CN106754652B - IPS cell differentiation at ectoderm progenitor cells serum-free induced medium and abductive approach - Google Patents

The invention discloses a kind of iPS cell differentiations into the serum-free induced medium and abductive approach of ectoderm progenitor cells.The serum-free induced medium is by adding final concentration of 8 μM of Indomethacin in every 500ml DMEM/F12 basal medium, the glutamine of percent by volume 1%, the nonessential amino acid of percent by volume 1%, 10 μM of SB431542, the EGF of 12ng/ml, 0.1mmol/ml β mercaptoethanol, the hydrocortisone of 0.4 μ g/ml, the insulin of 60 μ g/ml, the transferrins of 100 μ g/ml, the sodium selenite of 10ng/ml, the penicillin of 0.1mg/ml and the streptomysin of 0.1mg/ml are prepared.IPS cell directional can be induced to differentiate at the serum-free induced medium and abductive approach of ectoderm progenitor cells by using above-mentioned iPS cell differentiation by ectoderm progenitor cells, to be induced to differentiate into each cell of adult for iPS, especially skin relevant cell provides technical support.

IPS cell differentiation is at the serum-free induced medium of ectoderm progenitor cells and induction Method

Technical field

The present invention relates to field of tissue engineering technology, more particularly, to a kind of iPS cell differentiation at ectoderm progenitor cells without blood Clear induced medium and abductive approach.

Background technique

Skin tissue engineering is research hotspot in recent years, but the relevant cell of skin belongs to terminally differentiated cells, It is difficult to carry out cultivating amplification on a large scale in vitro.Seed cell carrys out source problem and hinders the development of organizational project, self kind Daughter cell limited source, there are rejection, embryonic stem cells there is ethics problem again for allogeneic seed cell.Therefore from urine The induction of urine cell is become iPS cell (inductive pluripotent stem cells) by middle collection urine cell, and iPS cell induces again to be become The seed source that skin tissue engineering can be solved in keratinocyte, fibroblast, sweat gland cells, hair papilla cell etc. is asked Topic.Cell is obtained from urine, it is convenient and efficient, do not have to obtain by wound, it is from a wealth of sources.

Each cell induction scheme that iPS cell is induced to differentiate into skin at present mainly has the simple cell factor to induce, altogether Culture induction, embryoid body induction etc..However simple cell factor induced efficiency is low, there are immunogenicity, embryoids for co-culturing, inducing There are non-directionals to induce feature for body, ultimately forms three germinal layers, cannot efficiently obtain some germinal layer.

Summary of the invention

Based on this, it is necessary to provide a kind of iPS cell differentiation into the serum-free induced medium of ectoderm progenitor cells and lure Guiding method.

A kind of iPS cell differentiation is the serum-free induced medium of ectoderm progenitor cells, the serum-free induced medium It is by adding final concentration of 5-10 μM of Indomethacin, percent by volume in every 500ml DMEM/F12 basal medium 1% glutamine, the nonessential amino acid of percent by volume 1%, 5-20 μM SB431542,5-18ng/ml EGF, 0.05-0.2mmol/ml β mercaptoethanol, the hydrocortisone of 0.1-1 μ g/ml, the insulin of 20-100 μ g/ml, 50-150 μ g/ The transferrins of ml, the sodium selenite of 4-15ng/ml, the penicillin of 0.05-0.1mg/ml and the strepto- of 0.05-0.1mg/ml Element is prepared.

The serum-free induced medium is by training on the basis every 500ml DMEM/F12 in one of the embodiments, Support base in add final concentration of 8-10 μM of Indomethacin, the glutamine of percent by volume 1%, percent by volume 1% it is non- Essential amino acid, the EGF of 8-12 μM SB431542,10-15ng/ml, 0.08-0.12mmol/ml β mercaptoethanol, 0.1-0.5 The hydrocortisone of μ g/ml, the insulin of 50-80 μ g/ml, the transferrins of 80-120 μ g/ml, 8-12ng/ml selenous acid The streptomysin of sodium, the penicillin of 0.08-0.1mg/ml and 0.08-0.1mg/ml is prepared.

The serum-free induced medium is by training on the basis every 500ml DMEM/F12 in one of the embodiments, Support base in add final concentration of 8 μM of Indomethacin, the glutamine of percent by volume 1%, percent by volume 1% it is nonessential Amino acid, the EGF of 10 μM SB431542,12ng/ml, 0.1mmol/ml β mercaptoethanol, 0.4 μ g/ml hydrocortisone, The insulin of 60 μ g/ml, the transferrins of 100 μ g/ml, the sodium selenite of 10ng/ml, the penicillin of 0.1mg/ml and The streptomysin of 0.1mg/ml is prepared.

A kind of iPS cell differentiation is the abductive approach of ectoderm progenitor cells, using above-mentioned serum-free induced medium to iPS Cell carries out induction differentiation culture.

The time of the induction differentiation culture is 7-10 days in one of the embodiments,.

The iPS cell differentiation further includes luring at the abductive approach of ectoderm progenitor cells in one of the embodiments, Before leading differentiation, the step of iPS cell is formed into unicellular embryoid body by the method for culture plate and centrifugation.

It is described in one of the embodiments, iPS cell is formed into unicellular group by the method for culture plate and centrifugation to intend The step of idiosome is that 0.5-1 × 10 are added according to each cultivation plate hole⁶A iPS cell, addition EB form culture medium, then with 400- 700r/min low-speed centrifugal after five minutes, is put into 5%CO², cultivate 30-50 hours in 37 DEG C of environment, it is quasi- to form the unicellular group Idiosome.

The culture plate is AggreWell in one of the embodiments,^TM800 culture plates.

The iPS cell differentiation further includes as follows at the abductive approach of ectoderm progenitor cells in one of the embodiments, The step of iPS cell recovery and unicellular formation: the iPS cell of P30 is recovered, with mTeSR1 without stroma cell culture, It is unicellular with the digestion of Accutase digestive juice, then will be described unicellular according to each cultivation plate hole in 70-80% convergence degree 0.5-1×10⁶A iPS cell is added in the cultivation plate hole to form the unicellular embryoid body.

By using the iPS cell differentiation of above-mentioned special component and concentration at the serum-free Fiber differentiation of ectoderm progenitor cells Base and abductive approach, iPS cell directional can be induced to differentiate into outer by each ingredient coordinated in the serum-free induced medium Endodermal progenitor cell, to be induced to differentiate into each cell of adult for iPS, especially skin relevant cell provides technology branch It holds.The serum-free induced medium and abductive approach do not use serum simultaneously, to efficiently avoid animal sources venereal disease Substance bring risk, improves the safety of clinical application.And compared with traditional embryoid body culture medium containing fetal calf serum Deng significantly improving the induction differentiation efficiency of the outside endodermal progenitor cell of iPS cell, high reliablity is applied widely.And pass through Each constituent concentration for advanced optimizing the serum-free Fiber differentiation can obtain significantly more induction iPS cell to ectoderm The efficiency and effect of progenitor cells differentiation.

Detailed description of the invention

Fig. 1 is the gene expression dose comparison result of the ectoderm progenitor cells of experimental group and control group, for each gene, Left side is experimental group in figure, and right side is control group；

Fig. 2 is the expression rate comparison result of the ectoderm progenitor cell surface NCAM albumen of experimental group and control group.

Specific embodiment

To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough Comprehensively.

Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases Any and all combinations of the listed item of pass.

Present embodiments provide the serum-free induction that a kind of efficient inducing pluripotent stem cells are divided into ectoderm progenitor cells Culture medium and abductive approach.IPS cell is first formed into unicellular group by the method for AggreWellTM800 culture plate and centrifugation Embryoid body, experimental group unicellular embryoid body of serum-free induced medium culture, control group are to continue with embryoid body culture medium It is cultivated.Then two groups of cell is detected by fluorescence real-time quantitative PCR and flow cytometer, comparative experiments group and The ectoderm gene of control group and the expression of albumen.Detailed process is as follows.

One, serum-free Fiber differentiation based component and its content

1. the serum-free induced medium of experimental group

2. the induced medium of control group

It is understood that in other embodiments, the ingredient of the serum-free induced medium of experimental group is not limited to described in table, As long as the serum-free induced medium satisfaction is final concentration of by adding in every 500ml DMEM/F12 basal medium 5-10 μM of Indomethacin, the glutamine of percent by volume 1%, the nonessential amino acid of percent by volume 1%, 5-20 μM The EGF of SB431542,5-18ng/ml, 0.05-0.2mmol/ml β mercaptoethanol, 0.1-1 μ g/ml hydrocortisone, 20- The blueness of the insulin of 100 μ g/ml, the transferrins of 50-150 μ g/ml, the sodium selenite of 4-15ng/ml, 0.05-0.1mg/ml The streptomysin of mycin and 0.05-0.1mg/ml are prepared, as long as further meeting is by every 500ml Final concentration of 8-10 μM of Indomethacin, the glutamine of percent by volume 1%, volume are added in DMEM/F12 basal medium The nonessential amino acid of percentage 1%, the EGF of 8-12 μM SB431542,10-15ng/ml, 0.08-0.12mmol/ml β mercapto Base ethyl alcohol, the hydrocortisone of 0.1-0.5 μ g/ml, the insulin of 50-80 μ g/ml, 80-120 μ g/ml transferrins, 8- The streptomysin of the sodium selenite of 12ng/ml, the penicillin of 0.08-0.1mg/ml and 0.08-0.1mg/ml be prepared i.e. It can.

Two, step

(1) preparation of experimental group culture medium

By 500ml basal medium DMEM/F12 be added Insulin 3 0mg, hydrocortisone 0.2mg, transferrins 50mg, 5 μ g of sodium selenite, β mercaptoethanol 0.05mmol, EGF6 μ g, 10 SB431542 μM (micromolecular inhibitor), Indomethacin 8uM, Nonessential amino acid 5ml (purchase from: hundred Aurion of Beijing wins Science and Technology Ltd., product number: BL1266), glutamine 5ml, Penicillin 50mg and streptomysin 50mg.

(2) control group culture medium

(main component: embryoid body (EB) basis of formation culture medium, embryoid body (EB) form Special tire ox blood to EB culture medium Clearly, glutamine, dual anti-, nonessential amino acid, 2 mercapto ethanol, producer: Sai Ye Biotechnology Co., Ltd, product number: MUXES-90051)。

(3) recovery cell: the iPS cell (source Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health) of P30 is selected It recovers, is unicellular, meter with the digestion of Accutase digestive juice when 80% convergence degree with mTeSR1 without stroma cell culture Number.

(4) formation of unicellular embryoid body: each AggreWell is pressed^TM1 × 10 is added in 800 cultivation plate holes⁶It is a thin Born of the same parents, are added EB and form culture medium, operation manual are used by product, by AggreWell^TM800 culture plates with 700r/min low speed from After heart 5min, it is put into 5%CO², cultivate in 37 DEG C of incubators, form unicellular embryoid body after cultivating 48h.

(5) induction differentiation: being transferred in 24 orifice plate of low adsorption of unicellular embryoid body will be formed, 24 orifice plates are divided into two Liquid is changed in group, experimental group and control group, every group of 12 holes every other day, cultivates to after 7 days, the gene expression dose of external endodermal progenitor cell It is detected with surface protein expression.

(6) identification of ectoderm progenitor cells:

It uses the digestive juice containing 0.25% trypsase and 0.02%EDTANa2 to handle 1-3 minutes in cell, makes cell detachment Culture substrate simultaneously becomes unicellular, and the culture medium containing 10% fetal calf serum is added and carries out neutralization digestion, and 1200rpm is centrifuged 5 minutes, With cold PBS buffer solution rinse cell, cell precipitation is collected in 1.5ml EP pipe, cell total rna extracts kit is used (RneasyMiniKit, QIAGEN74104) by its operation instruction lytic cell and extracts intracellular total serum IgE, utilizes ultraviolet point After light photometric determination RNA concentration and purity, using reverse transcription reagent box (ReverTraAceqPCRRTMasterMix, It TOYOBOFSQ-201 is) cDNA by 1 μ g total serum IgE reverse transcription.CDNA, primer are reacted into premix reagent with qPCR (THUNDERBIRDSYBRqPCRMix, TOYOBOQPS-201) mixing, it is real-time by the opticon2 fluorescence of Bio-Rad company Quantitative PCR apparatus expands target gene, using people GAPDH gene expression amount as reference.Each sample reaction is independent to repeat three It is secondary to eliminate systematic error.

Inducing cell mrna expression is identified in aforementioned manners, identifies ectoderm progenitor cells differentiation gene The expression of GBX2, HoxA1, SOX1 and ZIC1, as a result as shown in Figure 1.

(7) flow cytometry identifies cell surface marker Protein Detection:

Cell surface marker Protein Detection is identified using flow cytometry, concrete operations are as follows:

Take 1 × 10⁶A cell uses the digestive juice containing 0.25% trypsase and 0.02%EDTANa2 to handle 1-3 minutes, makes Cell detachment culture substrate simultaneously becomes unicellular, and the culture medium containing 10% fetal calf serum is added and carries out neutralization digestion, 1200rpm is centrifuged 5 minutes, and with cold PBS buffer solution rinse cell, cell is resuspended in 100 μ l PBS buffer solution after being centrifuged again In, the NCAM antibody (fluorescent component: FITC, producer: green skies Biotechnology Co., Ltd, product for carrying fluorescent marker is added Number: A0562) and mix, 4 DEG C are protected from light incubation 45 minutes.After antibody incubation by cell with twice of cold PBS buffer solution rinse to remove Unbonded antibody is removed, then cell is resuspended in 400 μ l PBS buffer solution, using stream type cell analyzer to cell surface The expression rate of NCAM albumen is detected.As a result as shown in Figure 2.

Three, result

The result shows that the high expression rate of ectoderm progenitor cells differentiation gene GBX2, HoxA1, SOX1 and ZIC1 illustrate iPS The directed differentiation of cell, the efficiency using the outside endodermal progenitor cell differentiation of the cell of experimental group are significantly higher than control group, illustrate benefit Efficiently ectoderm progenitor cells can be divided by inducing pluripotent stem cells with the method for the present embodiment.Whole process is not used simultaneously To serum, animal derived pathogen bring risk is effectively avoided, the safety of clinical application is improved.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.