CN103184187A - Method for directional differentiation of human induced pluripotent stem cells into corneal epithelioid cells - Google Patents
Method for directional differentiation of human induced pluripotent stem cells into corneal epithelioid cells Download PDFInfo
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Abstract
The invention provides a method for directional differentiation of human induced pluripotent stem cells into corneal epithelioid cells. The method comprises the steps of: A. on the basis of ocular surface tissues of patients, separating human conjunctival stromal fibroblasts and human conjunctival epithelium cells; B. leading gene Oct4, Sox2, c-Myc and Klf4 into the fibroblasts and the human conjunctival epithelium cells, conducting induction to generate iPSCs (induced pluripotent stem cells); and C. adding IV type collagen into the iPSCs, employing a KSFM cell medium and an SHEM cell medium to perform induction in turn, and carrying out directional differentiation of the iPSCs into corneal epithelioid cells. The method provided in the invention is fast, simple and efficient, and can obtain a lot of corneal epithelial stem cells.
Description
Technical field
The present invention relates to the stem cells technology field, particularly a kind of people's inductive pluripotent stem cells is to the method for corneal epithelium like cell directed differentiation.
Background technology
(Limbal Stem Cells, LSCs), namely limbal stem cell is positioned at the limbal epithelium stratum basale to corneal epithelial stem cells, is unique cell source of corneal epithelium.Corneal epithelial stem cells can be carried out self, to keep the height multiplication capacity of these stem cells.Corneal epithelial stem cells can generate of short duration expansion cell, and the latter moves to central cornea, finally enters end differentiation eventually, forms corneal epithelial cell.Corneal epithelial stem cells not only can be broken up, hyperplasia is epithelial cell, the more important thing is one barrier of corneal epithelial stem cells picture, can stop conjunctival epithelial cell to travel to anterior corneal surface, dynamic stability and the corneal transparency of keeping corneal epithelium are being brought into play important role.
Simple eye corneal epithelial stem cells lacks the patient, can adopt from the strong cornea edge tissue transplantation of body, and the transplanting behind the strong eye ex vivo expansion of stem cell.Lack the patient for the cornea of both eyes epithelial stem cell, can adopt the allosome limbal transplantation, but because rejection easily takes place in heteroplastic transplantation, clinical effectiveness is unsatisfactory.In recent years, having research to adopt transplants behind body oral mucosa epithelial cell amplification in vitro, but discover, the oral mucosa epithelial cell in vitro culture 3-4 week can not be divided into the corneal epithelial cell phenotype, also do not express a transcription factor PAX6 of superficial epithelium cell-specific, Saliva Orthana is expressed with the normal cornea epithelial cell and is also had larger difference.Transplant in the oral mucosa epithelial cell on eye surface and can not be divided into the corneal epithelial cell phenotype in vivo.Though postoperative cornea is in a short time kept transparency preferably, long term follow-up finds that cornea easily produces new vessel.This shows, the desirable cell source that corneal epithelium is rebuild remains the epithelium of autologous cornea stem cell, lack patient's treatment for the eyes stem cell, still await seeking and a kind ofly can be divided into normal cornea epithelial " seed cell ", this also is the emphasis of present corneal epithelial tissue engineering research.
Except adult stem cell, multipotential stem cell (pluripotent Stem Cells) comprises embryonic stem cell (Embryonic Stem Cells, ESCs) and inductive pluripotent stem cells (Induced Pluripotent StemCells, iPSCs)) also be used in external evoked generation corneal epithelial stem cells.Homma people such as (names) is incubated at the mouse embryonic stem cell on the IV Collagen Type VI, successfully induces epithelial stem cell, and is used for repairing the mouse corneal epithelial wound.Hiroki (name) etc. import mouse embryo stem cell with the PAX6 gene, successfully induce the epithelioid cell with corneal epithelial cell feature.There is research that human embryo stem cell is incubated on the IV Collagen Type VI recently, and personnel selection corneal limbus inoblast conditioned medium is cultivated altogether, successfully induce the epithelioid cell, but further the terminally differentiated cells of differentiation generation not only has the corneal epithelium phenotype, also has the skin phenotype.Because embryonic stem cell has infinite multiplication potential, make it become a kind of very potential seed cell source that the treatment corneal epithelial stem cells lacks.But, the differentiation regulation and control of embryonic stem cell, ethics problem, and the problems such as rejection of heteroplastic transplantation become the obstacle of human embryo stem cell research.Therefore, finding suitable seed cell source for the cornea stem cell transplantation, is present problem demanding prompt solution.
Summary of the invention
The invention provides a kind of fast, easy, efficient, and lower-cost people's inductive pluripotent stem cells is to the method for corneal epithelium like cell directed differentiation, to obtain corneal epithelial stem cells in a large number.
To achieve these goals, the invention provides following technical scheme:
A kind of people's inductive pluripotent stem cells is to the method for corneal epithelium like cell directed differentiation, and it comprises step:
A, based on the patient the eye table organization, separation of human conjunctiva matrix inoblast and people's conjunctival epithelial cell;
B, at described inoblast and people's conjunctival epithelial cell quiding gene Oct4, Sox2, c-Myc and Klf4 (gene title), induce to produce inductive pluripotent stem cells (iPSCs);
C, in described iPSCs, add the IV Collagen Type VI, adopt KSFM (Keratinocyte Serum-FreeMedium, KSFM) cell culture medium and SHEM (Supplement Hormonal EpithelialMedium, SHEM) the cell culture medium method of inducing in turn, to described inductive pluripotent stem cells iPSCs to corneal epithelium like cell directed differentiation.
Preferably, in described steps A, adopt people's conjunctiva matrix inoblast and people's conjunctival epithelial cell in eye table organization source to carry out the foundation of inductive pluripotent stem cells system;
Preferably, in described step B, adopt the method for alkaline phosphatase staining, immunofluorescence dyeing, teratoma experimental verification differentiation potential and gene chip analysis to carry out the clone evaluation.
Preferably, in described step C, also carry out corneal epithelium like cell phenotypic evaluation.
By implementing above technical scheme, have following technique effect: differentiation method provided by the invention is quick, easy, efficient, and cost is lower, can obtain corneal epithelial stem cells in a large number.
Embodiment
Technical scheme is for a better understanding of the present invention described embodiment provided by the invention below in detail.
The embodiment of the invention provides a kind of people's inductive pluripotent stem cells to corneal epithelium like cell directed differentiation method, and the method comprising the steps of:
A, based on the patient the eye table organization, separation of human conjunctiva matrix inoblast and people's conjunctival epithelial cell;
B, at described inoblast and people's conjunctival epithelial cell quiding gene Oct4, Sox2, c-Myc and Klf4, induce to produce iPSCs (Induced Pluripotent Stem Cells, iPSCs, inductive pluripotent stem cells;
C, in described inductive pluripotent stem cells, add the method that IV Collagen Type VI, KSFM cell culture medium and SHEM substratum are induced in turn, to described iPSCs to corneal epithelium like cell directed differentiation.
Further, in other embodiments, specifically comprise in the described steps A: adopt people's conjunctiva matrix inoblast and people's conjunctival epithelial cell in eye table organization source to carry out the foundation of inductive pluripotent stem cells system; Adopt the somatocyte in human eye table organization source to set up inductive pluripotent stem cells system, have more to the advantage of corneal epithelium like cell directed differentiation.Among the described step B, adopt the method for alkaline phosphatase staining, immunofluorescence dyeing, teratoma formation and gene chip analysis to carry out the evaluation of inductive pluripotent stem cells system.
Further, in other embodiments, in described step C, also carry out cell phenotype and identify.
In step B, inductive pluripotent stem cells (iPSCs) is induced the result:
Retrovirus has remarkable change since the 4th day cellular form after importing the multipotency gene, and most of cell proliferation is vigorous, and stature diminishes, and comparison rule becomes.About 24-30 days, iPSCs clones formation behind the virus infection, cell rounding, and the nucleus proportion increases, and cellular form is very similar to embryonic stem cell.
In described step B, the evaluation of inductive pluripotent stem cells system:
After the iPSCs clone forms, must identify whether the iPS cell of inducing has and the similar feature of embryonic stem cell, and namely versatility is identified.This experiment adopts the method for alkaline phosphatase staining, immunofluorescence dyeing, teratoma experimental verification differentiation potential and gene chip analysis that the iPS cell of inducing is identified respectively.
(1), the stem cell characteristic of iPS cell is identified in alkaline phosphatase staining
Undifferentiated ES cell height is expressed alkaline phosphatase, therefore generally with alkaline phosphatase staining the stem cell characteristic of iPS cell is carried out Preliminary detection.And can calculate iPS according to clone's number that dyeing shows and induce efficient.Retrovirus infected about 30 days, no matter be conjunctiva matrix inoblast (HumanConjunctival Stromal Fibroblast, HCjSF) and people's conjunctival epithelial cell (HumanConjunctival Epithelium, HCjE) all form many iPSCs clones, through alkaline phosphatase staining, these clone's major parts are positive, and show that these iPS cells have good embryonic stem cell characteristic.Find that the iPSCs of matrix inoblast (HCjSF) induces efficient to be approximately 0.1-0.5% after calculating cloning efficiency, the iPSCs of conjunctival epithelial cell (HCjE) induces efficient then apparently higher than HCjSF, can reach 1-2%.
(2), immunofluorescence dyeing detects the expression of embryonic stem cell mark
Undifferentiated human embryo stem cell is expressed some special stem cell markers, such as multipotential stem cell gene SSEA4, TRA-1-81, NANOG, OCT4, LIN28 (gene title) etc.By immunofluorescence dyeing, detected the expression of these stem cell markers in the iPS cell of inducing.The HCjSF-iPSCs that this experiment is induced and HCjE-iPSCs all express human embryo stem cell marks such as SSEA4, TRA-1-81, NANOG, OCT4, LIN28, and do not express muroid embryonic stem cell specific gene SSEA-1 (gene title), HCjSF-iPSCs that this experiment induces and HCjE-iPSCs are described similar to human embryo stem cell aspect the expression of embryonic stem cell mark, the embryonic stem cell characteristic is good.
(3), the versatility differentiation potential of tire knurl experimental verification inductive pluripotent stem cells (iPSC)
Inductive pluripotent stem cells (iPSC) is injected in the immunodefiiciency mouse body can forms teratoma, the histocyte that contains 3 kinds of germinal layer types, illustrate that the HCjSF-iPSCs that this experiment induces is similar to human embryo stem cell in versatility with HCjE-iPSCs, the embryonic stem cell characteristic of inductive pluripotent stem cells (iPSC) is good.
(4), gene chip is analyzed HCjSF-iPSCs and the similarity degree of ESCs on full gene expression profile
This experiment is chosen HCjSF-iPSCs and is carried out the gene chip analysis.Extract earlier total RNA (Yeast Nucleic Acid) of HCjSF-iPSCs, HCjSF and ESCs respectively, carry out with software the gained data being handled after gene chip analyzes.The gene chip analytical results shows that the HCjSF-iPSCs that this experiment is induced and ESCs have similarity highly at full gene expression profile, and both homologies are near 99%.In contrast, HCjSF-iPSCs and the parent cell HCjSF similarity degree on full gene expression profile is obviously not as HCjSF-iPSCs and ESC, and both homologies only are 46%.In dendrogram, HCjSF-iPSCs and ESCs belong to same cluster, and the parent cell HCjSF of iPSCs then belongs to another cluster.These results show that HCjSF-iPSCs that this experiment is induced and ESCs have higher similarity at full gene expression profile, the cell reprogrammed in order, the embryonic stem cell characteristic is obvious.
In described step C, HCjSF-iPSCs identifies to directed differentiation and the cell phenotype of corneal epithelial stem cells
(1), induces the situation of HCjSF-iPSCs directed differentiation with the SHEM conditioned medium at the IV Collagen Type VI
Corneal epithelial stem cells is located immediately on the basilar membrane, and the IV Collagen Type VI is the important component of basilar membrane, may play an important role to keeping the corneal epithelial stem cells characteristic.The micro-factor contained in the SHEM substratum is conducive to keep the epithelial stem cell characteristic, the cloning that generally is applied to epithelial stem cell is cultivated, and also is a kind of substratum that is widely used in corneal limbus and people's conjunctival epithelial cell cloning cultivation in the present field of ophthalmology.Therefore, the HCjSF-iPSCs that induces gained is inoculated in the culture plate of 0.5mg/ml IV Collagen Type VI bag quilt, treats cell attachment and long after a certain size, induce HCjSF-iPSCs to break up for 1 week with the SHEM substratum, during the variation of routine observation cellular form.Along with the prolongation of condition incubation time, it is flat that cell becomes gradually, and original embryonic stem cell form fades away.After 1 week, these cells are carried out immunofluorescence dyeing, the cell phenotype after the detection directed differentiation, and iPSCs enters the ability of end differentiation eventually.Noble cells is expressed albumen p63, ABCG2 and the K19 (gene title) of some epithelial stem cell mark, but does not see the expression of the whole last corneal epithelial cell specificity marker things that break up such as K12 (gene title).Point out this method can induce HCjSF-iPSCs to break up to the epithelial stem cell direction to a certain extent.
(2), induce the situation of HCjSF-iPSCs directed differentiation with the KSFM conditioned medium at the IV Collagen Type VI
The KSFM substratum is a kind of keratinization serum free medium, be mainly used in cultivating keratinocyte, the mouse corneal epithelial stem cells is that TKE2 (clone title) can keep the stem cell characteristic in the KSFM substratum, and corneal epithelial cell and people's conjunctival epithelial cell also can be bred in the KSFM substratum.Therefore, the HCjSF-iPSCs that induces gained is inoculated in the culture plate of 0.5mg/ml IV Collagen Type VI bag quilt, treats cell attachment and long after a certain size, induce HCjSF-iPSCs to break up for 1 week with the KSFM substratum, during the variation of routine observation cellular form.The HCjSF-iPSCs differentiation is cloned peripheral cytodifferentiation early in order when just beginning, and cellular form is very similar to the corneal epithelial cell in being incubated at the KSFM substratum.Yet along with the prolongation of time, these noble cellss are dead gradually, fail to continue differentiation state and effective propagation.Above result shows that iPSCs cultivates at KSFM and concentrates propagation to be suppressed.Even so, this result can illustrate to a certain extent still that the KSFM substratum has and induces HCjSF-iPSCs to enter the eventually potentiality of end differentiation.
The application examples of aforesaid method is provided below, and this application examples comprises step:
(1), people's conjunctiva matrix inoblast (Human Conjunctival Stromal Fibroblast, HCjSF) and people's conjunctival epithelial cell (Human Conjunctival Epithelium, cultivation HCjE).
(2) injure the surperficial biopsy of eye that full-shape film limbal stem cell that serious diseases of eye surface such as thermal burn causes lacks the patient from Stevens-Johnson syndrome, eye chemistry, obtain 3 * 3mm size conjunctival tissue, place the KSFM substratum to cultivate a week whole tissue block (epithelium up), treat to cut out central tissue's piece after epithelial cell grows, continue in the KSFM substratum and cultivate one week of epithelial cell.
Reagent preparation: inoblast substratum: DMEM substratum, 10%FBS, 100U/ml mycillin, 100U/ml amphotericin B.KSFM substratum (500ml): 500ml KSFM substratum, 0.9ml ox pituitary gland extract (bovine pituitary extract, BPE), 7.5 μ g Urogastrons (EGF), 100U/ml PS, 100U/ml amphotericin B.IPS cell induction flow process comprises:
(1) induces required 4 kinds of retrovirus (OCT4, SOX2, c-MYC and KLF4) by liposome transfection method packing, pack pMX-GFP fluorescence virus simultaneously in contrast.
(2), infect the day before yesterday with HCJSF and HCjE digestion back counting, be inoculated in 12 orifice plates, 2 * 104/ holes.
(3), infected the same day (Day 0, is designated as D0), collect 48h virus supernatant after, in the 15ml centrifuge tube, every pipe adds the 2.5-3ml fresh culture to every 10cm plate virus supernatant again with 0.45 μ m membrane filtration, adding final concentration 6-8 μ g/ml polybrene, mixing.HCjSF and HCjE substratum are sopped up, add viral supernatant, SOX2, KLF4, OCT4, four kinds of viruses of c-MYC (KOSM) equal-volume is used pMX-GFP virus infection HCjSF and HCjE in contrast simultaneously.
(4), second day (Day 1, is designated as D1) does viral secondary infection, same viral the infecting for the first time of step.D2, secondary infection is intact, is changed to iPS-1 substratum (no VPA).Usually, GFP infects efficient can be more than 70% at D2, D5, and the recovery mouse becomes fiber trophocyte (MEF) to 10cm plate, 1-1.25 * 106/ plate.D6 is changed to HCJSF that iPS-1 substratum KOSM infects or HCjE with MEF trophocyte's substratum and counts after with 0.05% trysinization, and inoculation 1-10 * 104/10cm plate is to the trophocyte.D7 is changed to iPS-1 substratum+Valproic acid (valproic acid, VPA) (VPA handled 7-10 days continuously usually).D14-17 is changed to the iPS-2 substratum.D24-30 observes and Taking Pictures recording iPSCs clone forms, and can screen afterwards and cultivate.The reagent preparation:
IPS-1 substratum (500ml): 385ml DMEM/F12,50ml Defined FBS (Hyclone), 50ml KSR, 5ml L-GlutaMax (100 *), 5ml NEAA 10mM (100 *), 1ml2-Mecaptoenthanol 55mM (buying from GIBCO), 500 μ l bFGF, 8 μ g/ml (1000 *).(microbiotic is optional to be added, and by a false add of normal content, uses in substratum 2-3 week)
IPS-2 substratum (500ml): 385ml DMEM/F12,100ml KSR, 5ml L-GlutaMax (100 *), 5ml NEAA 10mM (100 *), 1ml 2-Mecaptoenthanol 55mM (GIBCO), 500ulbFGF 8 μ g/ml (1000 *).(microbiotic is optional to be added, and by a false add of normal content, uses in substratum 2-3 week).
Prostatropin (bFGF) solution (8 μ g/ml): 10 μ g bFGF, 1.25ml 0.1%BSA in PBS.(0.22 μ M filters ,-20 ℃ of preservations).
Valproic acid (valproic acid, VPA solution 200 *): VPA is added in the DMEM/F12 substratum, be made into 200mM solution.(0.22 μ m filters ,-20 ℃ of ℃ of preservations).
The cultivation of iPS cell with go down to posterity, comprising:
Need go down to posterity when (1), treating iPSCs clone length to a certain size.4 ℃ of dissolvings the day before yesterday Geltrex goes down to posterity.According to the experiment consumption, every 1ml Geltrex (buying from GIBCO) adds the DMEM/F12 substratum (the substratum title is bought from GIBCO) of 4 ℃ of preservations of 29ml, is made into Geltrex solution, mixing.According to dull and stereotyped size, add an amount of Geltrex solution (such as the 1ml/35mm hole); Place 37 ℃ to place 1 hour the culture plate that Geltrex solution is housed, wait for that Geltrex solution forms Geltrex glue.Then culture plate is taken out and placed super clean bench 1 hour, namely can be used for the iPS passage.Iuntercellular shone ultraviolet 30 minutes, on spirit lamp glass was carefully held drawing-down, curved to make the round end suction pipe.With the round end suction pipe iPSCs that chooses clone is separated with the MEF trophoderm under the mirror and be cut into several fritters, sucking-off is put into the plate that Geltrex is coated with confluent monolayer cells is housed.General iPS cell needs every day changes fresh culture, to guarantee that cell state is good, keeps highly dedifferenting state.At a small amount of differentiation occurring, scrape off noble cells as early as possible, occur if break up serious and continue, abandon.Only stay the clone of the similar ES cell of form stable.Induce the iPS cell to the corneal epithelial stem cells directed differentiation, comprising:
(1) with IV Collagen Type VI bag by culture plate:
1, preparation 0.5mg/ml IV Collagen Type VI solution.
2, add an amount of IV Collagen Type VI solution bag by culture plate according to the culture plate area.(adding 200 μ l IV Collagen Type VI solution as the 2cm2 hole), 4 ℃ of overnight incubation.
3, before second day inoculating cell, sop up unnecessary IV Collagen Type VI solution, wash 1 time with 1 * PBS, namely can be used for cell inoculation.
The reagent preparation:
IV Collagen Type VI solution: the IV Collagen Type VI is dissolved in 0.25% acetic acid, is made into 0.5mg/ml IV Collagen Type VI solution.4 ℃ dissolved 3 hours, intermittent vibration.
(2) conditioned medium is to iPS cell directional induced differentiation:
With the iPS cell inoculation in the culture plate of IV Collagen Type VI bag quilt.
Treat that the clone is long after a certain size, induce in turn with SHEM substratum, KSFM substratum and this two kinds of substratum respectively and (induced 2 days with the KSFM substratum earlier, changing the SHEM substratum again into induced 2 days, and change the KSFM substratum into and induced 2 days) method induces the iPS cell to carry out directed differentiation so repeatedly.
Cell phenotype after the iPS cell directional differentiation is identified: induce in the process to cellular form observations of taking pictures continuously, to record the cellular form change procedure.Conditioned medium is fixed cell after inducing for 1 week, carries out immunofluorescence dyeing, detects differentiation effect from protein level.
More than a kind of people's inductive pluripotent stem cells that the embodiment of the invention is provided be described in detail to corneal epithelium like cell directed differentiation method, for one of ordinary skill in the art, thought according to the embodiment of the invention, part in specific embodiments and applications all can change, in sum, this description should not be construed as limitation of the present invention.
Claims (3)
1. people's inductive pluripotent stem cells is characterized in that to the method for corneal epithelium like cell directed differentiation, comprises step:
A, based on the patient the eye table organization, separation of human conjunctiva matrix inoblast and people's conjunctival epithelial cell;
B, at described inoblast and people's conjunctival epithelial cell quiding gene Oct4, Sox2, c-Myc and Klf4, produce inductive pluripotent stem cells iPSCs;
C, the method that in described iPSCs, adds the IV Collagen Type VI, adopts KSFM cell culture medium and SHEM cell culture medium to induce in turn, to described inductive pluripotent stem cells iPSCs to corneal epithelium like cell directed differentiation.
2. the method for claim 1 is characterized in that, described steps A specifically comprises: adopt people's conjunctiva matrix inoblast and people's conjunctival epithelial cell in eye table organization source to carry out the foundation of inductive pluripotent stem cells system; Among the described step B, adopt the method for alkaline phosphatase staining, immunofluorescence dyeing, teratoma formation and gene chip analysis to carry out the evaluation of inductive pluripotent stem cells system.
3. the method for claim 1 is characterized in that, in described step C, also carries out corneal epithelium like cell phenotypic evaluation.
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| CN107208052A (en) * | 2015-01-15 | 2017-09-26 | 国立大学法人大阪大学 | From multipotential stem cell to the induction of corneal epithelial cell |
| US11066641B2 (en) | 2015-01-15 | 2021-07-20 | Osaka University | Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells |
| CN109563485A (en) * | 2016-08-10 | 2019-04-02 | 加图立大学校产学协力团 | Broken up by what is induced multi-potent stem cell come the method and system of cultured corneal epithelium cell |
| CN110468094A (en) * | 2018-05-10 | 2019-11-19 | 澳门大学 | Corneal epithelial cell and corneal stent and its preparation method and application |
| CN110468094B (en) * | 2018-05-10 | 2021-04-13 | 澳门大学 | Corneal epithelial cells and corneal scaffold, and preparation method and application thereof |
| CN109517051A (en) * | 2018-12-03 | 2019-03-26 | 洛阳轩智生物科技有限公司 | Human epidermal stem cell induction is the method for corneal epithelial cell |
| CN109694855A (en) * | 2018-12-03 | 2019-04-30 | 洛阳轩智生物科技有限公司 | The method that human epidermal stem cell induction is corneal epithelial cell efficiency is improved using transgenic technology |
| CN109517051B (en) * | 2018-12-03 | 2020-10-16 | 拉菲尔(深圳)投资咨询有限公司 | Method for inducing human epidermal stem cells into corneal epithelial cells |
| CN109694855B (en) * | 2018-12-03 | 2020-12-29 | 北京仁源欣生生物科技有限公司 | Method for improving efficiency of inducing human epidermal stem cells into corneal epithelial cells by using transgenic technology |
| CN113736735A (en) * | 2020-05-27 | 2021-12-03 | 深圳华大生命科学研究院 | Method and kit for inducing limbal stem cells in vitro |
| CN113736735B (en) * | 2020-05-27 | 2024-02-20 | 深圳华大生命科学研究院 | Method and kit for in vitro induction of limbal-like stem cells |
| CN112410373A (en) * | 2020-11-27 | 2021-02-26 | 同济大学 | Method for establishing induced pluripotent stem cells from human amniotic epithelial cells |
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