CN101712947A - Preparation method and application of mesenchymal stem cells deriving from embryonic stem cells - Google Patents

Preparation method and application of mesenchymal stem cells deriving from embryonic stem cells Download PDF

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Publication number
CN101712947A
CN101712947A CN200910154572A CN200910154572A CN101712947A CN 101712947 A CN101712947 A CN 101712947A CN 200910154572 A CN200910154572 A CN 200910154572A CN 200910154572 A CN200910154572 A CN 200910154572A CN 101712947 A CN101712947 A CN 101712947A
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stem cell
embryonic stem
stem cells
nutrient solution
mescenchymal
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CN200910154572A
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张铭
谭舟
吴蓉蓉
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses a preparation method and application of mesenchymal stem cells deriving from embryonic stem cells. The preparation method comprises the following steps of: (1) culturing the embryonic stem cells in a embryonic stem cell culture solution; (2) differentiating the embryonic stem cells into the mesenchymal stem cells in a differentiation culture solution; and (3) culturing the mesenchymal stem cells deriving from the embryonic stem cells in a mesenchymal stem cell culture solution, and applying the mesenchymal stem cells to repair of organs losing functions or pathological tissues or medicine screening. The invention has the advantages that: (1) the mesenchymal stem cells deriving from the embryonic stem cells are novel stem cells, have both advantages of the embryonic stem cells and the mesenchymal stem cells, and have wide sources and good biosafety; (2) the application of the mesenchymal stem cells deriving from the embryonic stem cells to repair damaged tissues or organs has simple method, strong maneuverability and good biocompatibility; and (3) the application of the mesenchymal stem cells deriving from the embryonic stem cell to screen medicines has the advantages of high pass, low cost, simple operation and wide application range.

Description

Derive from the preparation method and its usage of the mescenchymal stem cell of embryonic stem cell
Technical field
The present invention relates to a kind of preparation method and its usage that derives from the mescenchymal stem cell of embryonic stem cell.
Background technology
Stem cell is the cell that a class has self and differentiation potential.It comprises embryonic stem cell and adult stem cell.The growth of stem cell is subjected to the influence of multiple inherent mechanism and microenvironment factor.Embryonic stem cell can be successfully in vitro culture at present, and directional induction is divided into various functioning cells under specific culture condition, and then the various histoorgans of regenerating, be used to repair the tissue and the organ of impaired or pathology, difficulty is big, cost is high yet embryonic stem cell is cultivated, exist potential to become the knurl risk, and have the possibility of transplant rejection, limited its application in medical research.Adult stem cell such as mescenchymal stem cell can laterally be divided into the cell and the tissue of other types, mescenchymal stem cell does not become the knurl risk, and can effectively regulate immunity system, escape immunological rejection, for the widespread use of stem cell provides the foundation, but because of the donor source deficiency, the separating and purifying technology instability has influenced its widespread use in medical research to adult stem cell again.Thereby seek a kind of safe and effective, wide material sources, easy and simple to handle, cultivate the low novel stem cell of cost for the research of stem cell with use very crucial.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art a kind of preparation method and its usage that derives from the mescenchymal stem cell of embryonic stem cell is provided.
The preparation method who derives from the mescenchymal stem cell of embryonic stem cell may further comprise the steps:
1) in the embryonic stem cell nutrient solution, embryonic stem cell is cultivated 10 6～10 7Individual embryonic stem cell;
2) use differentiation culture liquid pre-treatment embryonic stem cell 1～2 hour, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 12～24 hours, and continued to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again;
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 8～10 9Individual cell.
Consisting of of described embryonic stem cell nutrient solution: 80ml knock-out DMEM, 20ml SR, 2mML-glutamine, 0.1mM beta-mercaptoethanol, 10ng/ml bFGF, the leukocyte inhibitory factor LIF of 1000U/ml, 0.1mM MEM.
Consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution+10～100 μ MRho coupling kinase inhibitor, molecular formula is C 14H 21N 3O2HClH 2O.
Consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.
The mescenchymal stem cell that derives from embryonic stem cell is used for tissue or organ damage reparation or drug screening.The beneficial effect that the present invention has is:
1) mescenchymal stem cell that derives from embryonic stem cell is a kind of novel stem cell, and it has the advantage of embryonic stem cell and mescenchymal stem cell concurrently, wide material sources, and biological safety is good.
2) it is simple, workable to use the method for the mescenchymal stem cell damaged tissue repair derive from embryonic stem cell or organ, good biocompatibility.
3) use the mescenchymal stem cell screening of medicaments that derives from embryonic stem cell and can realize high-throughput, low cost, simple to operate, applied widely.
Description of drawings
Fig. 1 detects through the fluidic cell detector, the mescenchymal stem cell in embryonic stem cell source shows adult mescenchymal stem cell feature synoptic diagram, the mescenchymal stem cell in embryonic stem cell source shows following surface molecular feature as shown in the figure: the CD34 feminine gender, the CD45 feminine gender, the CD73 positive, the CD105 positive, the CD166 positive, STRO-1 feminine gender;
Fig. 2 is that transaminase level obviously reduces in the liver injury mice serum after the mesenchymal stem cell transplantation treatment in embryonic stem cell source, albumin and the total protein level synoptic diagram that obviously raises.
Embodiment
The present invention has utilized embryonic stem cell to induce and has obtained a kind of novel mesenchyme sample stem cell.Particularly, the mescenchymal stem cell in embryonic stem cell source had both had that adult mescenchymal stem cell suitability is strong, immunomodulatory outstanding effect advantage, was not subjected to adult mescenchymal stem cell source and cell count quantitative limitation again.The stem-cell therapy tissue injury method in utilization embryonic stem cell source is simple, workable, good biocompatibility.
Embodiment 1
1) in the embryonic stem cell nutrient solution, embryonic stem cell is cultivated 10 6Individual embryonic stem cell; Consisting of of described embryonic stem cell nutrient solution: 80ml knock-out DMEM, 20ml SR, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 10ng/ml bFGF, the leukocyte inhibitory factor LIF of 1000U/ml, 0.1mM MEM.
2) use differentiation culture liquid pre-treatment embryonic stem cell 1 hour, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 12 hours, continue to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again; Consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution+10 μ M Rho coupling kinase inhibitor, molecular formula is C 14H 21N 3O2HClH 2O.
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 8Individual cell; Consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.
The mescenchymal stem cell in the embryonic stem cell source that obtains as mentioned above has the surface molecular feature (Fig. 1) of mescenchymal stem cell.
Embodiment 2
1) in the embryonic stem cell nutrient solution, embryonic stem cell is cultivated 10 7Individual embryonic stem cell; Consisting of of described embryonic stem cell nutrient solution: 80ml knock-out DMEM, 20ml SR, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 10ng/ml bFGF, the leukocyte inhibitory factor LIF of 1000U/ml, 0.1mM MEM.
2) use differentiation culture liquid pre-treatment embryonic stem cell 2 hours, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 24 hours, continue to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again; Consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution 100 μ M Rho coupling kinase inhibitor, molecular formula is C 14H 21N 3O2HClH 2O.
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 9Individual cell; Consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.
Embodiment 3
1) in the embryonic stem cell nutrient solution, embryonic stem cell is cultivated 10 7Individual embryonic stem cell; Consisting of of described embryonic stem cell nutrient solution: 80ml knock-out DMEM, 20ml SR, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 10ng/ml bFGF, the leukocyte inhibitory factor LIF of 1000U/ml, 0.1mM MEM.
2) use differentiation culture liquid pre-treatment embryonic stem cell 1 hour, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 12～24 hours, and continued to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again; Consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution+10 μ M Rho coupling kinase inhibitor, molecular formula is C 14H 21N 3O2HClH 2O.
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 9Individual cell; Consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.
Embodiment 4
1) embryonic stem cell is cultivated required cell count 10 7Used substratum is: 80ml knock-outDMEM (Sigma), 20ml SR (GIBCO), 2mM L-glutaminate (GIBCO), 0.1mM beta-mercaptoethanol (Sigma), 10ng/ml bFGF (Sigma), the leukocyte inhibitory factor LIF (Hyclone) of 1000U/ml, 0.1mM MEM (Hyclone).
2) use differentiation culture liquid pre-treatment embryonic stem cell 2 hours, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 24 hours, continue to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again; Consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution 10 μ M Rho coupling kinase inhibitor, molecular formula is C14H21N3O2HClH2O.
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 9Individual cell; Consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.Adopt intravenous mode to carry out cellular transplantation therapy.Adopt CCl4 inductive mouse liver injury model, contrasted the level of transaminase (ALT), albumin (ALB) and total protein (TP) in the mouse blood of treatment front and back respectively, determine the effect (Fig. 2) of novel stem cell through vein transplantation treatment liver injury.

Claims (5)

1. preparation method who derives from the mescenchymal stem cell of embryonic stem cell is characterized in that may further comprise the steps:
1) in the embryonic stem cell nutrient solution, embryonic stem cell is cultivated 10 6～10 7Individual embryonic stem cell;
2) use differentiation culture liquid pre-treatment embryonic stem cell 1～2 hour, adopt tryptic digestion to make embryonic stem cell be dispersed into one embryonic stem cell, in differentiation culture liquid, cultivated 12～24 hours, and continued to cultivate the mescenchymal stem cell that obtains the embryonic stem cell source with the mescenchymal stem cell nutrient solution again;
3) mescenchymal stem cell to 10 in enlarged culturing embryonic stem cell source in the mescenchymal stem cell nutrient solution 8～10 9Individual cell.
2. a kind of preparation method who derives from the mescenchymal stem cell of embryonic stem cell according to claim 1, it is characterized in that: the consisting of of described embryonic stem cell nutrient solution: 80ml knock-out DMEM, 20ml serum substitute SR, the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 10ng/ml Prostatropin bFGF, the leukocyte inhibitory factor LIF of 1000U/ml, the nonessential amino acid MEM of 0.1mM.
3. a kind of preparation method who derives from the mescenchymal stem cell of embryonic stem cell according to claim 1, it is characterized in that: the consisting of of described differentiation culture liquid: 10% serum+90%DMEM nutrient solution+10～100 μ M Rho coupling kinase inhibitor, molecular formula is C 14H 21N 3O2HClH 2O.
4. a kind of preparation method who derives from the mescenchymal stem cell of embryonic stem cell according to claim 1 is characterized in that: the consisting of of described mescenchymal stem cell nutrient solution: 15% serum+85%DMEM nutrient solution.
5. the purposes of the mescenchymal stem cell that derives from embryonic stem cell of method preparation according to claim 1 is characterized in that: be used for tissue or organ damage reparation or drug screening.
CN200910154572A 2009-11-12 2009-11-12 Preparation method and application of mesenchymal stem cells deriving from embryonic stem cells Pending CN101712947A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886060A (en) * 2010-07-20 2010-11-17 东北农业大学 Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells
CN102021143A (en) * 2010-11-24 2011-04-20 浙江大学 Pretreatment method for improving migration capability of mesenchymal stem cells
CN105154393A (en) * 2014-12-22 2015-12-16 浙江大学 Method for differentiation of embryonic stem cells (ESC) into mesenchymal stem cells (MSC)
CN105441387A (en) * 2014-09-03 2016-03-30 黄福来 Special culture medium and special culture method for sub-totipotent stem cells
CN106562995A (en) * 2016-10-26 2017-04-19 沈会强 Preparation method for preparing swan embryos and application of swan embryos
WO2019144605A1 (en) * 2018-01-26 2019-08-01 皓昇莱生物制药有限公司 High performance method for differentiation of hpscs into mscs

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886060A (en) * 2010-07-20 2010-11-17 东北农业大学 Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells
CN102021143A (en) * 2010-11-24 2011-04-20 浙江大学 Pretreatment method for improving migration capability of mesenchymal stem cells
CN102021143B (en) * 2010-11-24 2012-07-25 浙江大学 Pretreatment method for improving migration capability of mesenchymal stem cells
CN105441387A (en) * 2014-09-03 2016-03-30 黄福来 Special culture medium and special culture method for sub-totipotent stem cells
CN105154393A (en) * 2014-12-22 2015-12-16 浙江大学 Method for differentiation of embryonic stem cells (ESC) into mesenchymal stem cells (MSC)
CN106562995A (en) * 2016-10-26 2017-04-19 沈会强 Preparation method for preparing swan embryos and application of swan embryos
WO2019144605A1 (en) * 2018-01-26 2019-08-01 皓昇莱生物制药有限公司 High performance method for differentiation of hpscs into mscs

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Open date: 20100526