CN104651298B - Inducing embryo stem cell directed differentiation is the culture medium and method of keratinocyte - Google Patents
Inducing embryo stem cell directed differentiation is the culture medium and method of keratinocyte Download PDFInfo
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- CN104651298B CN104651298B CN201310596606.1A CN201310596606A CN104651298B CN 104651298 B CN104651298 B CN 104651298B CN 201310596606 A CN201310596606 A CN 201310596606A CN 104651298 B CN104651298 B CN 104651298B
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Abstract
It include Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid the present invention provides inducer described in the inducer that a kind of inducing embryo stem cell directed differentiation is keratinocyte.Preferably, in the inducer, the content ratio of Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid is (1-50g): (0.1-5mol): (50-600mol).Inducing embryo stem cell directed differentiation provided by the invention is the inducer of keratinocyte, it is compounded with Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid, the efficiency (up to 77.93% that hESC Induction of committed differentiation is keratinocyte can be effectively improved, significantly larger than other formula inducers) an efficiently induction approach of the hESC to keratinocyte differentiation is provided, seed cell is provided for Oral Tissue-engineered Mucosa building.
Description
Technical field
It is dry thin that the present invention relates to a kind of inducers of inducing embryo stem cell directed differentiation more particularly to a kind of inducing embryo
Born of the same parents' directed differentiation is inducer, culture medium and the method for keratinocyte.
Background technique
Mucous membrane of mouth is the first barrier that human body resists extraneous various injurious factors, is had various under protection mucous membrane of mouth
Tissue, feeling, temperature adjust and the highly important functions such as secretion.Due to surgical operation, tooth kind before periodontal surgery, dentures repai
Plant art, congenital or the facial deformity prosthesis day after tomorrow, wound and tumor resection etc. may cause oral cavity mucous membrane tissue defect or
Insufficiency generally requires to carry out oral cavity mucous membrane tissue reconstruction.Auto-skin grafting or mucous membrane of mouth is widely used in clinic at present
Valve skin grafing and mending defect.But skin epidermis original structure function and angling, secretion, hair life are still kept after transplanting skin many years
The features such as long, lack the moist smooth feeling of normal mucosa, so that patient is felt more uncomfortable, clinical needs are far from satisfying, to limit
Clinical application is made.And the above method will open up the second operation, and new wound and pain can be brought to patient.Therefore, it seeks
Looking for a kind of ideal mucous membrane of mouth substitute is clinically urgent need to solve the problem.
Oral mucosa keratinocyte category stratified squamous epithelium, based on keratinocyte.Inducing embryo stem cell (hESC) orientation point
The key for turning to keratinocyte is to prevent it from breaking up to nerve cell direction at hESC differentiation initial stage, is lured by suitable inducer
It leads to form keratinocyte progenitor cells, and further breaks up, maintain the phenotype of epithelial cell.The prior art mainly uses different groups
Point inducer, bon e formation egg 4(bone morphogenetic protein 4, BMP4 is such as used alone), be used alone it is anti-
Bad hematic acid (ascorbic acid, AA) is used in combination BMP4 and vitamin A acid (retinoic acid, RA), BMP4 is used in combination
And AA, the expression rate of cytokeratin 14 is respectively 5.4%, 8.9%, 35% when inducing the 35th day using these inducers being formulated
With 59%.These cells are maintained in phenotype, and screening purifying, terminal differentiation etc. still is apparent not enough, and need to be optimized induction point
Change method.
Summary of the invention
It is keratinocyte the purpose of the present invention is to overcome the above shortcomings and to provide a kind of inducing embryo stem cell directed differentiation
Inducer, culture medium and method, can effectively improve hESC Induction of committed differentiation be keratinocyte efficiency, be organizational project
Change mucous membrane of mouth building and seed cell is provided.
The first aspect of the invention is to provide the inducer that a kind of inducing embryo stem cell directed differentiation is keratinocyte,
The inducer includes Bone Morphogenetic Protein 4(BMP4), vitamin A acid (RA) and ascorbic acid (AA).
In one preferred embodiment, in the inducer, Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid contain
Amount is than being (1-50g): (0.1-5mol): (50-600mol).
It is further preferred that the content ratio of Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid is (5-45g): (0.2-
4.5mol): (100-500mol).
It is further preferred that the content ratio of Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid is (10-40g): (0.4-
4mol): (150-450mol).
It is further preferred that the content ratio of Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid is (20-35g): (0.5-
3mol): (200-400mol).
It is further preferred that the content ratio of Bone Morphogenetic Protein 4, vitamin A acid and ascorbic acid is (25-30g): (1-2mol):
(250-350mol).
The second aspect of the invention is to provide the culture medium that a kind of inducing embryo stem cell directed differentiation is keratinocyte,
The culture medium contains inducer described in above-mentioned first aspect.
In one preferred embodiment, the inducing embryo stem cell directed differentiation is in the culture medium of keratinocyte
Contain:
4 1-50ng/ml of Bone Morphogenetic Protein, more preferably 5-45 ng/ml, more preferably 10-40 ng/ml, more preferably
20-35 ng/ml, more preferably 20-35 ng/ml；
Vitamin A acid 0.1-5 uM, more preferably 0.2-4.5 uM, more preferably 0.4-4 uM, more preferably 0.5-3 uM,
More preferably 1-2 uM；
Ascorbic acid 0.05-0.6mmol/L, more preferably 0.1-0.5 mmol/L, more preferably 0.15-0.45 mmol/
L, more preferably 0.2-0.4 mmol/L, more preferably 0.25-0.35 mmol/L, such as 0.3 mmol/L, 0.32 mmol/L
Or 0.28 mmol/L.
In one preferred embodiment, the inducing embryo stem cell directed differentiation is the culture medium packet of keratinocyte
Include the differential medium for embryonic stem cell culture.
Preferably, the differential medium for embryonic stem cell culture contains DMEM/F12 culture solution and N2 addition
Agent:
The concentration of volume percent of DMEM/F12 culture solution is 95-99.9%, more preferably 96-99.5%, 97-99.2t%,
98-99%；
The concentration of volume percent of N2 additive be 0.1-5%, more preferably 0.5-4%, more preferably 0.8-3%, more preferably
For 1-2%.
In a further preferred embodiment, the inducing embryo stem cell directed differentiation is the training of keratinocyte
Feeding base is formulated by the following method: inducer described in first aspect of the present invention being added to the embryo that is used for and is done
In the differential medium of cell culture.
The third aspect of the invention is to provide a kind of method that inducing embryo stem cell directed differentiation is keratinocyte, adopts
It is the culture medium of keratinocyte with the inducing embryo stem cell directed differentiation, comprising the following steps:
Step 1, using embryonic stem cell after passage (the 2-7 days cells after preferably passing on, for example, the 3rd day after passing on,
4th day, the 5th day or the 6th day cell) in the middle patch that the culture medium inducing embryo stem cell directed differentiation is keratinocyte
Wall culture 5-10 days (preferably 6-9, more preferably 7-8), liquid was changed daily.
In one preferred embodiment, further include pre-treatment step: embryonic stem cell strain is carried out in the medium
Culture, passage.
In one preferred embodiment, further include post-processing step: the cell that step 1 processing is obtained is with neutral egg
After white enzymic digestion, it is seeded in the coated culture dish of type i collagen, it is (more excellent with the continuation of keratinocyte culture solution adhere-wall culture 3-6 weeks
Be selected as 4-5 weeks), the next day change liquid, cell log growth period pass on.
Inducing embryo stem cell directed differentiation provided by the invention is the inducer of keratinocyte, is compounded with Bone Morphogenetic Protein
4, vitamin A acid and ascorbic acid can effectively improve efficiency (the Keratin 14 expression that hESC Induction of committed differentiation is keratinocyte
Rate is up to 77.93%, significantly larger than other formula inducers) provide an efficiently induction way of the hESC to keratinocyte differentiation
Diameter provides seed cell for Oral Tissue-engineered Mucosa building.
Detailed description of the invention
Fig. 1 is single morphologic detection result of the hESC after inducer provided by the invention induction differentiation；
Fig. 2 is hESC single stream Schwann Cells testing result after inducer provided by the invention induction differentiation；
Fig. 3 is hESC single immunization fluorescent staining result after inducer provided by the invention induction differentiation.
Specific embodiment
With reference to the accompanying drawings, the present invention is further illustrated in conjunction with specific embodiments, to more fully understand this hair
It is bright.
The routine culture of 1.hESC: this research uses the H9 human embryo stem cell strain of International Health Organization registration
(agreement no.11-W0039, WiCell Research Institute, Madison, WI).HESC on matrigel with
MTeSR1 culture medium culture is passed on for 5 days.
2.hESC is to keratinocyte induction scheme:
2.1 experimental group inducing embryo stem cell directed differentiations for the culture medium of keratinocyte preparation: the present invention is provided
Inducing embryo stem cell directed differentiation be keratinocyte inducer be added to the differentiation culture for embryonic stem cell culture
In base both, wherein the inducing embryo stem cell directed differentiation be keratinocyte culture medium in contain: 25ng/ml bone shape
It is described for embryonic stem cell culture at albumen 4(BMP4), 1 uM vitamin A acid (RA) and 0.3mmol/L ascorbic acid (AA)
The DMEM/F12 culture that differential medium contains the N2 additive that concentration of volume percent is 1%, concentration of volume percent is 99%
Liquid.
2.2 experimental group abductive approach: the 4th day after passing on hESC adhere-wall culture 7 in the inducer of above-mentioned formula is used
Day, change liquid daily, the 7th day by cell with neutral proteinase (Dispase) digestion after, take 1/3 cell inoculation to type i collagen packet
The culture dish of quilt with keratinocyte culture solution continuation adhere-wall culture 4 weeks, the next day change liquid, cell log growth period passage.
The setting of 2.3 control groups: the preparation of control group culture medium: control inducer is added to and is used for embryonic stem cell training
In feeding differential medium both, wherein control inducer is made of Bone Morphogenetic Protein 4 and ascorbic acid, control group culture medium
In contain 25ng/ml Bone Morphogenetic Protein 4(BMP4) and 0.3mmol/L ascorbic acid (AA).Embryonic stem cell is used in control group
The same experimental group of the differential medium of culture, and the same experimental group of control group abductive approach.
3. effect detection of the invention:
3.1 morphologic detections, as a result as (A2, A 7, A 14, A 21, A 28 and A 35 are respectively cellular control unit to Fig. 1
Break up photo under the 2nd, 7,14,21,28 and 35 day inverted microscope mirror, B2, B 7, B 14, B 21, B 28 and B 35 are experiment
Group the 2nd, 7,14,21,28 and 35 day photo of cell differentiation, scale show 0.1mm) shown in:
Control group induction differentiation 1-7d, clone's area are gradually increased, and differentiation is started to be in progress to center from clone edge, differentiation
Cell is grown in oval monolayer adherence, and paving stone shape, neoblast still cloning aggregation growth, differentiation is presented in partial region
Demarcate between cell and neoblast obvious.1:3 passage in 7th day is inoculated in the culture dish of type i collagen paving quilt and is trained with DSFM
Support, for 24 hours in adherent still grown with cloned version thereof.10th day, accelerated cellular proliferation, Mitotic index was common, and cell quantity obviously increases
More, the 14th day, cell was grown into fusion in flakes, and paving stone shape cell is seen around clone, and center still keeps neoblast layer
It is stacked.16th day, cell fusion passed on according to the above method, and the 18th day, cell was adherent, and the cell of full extension is in flat polygonal
Shape, size shape are different.35th day, see cell fusion, neoblast is intensively in heaps, and noble cells is dispersed in growth in paving stone
Shape.
Experimental group induces 1-7d, and ES clonal growth rate is greater than experiment A group, sees noble cells more than A under the 2nd clear water surface
Group, cell are in oval, the uniform paving stone shape of form, fusion are grown into flakes, between differentiation and neoblast
Boundary is unobvious.Cell is dense laminated within 7th day, and propagating method is with A group, and interior attached cell is more than A group for 24 hours.Cell accelerates within 9th day
Gradually in flakes, nuclear fission is in uniform paving stone shape as common to proliferation for fusion.Cell is grown into and is fused within 14th day
Piece, clone's surrounding are shown in streak cell, and center is uniform paving stone shape cell, Monolayer growth of cells.Cell growth speed
Rate slows down, hence it is evident that is less than A group.19th day cell fusion is passed on A group.Cell is gradually adherent within 21st day, is the strong circle of refractivity
Shape cell.Subsequent cell gradually stretches out short and small pseudopodium, cytoplasm expansion, single cells grown.35th day, cytosis was in size shape
The uniform paving stone shape of state, has no neoblast.
3.2 stream Schwann Cells technology detections, as a result as (ES is the CK14 expression rate of hESC before inducing to Fig. 2, and A is control component
Change the 35th day cell CK14 positive rate, B is that experimental group breaks up the 35th day cell CK14 positive rate) shown in:
It is 77.93% ± 4.08(n=3 that experimental group, which breaks up the 35th day cell CK14 positive rate), cellular control unit positive rate is
(9.97 ± 2.16) %(n=3), it is examined through Student ' s t, difference is statistically significant (t=28.9, P < 0.01).
3.3 immunofluorescence dyeings detection, as a result as (A is that cellular control unit breaks up CK15 dyeing in the 35th day to Fig. 3, and B is real
Test the CK15 dyeing in the 35th day of group cell differentiation；C is the P63 dyeing in the 35th day of experimental group cell differentiation, and D, which shines for C with nuclear staining, to be superimposed；
E is that cellular control unit breaks up P63 dyeing in the 35th day, and F is shown in E is superimposed with nuclear staining photograph):
Break up the 35th day, experimental group CK15, P63 luciferase expression amount is all remarkably higher than control group.
The present invention is by AA, BMP4 and RA combined induction embryonic stem cell, cell height expression keratinocyte label at the 35th day
Object p63 albumen, Keratin 14,15, the cell paving stone shape uniform in size and form are close in appearance and biological characteristics
Human keratinocyte.It is up to 77.93% through Flow Cytometry detection Keratin 14 expression rate, significantly larger than other formula inducers.
Inducer provided by the invention improves the efficiency that human embryo stem cell for directional is induced to differentiate into keratinocyte, provides one efficiently
Induce approach of the hESC to keratinocyte differentiation.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (5)
1. a kind of inducing embryo stem cell directed differentiation be keratinocyte culture medium, which is characterized in that the culture medium be by
Inducing embryo stem cell directed differentiation is that the inducer of keratinocyte is added to the differential medium for embryonic stem cell culture
What middle preparation obtained, the inducing embryo stem cell directed differentiation is to contain 25ng/mL bon e formation in the culture medium of keratinocyte
4,1 μM of vitamin A acids of albumen and 0.3mmol/L ascorbic acid.
2. the culture medium that inducing embryo stem cell directed differentiation according to claim 1 is keratinocyte, which is characterized in that
The differential medium for embryonic stem cell culture contains N2 additive, the percent by volume that concentration of volume percent is 1%
The DMEM/F12 culture solution that concentration is 99%.
3. a kind of method that inducing embryo stem cell directed differentiation is keratinocyte, which is characterized in that use claims 1 or 2
The inducing embryo stem cell directed differentiation is the culture medium of keratinocyte, comprising the following steps:
It step 1, is angle in inducing embryo stem cell directed differentiation of any of claims 1 or 2 by the embryonic stem cell after passage
Change in the culture medium of cell adhere-wall culture 5-10 days, changes liquid daily.
4. according to the method described in claim 3, it is characterized in that, further including pre-treatment step before step 1: by embryo
Stem cell strain is cultivated in mTeSR1 culture medium, passage.
5. according to the method described in claim 3, it is characterized in that, further including post-processing step: step 1 processing being obtained thin
It after born of the same parents are with neutral protein enzymic digestion, is seeded in the coated culture dish of type i collagen, adhere-wall culture is continued with keratinocyte culture solution
3-6 weeks, the next day change liquid, cell log growth period pass on.
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| CN105087473A (en) * | 2015-08-14 | 2015-11-25 | 上海交通大学医学院附属第九人民医院 | Method for screening extracellular matrixes for in-vitro induced directional differentiation of human embryonic stem cells into keratinocytes |
| CN106399229A (en) * | 2016-10-25 | 2017-02-15 | 浙江译美生物科技有限公司 | Inductive agent and method for preparing epidermal stem cells by adoption of inductive agent |
| CN111088213B (en) * | 2018-10-24 | 2022-04-08 | 澳门大学 | Method for inducing stem cells to gradually differentiate to form keratinocytes and keratinocytes |
| CN111254114B (en) * | 2020-03-24 | 2021-12-07 | 山东兴瑞生物科技有限公司 | Culture method for converting human oral mucosa stem cells into astrocytes |
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| US20070042491A1 (en) * | 2005-08-18 | 2007-02-22 | Karp Jeffrey M | Amplification of cell populations from embryonic stem cells |
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| US20070042491A1 (en) * | 2005-08-18 | 2007-02-22 | Karp Jeffrey M | Amplification of cell populations from embryonic stem cells |
| CN101711890A (en) * | 2009-01-15 | 2010-05-26 | 中国人民解放军军事医学科学院基础医学研究所 | Extracellular matrix gel model used for researching development and differentiation of embryonic stem cells |
| CN103361307A (en) * | 2013-06-24 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | Induced differentiation method of C3H10T1/2 mesoderm pluripotent embryonic stem cell strain |
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