium, all cells
It is consistently placed at 37 DEG C, 5%CO2Cell culture incubator in culture.
Embodiment 2. viral vector infection pig ADSCs
Method according to embodiment 1, inoculates the pig ADSCs of low generation, 37 DEG C, 5%CO in Tissue Culture Flask2's
Cultivate under conventional culture conditions to degree of converging reach 80～90% when, with 0.25% trypsin-EDTA（Gibco company）In 37 DEG C
After digesting for single cell suspension, with the vial supernatant infection 1 × 10 collected5Individual ADSCs, infection multiplicity（MOI）For 3（Used
The titre of virus is 5～10 × 106IU/mL, carries four kinds of viruses of multipotency sex factor cDNA by 1:1:1:1 mixing）, Ran Houjie
Plant in 6 well culture plates, continue in 37 DEG C, 5%CO2Conventional culture conditions under cultivate.Described vial supernatant be by with
The medicine of the cDNA comprising people Oct4, Sox2, Klf4 and c-Myc can induce（RevTet-On）Type Lentiviral
（SiDanSai company）Transfect 293T cell according to a conventional method（Fugene HD, Roche company）Obtain（Referring to《Molecular cloning
Experiment guide》, J.Sambrook et al. writes（2008））.
The continuation culture of embodiment 3. infection cell and colony screening
The 2nd day after infection, using DPBS buffer solution for cleaning culture hole twice, more metainfective ADSCs is used
0.25% trypsin-EDTA（Gibco company）After digesting for single cell suspension in 37 DEG C, by 2,500 cell/cm2Close
Degree is inoculated in 6 new well culture plates, inoculates 4 culture hole altogether, culture plate ware face is coated matrigel in advance（BD
Pharmingen company）, it is continuing with ADSCs complete medium.Here, in the Duplicate Samples of 4 plate holes, 1 plate hole is used for alkalescence
Phosphatase（AP）Dyeing（SiDanSai company）, count positive colony number, another 3 plate holes are selected for clone, and experiment is repeated
Three times.The 3rd day after infection, ADSCs complete medium is replaced by the pig iPSCs serum-free described in embodiment 1 complete
Culture medium, continues in 37 DEG C, 5%CO2Conventional culture conditions under cultivate.Cultivate to the 5th day about, have typical cell collection
Drop out existing, after ESCs like cell clone occurs on the about the 6th～8 day, pig iPSCs Serum-free complete medium is added 0.5 μM
The GSK3 signal pathway inhibitor CHIR99021 of MEK signal pathway inhibitor PD0325901 and 3 μM, during processing reprogramming
Cell.
It is important to note that metainfective ADSCs is inoculated in Tissue Culture Plate with too high or too low density,
And add PD0325901 and CHIR99021 too early or too late, also include simply using one of little molecules in inhibiting
Agent, the formation rate of pig iPSCs positive colony all can substantially reduce.
Cultivate to the 10th day about, will be used for cloning 3 plate holes selected, using glass needle segmenting edge is smooth, cell
The single clone of the typical ESCs sample that core is clear, form is compacted, then draws the clone that these separate by a formula with glass tubing
Difference is inoculated in the corresponding hole of two piece of 96 well culture plate, and a clone is inoculated in each hole, and mice tire is used in culture plate ware face in advance
The feeder layer cells of youngster fibroblast preparation are coated, 37 DEG C, 5%CO2Conventional culture conditions under cultivate.In 96 well culture plates
After culture about a week, choose one piece of culture plate therein and carry out AP dyeing.The positive clone of AP is used TryPLE Express
（Gibco company）After digesting for single cell suspension in 37 DEG C, by 1:The ratio of 6-12 passes on, successively from 96 holes, 24 holes, 12 hole trainings
Foster plate, expands to 6 well culture plates.In these iPSCs candidate clones, we have selected 2 and are further identified.
Do not carry out cloning that 16 well culture plate plate holes of picking, directly carry out AP dyeing, according to colony morphology and AP dye
The colony count to typical ESCs for the color result（Taken pictures under microspur using Single-sens reflex camera, then use " ImageJ " soft
Part counts）, calculate induced efficiency formula：Induced efficiency=infected cell quantity × 100% of AP positive colony quantity ÷.Through test
Find after repeating statistical analysiss, inoculate 2.5 × 104Individual metainfective pig ADSCs, can form 382 AP positive colonies, that is, I
The overall efficiency of this method be of about 1.53%, with the same period setting matched group fibroblast about 0.25% efficiency compared with
More than 6 times.
Embodiment 4.Real-Time PCR identifies the expression of versatility gene in pig iPSCs clone
Using RNeasy Mini（QIAGEN company）Test kit, illustrates to extract pig iPSCs total serum IgE according to manufacturer；With
QuantiTect Reverse Transcription（QIAGEN company）Test kit carries out reverse transcription, and uses FastStart
SYBR Green Master（Rox）Test kit（Roche company）, StepOnePlus quantitative real time PCR Instrument（Applied
Biosystems company）Carry out Real-Time PCR.All above-mentioned PCR conditions all using Standard PCR condition, according to manufacturer
Explanation is carried out.
Using the present processes obtain pig iPSCs clone in, not only endogenic Oct4, Sox2, Nanog,
Dnmt3b, Tert gene is activated, and Lin28, Esrrb, Utf1, Dppa5 gene associating with cell reprogramming degree is also abundant
Up-regulated expression, and the expression or relatively low that these multipotency sex factors all not can detect that in the ADSCs without induction reprogramming
Expression.
Embodiment 5. protein immunization fluorescence staining identifies the expression of ESCs label in pig iPSCs clone
After DPBS buffer solution cultivates the pig iPSCs clone of 2～3 days, using 4% paraformaldehyde（Solarbio company）
Fixing；Using 0.5%Triton X-100（Solarbio company）Permeabilized cells（Limit core internal labeling analyte detection）, then with containing 1%
BSA（Sigma company）DPBS buffer blind process；After period cyclic washing, sequentially add one and resist（Anti- Oct4 and Nanog
Antibody be purchased from Abcam company, anti-Sox2 antibody be purchased from Cell Signaling company, the antibody of anti-SSEA-3 and SSEA-4
Purchased from Developmental Studies Hybridoma Bank company）, two resist（Alexa Fluor594 is purchased from
Molecular Probes company）Incubation；Finally use DAPI dyestuff（Sigma company）Positioning nucleus, using conventional fluorescence
Micro- sem observation.Result display pig iPSCs clone high expression ESCs label Oct4, Sox2, Nanog, SSEA3 and SSEA4 egg
In vain.
Embodiment 6. identifies that pig iPSCs is cloned in the ability that differentiation in vivo forms three embryonic tissue structures
By confirming whether institute detection pig iPSCs clone has the characteristic of for a long time maintenance Multidirectional Differentiation of Cells potential, Wo Menji
Test in the monster neoplasia that the cell passing on more than 10 generations to be carried out.
After pig iPSCs clone is digested for single cell suspension in 37 DEG C using TryPLE Express, renewed vaccination is to newly
Culture dish in, be placed in 37 DEG C, 5%CO2Incubator stands 45 minutes, takes the not adherent cell in upper strata（Feeder layer cells are adherent
Hurry up, therefore feeder layer cells can be separated with pig iPSCs）；Count, take 5,000,000 pig iPSCs single-cell suspensions to contain 15% in 300 μ L
KnockOutTMIn the DMEM/F-12 of SR, NOD/SCID congenital immune deficiency mice is carried out with the aseptic note of back leg root muscle
Penetrate；After injected in mice, it is placed in middle raising between SPF level laminar flow, period does not stop to feed the doxycycline of 2mg/mL（It is dissolved in aseptic sugarcane
Sugar juice）, change within about 3～5 days time water, gradually have the lump of grain of rice size to grow, grow up to after can take after tumor mass and medicine is removed
Fall；After withdrawal, teratoma is taken out with eye scissorss, surgical forcepss, is fixed in 4% paraformaldehyde by normal nursing 3～4 weeks；After fixation
Teratoma, through paraffin embedding, section, and hematoxylin-eosin（Hematoxylin-Eosin）Teratoma differentiation is observed in dyeing
Situation.Result shows, pig iPSCs successfully differentiates the organizational structure of the dissimilar cell with three germinal layers, such as entoderm
Enteric epithelium structure, mesoblastic lipid structure and ectodermic Eponychium structure,
By upper result prove further using the present processes can effective acquisition pig iPSCs, and there is the spy of ESCs
Property.
Last should be noted that above example is only in order to illustrate technical scheme rather than to present invention guarantor
The restriction of shield scope, although being explained in detail to the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (3)
1. a kind of method of effective acquisition pig inductive pluripotent stem cells, for by the fat stem cell from pig（ADSCs）
Induction reprogramming is inductive pluripotent stem cells（iPSCs）It is characterised in that comprising the steps：
（1）The cDNA of multipotency sex factor is imported ADSCs, wherein institute by medicine derivable RevTet-On type expression vector
The multipotency sex factor stated is Oct4, Sox2, Klf4, c-Myc gene；
（2）No raising in coating systems, using serum-free medium or add the culture medium culturing step for serum additive
（1）The ADSCs obtaining；Described serum-free medium also comprises：40% DMEM/F-12 culture medium, 40% Neurobasal training
Foster base, 1% N-2 additive, 1% B-27 additive, 1% GlutaMAX additive, 0.1mM beta -mercaptoethanol, 1000U/mL
Leukaemia inhibitory factor, 2 μ g/mL doxycyclines；Matrigel is coated on culture dish, by the ADSCs of culture with 2500 in advance
Cell/cm2Density be inoculated on culture dish；
（3）After embryonic stem cell-like cell clone occurs, described culture medium is added the MEK signal that working concentration is 0.5 μM
Pathway inhibitor and the GSK3 signal pathway inhibitor that working concentration is 3 μM, continuation culture 3 days, then picking cell clone simultaneously expand
Big culture；And
（4）The versatility of identification of cell clone, including the table of the alkaline phosphatase activitieses of detection cell, endogenous versatility gene
Reach, embryonic stem cell（ESCs）The expression of label, differentiation in vivo form teratomatous ability.
2. method according to claim 1 it is characterised in that：Described step（2）In, described generation serum additive is volume
Percentage ratio is 15% serum substitute or 5mg/mL high fat bovine serum albumin.
3. method according to claim 1 is it is characterised in that what described leukaemia inhibitory factor was originated for mice.
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| CN106544315A (en) * | 2016-10-12 | 2017-03-29 | 广东艾时代生物科技有限责任公司 | A kind of method that fat mesenchymal stem cell induces into pluripotent stem cell |
| CN106978361B (en) * | 2017-04-06 | 2020-08-11 | 广东工业大学 | Anti-inflammatory effect of yeast oxidative stress metabolite and application thereof |
| CN107245474A (en) * | 2017-06-07 | 2017-10-13 | 北京呈诺医学科技有限公司 | A kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free |
| CN107723273A (en) * | 2017-10-27 | 2018-02-23 | 广西大学 | A kind of preparation method of the induction goat multipotential stem cell of micromolecular compound completely |
| CN108441517A (en) * | 2018-03-28 | 2018-08-24 | 长春博邦企业管理咨询有限公司 | A kind of preparation method of people's induced multi-potent stem cell |
| CN109666650A (en) * | 2019-01-04 | 2019-04-23 | 北京航空航天大学 | A kind of cell reprogramming method |
| CN113025562B (en) * | 2021-03-18 | 2022-11-29 | 浙江大学 | Application of R406 in promotion of somatic cell reprogramming, reprogramming culture medium and method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433299A (en) * | 2011-11-17 | 2012-05-02 | 安徽农业大学 | Method for separating, culturing and purifying mouse adipose-derived stem cells |
| WO2012087965A2 (en) * | 2010-12-22 | 2012-06-28 | Fate Therapauetics, Inc. | Cell culture platform for single cell sorting and enhanced reprogramming of ipscs |
| CN102851314A (en) * | 2011-07-01 | 2013-01-02 | 中国科学院上海药物研究所 | Preparation method for induced multipotential stem cells and culture medium for preparing induced multipotential stem cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173490B (en) * | 2011-10-21 | 2015-01-07 | 中国科学院广州生物医药与健康研究院 | Method for improving inductive generation efficiency of induced pluripotent stem cells |
-
2013
- 2013-11-15 CN CN201310576436.0A patent/CN103667349B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012087965A2 (en) * | 2010-12-22 | 2012-06-28 | Fate Therapauetics, Inc. | Cell culture platform for single cell sorting and enhanced reprogramming of ipscs |
| CN102851314A (en) * | 2011-07-01 | 2013-01-02 | 中国科学院上海药物研究所 | Preparation method for induced multipotential stem cells and culture medium for preparing induced multipotential stem cells |
| CN102433299A (en) * | 2011-11-17 | 2012-05-02 | 安徽农业大学 | Method for separating, culturing and purifying mouse adipose-derived stem cells |
Non-Patent Citations (3)
| Title |
|---|
| FEEDER-FREE DERIVATION OF INDUCED PLURIPOTENT STEM CELLS FROM ADULT HUMAN ADIPOSE SETM CELLS;NING SUN ET AL;《PNAS》;20090915;第106卷(第37期);15720-15725 * |
| PROMOTION OF REPROGRAMMING TO GROUND STATE PLURIPOTENCY BY SIGNAL INHIBITION;JOSE SILVA ET AL.;《PLOS BIOL》;20081021;第6卷(第10期);2237-2247 * |
| 限定性因子诱导胎猪成纤维细胞重编程为多能性细胞;殷慧群 等;《生物化学与生物物理进展》;20100630;第37卷(第6期);607-612 * |
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