CN102465111A - Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells - Google Patents
Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells Download PDFInfo
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- CN102465111A CN102465111A CN2010105504872A CN201010550487A CN102465111A CN 102465111 A CN102465111 A CN 102465111A CN 2010105504872 A CN2010105504872 A CN 2010105504872A CN 201010550487 A CN201010550487 A CN 201010550487A CN 102465111 A CN102465111 A CN 102465111A
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Abstract
The invention provides a test method for induced differentiating human embryo stem cells to retina pigment epithelial cells, which comprises the following steps that: A) 0.25% pancreatic enzyme and 0.1mg/mL collagenase iv are mixed into phosphate buffered saline (PBS) solution containing 1mM calcium chloride (CaCl2) and 20% KSR, the solution is used for digesting embryo stem (ES) clones, so the ES becomes an ES cell group with 5 to 10 cells; B) the ES clone is incubated in a culture dish which is wrapped by gelatin in a short time so as to remove the feeder layer cells; and C) the ES cell group is cultured in a continuous differential culture medium with the concentration of 6.7*102 cell groups per liters in a non-adhesive bacteria culture dish. The test method has the advantages such as simple and convenient method, good repeatability, high successful rate, good omnisexuality, rich and economical source and the like.
Description
Technical field
The present invention relates to a kind of embryo stem cell for directional differentiation technique, particularly a kind of vitro human embryonic stem cell is induced to differentiate into the TP of retinal pigment epithelium.
Background technology
Human embryo stem cell (human Embnyonic Stem Cell, ES cell) is to belong to all-round liver cell, has the potential of the various human tissue cells of three germinal layers of differentiation.It is single how to induce the ES cell directional to be divided into, and specific cell is the focus of studying at present, and it will bring new hope for the replacement therapy of organizational project and organ.At present, external uncut jade animal (mouse, rat, cavy etc.) the ES cytodifferentiation of inducing is polytype cells such as neurone, hematopoietic cell, the willing cell of one-tenth, chondrocyte, myocardial cell, blood vessel pork skin.To be tied to form power low because people ES cell is built, and knows that the Thomson of the U.S. in 1998 just sets up people's ES clone first, thus the directed differentiation research of relevant people ES cell, at present still seldom.
Summary of the invention
The object of the present invention is to provide a kind of method, be specially a kind of TP that the vitro human embryonic stem cell is induced to differentiate into retinal pigment epithelium.
To achieve these goals, technical scheme provided by the present invention is:
A kind of vitro human embryonic stem cell is induced to differentiate into the TP of retinal pigment epithelium, it is characterized in that, concrete steps are:
A) the collagenase iv of 0.25% pancreatin and 0.1mg/mL is formulated in contains 1mM CaCl
2In the PBS solution of 20%KSR,, make it become the ES cell mass of 5-10 cell with its digestion ES clone.Be suspended in after the digestion in the serum free medium that contains WNT suppressor factor DKK1 (100ng/mL) and NODAL suppressor factor LEFTY-A (500ng/mL).
B) with ES clone short incubation period in the petridish that gelatin encapsulates to remove feeder layer cells
C) the ES cell mass in non-adhesion Micro-Organism Culture Dish with every milliliter 6.7 * 10
2The concentration of individual cell mass is being cultivated in the division culture medium continuously:
As preferred version of the present invention, continuous division culture medium comprises described in the step C:
(1) DMEM/F12,20%KSR, 0.1mM NMAA, 2mM L-glutamine substratum;
(2) G-MEM, 20%KSR, 0.1mM NMAA, 1mM pyruvate, 0.1mM BME substratum;
(3) G-MEM, 15%KSR, 0.1mM NMAA, 1mM pyruvate, 0.1mM BME substratum;
As preferred version of the present invention, the ES cell mass was cultivated 2 days in said (1) substratum; In said (2) substratum, cultivated 4 days; In said (3) substratum, cultivated 8 days; In said (4) substratum, cultivated 6 days; Then, add the suppressor factor DKK-1 (100ng/mL) of WNT and the suppressor factor LEFTY-A (500ng/mL) of NODAL.
Further; Every square centimeter of 10-15 ES cell cluster is layered on 8 holes of containing poly-lysine, ln and fibronectin again and cultivates on the slide, is containing the ES division culture medium of 10%KSR (G-MEM, 0.1mM NMAA; 1mM pyruvate, 0.1mM BME) the middle cultivation.
The invention has the beneficial effects as follows: have that method is easy, good reproducibility, success ratio be high, good perfection, the economic dispatch advantage is enriched in the source.
Embodiment
Below in conjunction with accompanying drawing preferred embodiment of the present invention is set forth in detail, thereby protection scope of the present invention is made more explicit defining so that advantage of the present invention and characteristic can be easier to it will be appreciated by those skilled in the art that.
A kind of vitro human embryonic stem cell is induced to differentiate into the TP of retinal pigment epithelium, and concrete steps are: A) the collagenase iv of 0.25% pancreatin and 0.1mg/mL is formulated in and contains 1mM CaCl
2In the PBS solution of 20%KSR,, make it become the ES cell mass of 5-10 cell with its digestion ES clone.Be suspended in after the digestion in the serum free medium that contains WNT suppressor factor DKK1 (100ng/mL) and NODAL suppressor factor LEFTY-A (500ng/mL); B) with ES clone short incubation period in the petridish that gelatin encapsulates to remove feeder layer cells; C) ES cell mass concentration with every milliliter of 6.7 * 102 cell masses in non-adhesion Micro-Organism Culture Dish is cultivated in following continuous division culture medium: (1) at DMEM/F12,20%KSR, and 0.1mM NMAA cultivated 2 days in the 2mM L-glutamine substratum; (2) at G-MEM, 20%KSR, 0.1mM NMAA, 1mM pyruvate cultivated 4 days in the 0.1mM BME substratum; (3) at G-MEM, 15%KSR, 0.1mM NMAA, 1mM pyruvate cultivated 8 days in the 0.1mM BME substratum; (4) at G-MEM, 10%KSR, 0.1mM NMAA, 1mM pyruvate cultivated 6 days in the 0.1mM BME substratum; After 20 days, add the suppressor factor DKK-1 (100ng/mL) of WNT and the suppressor factor LEFTY-A (500ng/mL) of NODAL.Every square centimeter of 10-15 ES cell cluster is layered on 8 holes of containing poly-lysine, ln and fibronectin again and cultivates on the slide; Containing the ES division culture medium (G-MEM of 10%KSR; 0.1mM NMAA, 1mM pyruvat e, 0.1mMBME) the middle cultivation.
At the 25th day, can observe Rx (neural retina progenitor cell mark) positive colony; At the 50th day, with abundant Mitf (RPE progenitor cell mark) and Pax6 positive colony (30.6 ± 4.7%) occurring; At the 60th day, there is the pigment cell of flakey and sexangle appearance to occur; At the 120th day, anti-ZO-1 antibody showed that the pigment cell that derives from people ES cell forms connection closely.
The above; Be merely embodiment of the present invention; But protection scope of the present invention is not limited thereto; Any those of ordinary skill in the art are in the technical scope that the present invention disclosed, and variation or the replacement that can expect without creative work all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (4)
1. a vitro human embryonic stem cell is induced to differentiate into the TP of retinal pigment epithelium, it is characterized in that concrete steps are:
A) the collagenase iv of 0.25% pancreatin and 0.1mg/mL is formulated in contains 1mM CaCl
2In the PBS solution of 20%KSR,, make it become the ES cell mass of 5-10 cell with its digestion ES clone.Be suspended in after the digestion in the serum free medium that contains WNT suppressor factor DKK1 (100ng/mL) and NODAL suppressor factor LEFTY-A (500ng/mL);
B) with ES clone short incubation period in the petridish that gelatin encapsulates to remove feeder layer cells;
C) the ES cell mass in non-adhesion Micro-Organism Culture Dish with every milliliter 6.7 * 10
2The concentration of individual cell mass is being cultivated in the division culture medium continuously.
2. a kind of vitro human embryonic stem cell according to claim 1 is induced to differentiate into the TP of retinal pigment epithelium, it is characterized in that, continuous division culture medium comprises described in the step C:
(1) DMEM/F12,20%KSR, 0.1mM NMAA, 2mM L-glutamine substratum;
(2) G-MEM, 20%KSR, 0.1mM NMAA, 1mM pyruvate, 0.1mM BME substratum;
(3) G-MEM, 15%KSR, 0.1mM NMAA, 1mM pyruvate, 0.1mM BME substratum;
3. a kind of vitro human embryonic stem cell according to claim 2 is induced to differentiate into the TP of retinal pigment epithelium, it is characterized in that,
In said (1) substratum, cultivated 2 days;
In said (2) substratum, cultivated 4 days;
In said (3) substratum, cultivated 8 days;
In said (4) substratum, cultivated 6 days;
Then, add the suppressor factor DKK-1 (100ng/mL) of WNT and the suppressor factor LEFTY-A (500ng/mL) of NODAL.
4. a kind of vitro human embryonic stem cell according to claim 3 is induced to differentiate into the TP of retinal pigment epithelium; It is characterized in that; Every square centimeter of 10-15 ES cell cluster is layered on 8 holes of containing poly-lysine, ln and fibronectin again and cultivates on the slide, is containing the ES division culture medium of 10%KSR (G-MEM, 0.1mM NMAA; 1mM pyruvate, 0.1mM BME) the middle cultivation.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178452A (en) * | 2014-08-26 | 2014-12-03 | 同济大学 | Retina pigment epithelial cell culture medium and application thereof |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
| CN105814194A (en) * | 2013-12-11 | 2016-07-27 | 辉瑞有限公司 | Method for producing retinal pigment epithelial cells |
| CN106609256A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for inducing human embryonic stem cells to be differentiated into retinal pigment epitheliums (RPE) in vitro |
| CN106609263A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for efficiently inducing pluripotent stem cells to be differentiated to retinal pigment epitheliums (RPE) |
| CN106609257A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro |
| CN109423480A (en) * | 2017-12-21 | 2019-03-05 | 中山大学中山眼科中心 | A kind of mechanizable abductive approach of simple and efficient that human pluripotent stem cells are divided into retinal tissue |
| CN110628696A (en) * | 2019-08-28 | 2019-12-31 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
-
2010
- 2010-11-19 CN CN2010105504872A patent/CN102465111A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105814194A (en) * | 2013-12-11 | 2016-07-27 | 辉瑞有限公司 | Method for producing retinal pigment epithelial cells |
| CN104178452A (en) * | 2014-08-26 | 2014-12-03 | 同济大学 | Retina pigment epithelial cell culture medium and application thereof |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
| CN106609256A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for inducing human embryonic stem cells to be differentiated into retinal pigment epitheliums (RPE) in vitro |
| CN106609263A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for efficiently inducing pluripotent stem cells to be differentiated to retinal pigment epitheliums (RPE) |
| CN106609257A (en) * | 2015-10-22 | 2017-05-03 | 同济大学 | Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro |
| CN109423480A (en) * | 2017-12-21 | 2019-03-05 | 中山大学中山眼科中心 | A kind of mechanizable abductive approach of simple and efficient that human pluripotent stem cells are divided into retinal tissue |
| CN110628696A (en) * | 2019-08-28 | 2019-12-31 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
| CN110628696B (en) * | 2019-08-28 | 2021-09-07 | 郑州大学 | Small molecule composition for directionally inducing cell differentiation and preparation method of retinal pigment epithelial cells |
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Application publication date: 20120523 |