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CN106754653B - IPS cell differentiation at entoderm progenitor cells serum-free induced medium and abductive approach - Google Patents

  • ️Fri Sep 27 2019
IPS cell differentiation at entoderm progenitor cells serum-free induced medium and abductive approach Download PDF

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Publication number
CN106754653B
CN106754653B CN201710128090.6A CN201710128090A CN106754653B CN 106754653 B CN106754653 B CN 106754653B CN 201710128090 A CN201710128090 A CN 201710128090A CN 106754653 B CN106754653 B CN 106754653B Authority
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culture medium
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medium
ips cell
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2017-03-06
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CN106754653A (en
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车七石
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Guangzhou Rainhome Pharm and Tech Co Ltd
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Guangzhou Rainhome Pharm and Tech Co Ltd
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2017-03-06 Application filed by Guangzhou Rainhome Pharm and Tech Co Ltd filed Critical Guangzhou Rainhome Pharm and Tech Co Ltd
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2017-05-31 Publication of CN106754653A publication Critical patent/CN106754653A/en
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Abstract

The invention discloses a kind of iPS cell differentiations into the serum-free induced medium and abductive approach of entoderm progenitor cells.The serum-free induced medium includes first stage culture medium, second stage culture medium and phase III culture medium.IPS cell directional can be induced to differentiate at the serum-free induced medium and abductive approach of entoderm progenitor cells by using above-mentioned iPS cell differentiation by entoderm progenitor cells, to provide technical support for the iPS each cell for being induced to differentiate into adult.The serum-free induced medium and abductive approach do not use serum simultaneously, to efficiently avoid animal derived pathogen bring risk, improve the safety of clinical application.

Description

IPS cell differentiation is at the serum-free induced medium of entoderm 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 entoderm progenitor cells without blood Clear induced medium and abductive approach.

Background technique

Organizational project is research hotspot in recent years, but seed cell is difficult to carry out cultivating on a large scale in vitro to expand Increase, seed cell carrys out source problem and hinders the development of organizational project.Self seed cell limited source, allogeneic seed cell There are rejection, there is ethics problem in embryonic stem cell again, therefore urine cell is collected from urine, and urine cell is induced As iPS cell (inductive pluripotent stem cells), iPS cell is induced again as each adult cell, can solve organizational project Seed source problem.Cell is obtained from urine, it is convenient and efficient, do not have to obtain by wound, it is from a wealth of sources.

IPS cell, which is induced to differentiate into adult cell induction scheme mainly, at present has the induction of the simple cell factor, co-cultivation to lure Lead, embryoid body culture medium induction etc..However simple cell factor induced efficiency is low, there are immunogenicity, embryoids for co-culturing, inducing There are three germinal layer inductions of non-directional in body, directed differentiation rate is low.Each cell origin of adult is in the not homeomorphism of germinal layer Layer, therefore, efficiently obtain certain adult cell, need efficiently and directionally and obtain a certain 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 entoderm progenitor cells and lure Guiding method.

A kind of serum-free induced medium of iPS cell differentiation at entoderm progenitor cells, including first stage culture medium, Two-stage culture medium and phase III culture medium；Wherein, the first stage culture medium is to add in every 500ml basal medium There are the HGF of the Activin A and final concentration of 5-18ng/ml of final concentration of 80-160ng/ml；The second stage culture medium For the BMP-4 for being added with final concentration of 8-25ng/ml in every 500ml basal medium；The phase III culture medium is every The FGF-2 of HGF and final concentration of 15-28ng/ml in 500ml basal medium added with final concentration of 5-18ng/ml；Institute Stating basal medium is the β sulfydryl second that final concentration of 0.05-0.2mmol/l is added in every 500ml DMEM/F12 culture medium Hydrocortisone, the final concentration of 25- of alcohol, the Wnt3a of final concentration of 75-180 μ g/ml, final concentration of 0.15-0.7 μ g/ml The insulin of 100 μ g/ml, the sodium selenite of final concentration of 5-20ng/ml and final concentration of 85-180 μ g/ml turn iron egg It is white.

The first stage culture medium is in every 500ml basal medium added with dense eventually in one of the embodiments, Degree is the HGF of the Activin A and final concentration of 8-12ng/ml of 80-120ng/ml；The second stage culture medium is every The BMP-4 of final concentration of 15-25ng/ml is added in 500ml basal medium；The phase III culture medium is every 500ml The FGF-2 of HGF and final concentration of 15-25ng/ml in basal medium added with final concentration of 8-12ng/ml；The base Basal culture medium is β mercaptoethanol, the end that final concentration of 0.08-0.12mmol/l is added in every 500ml DMEM/F12 culture medium Concentration is the Wnt3a of 80-120 μ g/ml, the hydrocortisone of final concentration of 0.2-0.6 μ g/ml, final concentration of 50-80 μ g/ml Insulin, the sodium selenite of final concentration of 8-12ng/ml and the transferrins of final concentration of 85-120 μ g/ml.

The first stage culture medium is in every 500ml basal medium added with dense eventually in one of the embodiments, Degree is the HGF of the Activin A and final concentration of 10ng/ml of 100ng/ml；The second stage culture medium is every 500ml base The BMP-4 of final concentration of 20ng/ml is added in basal culture medium；The phase III culture medium is every 500ml basal medium In added with final concentration of 10ng/ml HGF and final concentration of 20ng/ml FGF-2；The basal medium is every Added with the β mercaptoethanol of final concentration of 0.1mmol/l, final concentration of 100 μ g/ml in 500ml DMEM/F12 culture medium Wnt3a, the hydrocortisone of final concentration of 0.4 μ g/ml, the insulin of final concentration of 60 μ g/ml, final concentration of 10ng/ml The transferrins of sodium selenite and final concentration of 100 μ g/ml.

A kind of iPS cell differentiation is at the abductive approach of entoderm progenitor cells, successively above-mentioned first stage culture medium, second-order Section culture medium and phase III culture medium are divided into three phases to iPS cell and carry out induction differentiation.

The three phases are respectively the 1st day to the 3rd day for inducing differentiation, the 4th day to the in one of the embodiments, 7 days and the 8th day to the 11st day, in the culture of each stage, culture medium was changed every other day.

The iPS cell differentiation further includes luring at the abductive approach of entoderm 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- After 700r/min low-speed centrifugal 4-10 minutes, it is put into 5%CO2, cultivate 40-65 hours in 37 DEG C of environment, formed described unicellular Group's embryoid body.

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 entoderm 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 entoderm progenitor cells Base and abductive approach, in iPS cell directional can be induced to differentiate by each ingredient coordinated in the serum-free induced medium Endodermal progenitor cell, to provide technical support for the iPS each cell for being induced to differentiate into adult.The training of serum-free induction simultaneously It supports base and abductive approach does not use serum, to efficiently avoid animal derived pathogen bring risk, improve The safety of clinical application.And compared with traditional embryoid body culture medium etc. containing fetal calf serum, significantly improve iPS cell to The induction differentiation efficiency of entoderm progenitor cells, high reliablity are applied widely.And by advanced optimizing serum-free induction Each constituent concentration of culture can obtain the efficiency and effect of significantly more induction iPS cells towards endodermal progenitor cells differentiation.

Detailed description of the invention

Fig. 1 is the qualification result figure of the immunofluorescence dyeing SOX17 expression of the entoderm progenitor cells of experimental group and control group；

Fig. 2 is the comparison result of SOX17 positive rate in the entoderm progenitor cells 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.

IPS cell is first formed unicellular group by the method for AggreWellTM800 culture plate and centrifugation and intended by the present embodiment Idiosome, experimental group form definitive endoderm with unicellular embryoid body of serum-free induced medium culture, and control group is to continue with use Embryoid body culture medium is cultivated, and immunofluorescence method identification of cell, the entoderm of comparative experiments group and control group are then passed through The expression of SOX17.Detailed process is as follows.

One, culture medium

1. experimental group

2. control group culture medium

Title Working concentration
EB culture medium 500ml

It is understood that in other embodiments, the ingredient of experimental group culture medium is not limited in table, as long as meeting the first stage Culture medium is the ActivinA and final concentration of 5- that final concentration of 80-160ng/ml is added in every 500ml basal medium The HGF of 18ng/ml；Second stage culture medium is the BMP- that final concentration of 8-25ng/ml is added in every 500ml basal medium 4；Phase III culture medium is in every 500ml basal medium added with the HGF of final concentration of 5-18ng/ml and final concentration of The FGF-2 of 15-28ng/ml；Basal medium is that final concentration of 0.05- is added in every 500ml DMEM/F12 culture medium The β mercaptoethanol of 0.2mmol/l, the Wnt3a of final concentration of 75-180 μ g/ml, final concentration of 0.15-0.7 μ g/ml hydrogenation can The insulin of loose, final concentration of 25-100 μ g/ml, final concentration of 5-20ng/ml sodium selenite and final concentration of 85- The transferrins or further satisfaction first stage culture medium of 180 μ g/ml is to add in every 500ml basal medium There are the HGF of the Activin A and final concentration of 8-12ng/ml of final concentration of 80-120ng/ml；Second stage culture medium is every The BMP-4 of final concentration of 15-25ng/ml is added in 500ml basal medium；Phase III culture medium is every basis 500ml The FGF-2 of HGF and final concentration of 15-25ng/ml in culture medium added with final concentration of 8-12ng/ml；Basal medium For the β mercaptoethanol, final concentration of for being added with final concentration of 0.08-0.12mmol/l in every 500ml DMEM/F12 culture medium The Wnt3a of 80-120 μ g/ml, the hydrocortisone of final concentration of 0.2-0.6 μ g/ml, final concentration of 50-80 μ g/ml pancreas islet The transferrins of the sodium selenite of plain, final concentration of 8-12ng/ml and final concentration of 85-120 μ g/ml.

Two, step

(1) preparation of basal medium:

By every 500ml DMEM/F12 culture medium be added Insulin 3 0mg, hydrocortisone 0.2mg, transferrins 50mg, 5 μ g of sodium selenite, 50 μ g of β mercaptoethanol 0.05mmol and Wnt3a are prepared.

(2) first stage culture medium:

By the basal medium 500ml of step 1,50 5 μ g of μ g, HGF of Activin A is added.

(3) second stage culture medium:

By the basal medium 500ml of step 1,10 μ g of BMP-4 is added.

(4) phase III culture medium:

By the basal medium 500ml of step 1,5 10 μ g of μ g, FGF-2 of HGF is added.

(5) control group culture medium: EB culture medium (main component: embryoid body (EB) basis of formation culture medium, embryoid body (EB) Formed dedicated fetal calf serum, glutamine, dual anti-, nonessential amino acid (purchase from: hundred Aurion of Beijing wins Science and Technology Ltd., Product number: BL1266), 2 mercapto ethanol, producer: Sai Ye Biotechnology Co., Ltd, product number: MUXES-90051)

(6) 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.

(7) formation of unicellular embryoid body: each AggreWell is pressedTM1 × 10 is added in 800 cultivation plate holes6A iPS Cell, is added EB and forms culture medium, operation manual is used by product, by AggreWellTM800 culture plates are with 700r/min low speed After being centrifuged 5min, it is put into 5%CO2, cultivate in 37 DEG C of incubators, form unicellular embryoid body after cultivating 48h.

(8) induction differentiation: unicellular embryoid body is transferred in 24 orifice plate of low adsorption, and 24 orifice plates are divided into two groups, experiment Liquid is changed in group and control group, every group of 12 holes every other day, cultivates to after 11 days, the gene expression dose of external endodermal progenitor cell and surface Protein expression level is detected.

Experimental group abductive approach: start being denoted as first day first day (Day 1) for induction.First stage (Day 1-3): it uses First stage culture medium culture iPS cell.Second stage (Day 4-7): it is cultivated with second stage culture medium.Phase III (Day8-11): being cultivated with phase III culture medium.

Observation discovery: by the induction in second, third stage, form changes, cell quantity increases, and determines row entoderm Cell basically forms.

The abductive approach of control group: whole process is all to carry out Fiber differentiation with EB culture medium.

Three, the identification of entoderm progenitor cells

Immunofluorescence dyeing:

(1) culture medium abandoned in orifice plate is inhaled, is careful not to encounter cell.

(2) it is primary that 1 × PBS cleaning cell is added, 5min inhales abandoning PBS.

(3) paraformaldehyde (4%PFA) for adding 4%, is put into 37 DEG C, 5%CO220min is incubated in incubator.This step Super-clean bench operation can be taken out after rapid.

(4) it inhales and abandons 4% paraformaldehyde, be careful not to encounter cell.Cell 3 times are cleaned with 1 × PBS, each 5min.

(5) it inhales and abandons PBS.0.1%Triton punching is added, acts on 10min.

(6) it inhales and abandons 0.1%Triton, clean cell 3 times with 1 × PBS, each 3min.

(7) 5%BSA is added, room temperature closes 1h.

(8) before terminating closing, according to the ratio of 1:100, primary antibody is diluted with PBS.

(9) it inhales and abandons confining liquid, it is primary to wash cell with PBS.

(10) primary antibody diluted, 4 DEG C of overnight incubations are added.

(11) it inhales and abandons primary antibody.Cell 3 times are cleaned with 1 × PBS, each 3min.

(12) according to the dilution proportion fluorescence secondary antibody of 1:500, (ingredient: FITC, producer: Amy victory Science and Technology Ltd. is produced Product number: A22110), secondary antibody is added into orifice plate, is incubated at room temperature 1h.

(13) it inhales and abandons secondary antibody.Cell 3 times are cleaned with 1 × PBS, each 3min.

(14) Hochest (pure water 1:10000 dilution) redyes nucleus, and room temperature acts on about 2min.

(15) it inhales and abandons Hochest dye liquor, clean cell 3 times with 1 × PBS, each 5min.

(16) inverted fluorescence microscope is taken a picture, the good Cell Name of remarks, multiple, time etc..

(17) statistical analysis method is used, two groups of positive rate is compared.

Four, result

As depicted in figs. 1 and 2, the results showed that, the entoderm SOX17 expression rate of experimental group significantly improves, and illustrates that iPS is thin The directed differentiation of the inside endodermal progenitor cell of born of the same parents, further explanation utilize serum-free induced medium and abductive approach of the invention, The differentiation of iPS cells towards endodermal progenitor cells can efficiently be induced.Whole process does not use serum simultaneously, effectively avoids Animal derived pathogen bring risk, improves the safety of clinical application.

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.

Claims (7)

1. a kind of for inducing iPS cell differentiation at the kit of entoderm progenitor cells, which is characterized in that trained including the first stage Support base, second stage culture medium and phase III culture medium, the first stage culture medium, the second stage culture medium and The phase III culture medium is serum free medium；Wherein, the first stage culture medium is every 500ml basal medium In added with final concentration of 80-160ng/ml Activin A and final concentration of 5-18ng/ml HGF；The second stage Culture medium is the BMP-4 that final concentration of 8-25ng/ml is added in every 500ml basal medium；The phase III culture medium For the FGF- of HGF and final concentration of 15-28ng/ml in every 500ml basal medium added with final concentration of 5-18ng/ml 2；The basal medium is the β sulfydryl that final concentration of 0.05-0.2mmol/l is added in every 500ml DMEM/F12 culture medium Hydrocortisone, the final concentration of 25- of ethyl alcohol, the Wnt3a of final concentration of 75-180 μ g/ml, final concentration of 0.15-0.7 μ g/ml The insulin of 100 μ g/ml, the sodium selenite of final concentration of 5-20ng/ml and final concentration of 85-180 μ g/ml turn iron egg It is white.

2. as described in claim 1 for inducing iPS cell differentiation at the kit of entoderm progenitor cells, which is characterized in that The first stage culture medium be every 500ml basal medium in added with final concentration of 80-120ng/ml Activin A with And the HGF of final concentration of 8-12ng/ml；The second stage culture medium is that final concentration is added in every 500ml basal medium For the BMP-4 of 15-25ng/ml；The phase III culture medium is that final concentration of 8- is added in every 500ml basal medium The FGF-2 of the HGF of 12ng/ml and final concentration of 15-25ng/ml；The basal medium is every 500ml DMEM/F12 training It supports and is added with the β mercaptoethanol of final concentration of 0.08-0.12mmol/l, the Wnt3a of final concentration of 80-120 μ g/ml, end in base Concentration is the hydrocortisone of 0.2-0.6 μ g/ml, the insulin of final concentration of 50-80 μ g/ml, final concentration of 8-12ng/ml The transferrins of sodium selenite and final concentration of 85-120 μ g/ml.

3. as claimed in claim 2 for inducing iPS cell differentiation at the kit of entoderm progenitor cells, which is characterized in that The first stage culture medium be every 500ml basal medium in added with final concentration of 100ng/ml Activin A and The HGF of final concentration of 10ng/ml；The second stage culture medium is in every 500ml basal medium added with final concentration of The BMP-4 of 20ng/ml；The phase III culture medium is in every 500ml basal medium added with final concentration of 10ng/ml The FGF-2 of HGF and final concentration of 20ng/ml；The basal medium is to be added in every 500ml DMEM/F12 culture medium The β mercaptoethanol of final concentration of 0.1mmol/l, the Wnt3a of final concentration of 100 μ g/ml, final concentration of 0.4 μ g/ml hydrogenation can The insulin of loose, final concentration of 60 μ g/ml, the sodium selenite of final concentration of 10ng/ml and final concentration of 100 μ g/ml Transferrins.

4. a kind of iPS cell differentiation is at the abductive approach of entoderm progenitor cells, which is characterized in that successively use claim 1-3 Any one of described in first stage culture medium, second stage culture medium and phase III culture medium three are divided into iPS cell Stage carries out induction differentiation.

5. iPS cell differentiation as claimed in claim 4 is at the abductive approach of entoderm progenitor cells, which is characterized in that described three A stage is respectively the 1st day to the 3rd day, the 4th day to the 7th day and the 8th day to the 11st day for inducing differentiation, is trained in each stage When supporting, culture medium is changed every other day.

6. iPS cell differentiation as claimed in claim 5 is at the abductive approach of entoderm progenitor cells, which is characterized in that further include Before induction differentiation, the step of iPS cell is formed into unicellular embryoid body by the method for culture plate and centrifugation.

7. iPS cell differentiation as claimed in claim 6 is at the abductive approach of entoderm progenitor cells, which is characterized in that further include The step of following iPS cell recovery and unicellular formation: the iPS cell of P30 is recovered, and is trained with mTeSR1 without stroma cell It supports, is unicellular with the digestion of Accutase digestive juice, then will be described unicellular according to each culture in 70-80% convergence degree Plate hole 0.5-1 × 106A iPS cell is added in the cultivation plate hole to form the unicellular embryoid body.

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