CN109563485A - Broken up by what is induced multi-potent stem cell come the method and system of cultured corneal epithelium cell - Google Patents
Broken up by what is induced multi-potent stem cell come the method and system of cultured corneal epithelium cell Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Abstract
The present invention provides through the methods that the differentiation induced multi-potent stem cell carrys out cultured corneal epithelium cell comprising: prepare the process of the basal medium comprising Dahl Burke Improved Eagle Medium/F12 (DMEM F12), L-AA, sodium selenite and sodium chloride；Addition is for cultivating the process of the additive induced multi-potent stem cell in the basal medium；The process of no feeder layer culture medium is prepared by applying vitronectin recombinant human protein on the basal medium；The process for inducing multi-potent stem cell and then being cultivated is added in the no feeder layer culture medium；The process for creating the additive of stem cell growth environment is added in the no feeder layer culture medium；Add bone morphogenetic protein 4 and keratinocyte growth factor successively in no feeder layer culture medium in order to induce and described induce multi-potent stem cell the process of corneal epithelial cell of being divided into；And in PI culture solution cultivate by the cell for inducing multi-potent stem cell differentiation process.
Description
Technical field
The present invention relates to the differentiation by inducing multi-potent stem cell come the method and system of cultured corneal epithelium cell.
Background technique
Corneal epithelial cell is located at the outermost of cornea, is to play weight in composition cornea and in terms of maintaining the transparency of cornea
The cellular layer to be acted on.The wound that the deformation of corneal epithelial cell or shortage are likely to decrease anterior corneal surface restores and remains transparent
The function of property, so as to cause corneal degeneration.
Corneal epithelial cell by corneal limbus corneal epithelium progenitor cells (corneal epithelial progenitor
Cells or limbal stem cells) differentiation, if the corneal epithelial cell in corneal epithelium deforms or lacks, begin
It is supplemented eventually.But if for genetic reasons or the acquired disposition of burn or disease etc and lead to the dry thin of corneal limbus
Born of the same parents lack, then can not form corneal epithelial cell, therefore, it is impossible to supplement corneal epithelium, finally make corneal degeneration.
In order to treat the corneal epithelium disease generated by this reason, many corneal epithelial regeneration sides are implemented
The research in face.Specifically, in order to treat corneal epithelium disease, it is intended to cross corneal transplant edge, but unmet effect.And
And also attempted to utilize mescenchymal stem cell (mesenchymal stem cell), adult stem cell and mouth epithelial cells
(oral mucosal cell) is treated, but be divided into corneal epithelial cell to be divided into corneal epithelial cell using it
Differentiation capability still reach to less than expectation, to be difficult to expect big effect.
Therefore, currently, it is badly in need of that the technology of the therapeutic effect for corneal epithelial cell exception can be improved.
Summary of the invention
Technical problem
It is an object of the present invention to solve problem of the prior art as described above and previous technical problem.
The present inventor is after passing through duplicate Depth Study and kinds of experiments, by confirming following situation come real
Completion of the invention is showed.That is, as by induce multi-potent stem cell (Induced Pluripotent Stem Cell,
IPSC the method that differentiation) carrys out cultured corneal epithelium cell, by applying vitronectin recombinant human protein on basal medium
(Vitronectin Recombinant Human Protein) prepares no feeder layer culture medium (feeder free
Culture medium), and in no feeder layer culture medium culture induce multi-potent stem cell after, successively add Bones morphology
Albumen 4 (BMP4) and keratinocyte growth factor (KGF) are divided into corneal epithelial cell to induce to induce multi-potent stem cell,
In this case, when corneal transplant, it can cultivate and be immunoreacted low corneal epithelial cell, and improve and be divided into corneal epithelial cell
Differentiation capability.
Solution to problem
It is therefore, of the invention to be broken up by what is induced multi-potent stem cell come the method for cultured corneal epithelium cell,
It is characterized in that, comprising: prepare comprising Dahl Burke Improved Eagle Medium/F12 (DMEM F12), L-AA, sub- selenium
The process of the basal medium of sour sodium (Sodium selenite) and sodium chloride；Addition is for training in the basal medium
Support the process of the additive induced multi-potent stem cell；By on the basal medium apply vitronectin recombinant human protein come
The process without feeder layer culture medium of preparation；Addition is induced multi-potent stem cell and then is cultivated in the no feeder layer culture medium
Process；The process for creating the additive of stem cell growth environment is added in the no feeder layer culture medium；In no feeding
It supports and successively adds bone morphogenetic protein 4 and keratinocyte growth factor in layer culture medium to induce the induced multi-potent dry
Cell differentiation is the process of corneal epithelial cell；And it is cultivated in PI culture solution and induces multi-potent stem cell the thin of differentiation by described
The process of born of the same parents.
It is described for cultivate the additive induced multi-potent stem cell to may include people's Holo-transferrin in a concrete example
(Holo transferrin), basic fibroblast growth factor (bFGF), transforminggrowthfactor-β1 (TGF β 1) and pancreas islet
Element.
In a concrete example, the additive for creating stem cell growth environment may include epidermal growth because
Sub (EGF) and insulin.
In a concrete example, in the mistake for successively adding the bone morphogenetic protein 4 and keratinocyte growth factor
Cheng Zhong, can add bone morphogenetic protein 4 can add keratinocyte growth factor, in detail after processing about 2 days to 4 days
Ground, can add bone morphogenetic protein 4 can add keratinocyte growth factor after processing about 3 days.
In a concrete example, in the mistake for successively adding the bone morphogenetic protein 4 and keratinocyte growth factor
Cheng Zhong can add keratinocyte growth factor, handle about 2 days to 4 days, in detail, can add keratinocyte growth factor,
Processing about 3 days.
In a concrete example, in the PI culture solution, Panserin culture solution: Yi Sikefu culture solution (Iscove '
S Medium) weight ratio can for 2:1 to 1:2 can be in detail 1:1.
In a concrete example, can be cultivated in PI culture solution by the cell for inducing multi-potent stem cell differentiation 1 week to 3
Week.
The present invention also provides the differentiation by inducing multi-potent stem cell come the system of cultured corneal epithelium cell.
The system can include: without feeder layer culture medium, by including DMEM F12, L-AA, sodium selenite
And addition induces multi-potent stem cell culture additive in the basal medium of sodium chloride, and applies vitronectin recombinant human protein
To prepare；It induces multi-potent stem cell, is cultivated in the no feeder layer culture medium；For creating the addition of stem cell growth environment
Agent is added in the no feeder layer culture medium；Bone morphogenetic protein 4 and keratinocyte growth factor, comprising described
Cultivated in no feeder layer culture medium induce multi-potent stem cell and the culture medium of the additive for creating stem cell growth environment
In successively add to induce to induce multi-potent stem cell and be divided into corneal epithelial cell；And PI culture solution, for cultivating by institute
State the cell for inducing multi-potent stem cell differentiation.
Detailed description of the invention
Fig. 1 is to shoot inducing multi-potent stem cell without what is cultivated in feeder layer culture medium in embodiment 1 as time goes by
Photo.
Fig. 2 is the shooting photo induced multi-potent stem cell cultivated in the feeder layer culture medium of comparative example 17 days.
Fig. 3 and Fig. 4 be using undifferentiated iPSC mark come in embodiment 1 without the induction cultivated in feeder layer culture medium
Multipotential stem cell carries out immunostaining, and uses the result of protein expression display (protein expression array)
Shoot photo.
Fig. 5 to Fig. 8 is the shooting photograph of the corneal stem cells label of embodiment 1 and the expression status of corneal epithelial cell label
Piece.
Fig. 9 is by the bat of gas lift (Air Lift) experimental result of the cell for inducing multi-potent stem cell differentiation of embodiment 1
Take the photograph photo.
Specific embodiment
Hereinafter, being illustrated referring to the embodiment of the present invention, but it is used to be easier to understand the present invention, scope of the invention
It is not limited to this.
Embodiment 1
It induces multi-potent stem cell, has prepared comprising DMEM F12, L-AA, sodium selenite and sodium chloride in order to cultivate
Basal medium, and be adjusted to pH7.4.In basal medium, as cultivating the additive induced multi-potent stem cell,
It is added to turn of people's Holo-transferrin of 10 μ g/ml, the basic fibroblast growth factor of 100ng/mL, 1.74ng/ml
Change grouth factor beta 1 and the insulin of 20 μ g/ml.
Using feeder layer (feeder) to cultivate stem cell, when transplanting differentiated corneal epithelial cell,
Immune response is possible to cause problem, thus in order to prepare without using feeder layer without feeder layer culture medium, in basal medium
On be coated with after vitronectin recombinant human protein, cultivated and induced multi-potent stem cell 7 days.
Vitronectin recombinant human protein is coated with by following procedure.On the basis of 6 orifice plates culture vessel, in Du of 9ml
Primary section's phosphate buffer (DPBS) (phosphate buffer (Phosphate Buffered Saline) of no Ca2+ and Mg2+)
The vitronectin recombinant human protein (50 μ g/ml) of 60 μ l of middle investment is diluted, and is respectively put into 1.5ml in each hole,
After keeping not dry, under frozen state (about 4 DEG C), react 12 hours.Later, at room temperature, it reacts 1 hour, and
It is washed using phosphate buffer (PBS).
In order to which cultured induce multi-potent stem cell is divided into corneal epithelial cell, firstly, in no feeder layer culture medium
In, as the additive for creating stem cell growth environment, it is added to the epithelical cell growth factor and 5 μ g/ml of 10ng/ml
Insulin.
Then, for inducing ectodermal progenitor cells (ectoderm progenitors) in the early stage, it is added to 100ng/ml
Bone morphogenetic protein 4, processing 3 days after, in order to be divided into corneal epithelial cell, the keratinization for being added to 200ng/ml is thin
The intracellular growth factor is handled 3 days.
Then, in the PI culture solution for mixing Panserin culture solution and Yi Sikefu culture solution with the weight ratio of 1:1, training
It has supported by induce multi-potent stem cell differentiation cell about 1 week to 3 weeks.
Comparative example 1
It induces multi-potent stem cell, has prepared comprising DMEM F12, L-AA, sodium selenite and sodium chloride in order to cultivate
Basal medium, and be adjusted to pH7.4.In basal medium, as cultivating the additive induced multi-potent stem cell,
It is added to turn of people's Holo-transferrin of 10 μ g/ml, the basic fibroblast growth factor of 100ng/mL, 1.74ng/ml
Change grouth factor beta 1 and the insulin of 20 μ g/ml.As feeder layer, it is added to 25000 cells/cm2 and uses mitomycin C
Mouse embryonic fibroblasts (mouse embryonic fibroblast, the MEF) raising of (mitomycin C, MMC) processing
After layer, cultivates and induced multi-potent stem cell.
Comparative example 2
It induces multi-potent stem cell, has prepared comprising DMEM F12, L-AA, sodium selenite and sodium chloride in order to cultivate
Basal medium, and be adjusted to pH7.4.In basal medium, as cultivating the additive induced multi-potent stem cell,
It is added to turn of people's Holo-transferrin of 10 μ g/ml, the basic fibroblast growth factor of 100ng/mL, 1.74ng/ml
Change grouth factor beta 1 and the insulin of 20 μ g/ml.
It applies vitronectin recombinant human protein on basal medium to prepare without in feeder layer culture medium, has cultivated and lured
Lead multipotential stem cell.
In order to which cultured induce multi-potent stem cell is divided into corneal epithelial cell, firstly, in no feeder layer culture medium
In, as the additive for creating stem cell growth environment, it is added to the epithelical cell growth factor and 5 μ g/ml of 10ng/ml
Insulin.
Then, it is added to the bone morphogenetic protein 4 of 100ng/ml, after processing 2 days to 14 days, with the weight of 1:1
Than having cultivated by inducing multi-potent stem cell differentiation in the PI culture solution of mixing Panserin culture solution and Yi Sikefu culture solution
Cell about 1 week to 3 weeks.
Experimental example 1
It observes in embodiment 1 without inducing multi-potent stem cell of cultivating in feeder layer culture medium and in the feeding of comparative example 1
Support the cultivation conditions induced multi-potent stem cell cultivated in layer culture medium.The shooting photo of its result is shown in Fig. 1 and Fig. 2.
Fig. 1 is to shoot inducing multi-potent stem cell without what is cultivated in feeder layer culture medium in embodiment 1 as time goes by
Photo, Fig. 2 is the shooting photo induced multi-potent stem cell cultivated in the feeder layer culture medium of comparative example 17 days.
Referring to FIG. 1 and FIG. 2, inducing multi-potent stem cell without what is cultivated in feeder layer culture medium in embodiment 1 is confirmed
It is induced multi-potent stem cell compared to what is cultivated in the feeder layer culture medium of comparative example 1, clearly formation bacterium colony, it is seen that embodiment 1
Induce multi-potent stem cell by size and in the form of uniform state cultivated.
Experimental example 2
Using SOX2, OCT4A, SSEA4, TRA-1-81 and the TRA1-60S marked as undifferentiated iPSC come immunostaining
In being induced multi-potent stem cell without what is cultivated in feeder layer culture medium for embodiment 1, and displayed using protein expression to carry out
Confirmation.It the results are shown in Fig. 3 and Fig. 4.
Firstly, referring to Fig. 3, confirm it is cultured induce multi-potent stem cell it is dry with the presence or absence of showing to have in (iPSC)
The expression of protein labeling TRA-1-81, TRA1-60S, SSEA4, OCT4A of cell characteristics.
Then, referring to Fig. 4, in order to which the protein isolate matter in cultured induce multi-potent stem cell and comparison protein are expressed
Form implements protein array (protein array) to stem cell labeling, confirms the label as stem cell
The expression of SOX2, OT 3/4.
Experimental example 3
For the cell for inducing multi-potent stem cell differentiation by embodiment 1 and inducing multi-potent stem cell point by comparative example 2
The cell of change before cultivating in PI culture solution and after culture 3 weeks, is utilized respectively corneal stem cells label (ABCG2, △
Np63, Pax6, CK14) and corneal epithelial cell label (CK3, CK12) analyze expression status.
It for comparative example 2 the results show that even if handling most 2 weeks using bone morphogenetic protein 4, and pass through
The expression of atomization, corneal stem cells and corneal epithelial cell label is also restricted.Conversely, being observed for embodiment 1
CK14, Pax6 as corneal limbus label are expressed, dry compared to cornea thin after being handled with keratinocyte growth factor
The expression of born of the same parents' label, corneal epithelial cell label is stronger.The corneal stem cells label and corneal epithelial cell of embodiment 1 will be shot
The shooting photo of the expression status of label is shown in Fig. 5 into Fig. 8.
Experimental example 4
It implements and is formed for confirming by the corneal epithelial cell layer of the cell for inducing multi-potent stem cell differentiation of embodiment 1
The gas lift (Air Lift) of ability is cultivated, and is observed by electron microscope.It the results are shown in Fig. 9, thus may be used
Confirm the cellular layer for being formed with multilayer.
More than, it is illustrated referring to the embodiment of the present invention, but for the ordinary skill of the technical field of the invention
For personnel, it is based on the content, a variety of applications and deformation can be carried out within the scope of the present invention.
Practicability
As described above, of the invention broken up by what is induced multi-potent stem cell come the method for cultured corneal epithelium cell
And system is immunoreacted low corneal epithelial cell when can provide corneal transplant.
The differentiation capability that corneal epithelial cell is divided by inducing multi-potent stem cell can be improved in method and system of the invention.
The corneal epithelial cell provided by method and system of the invention can for cell differentiation basic research and hair
Interpretation of the cause, onset and process of an illness system, which is found out, is used as model in research process.
Therapeutic agent for replacing organ transplantation can be used as by the corneal epithelial cell that method and system of the invention provide
Developmental research model.
Method and system of the invention are applicable as that be divided into a variety of bodies dirty by inducing multi-potent stem cell for being suitable for
The platform technology of the differentiation technique of device.
Claims (11)
1. a kind of broken up by what is induced multi-potent stem cell come the method for cultured corneal epithelium cell, which is characterized in that packet
It includes:
Add bone morphogenetic protein 4 and keratinocyte growth factor successively in no feeder layer culture medium in order to induce induction
Pluripotent stem cell differentiation is the process of corneal epithelial cell；And
Cultivate the process by the cell for inducing multi-potent stem cell differentiation.
2. the method according to claim 1, wherein being gone back before being induced to differentiate into the corneal epithelial cell
Include:
The process without feeder layer culture medium of preparation；
The process for inducing multi-potent stem cell and then being cultivated is added in the no feeder layer culture medium；And
The process for creating the additive of stem cell growth environment is added in the no feeder layer culture medium.
3. according to the method described in claim 2, it is characterized in that, the process of the preparation without feeder layer culture medium includes:
Prepare the process of the basal medium comprising DMEM F12, L-AA, sodium selenite and sodium chloride；
Addition is for cultivating the process of the additive induced multi-potent stem cell in the basal medium；And
The process of no feeder layer culture medium is prepared by applying vitronectin recombinant human protein on the basal medium.
4. the method according to claim 1, wherein being cultivated in PI culture solution described by inducing multi-potent stem cell
The cell of differentiation.
5. according to the method described in claim 3, it is characterized in that, described for cultivating the additive packet induced multi-potent stem cell
Holo-transferrin containing people, basic fibroblast growth factor, transforminggrowthfactor-β1 and insulin.
6. according to the method described in claim 2, it is characterized in that, described for creating the additive packet of stem cell growth environment
Containing epithelical cell growth factor and insulin.
7. the method according to claim 1, wherein successively adding bone morphogenetic protein 4 and cutin described
During changing Porcine HGF, bone morphogenetic protein 4 is added, after processing 2 days to 4 days, addition keratinocyte is raw
The long factor.
8. the method according to the description of claim 7 is characterized in that successively adding bone morphogenetic protein 4 and cutin described
During changing Porcine HGF, keratinocyte growth factor is added, is handled 2 days to 4 days.
9. according to the method described in claim 4, it is characterized in that, in the PI culture solution, Panserin culture solution: Yi Si
The weight ratio of Ke's husband's culture solution is 2:1 to 1:2.
10. according to the method described in claim 4, it is characterized in that, being cultivated in PI culture solution dry thin by the induced multi-potent
Cell 1 week to 3 weeks of born of the same parents' differentiation.
11. a kind of broken up by what is induced multi-potent stem cell come the system of cultured corneal epithelium cell, which is characterized in that packet
It includes:
Without feeder layer culture medium, by the basal medium comprising DMEM F12, L-AA, sodium selenite and sodium chloride
Middle addition applies vitronectin recombinant human protein for cultivating the additive induced multi-potent stem cell to prepare；
It induces multi-potent stem cell, is cultivated in the no feeder layer culture medium；
For creating the additive of stem cell growth environment, added in the no feeder layer culture medium；
Bone morphogenetic protein 4 and keratinocyte growth factor, the induction cultivated in comprising the no feeder layer culture medium
It successively adds in the culture medium of multipotential stem cell and the additive for creating stem cell growth environment in order to induce induced multi-potent
Stem cell is divided into corneal epithelial cell；And
PI culture solution, for cultivating by the cell for inducing multi-potent stem cell differentiation.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160101953A KR102015815B1 (en) | 2016-08-10 | 2016-08-10 | Method for Culturing Cornea Epithealial Cell by Inducing Differentiation of Induced Pluripotent Stem Cell and System for the Same |
| KR10-2016-0101953 | 2016-08-10 | ||
| PCT/KR2017/008465 WO2018030719A1 (en) | 2016-08-10 | 2017-08-04 | Method and system for culturing corneal epithelial cell by inducing differentiation of induced pluripotent stem cell |
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| CN109563485A true CN109563485A (en) | 2019-04-02 |
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| Country | Link |
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| KR (1) | KR102015815B1 (en) |
| CN (1) | CN109563485A (en) |
| WO (1) | WO2018030719A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337459A (en) * | 2021-06-02 | 2021-09-03 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Method for improving differentiation efficiency of pluripotent stem cells |
| 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 |
| CN117487659A (en) * | 2023-11-10 | 2024-02-02 | 世联生物工程无锡有限公司 | Snail stem cell culture environment regulation and control system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112608898B (en) * | 2020-12-30 | 2022-11-11 | 江苏艾尔康生物医药科技有限公司 | Corneal endothelial cell induction method |
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| CN103492555A (en) * | 2011-04-20 | 2014-01-01 | 国立大学法人大阪大学 | Ips cell having differentiation propensity for corneal epithelium |
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| WO2016114285A1 (en) * | 2015-01-15 | 2016-07-21 | 国立大学法人大阪大学 | Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells |
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| RU2018128383A (en) * | 2012-03-07 | 2019-03-14 | Янссен Байотек, Инк. | ENVIRONMENT OF A DEFINED COMPOSITION FOR REPRODUCTION AND UPDATE OF PLURIPOTENT STEM CELLS |
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- 2016-08-10 KR KR1020160101953A patent/KR102015815B1/en active IP Right Grant
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- 2017-08-04 WO PCT/KR2017/008465 patent/WO2018030719A1/en active Application Filing
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| WO2018030719A1 (en) | 2018-02-15 |
| KR102015815B1 (en) | 2019-08-29 |
| KR20180017703A (en) | 2018-02-21 |
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