CN103667349A - 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 a kind of method of effective acquisition pig inductive pluripotent stem cells.
Background technology
Stem cell is human body and various histiocytic initial source thereof, and its most significant biological property is the ability of existing self and continuous propagation, has again the potential of Multidirectional Differentiation.Stem cell is divided into adult stem cell (Adult stem cells) and embryonic stem cell (Embryonic stem cells, ESCs) according to different sources.Adult stem cell comprises mesenchymal stem cells MSCs, pancreatic stem cells, neural stem cell, fat stem cell etc., in adult tissue, exists.
1981, first the separation of ESCs and cultivate succeeded in mouse, be study the most so far, the most ripe stem cell system.And people's stem cell for starting from 1998, American scientist James A.Thomson leads research team from human embryos tissue, to extract and turn out ESCs strain first, and confirm that this strain cell has myeloid-lymphoid stem cell feature, this research paper is published in top academic journal " Science " above.The application prospect of people ESCs cell research is mainly regenerative medicine field, usings people ESCs as seed cell in organizational engineering field, can be the transplantation treatment of cell, tissue or organ clinically a large amount of materials is provided.The Differentiation Induction in vitro strategies such as key molecule gene that can promote people ESCs directed differentiation by control people ESCs differentiation culture environment, transfection, can obtain specific histocyte type.This class cell, for transplantation treatment, will bring new hope to the treatment of the diseases such as diabetes, Parkinson's disease, Spinal injury, leukemia, myocardial damage, renal failure, liver cirrhosis.
Yet all the time, people ESCs research is faced with many difficult problems and dispute, mainly comprises the following aspects: the source difficulty of (1) donor ovocyte, people ESCs establishment efficiency is low.In addition, body-cell neucleus transplanting (Somatic cell nuclear transfer, SCNT) the immature of technology will need further to expend more human oocytes, so its source is difficult to be guaranteed; (2) immunological rejection, unless adopt SCNT technology, still there is immunological rejection with organizing in the various cells that patient differentiates people ESCs; (3) people ESCs has into knurl, has the possibility of the tumour of developing into after being transplanted in the body of acceptor, even if adopt SCNT technology, the counter-measures such as suicide gene are set to transplanted cells, also not necessarily can address this problem well.
For avoiding the ethics arguement of people ESCs and therapeutic cloning research, need to find a kind of alternative route, to the mankind's somatocyte is converted into pluripotent stem cell, for patient provides the autologous stem cells of " personalization ".2003, Gurdon research group of univ cambridge uk finds, the nucleus of the mouse chest cell of differentiation completely or one-tenth human peripheral lymphocyte is injected after Xenopus Oocytes, the differentiation mark of mammal nuclear is lost, in mammalian stem cell, the distinctive mark Oct4 of tool gene is high expression level, thereby prompting mammal nuclear can directly be expressed Oct4 gene by the reconstruct of Amphibians ovocyte nuclear vacuole institute.
2006, Yamanaka research group of Kyoto Univ Japan adopts outer-gene rotaring dyeing technology, from 24 factors, filter out Oct4, Sox2, Klf4, 4 versatility factors such as c-Myc gene, by retrovirus, above-mentioned 4 versatility factors are imported to mouse fetal inoblast or adult mice tail skin inoblast, under the culture condition of mouse ESCs, obtained pluripotent stem cell system, this clone is in cellular form, growth characteristics, gene expression profile, surface antigen marker, the epigenetics state of pluripotent stem cell specific gene, telomerase activation, form the aspects such as teratoma and mouse ESCs closely similar, therefore by its called after inductive pluripotent stem cells (Induced pluripotent stem cells, iPSCs).
Then, Yamanaka research group utilizes identical technology, and above-mentioned 4 same versatility factors are imported in human skin fibroblast, has successfully obtained people iPSCs.Primary human fibroblast's sample synovial cell and be derived from the fibroblastic clone of newborn infant and equally also can be reconstructed into as people iPSCs.Each side characteristic and the people ESCs of this class iPSCs are closely similar, and during the embryoid body cultivate forming in vitro and all can be divided into the different cell types of three germinal layers in the teratoma forming in Mice Body.Meanwhile, Thomson research group of winconsin university has also reported that successfully inducing fetal fibroblast reprogrammed is the iPSCs with people ESCs essential characteristic, difference is that they use slow virus as carrier, and has selected 4 genes such as Oct4, Sox2, Nanog, Lin28 to carry out transfection in 14 candidate genes.This important breakthrough that is called bio-science " milestone " by educational circles is expected to the ethics, the morals dispute that help scientist to walk around clone technology, for gate is opened in medical use.
Although scientists is having made significant achievement aspect people ESCs, from ESCs, iPSCs is to tissue and the organ that can transplant use in other words, and road is still hard and remote.From another angle, part scientist has started to find organ that can directly transplanting.But due to the limitation in human organ source, the mammiferous organ of other species, especially ungulates starts to enter in investigator's the visual field, and the pig typical example that is exactly one of them.Due to pig aspect organ morphology, volume and physiological function with people's similarity, and the life-span is longer, and uses at present mouse widely to compare, and is more conducive to the mankind and carries out thoroughly deeply and scientific experiment targetedly.Mouse ESCs uses example to tell us widely, on ESCs, can realize genetic modification very efficiently and reproduction is chimeric, and then produces and have specific trait animal offspring.Equally, use genetic engineering means to carry out genetic manipulation to the ESCs of pig, can cultivate the pig of medical prospect as the colony of anti-Hyperacute immunological rejection, be expected to realize the mankind's organ transplantation treatment.In the past few decades, the scientific research for pig has obtained gratifying progress.Yet, although many scientists once attempted the ESCs of separated pig, all end in failure, comprise ox, sheep etc., that obtain is only also class ESCs, these cells are the state of long term maintenance versatility and self in vitro.Therefore, the current genetic manipulation for pig cell is mostly based on somatic cell nuclear transfer technique.But, because ovocyte is not thorough for the reprogrammed of somatic cell nuclear, the offspring of generation often has deformity, and the nuclear transplantation production cycle is long, thereby on the low side in efficiency, waste time and energy.Thus, pig iPSCs becomes the selection that instantly has application prospect.
In addition, by people iPSCs be applied to clinical before, its medical effect of bringing into play awaits animal experiment assessment, safety problem knurl problem also need strictly to be detected.Because the life-span of mouse is shorter, and the raising of primate monkey is comparatively difficult, cost is very high, also relates to ethics morals problem.Thereby, except for organ transplantation, pig can candidate as another living model, also start to be subject to a lot of scientists and clinician's high praise, as carried out clinical efficacy, the security of monitoring analysis iPSCs in tissue regeneration medical research by setting up the pig model of the multiple heredopathia proterties of the mankind, for accelerating the clinical application of mankind iPSCs, provide science data.In addition, at pharmacy field, be to take mouse as laboratory animal carries out pharmacology, drug efficacy study for a long time, based on pig iPSCs, produce the popularization of the disease model obtaining, undoubtedly also by the reliability of greatly strengthening testing before clinical drug, improve the quality of medical experiment.
Current, there is the research group of a plurality of countries to obtain pig iPSCs, although a series of exogenous factors of verified transfection can be so that the fibroblastic epigenetic modification of pig and genetic transcription be reset to the state that approaches ESCs, but the induction efficiency of pig iPSCs is still very low, efficiency value is about 0.1-0.2%, and it is longer that positive iPSCs clone forms the required cycle, generally about more than 2 weeks.Meanwhile, be limited by pig ESCs is cultivated and lacks enough understanding and experience, the imperfection in reprogramming of somatic cells condition setting, the versatility level of the clone of setting up is also also insufficient, and these all will be an impediment to its subsequent applications industrially.Different cell types is different on reprogramming efficiency, and identical cell type reprogramming efficiency under different inductive conditions is also different, thereby finds more efficient pig iPSCs reprogrammed technical system, is to need the problem overcoming badly in research from now on.
Summary of the invention
The problem that above-mentioned induction pig iPSCs efficiency is low in order to overcome, the cycle is long, the object of the present invention is to provide the technological method of a kind of effective acquisition pig iPSCs, the method is under without feeder layer, serum-free culture condition, it is multipotential stem cell that ADSCs is induced to reprogrammed, and the specified phase in reprogrammed process, is used MEK signal pathway inhibitor PD0325901 and GSK3 signal pathway inhibitor CHIR99021 to process cell.
The method of a kind of effective acquisition pig inductive pluripotent stem cells of the present invention, being used for the fat stem cell that derives from pig (ADSCs) induction reprogrammed is inductive pluripotent stem cells, it is characterized in that, comprises the steps:
(1) cDNA of the versatility factor is imported to ADSCs, the wherein said versatility factor is Oct4, Sox2, Klf4, c-Myc gene;
(2) in without feeder layer system, the ADSCs that uses serum free medium or add the culture medium culturing step (1) for serum additive to obtain;
(3) occur, after embryonic stem cell like cell clone, in described substratum, adding MEK signal pathway inhibitor PD0325901 and GSK3 signal pathway inhibitor CHIR99021, continue to cultivate 3 days, then picking cell clone enlarged culturing;
(4) identification of cell clone's versatility, comprises and detects the alkaline phosphatase activities of cell, be differentiated to form teratomatous ability in the expression of the expression of endogenous versatility gene, embryonic stem cell (Embryonic stem cells, ESCs) marker, body.
According to the further feature of method of the present invention, in described step (1), the cDNA of the versatility factor imports described ADSCs by virus vector.Preferably, described virus vector is lentiviral vectors.More preferably, described lentiviral vectors is the derivable RevTet-On type of medicine expression vector.RevTet-On type Lentiviral makes the expression of the external source versatility factor in iPSCs that reprogrammed obtains have Modulatory character at any time, as iPSCs carried out after the genetic manipulations such as genetic modification, can select to keep original state, can be divided into normal somatocyte again, be convenient to carry out as required corresponding utilization and process.
According to method of the present invention, in step (2), adopt without feeder layer system, can improve positive iPSCs clone's purity.Preferably, for described without feeder layer system, need to be on culture dish coated matrigel in advance.Matrigel can be assembled the three dimensional matrix that formation has biologic activity automatically, and structure, composition, physical property and the function of analogue body inner cell basilar membrane, be conducive to the cultivation of cell in vitro.
According to method of the present invention, in step (2), preferably adopt the substratum of serum-free, it is formulated by each component for chemically defined property, composition is clear, clear and definite, is convenient to carry out further optimize improvement, transformation.Preferably,
According to the further feature of method of the present invention, the serum free medium in described step (2) can add for serum additive, preferably 15%(volume percent) serum substitute (KnockOut for example
tMsR) or the high fat bovine serum albumin of 5mg/mL (for example
iI).For serum additive, can avoid not clear and definite component and batch difference in serum to bring labile factor to iPSCs cultivation conditions.
According to the further feature of method of the present invention, described serum free medium also comprises: 40%DMEM/F-12 substratum, 40%
substratum, 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 that mouse is originated.The present invention also can adopt the leukaemia inhibitory factor of other source of species, but the leukaemia inhibitory factor in mouse source is relatively more cheap on cost.
According to method of the present invention, the inoculum density of the ADSCs cultivating in described step (2) is one of key factor of the present invention.If cell inoculum density is too low, the cell radix of reprogrammed is very little, can reduce positive iPSCs clone's yield, is unfavorable for that behavior is done in the secretion of the useful factor of iuntercellular mutually simultaneously; Otherwise, if cell inoculum density is too high, spacing is too small, different unicellular propagation and the cell clone that comes can come in contact to such an extent as to converge to together during reprogrammed, thereby cause the iuntercellular of different reprogrammed levels to produce crossed contamination, cannot obtain the positive iPSCs clone of complete reprogrammed.Preferably, the ADSCs cultivating in described step (2) is with 2,500 cell/cm
2density be inoculated on culture dish.Experiment shows, the ADSCs under this inoculum density can guarantee successfully to complete reprogrammed process, obtains simple positive iPSCs clone.
According to the further feature of method of the present invention, the MEK signal pathway inhibitor adding in described step (3) can be the PD0325901 product (article number is S1036) of Selleckchem company; The GSK3 signal pathway inhibitor adding can be the CHIR99021 product (article number is S2924) of Selleckchem company.Experiment shows, the GSK3 signal pathway inhibitor of the MEK of 0.5 μ M and 3 μ M, not only can promote the generation of cell reprogrammed, at the later stage of induction reprogrammed, mek inhibitor can also impel not by cell generation apoptosis reprogrammed and that part reprogrammed then move towards is again broken up.
Compared with prior art, the present invention has following beneficial effect:
Disclosed other are different for obtaining the strategy of pig iPSCs from prior art, method of the present invention has been carried out comprehensive optimization as compared with the past, on the cell material of source, chosen the adult stem cell ADSCs that is easy to efficient reprogrammed, in reprogrammed environment, used saferly without feeder layer, serum-free system, at the later stage of reprogrammed process, applied the pathway inhibitor of shielding cytodifferentiation signal.Therefore, ADSCs used not only can induce and form pig iPSCs, and the efficiency of induction reprogrammed is significantly greater than conventional inoblast, pig ADSCs transcribes after the versatility factor, under without feeder layer, serum-free condition, can induce as iPSCs efficient, high-purityly, efficiency is about 1.53% left and right, compare with inoblast, the efficiency of induction reprogrammed improves over 6 times, and cell completes the required cycle shortening of reprogrammed process more than at least 4 days; The expression of these iPSCs versatility markers that produce and ESCs(are with reference to mouse, people) close, the expression of exogenous factor can be by complete silence simultaneously; The specified phase of rearranging at ADSCs is used specific signal pathway inhibitor to process, obviously improved the quality of pig iPSCs, the some genes associated with reprogrammed level, the expression in epigenetic modification site demonstrate cell and have reached abundant reprogrammed state, for the research in this pig multipotential stem cell field provides good platform.In addition, with respect to being difficult at present, directly from the embryo of pig, extract ESCs, and required higher human and material resources, time cost in leaching process, the method will be passed through the iPSCs of effective acquisition pig, for genetic manipulation provides great convenience, production and the popularization of pig, human genetic disease's swine model for the organ transplantation that promotion is set up based on pig iPSCs, serve the clinical practice of physianthropy.
Accompanying drawing explanation
Fig. 1 is the pig iPSCs aspect graph that ADSCs and induction reprogrammed thereof obtain.
Fig. 2 is the schematic diagram of pig iPSCs induction reprogrammed process.
Fig. 3 is alkaline phosphatase staining result and the reprogramming efficiency analysis chart thereof of pig iPSCs.
Fig. 4 is versatility gene and the ESCs marker representation evaluation figure of pig iPSCs.
Fig. 5 is the teratoma differentiation evaluation figure of pig iPSCs.
Embodiment
1. define and technology:
Except as otherwise noted, practice of the present invention will be used the conventional art of molecular biology, cytobiology, and it belongs to art technology scope.Referring to < < molecular cloning experiment guide > >, the people such as J.Sambrook write (2008); < < fine works molecular biology experiment guide > >, the people such as F.M.Ausubel write (2008); < < animal cell culture---basic fundamental guide > >, the people such as R.I.Freshney write (2008); < < stem cell handbook > >, the people such as R.Lanza write (2013).
Some terms that use in the present invention have the implication of following definition: all Digital IDs, for example pH, temperature, time, concentration and molecular weight, comprise scope, is all approximation.Understand, although not always clear and definite narration all adds term " about " before all Digital IDs.Also will understand, although not always clear and definite narration, the reagent of describing in the present invention is only example, and its Equivalent is known in the art.
" inductive pluripotent stem cells (iPSCs) " of the present invention is such cell, it is under embryonic stem cell (ESCs) culture condition, express in cellular form, growth characteristics, surface marker with ESCs, that inside and outside can be differentiated to form the aspects such as weave construction that comprise three germinal layer cells is closely similar, and also quite similar at aspects such as genomic DNA methylation level mode, gene expression profile, chromatin states.
Fat stem cell of the present invention (ADSCs) is from mammiferous ADSCs, and it is that separation and Extraction obtains from the back of the body, abdominal subcutaneous adipose tissues, has plasticity-.Fatty tissue is positioned at skin below, belong to a kind of loose connective tissue, distribute in a large number in animal body, according to the difference of adipocyte structure and function, fatty tissue is divided into white (yellow) fatty tissue, brown adipose tissue, and experiment is drawn materials and used is belonged to the former.Fatty tissue, except the adipocyte by trooping in a large number forms, is also rich in the ADSCs with self-renewal capacity, can participate in the injury repairing of the tissues such as skin.
The ADSCs cultivating is that fibroblast-like short fusiformis is adherent, vortex shape growth, and form is full and refractivity is very strong; Secrete some adhesion molecules, extracellular matrix proteins, STEM CELL FACTOR and somatomedin, express mescenchymal stem cell specific marker thing CD44, CD90, CD29; Under certain inductive condition, can realize plasticity-, show as laterally or differentiation capability longitudinally, as to adipocyte, osteocyte, Chondrocyte Differentiation.
The ADSCs of various animal-origins all can be easily separated from animal, is convenient to process, and to its research, also can not relate to ethics morals problem.For example from the postoperative fatty tissue waste of physianthropy beauty treatment related surgical, separation obtains, or extracts from fatten the fatty tissue of domestic animal.
Term of the present invention " induction reprogrammed " refers to somatocyte is dedifferented to the process into multipotent stem cells.Preferably, by being described ADSCs by maintaining the required versatility factor cDNA importing somatocyte of stem cell versatility, can be dedifferentiated into as multipotent stem cells by inductor cell.Wherein, preferably, the described versatility factor comprises Oct4, Sox2, Klf4, and c-Myc gene.Most preferably, described versatility factor behaviour source Oct4, Sox2, Klf4, and c-Myc gene.Particularly, the described versatility 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.
It can be multiple technologies well known to those skilled in the art that described versatility factor cDNA is imported to somatic method, comprises the various methods that DNA proceeded to cell such as virus infection, liposome transfection, electroporation.Preferably, use the virus vector that comprises cDNA to carry out transfection, described virus vector comprises the multiple virus vector such as lentiviral vectors, retroviral vector.Preferably lentiviral vectors (for example can be subject to the certain drug RevTet-On type carrier of regulating and expressing at any time), as be shown in the examples.
" without feeder layer, serum-free culture condition " of the present invention is the optimization process on conventional stem cell cultivation conditions basis, this area, and comprises some suitable each concrete clones, but do not affect the modification of cell essential property.Cultural method and culture condition are referring to < < stem cell handbook > >, and the people such as R.Lanza write (2013).
The process of the technological method preferred embodiment of effective acquisition pig inductive pluripotent stem cells of the present invention is as follows, and Fig. 1 is the pig iPSCs aspect graph that ADSCs and induction reprogrammed thereof obtain; Fig. 2 is the schematic diagram of pig iPSCs induction reprogrammed process; Fig. 3 is alkaline phosphatase staining result and the reprogramming efficiency analysis chart thereof of pig iPSCs; Fig. 4 is versatility gene and the ESCs marker representation evaluation figure of pig iPSCs; Fig. 5 is the teratoma differentiation evaluation figure of pig iPSCs.
2. embodiment
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.
Given an example contriver's the standard laboratory practice of following implementation column, for example pattern of the present invention, and should not be interpreted as the present invention the scope that is defined in these embodiment.These embodiment, and those skilled in the art's open according to the present invention general level, technician will understand following only for example, can in being no more than scope of the present invention, carry out various changes, modification and transformation.Wherein related technology, unless stated otherwise, is all routine techniquess of the aspects such as molecular biology well known to those skilled in the art, cytobiology.
Preparation and the cultivation of embodiment 1. cells
The cultivation of 1.1 pork fat stem cells (ADSCs)
From the pig back of the body, subcutaneous abdomen, isolate fatty tissue, remove blood vessel, muscle residue, fully washing of DPBS damping fluid (Gibco company) washing, then with scissors for surgery, fatty tissue is fully shredded to almost without shearing resistance, be transferred in centrifuge tube, add 0.09% the type i collagen enzymic digestion liquid (Sigma company) that is equivalent to 2～3 times of fatty tissue cumulative volumes, be placed in vibration, digestion process in 37 ℃ of water-baths; After tissue suspension gelatinization, with pasteur pipet, repeatedly blow and beat the chip with dispersion tissue, 1, the centrifugal 5min of 200rpm room temperature, discard the ripe fatty tissue on centrifuge tube upper strata, the Digestive system in middle level, then with containing the DMEM/F-12 basic medium (HyClone company) of the 10% foetal calf serum cell of resuspended centrifuge tube bottom fully; With aperture, be the nylon mesh screen filtration cell suspension of 250 μ m, 80 μ m, 25 μ m successively, discard basic medium after centrifugal, with the cell precipitation thing of fresh basic medium repetitive scrubbing centrifuge tube bottom; Recentrifuge also discards after basic medium, with the abundant re-suspended cell throw out of pig ADSCs perfect medium, and is inoculated in Tissue Culture Flask, puts into 37 ℃, 5%CO
2cell culture incubator in cultivate.In described pig ADSCs perfect medium, comprise DMEM/F-12 substratum (HyClone company), 10% foetal calf serum (Gibco company), 10ng/mL Prostatropin (Peprotech company), 50 μ g/mL L-AAs (Sigma company), 2mM L-glutaminate (Gibco company).
The cultivation of 1.2 pig inductive pluripotent stem cells (iPSCs)
In pig iPSCs serum-free perfect medium, comprise 40%DMEM/F-12 substratum (Gibco company), 40%
substratum (Gibco company), 15% serum substitute (KnockOut
tMsR) or the high fat bovine serum albumin of 5mg/mL (
iI) (Gibco company), 1%N-2 additive (Gibco company), 1%B-27 additive (Gibco company), 1%
additive (Gibco company), 0.1mM beta-mercaptoethanol (Gibco company), 1000U/mL leukaemia inhibitory factor (Millipore company), 2 μ g/mL doxycyclines (Clontech company).In addition, the working concentration of the micromolecular inhibitor PD0325901 that processing cell clone is used is 0.5 μ M, and the working concentration of CHIR99021 is 3 μ M.
The cultivation of 1.3 other cells
293T cell, as the package cell line of slow virus, is used the substratum identical with inoblast, and all cells is placed on 37 ℃, 5%CO always
2cell culture incubator in cultivate.
Embodiment 2. viral vector infection pig ADSCs
According to the method described in embodiment 1, in Tissue Culture Flask, inoculate the pig ADSCs of low generation, 37 ℃, 5%CO
2cellar culture condition under when being cultured to degree of converging and reaching 80～90%, with 0.25% trypsinase-EDTA(Gibco company) after 37 ℃ of digestion are single cell suspension, with the viral supernatant liquor of collecting, infect 1 * 10
5individual ADSCs, infection multiplicity (MOI) for the titre of 3(virus used be 5～10 * 10
6iU/mL, carries four kinds of viruses of versatility factor cDNA and mixes by 1:1:1:1), be then inoculated in 6 well culture plates, continue at 37 ℃, 5%CO
2cellar culture condition under cultivate.Described viral supernatant liquor is that the medicine of the cDNA that comprises people Oct4, Sox2, Klf4 and c-Myc by use can be induced (RevTet-On) type Lentiviral (SiDanSai company) transfection 293T cell (Fugene HD according to a conventional method, Roche company) (referring to < < molecular cloning experiment guide > >, the people such as J.Sambrook write (2008)) obtaining.
Continuation cultivation and the colony screening of embodiment 3. cells infecteds
At metainfective the 2nd day, use DPBS buffer solution for cleaning culture hole twice, then by metainfective 0.25% trypsinase-EDTA(Gibco company for ADSCs) after 37 ℃ of digestion are single cell suspension, by 2,500 cell/cm
2density be inoculated in 6 new well culture plates, inoculate altogether 4 culture hole, culture plate ware face is coated matrigel (BD Pharmingen company) in advance, continues to use ADSCs perfect medium.Here in the Duplicate Samples of 4 plate holes, 1 plate hole, for alkaline phosphatase (AP) dyeing (SiDanSai company), is added up positive colony number, and another 3 plate holes are selected for clone, and experiment has repeated three times.At metainfective the 3rd day, ADSCs perfect medium is replaced by the pig iPSCs serum-free perfect medium described in embodiment 1, continue at 37 ℃, 5%CO
2cellar culture condition under cultivate.Be cultured to about the 5th day, having typical cell colony occurs, within approximately 6th～8 days, occur after ESCs like cell clone, in pig iPSCs serum-free perfect medium, add the MEK signal pathway inhibitor PD0325901 of 0.5 μ M and the GSK3 signal pathway inhibitor CHIR99021 of 3 μ M, process the cell in reprogrammed process.
It needs to be noted, metainfective ADSCs is inoculated in Tissue Culture Plate with too high or too low density, and too early or excessively late PD0325901 and the CHIR99021 of adding, also comprise and only use one of them micromolecular inhibitor, the rate of formation of pig iPSCs positive colony all can reduce greatly.
Be cultured to about the 10th day, by for cloning 3 plate holes selecting, use the single clone of the typical ESCs sample that glass needle segmenting edge is smooth, nucleus is clear, form is compacted, then with Glass tubing, drawing these clones that separate is inoculated in the hole that two 96 well culture plates are corresponding by same form difference, a clone of each hole inoculation, the feeder layer cells that culture plate ware face is prepared with mouse fetal inoblast is in advance coated, 37 ℃, 5%CO
2cellar culture condition under cultivate.At 96 well culture plates, cultivate after approximately one week, a culture plate of choosing wherein carries out AP dyeing.The clone of the AP positive is used to TryPLE Express(Gibco company) after 37 ℃ of digestion are single cell suspension, in the ratio of 1:6-12, go down to posterity, successively from 96 holes, 24 holes, 12 well culture plates, to 6 well culture plates amplifications.In these iPSCs candidate clones, we have selected 2 to do further evaluation.
Clone that 16 well culture plate plate holes of picking, directly carry out AP dyeing, according to clone's form and AP coloration result, to clone's counting of typical ESCs, (use Single-sens reflex camera to take pictures under microspur, then use " ImageJ " software statistics), calculate induction effectiveness formula: infected cell quantity * 100% of induction efficiency=AP positive colony quantity ÷.After repeating statistical study, test finds inoculation 2.5 * 10
4individual metainfective pig ADSCs, can form 382 AP positive colonies, and the overall efficiency of our this method is about 1.53%, compares over 6 times with the efficiency of the control group inoblast approximately 0.25% arranging the same period.
Embodiment 4.Real-Time PCR identifies the expression level of versatility gene in pig iPSCs clone
Use RNeasy Mini(QIAGEN company) test kit, extracts the total RNA of pig iPSCs according to manufacturers's explanation; With QuantiTect Reverse Transcription(QIAGEN company) test kit carries out reverse transcription, and use FastStart SYBR Green Master(Rox) test kit (Roche company), StepOnePlus quantitative real time PCR Instrument (Applied Biosystems company) carries out Real-Time PCR.All above-mentioned PCR conditions are all used conventional PCR condition, according to manufacturers's explanation, carry out.
In the pig iPSCs clone that use the application's method obtains, not only endogenic Oct4, Sox2, Nanog, Dnmt3b, Tert gene are activated, Lin28, the Esrrb associated with cell reprogrammed degree, Utf1, Dppa5 gene be abundant up-regulated expression also, and these versatility factors all do not have expression or the lower expression that can detect in the ADSCs without induction reprogrammed.
The expression of ESCs marker in pig iPSCs clone is identified in embodiment 5. protein immunization fluorescent dyes
After the pig iPSCs that the washing of DPBS damping fluid is cultivated 2～3 days clones, use 4% paraformaldehyde (Solarbio company) fixing; Use 0.5%Triton X-100(Solarbio company) penetrating cell (limit core internal labeling quality testing is surveyed), then Yong Han 1%BSA(Sigma company) DPBS damping fluid sealing treatment; During this time after repetitive scrubbing, add successively primary antibodie (antibody of anti-Oct4 and Nanog purchased from the antibody of Abcam company, anti-Sox2 purchased from the antibody of Cell Signaling company, anti-SSEA-3 and SSEA-4 purchased from Developmental Studies Hybridoma Bank company), two anti-(Alexa Fluor594 is purchased from Molecular Probes companies) to hatch; Finally use DAPI dyestuff (Sigma company) positioning cells core, use conventional fluorescence microscope.Result shows pig iPSCs clone high expression level ESCs marker Oct4, Sox2, Nanog, SSEA3 and SSEA4 albumen.
By confirming whether the detections pig iPSCs clone of institute has the characteristic of long term maintenance Multidirectional Differentiation of Cells potential, and the teratoma formation that our cell based on going down to posterity more than 10 generations carries out is tested.
Use TryPLE Express after 37 ℃ of digestion are for single cell suspension pig iPSCs clone, renewed vaccination, in new culture dish, is placed in 37 ℃, 5%CO
2standing 45 minutes of incubator, gets the not adherent cell in upper strata (feeder layer cells is adherent fast, therefore can feeder layer cells is separated with pig iPSCs); Counting, gets 5,000,000 pig iPSCs single-cell suspensions and contains 15%KnockOut in 300 μ L
tMin the DMEM/F-12 of SR, NOD/SCID innate immunity deficient mice is carried out to back leg root muscle aseptic injection; After injected in mice, be placed between SPF level laminar flow and raise, during do not stop the to feed doxycycline (being dissolved in aseptic sucrose solution) of 2mg/mL, within approximately 3～5 days, change time water, have gradually the lump of grain of rice size to grow, until knurl block length, after can getting, medicine is removed greatly; After withdrawal, normal nursing 3～4 weeks, takes out teratoma with eye scissors, surgical forceps, is fixed in 4% paraformaldehyde; Teratoma after fixing, through paraffin embedding, section, and teratoma differentiation situation is observed in hematoxylin-eosin (Hematoxylin-Eosin) dyeing.Result demonstration, pig iPSCs successfully differentiates the weave construction of the dissimilar cell with three germinal layers, as endoblastic enteric epithelium structure, mesoblastic lipid structure and ectodermic Eponychium structure,
By upper result, further prove and use the application's the method can effective acquisition pig iPSCs, and there is the characteristic of ESCs.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (10)
1. a method for effective acquisition pig inductive pluripotent stem cells, for being inductive pluripotent stem cells (iPSCs) by fat stem cell (ADSCs) the induction reprogrammed that derives from pig, is characterized in that, comprises the steps:
(1) cDNA of the versatility factor is imported to ADSCs, the wherein said versatility factor is Oct4, Sox2, Klf4, c-Myc gene;
(2) in without feeder layer system, the ADSCs that uses serum free medium culturing step (1) to obtain;
(3) occur, after embryonic stem cell like cell clone, in described substratum, adding MEK signal pathway inhibitor and GSK3 signal pathway inhibitor, continue to cultivate 3 days, then picking cell clone enlarged culturing; And
(4) identification of cell clone's versatility, comprises and detects the alkaline phosphatase activities of cell, be differentiated to form teratomatous ability in the expression of the expression of endogenous versatility gene, embryonic stem cell (ESCs) marker, body.
2. method according to claim 1, is characterized in that, in described step (1), the cDNA of the versatility factor imports described ADSCs by virus vector.
3. method according to claim 2, is characterized in that, described virus vector is lentiviral vectors.
4. according to the method in claim 2 or 3, it is characterized in that, described lentiviral vectors is the derivable RevTet-On type of medicine expression vector.
5. method according to claim 1, is characterized in that, in described step (2) without feeder layer system need to be on culture dish coated matrigel in advance.
6. method according to claim 1, is characterized in that: in described step (2), add for serum additive, preferably 15%(volume percent in described serum free medium) serum substitute or the high fat bovine serum albumin of 5mg/mL.
7. according to the method for claim 1 or 6, it is characterized in that, described serum free medium also comprises: 40%DMEM/F-12 substratum, 40%
substratum, 1%N-2 additive, 1%B-27 additive, 1%
additive, 0.1mM beta-mercaptoethanol, 1000U/mL leukaemia inhibitory factor, 2 μ g/mL doxycyclines.
8. method according to claim 7, is characterized in that, described leukaemia inhibitory factor is that mouse is originated.
9. method according to claim 1, is characterized in that, the ADSCs cultivating in described step (2) is with 2,500 cell/cm
2density be inoculated on culture dish.
10. method according to claim 1, is characterized in that, in described step (3), the working concentration of MEK signal pathway inhibitor is 0.5 μ M; The working concentration of described GSK3 signal pathway inhibitor is 3 μ M.
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