CN1445363A - Method for directional differentiating human embryonic stem cell to epidermis stem cell by induction in vitro - Google Patents
Method for directional differentiating human embryonic stem cell to epidermis stem cell by induction in vitro Download PDFInfo
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- CN1445363A CN1445363A CN03113586A CN03113586A CN1445363A CN 1445363 A CN1445363 A CN 1445363A CN 03113586 A CN03113586 A CN 03113586A CN 03113586 A CN03113586 A CN 03113586A CN 1445363 A CN1445363 A CN 1445363A
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Abstract
An in-vitro inducing method for directionally differentiating the human embryo stem cells to epidermal stem cells includes such steps as culturing human ES cells, preparing ammiotic membrane, axenic separation, and induced differentating. Its advantages are simple process, high repeatability, and high scuccessful rate.
Description
The present invention relates to a kind of embryo stem cell for directional differentiation technique.
Human embryo stem cell (human Embnyonic Stem Cell, ES cell) is to belong to myeloid-lymphoid stem cell, has the potential of the various human tissue cells that are divided into three germinal layers.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 nurture 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 endothelium.To be tied to form power low because people ES cell is built, and just sets up people's ES clone first up to the Thomson of the U.S. in 1998, thus the directed differentiation research of relevant people ES cell, at present still seldom.We once successfully set up Chinese ES clone (number of patent application 03113529.3) and personnel selection amnion directional induction mouse ES cells is divided into epidermal stem cells (number of patent application 02143491.3), and it is the method for epidermal stem cells that the present invention sets up inducing in vitro human ES cytodifferentiation based on previous work.
The objective of the invention is to set up convenient, the simple and reliable method directional induction human embryo stem cell for directional of a cover and be divided into epidermal-like stem cells.
Technical scheme of the present invention comprises the steps:
1, the cultivation of people ES cell is people's ES clone CH
2(the special-purpose H-EMDM of ES cell includes foetal calf serum 20%, glutamine 1～4 μ mol/L, non-essential amino acid 0.05～0.3 μ mol/L, LIF10～20ng/ml, beta-mercaptoethanol 0.05～0.3mmol/L) at the complete culture solution of people ES cell in cultivation.
2, the chorion normal saline flushing amnion that contains gentamicin of full-term pregnancy c-section placenta is removed in the separation of the pure property of the preparation of amnion, the epithelial surface of amnion upwards is tiled on the nitrocellulose membrane, put into 37 ℃ of incubator 3～4h, the ES cell training liquid that adds no LIF then spends the night.
3, induce people ES cytodifferentiation behaviour epidermal-like stem cells to get people ES cell outward, with the digestion of 0.5～0.125%EDTA Digestive system, by 5 * 10
3～6Cell/be inoculated in 6 orifice plates that are covered with amnion was cultivated 3～6 days with the ES cell of no LIF training liquid, changed half every 24 and trained liquid, cultivate the 2nd～6 day after, the cytodifferentiation that is attached on the amnion epithelial surface is an epithelium sample stem cell.
The invention has the advantages that:
1, method is easy, and good reproducibility, the external evoked ES cell directional that the success ratio height is set up with us are divided into the epidermal stem cells method can efficiently to induce people ES cytodifferentiation be epidermal-like stem cells.
2, good perfection sticks at the amnion surface because the cell after the differentiation is clone's shape, and undifferentiated ES physiological status of cells is bad, and is removed from the amnion surface washing by PBS.
3, the source is enriched economic placenta and can freely be obtained from Obstetric and Gynecologic Department, and the source is abundant, and the amnion of a placenta can enough be induced the ES cytodifferentiation of ten 6 orifice plates, and cost is low, conveniently promotes the use of.
Provide embodiment below.
1, the cultivation of people ES cell is people's ES clone CH
2(No.2 Hospital Attached to Zhongshan Univ stem cell center provides) cultivates complete culture solution (the special-purpose H-EMDM of ES cell at people ES cell, include foetal calf serum 20%, glutamine 1～4 μ mol/L, non-essential amino acid 0.05～0.3 μ mol/L, LIF10～20ng/ml, beta-mercaptoethanol 0.05～0.3mmol/L) 1～3 day.
2, the chorion normal saline flushing amnion that contains gentamicin of full-term pregnancy c-section placenta is removed in the separation of the pure property of the preparation of amnion, the epithelial surface of amnion upwards is tiled on the nitrocellulose membrane, be cut into the circle of the about 3.5cm of diameter, epithelial surface upwards is laid on 6 well culture plates, put into 37 ℃ of incubator 3～4h, the ES cell training liquid that adds no LIF then spends the night.
3, induce people ES cytodifferentiation behaviour epidermal-like stem cells to get people ES cell outward, with the digestion of 0.5～0.125%EDTA Digestive system, by 5 * 10
3～6Cell/be inoculated in 6 orifice plates that are covered with amnion, ES cell training liquid with no LIF was cultivated 3～6 days, every 24 change half the training liquid, cultivate the 2nd～6 day after, the cytodifferentiation that is attached on the amnion epithelial surface is an epithelium sample stem cell, cell is the colony growth, arrange closely, rounded or oval, sharpness of border, immunohistochemistry shows that these colonies all present the epidermal stem cells specific marker, as β
1Whole and plain, cytokeratin 15 (Cytokeratin15, CK
15) and cytokeratin 19 strong positives.
Claims (2)
1, a kind of external evoked human embryo stem cell for directional is divided into the method for epidermal stem cells, it is characterized in that comprising the steps:
A. the cultivation of people ES cell is people's ES clone CH
2Complete culture solution at people ES cell is cultivated;
B. the chorion normal saline flushing amnion that contains gentamicin of full-term pregnancy c-section placenta is removed in the separation of the pure property of the preparation of amnion, the epithelial surface of amnion upwards is tiled on the nitrocellulose membrane, put into 37 ℃ of incubator 3～4h, the ES cell training liquid that adds no LIF then spends the night;
C. induce people ES cytodifferentiation behaviour epidermal-like stem cells to get people ES cell outside, with the digestion of 0.5～0.125%EDTA Digestive system, by 5 * 10
3～6Cell/be inoculated in 6 orifice plates that are covered with amnion was cultivated 3～6 days with the ES cell of no LIF training liquid, changed half every 24 and trained liquid, cultivate the 2nd～6 day after, the cytodifferentiation that is attached on the amnion epithelial surface is an epithelium sample stem cell.
2, external evoked human embryo stem cell for directional according to claim 1 is divided into the method for epidermal stem cells, it is characterized in that: nutrient solution includes foetal calf serum 20%, glutamine 1～4 μ mol/L, non-essential amino acid 0.05～0.3 μ mol/L, LIF10～20ng/ml, beta-mercaptoethanol 0.05～0.3mmol/L).
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| Application Number | Priority Date | Filing Date | Title |
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| CN03113586A CN1445363A (en) | 2003-01-17 | 2003-01-17 | Method for directional differentiating human embryonic stem cell to epidermis stem cell by induction in vitro |
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| CN03113586A CN1445363A (en) | 2003-01-17 | 2003-01-17 | Method for directional differentiating human embryonic stem cell to epidermis stem cell by induction in vitro |
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| CN03113586A Pending CN1445363A (en) | 2003-01-17 | 2003-01-17 | Method for directional differentiating human embryonic stem cell to epidermis stem cell by induction in vitro |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010127555A1 (en) * | 2009-05-05 | 2010-11-11 | 中国福利会国际和平妇幼保健院 | Method for culturing stem cells |
| CN103356709B (en) * | 2005-03-31 | 2015-09-30 | 斯丹姆涅恩有限公司 | Prepare the method for the medicine in order to treat acute wounds |
| 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 |
-
2003
- 2003-01-17 CN CN03113586A patent/CN1445363A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103356709B (en) * | 2005-03-31 | 2015-09-30 | 斯丹姆涅恩有限公司 | Prepare the method for the medicine in order to treat acute wounds |
| CN103356703B (en) * | 2005-03-31 | 2016-04-06 | 斯丹姆涅恩有限公司 | Prepare the method for the medicine in order to treat chronic infection wound |
| WO2010127555A1 (en) * | 2009-05-05 | 2010-11-11 | 中国福利会国际和平妇幼保健院 | Method for culturing stem cells |
| 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 |
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