CN104651298A - Inducing agent and medium for inducing directional differentiation of embryonic stem cell into keratinocytes - Google Patents
Inducing agent and medium for inducing directional differentiation of embryonic stem cell into keratinocytes Download PDFInfo
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- CN104651298A CN104651298A CN201310596606.1A CN201310596606A CN104651298A CN 104651298 A CN104651298 A CN 104651298A CN 201310596606 A CN201310596606 A CN 201310596606A CN 104651298 A CN104651298 A CN 104651298A
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Abstract
The invention provides an inducing agent for inducing directional differentiation of an embryonic stem cell into keratinocytes. The inducing agent comprises bone morphogenetic protein 4, retinoic acid and ascorbic acid. Preferably, in the inducing agent, the bone morphogenetic protein 4, retinoic acid and ascorbic acid are in a content ratio of (1-50g):(0.1-5 mol):(50-600mol). The inducing agent for inducing directional differentiation of the embryonic stem cell into keratinocytes combines the bone morphogenetic protein 4, retinoic acid and ascorbic acid, can effectively improve the efficiency for induced directional differentiation of hESC into keratinocytes (as high as 77.93%, and far higher than other formula inducing agents), provides an approach for efficiently inducing differentiation of hESC into keratinocytes, and provides seed cells for constructing tissue engineered oral mucosas.
Description
Technical field
The present invention relates to a kind of inductor of inducing embryo stem cell directed differentiation, particularly relating to a kind of inducing embryo stem cell directed differentiation is Keratinocytic inductor, substratum and method.
Background technology
Oral mucosa is the first barrier that human body resists extraneous various injurious factor, has various tissue under protection oral mucosa, sensation, temperature regulates and secretion etc. is very important function.Because surgical operation before periodontal surgery, dentures repai, tooth implantation, congenital or the facial deformity prosthesis day after tomorrow, wound and tumor resection etc. may cause defect or the functional defect of oral cavity mucous membrane tissue, often need to carry out oral cavity mucous membrane tissue reconstruction.Current clinical extensive employing auto-skin grafting or oral mucosa lobe skin grafing and mending defect.But cutify still keeps the feature such as skin epidermis original structure function and angling, secretion, hair growth for many years, lack the lubricious sense of humidity of normal mucosa, make patient feel more uncomfortable, can not meet clinical needs far away, thus limit clinical application.And aforesaid method all will open up second-hand visual area, new wound and misery can be brought to patient.Therefore, finding a kind of desirable oral mucosa surrogate is urgent need to solve the problem clinically.
Oral mucosa keratinocyte belongs to stratified squamous epithelium, based on keratinocyte.Inducing embryo stem cell (hESC) directed differentiation is Keratinocytic key is stop its neuralward cell direction to break up at the hESC differentiation initial stage, by suitable inductor induced synthesis keratinocyte progenitor cell, and breaks up further, maintains epithelial phenotype.Prior art mainly adopts the inductor of different components, as being used alone bone forming egg 4(bonemorphogenetic protein 4, BMP4) xitix (ascorbic acid, is used alone, AA), conbined usage BMP4 and vitamin A acid (retinoic acid, RA), conbined usage BMP4 and AA, when the inductor adopting these to fill a prescription induces the 35th day, the expression rate of CK14 is respectively 5.4%, 8.9%, 35% and 59%.These cells maintain in phenotype, and screening purifying, the aspects such as whole end differentiation still are apparent not enough, and need to be optimized method of inducing differentiation.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, a kind of inducing embryo stem cell directed differentiation is provided to be Keratinocytic inductor, substratum and method, effectively can improve hESC Induction of committed differentiation is Keratinocytic efficiency, provides seed cell for Oral Tissue-engineered Mucosa builds.
It is Keratinocytic inductor that first aspect of the present invention is to provide a kind of inducing embryo stem cell directed differentiation, and described inductor comprises bone morphogenetic protein 4(BMP4), vitamin A acid (RA) and xitix (AA).
In one preferred embodiment, in described inductor, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (1-50g): (0.1-5mol): (50-600mol).
Further preferably, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (5-45g): (0.2-4.5mol): (100-500mol).
Further preferably, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (10-40g): (0.4-4mol): (150-450mol).
Further preferably, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (20-35g): (0.5-3mol): (200-400mol).
Further preferably, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (25-30g): (1-2mol): (250-350mol).
It is Keratinocytic substratum that second aspect of the present invention is to provide a kind of inducing embryo stem cell directed differentiation, and described substratum contains the inductor described in above-mentioned first aspect.
In one preferred embodiment, described inducing embryo stem cell directed differentiation is contain in Keratinocytic substratum:
Bone morphogenetic protein 41-50ng/ml, is more preferably 5-45ng/ml, is more preferably 10-40ng/ml, is more preferably 20-35ng/ml, is more preferably 20-35ng/ml;
Vitamin A acid 0.1-5uM, is more preferably 0.2-4.5uM, is more preferably 0.4-4uM, is more preferably 0.5-3uM, is more preferably 1-2uM;
Xitix 0.05-0.6mmol/L, is more preferably 0.1-0.5mmol/L, is more preferably 0.15-0.45mmol/L, is more preferably 0.2-0.4mmol/L, is more preferably 0.25-0.35mmol/L, such as 0.3mmol/L, 0.32mmol/L or 0.28mmol/L.
In one preferred embodiment, described inducing embryo stem cell directed differentiation is that Keratinocytic substratum comprises the division culture medium cultivated for embryonic stem cell.
Preferably, the described division culture medium for embryonic stem cell cultivation contains DMEM/F12 nutrient solution and N2 additive:
The concentration of volume percent of DMEM/F12 nutrient solution is 95-99.9%, is more preferably 96-99.5%, 97-99.2t%, 98-99%;
The concentration of volume percent of N2 additive is 0.1-5%, is more preferably 0.5-4%, is more preferably 0.8-3%, be more preferably 1-2%.
In a further preferred embodiment, described inducing embryo stem cell directed differentiation for Keratinocytic substratum formulated by the following method: the inductor described in the present invention first aspect is joined described being used in the division culture medium that embryonic stem cell cultivates.
It is Keratinocytic method that 3rd aspect of the present invention is to provide a kind of inducing embryo stem cell directed differentiation, adopts the inducing embryo stem cell directed differentiation in claim 4-7 described in any one to be Keratinocytic substratum, comprises the following steps:
Step 1, employing go down to posterity after the embryonic stem cell (cell of 2-7 days after preferably going down to posterity, such as, after the going down to posterity cell of the 3rd day, the 4th day, the 5th day or the 6th day) substratum inducing embryo stem cell directed differentiation in claim 4-7 described in any one is Keratinocytic middle adherent culture 5-10 day (be preferably 6-9 day, be more preferably 7-8 day), every day changes liquid.
In one preferred embodiment, also comprise pre-treatment step: embryonic stem cell strain cultivated in the medium, go down to posterity.
In one preferred embodiment, also comprise post-processing step: step 1 is processed the cell that obtains with after neutral protein enzymic digestion, be seeded in the culture dish of type i collagen bag quilt, adherent culture 3-6 week (being more preferably 4-5 week) is continued with keratinocyte nutrient solution, the next day change liquid, go down to posterity vegetative period at cell log.
Inducing embryo stem cell directed differentiation provided by the invention is Keratinocytic inductor, be compounded with bone morphogenetic protein 4, vitamin A acid and xitix, effectively can improve hESC Induction of committed differentiation is that (Keratin 14 expression rate is up to 77.93% for Keratinocytic efficiency, far away higher than other formula inductors) provide an efficient induction hESC to the approach of keratinocyte differentiation, provide seed cell for Oral Tissue-engineered Mucosa builds.
Accompanying drawing explanation
Fig. 1 is the single morphologic detection result of hESC after inductor provided by the invention is differentiation-inducing;
Fig. 2 is hESC single stream Schwann Cells detected result after inductor provided by the invention is differentiation-inducing;
Fig. 3 is hESC single immunization fluorescent dye result after inductor provided by the invention is differentiation-inducing.
Embodiment
With reference to the accompanying drawings, the present invention is further illustrated in conjunction with specific embodiments, to understand the present invention better.
The cellar culture of 1.hESC: this research adopts the H9 human embryo stem cell strain (agreement no.11-W0039, WiCell Research Institute, Madison, WI) of WHO's registration.HESC with mTeSR1 culture medium culturing, goes down to posterity on matrigel for 5 days.
The cellar culture of 1.hESC: this research adopts the H9 human embryo stem cell strain (agreement no.11-W0039, WiCell Research Institute, Madison, WI) of WHO's registration.HESC with mTeSR1 culture medium culturing, goes down to posterity on matrigel for 5 days.
2.hESC is to keratinocyte induction scheme:
2.1 experimental group inducing embryo stem cell directed differentiation are the preparation of Keratinocytic substratum: be that Keratinocytic inductor both to join in the division culture medium cultivated for embryonic stem cell by inducing embryo stem cell directed differentiation provided by the invention, wherein, described inducing embryo stem cell directed differentiation is contain in Keratinocytic substratum: 25ng/ml bone morphogenetic protein 4(BMP4), 1uM vitamin A acid (RA) and 0.3mmol/L xitix (AA), the described division culture medium for embryonic stem cell cultivation contains the N2 additive that concentration of volume percent is 1%, concentration of volume percent is the DMEM/F12 nutrient solution of 99%.
2.2 experimental group induction methods: to adopt after going down to posterity the hESC of the 4th day adherent culture 7 days in the inductor of above-mentioned formula, every day changes liquid, after cell being digested with neutral protease (Dispase) in 7th, the culture dish that the cell getting 1/3 is seeded to type i collagen bag quilt continues adherent culture 4 weeks with keratinocyte nutrient solution, the next day change liquid, go down to posterity vegetative period at cell log.
2.3 control groups are arranged: the preparation of control group substratum: joined in the division culture medium cultivated for embryonic stem cell by contrast inductor and both obtained, wherein, contrast inductor is made up of bone morphogenetic protein 4 and xitix, containing 25ng/ml bone morphogenetic protein 4(BMP4 in control group substratum) and 0.3mmol/L xitix (AA).For the same experimental group of division culture medium that embryonic stem cell is cultivated in control group, and the same experimental group of control group induction method.
3. effect detection of the present invention:
3.1 morphologic detection, result as Fig. 1 (A2, A7, A14, A21, A28 and A35 be respectively cellular control unit differentiation the 2nd, 7,14,21,28 and 35 day inverted microscope mirror under photo, B2, B7, B14, B21, B28 and B35 are experimental group cytodifferentiation the 2nd, 7,14,21,28 and 35 days photos, scale display 0.1mm) shown in:
The differentiation-inducing 1-7d of control group, clone's area increases gradually, breaks up and is in progress to center by clone edge, noble cells is the growth of oval monolayer adherence, subregion presents paving stone shape, and undifferentiated cell still cloning assembles growth, and between noble cells and undifferentiated cell, boundary obviously.7th day 1:3 go down to posterity be inoculated in type i collagen paving quilt culture dish in cultivate with DSFM, adherently in 24h still to grow with cloned version thereof.10th day, accelerated cellular proliferation, Mitotic index was common, cell quantity showed increased, the 14th day, and cell grows gradually and merges in flakes, and clone sees paving stone shape cell around, and central authorities still keep undifferentiated cell layered laminate.16th day, cytogamy, went down to posterity as stated above, the 18th day, cell attachment, and the cell of full extension is flat polygon shape, and size shape differs.35th day, see cytogamy, undifferentiated cell is intensive in heaps, and noble cells is dispersed in growth in paving stone shape.
Experimental group induces 1-7d, and ES clonal growth speed is greater than experiment A group, and see under the 2nd clear water surface that noble cells is more than A group, cell is oval, the uniform paving stone shape of form, and growth is merged in flakes gradually, and between differentiation and undifferentiated cell, boundary is not obvious.Within 7th day, cell is dense laminated, and propagating method is with A group, and in 24h, attached cell is more than A group.Within 9th day, accelerated cellular proliferation merges in flakes gradually, nuclear fission as common, in uniform paving stone shape.14th day cell grows gradually and merges in flakes, and clone sees streak cell, paving stone shape cell homogeneous sized by central authorities, Monolayer growth of cells around.Cell growth rate slows down, and is significantly less than A group.19th day cytogamy, go down to posterity same A group.Within 21st day, cell is adherent gradually, is the round cell that refractivity is strong.Cell stretches out short and small pseudopodium gradually subsequently, and kytoplasm launches, single cells grown.35th day, cytosis, the paving stone shape homogeneous in size and form, had no undifferentiated cell.
3.2 stream Schwann Cells technology for detection, result is as shown in Fig. 2 (ES is the CK14 expression rate of hESC before induction, and A is that control group breaks up the 35th day cell CK14 positive rate, and B breaks up the 35th day cell CK14 positive rate for experimental group):
It is 77.93% ± 4.08(n=3 that experimental group breaks up the 35th day cell CK14 positive rate), cellular control unit positive rate is (9.97 ± 2.16) %(n=3,) check through Student ' st, difference has statistical significance t(=28.9, P < 0.01).
3.3 immunofluorescence dyeings detect, and (A is that cellular control unit breaks up CK15 dyeing in the 35th day to result such as Fig. 3, and B is the CK15 dyeing in the 35th day of experimental group cytodifferentiation; C is the P63 dyeing in the 35th day of experimental group cytodifferentiation, and D is that C superposes with nuclear staining photograph; E is that cellular control unit breaks up P63 dyeing in the 35th day, and F is that E is shown according to superposing with nuclear staining):
Break up the 35th day, experimental group CK15, P63 luciferase expression amount are all significantly higher than control group.
The present invention is by AA, BMP4 and RA combined induction embryonic stem cell, cell high expression level keratinocyte marker p63 albumen, Keratin 14,15 when the 35th day, cell is the homogeneous paving stone shape of size and form, all close to human keratinocyte in outward appearance and biological characteristics.Through Flow Cytometry detection angle protein 14 expression rate up to 77.93%, far away higher than other formula inductors.Inductor provided by the invention improves human embryo stem cell for directional and is induced to differentiate into Keratinocytic efficiency, provides one and efficiently induces hESC to the approach of keratinocyte differentiation.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. inducing embryo stem cell directed differentiation is a Keratinocytic inductor, it is characterized in that, described inductor comprises bone morphogenetic protein 4, vitamin A acid and xitix.
2. inductor according to claim 1, is characterized in that, in described inductor, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (1-50g): (0.1-5mol): (50-600mol).
3. inductor according to claim 2, is characterized in that, the content of bone morphogenetic protein 4, vitamin A acid and xitix is than being (20-35g): (0.5-3mol): (200-400mol).
4. an inducing embryo stem cell directed differentiation is Keratinocytic substratum, it is characterized in that, containing inductor according to claim 1 in described substratum, and the content of bone morphogenetic protein 4 is 1-50ng/ml in substratum, the content of vitamin A acid is 0.1-5uM, and the content of xitix is 0.05-0.6mmol/L.
5. substratum according to claim 4, is characterized in that, in described substratum, the content of bone morphogenetic protein 4 is 20-35ng/ml, and the content of vitamin A acid is 0.5-3uM, and the content of xitix is 0.2-0.4mmol/L.
6. substratum according to claim 4, is characterized in that, described substratum also comprises the division culture medium cultivated for embryonic stem cell.
7. substratum according to claim 6, it is characterized in that, the described division culture medium cultivated for embryonic stem cell contains DMEM/F12 nutrient solution and N2 additive, and the concentration of volume percent of DMEM/F12 nutrient solution is the concentration of volume percent of 95-99.9%, N2 additive is 0.1-5%.
8. inducing embryo stem cell directed differentiation is a Keratinocytic method, it is characterized in that, adopts the inducing embryo stem cell directed differentiation in claim 4-7 described in any one to be Keratinocytic substratum, comprises the following steps:
Step 1, adopt the substratum inducing embryo stem cell directed differentiation of embryonic stem cell in claim 4-7 described in any one after going down to posterity to be Keratinocytic middle adherent culture 5-10 day, every day changes liquid.
9. method according to claim 8, is characterized in that, also comprises pre-treatment step: embryonic stem cell strain cultivated in the medium, go down to posterity.
10. method according to claim 8, it is characterized in that, also comprise post-processing step: step 1 is processed the cell that obtains with after neutral protein enzymic digestion, be seeded in the culture dish of type i collagen bag quilt, adherent culture 3-6 week is continued with keratinocyte nutrient solution, the next day change liquid, go down to posterity vegetative period at cell log.
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| CN111088213A (en) * | 2018-10-24 | 2020-05-01 | 澳门大学 | Method for inducing stem cells to gradually differentiate to form keratinocytes and keratinocytes |
| CN111254114A (en) * | 2020-03-24 | 2020-06-09 | 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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