CN105296432A - Method for inducing T lymphocyte to form multipotential stem cell - Google Patents
Method for inducing T lymphocyte to form multipotential stem cell Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture, particularly to a method for inducing T lymphocyte to form a multipotential stem cell. The method comprises the following steps: transfecting T lymphocyte by virtue of sendai viruses containing a stem cell transcription factor gene to obtain an infected cell; mixing the infected cell with a culture layer cell, performing the primary culture by adopting a feed layer cell culture medium, then removing the feed layer cell culture medium, carrying out the secondary culture by adopting an embryonic stem cell culture medium, then carrying out the tertiary culture by adopting a mixed solution of a condition culture medium and a human embryonic stem cell culture medium, carrying out the fourth culture by adopting the human embryonic stem cell culture medium to finally obtain the multipotential stem cell. The sendai viruses are used as a transfection carrier, so that the genome of the virus can be prevented from being integrated into the genome of a donor cell, and the safety of the iPS cell is improved; the peripheral blood lymphocyte is used as the donor cell of the iPS cell, so that the source of the donor cell is simple; by adopting the method, the multipotential stem cell can be successfully obtained, and the conversion rate and purity of the multipotential stem cell are relatively high.
Description
Technical field
The present invention relates to technical field of cell culture, particularly a kind of method of T lymphocyte being induced into multipotential stem cell.
Background technology
Embryonic stem cell (embryonicstemcell, ESCs, be called for short ES, EK or ESC cell) be the class cell separated in body early embryo (before gastrula stage) or original sexual gland, it has the characteristic of vitro culture infinite multiplication, self and Multidirectional Differentiation.No matter in vitro or internal milieu, ES cell can be induced to differentiate into the nearly all cell type of body.Embryonic stem cell has using value in the field such as organizational project and regenerative medicine.
As the ES cell being referred to as " seed cell ", transplant for clinical histoorgan and lot of materials is provided.People ES cell is after immunological rejection gene knockout, and reorientation induction end-organ is to avoid interindividual transplant rejection.So just may solve the transplant rejection difficult problem between the allotype individuality that always annoying immune educational circles and medical circle.ES cell can be used for cell therapy, and cell therapy refers to in the human body cell directly transplanting of genetically engineered mistake or input patient body, reaches the object of curing and controlling disease.ES cell still stably can be bred in vitro and gone down to posterity after genetic manipulation.With ES cell for carrier, through in-vitro directed transformation, make the integration number of gene, site, expression degree and insert the stability of gene and screening operation etc. to carry out all on a cellular level, easy acquisition is stablized, satisfied transgenic ES cells system, for the incorperation and expression overcoming quiding gene in current gene therapy is difficult to control, and the cell being used as genetic manipulation in vitro not easily stably transfected and propagation go down to posterity and open new approach.
But, be difficult to obtain due to it and relate to responsive ethics problem, making research once be absorbed in awkward condition.In August, 2006, Yamanaka research group of Japan imports 4 kinds of transcription factor genes (Oct4, Sox2, c-Myc and Klf4) in l cell, be successfully ESCs sample inductive pluripotent stem cells (inducedpluripotentstemcells, iPSCs) by its reprogrammed.In the end of the year 2007, this revolutionary technology is achieved by Japanese Yamanaka group and Thomson group of the U.S. in succession in human cell.The research of iPSCs is a fundamental research with initiative therapeutic cloning, avoid ESCs to be for a long time difficult to obtain and the problem very easily causing dispute of ethic, for stem cell medical use opens up a new way, be considered to the great discovery of stem cell field and even whole field of biology.The many scientists comprising Univ Edinburgh UK IanWilmut professor all think that inductive pluripotent stem cells is only the research direction in stem cell future.IPSCs cell not only has important theory significance for stem-cell research, and has using value in fields such as cell therapy, tissue and organ regeneration, drug screening and evaluations.
But the security of iPSCs is the problem must considered before clinical application.Most virus vector for mediating goal gene can be incorporated in target cell genome, likely activated oncogene.For eliminating the potential safety hazard of iPSCs, researchist is seeking a kind of safer transcription factor lead-in mode always.
Summary of the invention
In view of this, the invention provides a kind of method of T lymphocyte being induced into multipotential stem cell.The method successfully can obtain multipotential stem cell, and security is high, and transformation efficiency and purity are all higher.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of method of T lymphocyte being induced into multipotential stem cell, comprise the steps:
By the Sendai virus transfection T lymphocyte containing stem cell transcription factor gene, obtain cells infected;
Cells infected is mixed with feeder layer cells, carry out the first cultivation, human embryonic stem cell medium is adopted to carry out the second cultivation, then the conditioned medium of feeder layer cells and the mixed solution of human embryonic stem cell medium is adopted to carry out the 3rd cultivation, finally adopt human embryonic stem cell medium to carry out the 4th cultivation, obtain multipotential stem cell.
First cultivation is specially: mixed with feeder layer cells by cells infected, adopts feeder layer cells substratum to cultivate, then abandons feeder layer cells substratum.
The present invention take Sendai virus as transfection carrier, take periphery blood T lymphocyte as donorcells, first by after cells infected and feeder layer cells mixed culture, human embryonic stem cell medium is adopted to cultivate, then the mixed solution of conditioned medium and human embryonic stem cell medium is adopted to cultivate, finally adopt human embryonic stem cell medium to cultivate, successfully obtain multipotential stem cell, the transformation efficiency of its multipotential stem cell and purity are all higher.
In embodiments more provided by the invention, stem cell transcription factor is one or more the mixture in OCT4, SOX2, C-MYC or KLF4.
As preferably, transfection is transfection 24 ~ 36h under 37 DEG C of conditions.
Preferably, transfection is transfection 24h under 37 DEG C of conditions.
In embodiments more provided by the invention, the formula of conditioned medium is: P2 is for the culture supernatant of MEF cell (mouse embryo fibroblasts).
In embodiments more provided by the invention, embryonic stem cell medium is human embryonic stem cell medium.
In embodiments more provided by the invention, the formula of human embryonic stem cell medium is: DMEM/F12, serum substitute, β-thioglycol, 1% non-essential amino acid, FGF, Basic Fibroblast Growth Factor.
As preferably, the volume ratio of conditioned medium and embryonic stem cell medium is 1:(1 ~ 1.5).
Preferably, the volume ratio of conditioned medium and embryonic stem cell medium is 1:1.
As preferably, what transfection adopted help, and to turn agent be polybrene.
As preferably, the first time of cultivating was 1 day, and the second time of cultivating was 9 ~ 11 days.
Preferably, the second time of cultivating was 9 days.
As preferably, the 3rd time of cultivating was 8 ~ 10 days, and the 4th time of cultivating was 7 ~ 15 days.
Preferably, the 3rd time of cultivating was 8 days, and the 4th time of cultivating was 7 days.
The invention provides a kind of method of T lymphocyte being induced into multipotential stem cell.The method comprising the steps of: by the Sendai virus transfection T lymphocyte containing stem cell transcription factor gene, obtains cells infected; Cells infected is mixed with feeder layer cells, carry out the first cultivation, human embryonic stem cell medium is adopted to carry out the second cultivation, then the mixed solution of conditioned medium and human embryonic stem cell medium is adopted to carry out the 3rd cultivation, finally adopt human embryonic stem cell medium to carry out the 4th cultivation, obtain multipotential stem cell.The present invention at least has one of following advantage:
1, the present invention selects Sendai virus as transfection carrier, avoids the genome conformity of virus in the genome of donorcells, improves the security of iPS cell;
2, the present invention selects periphery blood T lymphocyte as the donorcells of iPS cell, and donor cell sources is simple;
3, the present invention take Sendai virus as transfection carrier, take periphery blood T lymphocyte as donorcells, first by after cells infected and feeder layer cells mixed culture, human embryonic stem cell medium is adopted to cultivate, then the mixed solution of conditioned medium and human embryonic stem cell medium is adopted to cultivate, human embryo stem cell nutrient solution is finally adopted to cultivate, successfully can obtain multipotential stem cell, the transformation efficiency of its multipotential stem cell and purity are all higher, for iPS cell is applied to disease of immune system or tumor vaccine cells treatment establish certain theoretical basis.
Accompanying drawing explanation
Fig. 1 shows the form of the iPS cell of acquisition;
Fig. 2 shows alkaline phosphatase staining result.
Embodiment
The invention discloses a kind of method of T lymphocyte being induced into multipotential stem cell, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Terminological interpretation:
Induced multi-potent stem cells (inducedpluripotentstemcells, iPScells): by certain approach by the channel genes relevant with cells pluripotency in the somatocyte broken up, or the micromolecular compound adding some booster actions makes somatocyte dedifferente reprogrammed and gets back to embryonic stem cell state, and the cell obtained is iPS cell simultaneously.
Provided by the inventionly T lymphocyte is induced into agents useful for same, instrument in the method for multipotential stem cell and all can be buied by market.
The formula of conditioned medium is: when P2 is for MEF cell cultures, and the culture supernatant of the cultivation MEF cell of collection, is conditioned medium;
The formula of human embryonic stem cell medium is: 89% human embryo stem cell nutrient solution: knockout
tMdMEM/F12 (Gibco)+knockout
tMserumreplacement (KSR) (KnockOut serum substitute; Gibco)+β-thioglycol+1%NEAA (non-essential amino acid) (Sigma)+10ng/mLbasicFGF, recombinanthuman (Basic Fibroblast Growth Factor) (PeproTech);
Help and turn agent: polybrene.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 induced multi-potent stem cells
(1) total lymphocytic separation of T and sorting
1) transfer in centrifuge tube by the peripheral blood in anticoagulant tube, 400-500g (centrifugal force) centrifugal 5-10min, transfers to lower floor's hemocyte in 50mL centrifuge tube after centrifugal end, and the physiological saline adding two volumes dilutes;
2) a new sepmate centrifuge tube (purchased from STEMCELL company) is got, add Ficoll lymphocyte separation medium (purchased from STEMCELL company) 12mL, hemocyte after dilution is transferred to sepmate centrifuge tube, the centrifugal 15-20min of 600-800g;
3) after centrifugal, supernatant liquid is poured out, add physiological saline and carry out washing 1 time;
4) CD3 is used
+t lymphocyte sorts out from above-mentioned mononuclearcell by sorting test kit (purchased from STEMCELL company).
(2) preparation of feeder layer cells
1) by the MEF cell recovery in P2 generation;
2) when MEF cell (mouse embryo fibroblast stem cell) fusion rate reaches 80 ~ 90%, MEF cell is gone down to posterity.Then by 1-3 × 10
5cell be inoculated in six orifice plates;
3) ametycin of second day 5-20 μ g/mL carries out process 2 hours;
4) change MEF substratum into after 2 hours, obtain feeder layer cells.
(3) cultivation of Sendai virus transfection and iPS cell
1) operate according to Sendai virus reprogrammed test kit: the Sendai virus solution containing OCT4, SOX2, C-MYC and KLF4 is added in six orifice plates, liquor capacity finally complements to 2mL, finally add 2 helping of μ L5 ~ 20 μ g/mL and turn agent, after whole system mixing, transfer in a hole of pre-coated 6 good orifice plates, put into 37 DEG C of incubators after shaking up, spend the night.
2) virus infection is after 24 hours (the 1st day), is taped against in previously prepd feeder layer cells by the cell that yesterday infects.
3) the 2nd day, change DMEM high glucose medium into human embryonic cells nutrient solution, continue to cultivate.Within every 2 days later, change liquid once.
4) observe day by day, when cell grows to 5th ~ 7 days, cell aggregation little as seen, cellular form obviously there occurs change, and growth flocks together.
5) until the 10th day, because MEF cell is long for duration of service, therefore nutrient solution changes CM (conditionedmedium into, conditioned medium) 1:1 (volume ratio) mixed solution of the normal nutrient solution of+hESC (human embryo stem cell), to be supplied to cell sufficient nutrient.
6) treat that cell grows to the 18th day, can clone be chosen, the clone of embryonic stem cell sample is chosen on new MEF, continue cultivation 7 days by hESC (hESC) cultural method, amplification.IPS cell clone and normal hESC are morphologically as good as (Fig. 1) substantially.
The cell of above-mentioned acquisition is carried out the qualification (alkaline phosphatase activities detection) of iPS cell, operate according to Alkaline Phosphatase Kit specification sheets, concrete operation steps is as follows:
(1) sucking-off cell culture fluid, with PBS solution rinse 2 ~ 3 times, fixes 1 ~ 2min with 4% paraformaldehyde (PFA);
(2) sucking-off stationary liquid, washes 2 times by PBS solution;
(3) sucking-off PBS solution, with the rinse of TBST solution once;
(4) alkaline phosphatase reagent is prepared: [solution A (in vain): solution B (green): solution C=50ul:50ul:400ul];
(5) add enough staining reagents to make at the bottom of the enough coverage holes of staining fluid energy, room temperature lucifuge places 10 ~ 15min;
(6) sucking-off staining fluid, with the rinse of PBS damping fluid once, be finally stored in PBS solution, basis of microscopic observation coloration result (for indigo plant/purple, the cell dyeing of differentiation is colourless in undifferentiated cell dyeing).Result display blue cell is noble cells, namely obtains multipotential stem cell.
The preparation of embodiment 2 multipotential stem cell
(1) total lymphocytic separation of T and sorting
1) transfer in centrifuge tube by the peripheral blood in anticoagulant tube, 400-500g (centrifugal force) centrifugal 5-10min, transfers to lower floor's hemocyte in 50mL centrifuge tube after centrifugal end, and the physiological saline adding two volumes dilutes;
2) a new sepmate centrifuge tube (purchased from STEMCELL company) is got, add Ficoll lymphocyte separation medium (purchased from STEMCELL company) 12mL, hemocyte after dilution is transferred to sepmate centrifuge tube, the centrifugal 15-20min of 600-800g;
3) after centrifugal, supernatant liquid is poured out, add physiological saline and carry out washing 1 time;
4) CD3 is used
+t lymphocyte sorts out from above-mentioned mononuclearcell by sorting test kit (purchased from STEMCELL company).
(2) preparation of feeder layer cells
1) the MEF cell (mouse embryo fibroblast stem cell) in P2 generation is recovered;
2) when MEF cell confluency reaches 80 ~ 90%, MEF cell is gone down to posterity.Then by 1-3 × 10
5cell be inoculated in six orifice plates;
3) ametycin of second day 5-20 μ g/mL carries out process 2 hours;
4) change MEF substratum into after 2 hours, obtain feeder layer cells.
(3) cultivation of Sendai virus transfection and iPS cell
1) operate according to Sendai virus reprogrammed test kit: the Sendai virus solution containing OCT4, SOX2, C-MYC and KLF4 is added in six orifice plates, liquor capacity finally complements to 2mL, finally add 2 μ L to help and turn helping of agent 5 ~ 20 μ g/mL and turn agent, after whole system mixing, transfer in a hole of pre-coated 6 good orifice plates, put into 37 DEG C of incubators after shaking up, spend the night.
2) virus infection is after 24 hours (the 1st day), is taped against in previously prepd feeder layer cells by the cell that yesterday infects.
3) the 2nd day, change DMEM high glucose medium into human embryonic cells nutrient solution, continue to cultivate.Within every 2 days later, change liquid once.
4) observe day by day, when cell grows to 5th ~ 7 days, cell aggregation little as seen, cellular form obviously there occurs change, and growth flocks together.
5) until the 11st day, because MEF cell is long for duration of service, therefore nutrient solution changes CM (conditionedmedium into, conditioned medium) 1:1.2 (volume ratio) mixed solution of the normal nutrient solution of+hESC (human embryo stem cell), to be supplied to cell sufficient nutrient.
6) treat that cell grows to the 19th day, can clone be chosen, the clone of embryonic stem cell sample is chosen on new MEF, continue cultivation 10 days by hESC (hESC) cultural method, amplification.IPS cell clone and normal hESC are morphologically as good as substantially.
The cell of above-mentioned acquisition is carried out the qualification (alkaline phosphatase activities detection) of iPS cell,
Result display have successfully been obtained multipotential stem cell.
The preparation of embodiment 3 multipotential stem cell
(1) total lymphocytic separation of T and sorting
1) transfer in centrifuge tube by the peripheral blood in anticoagulant tube, 400-500g (centrifugal force) centrifugal 5-10min, transfers to lower floor's hemocyte in 50mL centrifuge tube after centrifugal end, and the physiological saline adding two volumes dilutes;
2) a new sepmate centrifuge tube (purchased from STEMCELL company) is got, add Ficoll lymphocyte separation medium (purchased from STEMCELL company) 12mL, hemocyte after dilution is transferred to sepmate centrifuge tube, the centrifugal 15-20min of 600-800g;
3) after centrifugal, supernatant liquid is poured out, add physiological saline and carry out washing 1 time;
4) CD3 is used
+t lymphocyte sorts out from above-mentioned mononuclearcell by sorting test kit (purchased from STEMCELL company).
(2) preparation of feeder layer cells
1) the MEF cell (mouse embryo fibroblast stem cell) in P2 generation is recovered;
2) when MEF cell confluency reaches 80 ~ 90%, MEF cell is gone down to posterity.Then by 1-3 × 10
5cell be inoculated in six orifice plates;
3) ametycin of second day 5-20 μ g/mL carries out process 2 hours;
4) change MEF substratum into after 2 hours, obtain feeder layer cells.
(3) cultivation of Sendai virus transfection and iPS cell
1) operate according to Sendai virus reprogrammed test kit: the Sendai virus solution containing OCT4, SOX2, C-MYC and KLF4 is added in six orifice plates, liquor capacity finally complements to 2mL, finally add 2 μ L to help and turn helping of agent 5 ~ 20 μ g/mL and turn agent, after whole system mixing, transfer in a hole of pre-coated 6 good orifice plates, put into 37 DEG C of incubators after shaking up, spend the night.
2) virus infection is after 24 hours (the 1st day), is taped against in previously prepd feeder layer cells by the cell that yesterday infects.
3) the 2nd day, change DMEM high glucose medium into human embryonic cells nutrient solution, continue to cultivate.Within every 2 days later, change liquid once.
4) observe day by day, when cell grows to 5th ~ 7 days, cell aggregation little as seen, cellular form obviously there occurs change, and growth flocks together.
5) until the 12nd day, because MEF cell is long for duration of service, therefore nutrient solution changes CM (conditionedmedium into, conditioned medium) 1:1.5 (volume ratio) mixed solution of the normal nutrient solution of+hESC (human embryo stem cell), to be supplied to cell sufficient nutrient.
6) treat that cell grows to the 20th day, can clone be chosen, the clone of embryonic stem cell sample is chosen on new MEF, continue cultivation 15 days by hESC (hESC) cultural method, amplification.IPS cell clone and normal hESC are morphologically as good as substantially.
The cell of above-mentioned acquisition is carried out the qualification (alkaline phosphatase activities detection) of iPS cell, result display have successfully been obtained multipotential stem cell.
The preparation method of the existing induced multi-potent stem cells of comparative example 1
(1) select human skin fibroblast as donorcells
Human skin fibroblast is provided by Ji'nan University's biological medicine study base, and substratum is DMEM+10%FBS;
(2) preparation of feeder layer cells
1) by the MEF cell recovery in P2 generation;
2) when MEF cell confluency reaches 80 ~ 90%, MEF cell is gone down to posterity.Then by 1-3 × 10
5cell be inoculated in six orifice plates;
3) ametycin of second day 5-20 μ g/mL carries out process 2 hours;
4) change MEF substratum into after 2 hours, obtain feeder layer cells.
(3) virus transfection and reprogrammed:
Use retroviral vector (containing four transcription factors: OCT-4, Sox2, c-Myc and Klf4) transfected fibroblast;
(4) cell cultures after transfection:
1) after gene transfer 2 days, be inoculated in six orifice plates with trypsin digestion and cell, containing mouse embryonic feeder confluent monolayer cells (MEF in six orifice plates, murineembryonicfibroblastcells) and substratum (DMEM+10%FBS), the inoculating cell density in every hole is 5 × 10
4/ mL;
2), after inoculating 24 hours, substratum is changed into ESC substratum, medium component is: knockoutDMEM+10%FBS+10%serumreplacementwithoutLIF (leukaemia inhibitory factor, leukemiainhibitoryfactor);
3), after transduceing 7 days, the celliform iPS cell clone of a small amount of ESC can be obtained.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. T lymphocyte is induced into a method for multipotential stem cell, it is characterized in that, comprise the steps:
By the Sendai virus transfection T lymphocyte containing stem cell transcription factor gene, obtain cells infected;
Described cells infected is mixed with feeder layer cells, carry out the first cultivation, human embryonic stem cell medium is adopted to carry out the second cultivation, then the conditioned medium of feeder layer cells and the mixed solution of human embryonic stem cell medium is adopted to carry out the 3rd cultivation, finally adopt human embryonic stem cell medium to carry out the 4th cultivation, obtain multipotential stem cell.
2. method according to claim 1, is characterized in that, described stem cell transcription factor is one or more the mixture in OCT4, SOX2, C-MYC or KLF4.
3. method according to claim 1, is characterized in that, described transfection is transfection 24 ~ 36h under 37 DEG C of conditions.
4. method according to claim 1, is characterized in that, the formula of described conditioned medium is: P2 is for the culture supernatant of MEF cell.
5. method according to claim 1, is characterized in that, the formula of described human embryonic stem cell medium is: DMEM/F12, serum substitute, β-thioglycol, 1% non-essential amino acid, FGF, Basic Fibroblast Growth Factor.
6. method according to claim 1, is characterized in that, the volume ratio of described conditioned medium and described embryonic stem cell medium is 1:(1 ~ 1.5).
7. method according to claim 1, is characterized in that, what described transfection adopted help, and to turn agent be polybrene.
8. method according to claim 1, is characterized in that, the described first time of cultivating was 1 day, and the described second time of cultivating was 9 ~ 11 days.
9. method according to claim 1, is characterized in that, the described 3rd time of cultivating was 8 ~ 10 days, and the described 4th time of cultivating was 7 ~ 15 days.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107435050A (en) * | 2017-06-20 | 2017-12-05 | 向孟清 | A kind of method by human or animal's somatic induction for NSC |
| CN108004203A (en) * | 2017-11-20 | 2018-05-08 | 广东艾时代生物科技有限责任公司 | A kind of culture medium and its method for induced multi-potent stem cell |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
| CN112961833A (en) * | 2021-03-08 | 2021-06-15 | 浙江大学 | Method for reprogramming immortalized lymphocyte cell line into induced pluripotent stem cell |
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- 2015-12-07 CN CN201510895176.2A patent/CN105296432B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107435050A (en) * | 2017-06-20 | 2017-12-05 | 向孟清 | A kind of method by human or animal's somatic induction for NSC |
| CN107435050B (en) * | 2017-06-20 | 2021-02-09 | 向孟清 | Method for inducing human or animal somatic cells into neural stem cells |
| CN108004203A (en) * | 2017-11-20 | 2018-05-08 | 广东艾时代生物科技有限责任公司 | A kind of culture medium and its method for induced multi-potent stem cell |
| CN109055306A (en) * | 2018-06-25 | 2018-12-21 | 佛山科学技术学院 | A kind of system and method for no feeder layer free serum culture spermatogonial stem cells into mouse |
| CN112961833A (en) * | 2021-03-08 | 2021-06-15 | 浙江大学 | Method for reprogramming immortalized lymphocyte cell line into induced pluripotent stem cell |
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