CN101338300B - Process for culturing human embryo sexual fold stem cell - Google Patents
Process for culturing human embryo sexual fold stem cell Download PDFInfo
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- CN101338300B CN101338300B CN2007101229997A CN200710122999A CN101338300B CN 101338300 B CN101338300 B CN 101338300B CN 2007101229997 A CN2007101229997 A CN 2007101229997A CN 200710122999 A CN200710122999 A CN 200710122999A CN 101338300 B CN101338300 B CN 101338300B
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Abstract
The invention discloses a culture method for a germ cell of a human embryo and a cell system prepared by the method. The method is realized by adding a PDGF and overcomes the defect of the prior vitro culture for the germ cell of the human embryo to lead the germ cell of the human embryo to be capable of being cultured in vitro for a long period and maintain the undifferentiated state of the germ cell of the human embryo; simultaneously the cultured germ cell of the human embryo can form a teratoma in the body of a small rat; the method of the invention firstly testifies the multi-potential performances of the EG cell of the human body in vivo in the world.
Description
Technical field
The present invention relates to cell culture processes, specially refer to a kind of cultural method of human embryo sexual fold stem cell.
Background technology
Human embryo sexual fold stem cell (EG embryonic germ) is from the isolated archeocyte of embryo's primordial germ ridge, is a kind of in the body early embryo multipotential stem cell, is the initiating cell that has self-replacation and have multidirectional differentiation potential.It has stable diploid caryogram, can be with original embryonism infinite multiplication, and can be divided into three kinds of embryonic germ layers by heteroplastic transplantation or suitable external naturally.
As a kind of embryo's derived stem cell, the surface marker of people EG cell and many differentiation potentials all are similar to human embryo stem cell (ES), this means that the EG cell also can be the same with the ES cell, people EG cell is the same with people ES cell, it all is embryo's derived stem cell with many differentiation potentials, under suitable condition, can directional induction be divided into various histocytes, even form complicated tissue and organ.Just because of this, it is expected to be used for cell, tissue and organ transplantation and Vectors in Gene Therapy for the many refractory diseases of the mankind provide unlimited cell source, can be used as the model and the medicaments sifting model of research Mammals developmental biology simultaneously.
1998, human ES cell was built and is tied to form after the merit soon, and Shamblott MJ separates from embryo sexual fold or the success of mesenteric mesaraic human embryo sexual fold stem cell vitro culture, has three study group to report this work respectively later on again.But up to the present the article of cultivating about people EG cells in vitro seldom.All reports all expose people EG cells in vitro multiplication capacity difference and easy spontaneous differentiation; Whether can form teratoma in vivo is the standard test that the check cell has or not multipotency, this experiment success is verified on human ES cell, but up till now, there is not the human EG cell of report proof can form teratoma, can not forms teratoma and reflect that people EG ability of cell proliferation is poor yet.
With people ES cell in recent years fast progress compare, human EG cell research progress but relatively slowly, one of them important reasons is not have good technology and culture systems to keep the propagation of people EG clone and keep undifferentiated state.Human ES cell can be cultivated for a long time and can pass very high algebraically and keep multipotency external, and the human EG cell of long-time vitro culture and keep its not differentiation state be very difficult, it is the important step that human embryo sexual fold stem cell is furtherd investigate and used that but human EG cells in vitro is cultivated, therefore, setting up good people EG cells in vitro culture condition is a urgent problem.
The present invention has overcome the defective of human embryo sexual fold stem cell vitro culture in the past, vitro culture human embryo sexual fold stem cell and keep its not differentiation state for a long time, the human embryo sexual fold stem cell of turning out simultaneously can form teratoma in the mouse body, in the world the multipotency of reference EG cell in vivo first.
Summary of the invention
The object of the present invention is to provide a kind of human embryo sexual fold stem cell nutrient solution.
Another object of the present invention is to provide a kind of cultural method of human embryo sexual fold stem cell.
Another purpose of the present invention is to provide a kind of human embryo sexual fold stem cell system according to method for preparing.
A kind of human embryo sexual fold stem cell nutrient solution provided by the invention is characterized in that, contains thrombocyte derivation somatomedin PDGF in the nutrient solution.
Wherein, described nutrient solution comprises: Knockout DMEM 80ml, Knockout serum replacement (serum substitute) 20ml, bFGF (Prostatropin) 400ng, LIF (leukaemia inhibitory factor) 1000ng, L-glutamine 2mM, Forskolin 10 μ M, non-essential amino acid 0.1mM, Sodium.alpha.-ketopropionate 1mM, beta-mercaptoethanol 0.1mM, PDGF (thrombocyte derivation somatomedin) 30-200ng/ml, preferred 50ng/ml.
The cultural method of a kind of human embryo sexual fold stem cell provided by the invention comprises,
The digestion of a, human embryo sexual fold stem cell;
B, postdigestive embryo sexual fold stem cell is inoculated in the culture dish that contains above-mentioned nutrient solution cultivates
C, postvaccinal embryo sexual fold stem cell cultivated go down to posterity.
It is characterized in that, be added with PDGF (thrombocyte derivation somatomedin) in the described nutrient solution.
Wherein, described nutrient solution comprises: Knockout DMEM 80ml, Knockout serum replacement20ml (serum substitute), bFGF (Prostatropin) 400ng, LIF (leukaemia inhibitory factor) 1000ng, L-glutamine 2mM, Forskolin 10 μ M, non-essential amino acid 0.1mM, Sodium.alpha.-ketopropionate 1mM, beta-mercaptoethanol 0.1mM, PDGF (thrombocyte derivation somatomedin) concentration is 30-200ng/ml, preferred 50ng/ml.
The digestion of described human embryo sexual fold stem cell is with 0.25% tryptic digestion 15min.
Specifically, the present invention is achieved in that
The cultural method of a kind of human embryo sexual fold stem cell provided by the invention may further comprise the steps:
The digestion of a, human embryo sexual fold stem cell
With 0.25% tryptic digestion 15min, stop digestion;
B, postdigestive embryo sexual fold stem cell is inoculated in cultivate in the culture dish that contains above-mentioned nutrient solution postdigestive embryo sexual fold stem cell is moved into suction pipe and is covered with in the culture dish that mitomycin handled, in culture dish, drip nutrient solution, place under 37 ℃, 5%CO2 condition and cultivate, changed nutrient solution in per 1～2 day later on, every day is the observation of cell growth conditions under inverted phase contrast microscope.
Wherein said nutrient solution comprises: Knockout DMEM 80ml, Knockout serum replacement (serum substitute) 20ml, sub-bFGF (basic fibroblast growth because of) 400ng, LIF (leukaemia inhibitory factor) 1000ng, L-glutamine 2mM, Forskolin 10 μ M, non-essential amino acid 0.1mM, Sodium.alpha.-ketopropionate 1mM, beta-mercaptoethanol 0.1mM, thrombocyte derivation somatomedin PDGF, in order to determine the optimum content of PDGF, the applicant has done a large amount of experiments, and the result specifically sees Fig. 1, the preferred 30-200ng/ml of the content of PDGF, most preferably 50ng/ml as seen from the figure.
C, postvaccinal embryo sexual fold stem cell cultivated go down to posterity
With mechanical process the clone is divided into some in per 15 days, and inoculated the cultivation of going down to posterity on the new culture dish.
Simultaneously, the present invention also provides a kind of human embryo sexual fold stem cell system according to method for preparing that knows clearly.
The applicant is by the method for gene chip, human embryo sexual fold stem cell EG cellular gene expression spectrum is studied, find that the expression level of thrombocyte derivation somatomedin pdgf receptor in the EG cell is very very high, therefore by in substratum, adding external source thrombocyte derivation somatomedin PDGF, overcome the defective of human embryo sexual fold stem cell vitro culture in the past, make human embryo sexual fold stem cell vitro culture and keep its not differentiation state for a long time, the human embryo sexual fold stem cell of turning out simultaneously can form teratoma in the mouse body, in the world the multipotency of reference EG cell in vivo first.
Therefore, our research may will advance people to people EG cell understanding greatly, for clinical cell therapy in the future provides the source competent multipotential cell.
Description of drawings
Fig. 1 PDGF content is to the influence of people EG cell proliferation.
The teratoma that Fig. 2 people EG cell forms in the mouse body.
Fig. 3 identifies that teratomatous HE techtology detects
Embodiment
1, the preparation of nutrient solution
According to following formulated nutrient solution: Knockout DMEM 80ml, serum substitute Knockoutserum replacement 20ml, Prostatropin bFGF 400ng, leukaemia inhibitory factor LIF1000ng, L-glutamine 2mM, Forskolin 10 μ M, non-essential amino acid 0.1mM, Sodium.alpha.-ketopropionate 1mM, beta-mercaptoethanol 0.1mM, PDGF40ng/ml.Wherein, Knock out DMEM, knock out serumreplacement, leukaemia inhibitory factor LIF purchase the company in Gibco, and L-glutamine, nonessential amino acid, Forskolin are purchased the company in Hyclone, and bFGF purchases the company in Peprotech.
2, the cultivation of human embryo sexual fold stem cell
With 0.25% tryptic digestion human embryo sexual fold stem cell (deriving from north doctor three institutes gives) 15min, stop digestion; Postdigestive embryo sexual fold stem cell is moved into suction pipe is covered with in the culture dish that mitomycin handled, in culture dish, drip nutrient solution, place under 37 ℃, 5%CO2 condition and cultivate, changed nutrient solution in later per 1～2 day, every day is the observation of cell growth conditions under inverted phase contrast microscope.
3, postvaccinal embryo sexual fold stem cell cultivation is gone down to posterity
With mechanical process the clone is divided into some in per 15 days, and inoculated the cultivation of going down to posterity on the new culture dish.
Embodiment 2
PDGF content is 30ng/ml, and all the other are with embodiment 1.
Embodiment 3
PDGF content is 200ng/ml, and all the other are with embodiment 1.
Embodiment 4
PDGF content is 80ng/ml, and all the other are with embodiment 1.
Embodiment 5
PDGF content is 150ng/ml, and all the other are with embodiment 1.
Experimental example 1
With the dried small cell of embodiment 1 prepared human embryo sexual fold 0.25% tryptic digestion, centrifugal, precipitation is resuspended with PBS, and the preparation cell density is 10
6The cell suspension of/ml is injected into SCID mouse back leg intramuscular with the 1ml syringe with cell suspension, and injection rate is 200 μ L, whether observed for 12 weeks continuously, detect tumour and form, detected result is seen Fig. 3, find finally to have formed teratoma, formed teratoma is seen Fig. 2.
As seen, the human embryo sexual fold stem cell that the present patent application is turned out finally can form teratoma in the mouse body,
Claims (1)
1. human embryo sexual fold stem cell nutrient solution, it is characterized in that described nutrient solution comprises: KnockoutDMEM 80ml, serum substitute 20ml, Prostatropin 400ng, leukaemia inhibitory factor 1000ng, L-glutamine 2mM, Forskolin 10 μ M, non-essential amino acid 0.1mM, Sodium.alpha.-ketopropionate 1mM, beta-mercaptoethanol 0.1mM, thrombocyte derivation growth factors 5 0ng/ml.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007101229997A CN101338300B (en) | 2007-07-06 | 2007-07-06 | Process for culturing human embryo sexual fold stem cell |
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| CN2007101229997A CN101338300B (en) | 2007-07-06 | 2007-07-06 | Process for culturing human embryo sexual fold stem cell |
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| CN101338300A CN101338300A (en) | 2009-01-07 |
| CN101338300B true CN101338300B (en) | 2010-06-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004070018A2 (en) * | 2003-02-04 | 2004-08-19 | Ludwig Institute For Cancer Research | Vegf-b and pdgf modulation of stem cells |
| CN1667119A (en) * | 2004-03-11 | 2005-09-14 | 中国人民解放军第二军医大学 | Adult stem cell and its culturing method and use |
| CN1865435A (en) * | 2005-05-20 | 2006-11-22 | 中国科学院上海生命科学研究院 | Human embryo stem cell and its culture method |
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2007
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004070018A2 (en) * | 2003-02-04 | 2004-08-19 | Ludwig Institute For Cancer Research | Vegf-b and pdgf modulation of stem cells |
| CN1667119A (en) * | 2004-03-11 | 2005-09-14 | 中国人民解放军第二军医大学 | Adult stem cell and its culturing method and use |
| CN1865435A (en) * | 2005-05-20 | 2006-11-22 | 中国科学院上海生命科学研究院 | Human embryo stem cell and its culture method |
Non-Patent Citations (4)
| Title |
|---|
| Liu S, et al,.Human embryonic germ cells isolation from early stagesof post-implantation embryos.Cell Tissue Res.318.2004,318525-531，具体参见第526页右栏倒数第2段. |
| Liu S,et al,.Human embryonic germ cells isolation from early stagesof post-implantation embryos.Cell Tissue Res.318.2004,318525-531,具体参见第526页右栏倒数第2段. * |
| Pan Y, et al,.In vitro neuronal differentiation of cultured humanembryonicgerm cells.Biochem Biophys Res Commun.327 2.2004,327(2),548-556, 具体参见第549页左栏倒数第1段. |
| Pan Y,et al,.In vitro neuronal differentiation of cultured humanembryonicgerm cells.Biochem Biophys Res Commun.327 2.2004,327(2),548-556, 具体参见第549页左栏倒数第1段. * |
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