CN104140951A - Method for developing and cultivating human induced pluripotent stem cells - Google Patents
Method for developing and cultivating human induced pluripotent stem cells Download PDFInfo
- Publication number
- CN104140951A CN104140951A CN201410382321.2A CN201410382321A CN104140951A CN 104140951 A CN104140951 A CN 104140951A CN 201410382321 A CN201410382321 A CN 201410382321A CN 104140951 A CN104140951 A CN 104140951A
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cells
- cells
- human
- induced multi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for developing and cultivating human induced pluripotent stem cells. The method is characterized by comprising the steps of a, preparing a human fibroblast-like cell feeding layer, seeding human fibroblast-like cells onto a petri dish, and processing 10mg/ml mitomycin C for three hours in advance to remove mitomycin C; b, reprogramming somatic cells, generating the human induced pluripotent stem cells, and transplanting the human induced pluripotent stem cells into the feeding layer prepared in the step a to be cultivated. By the adoption of the technical scheme, the influence of heterologous cells and heterologous protein on the induced pluripotent stem cells is avoided, transcription efficiency is high, the differentiation capacity of the induced pluripotent stem cells is not affected, the number of cultivation generations is large, life is long, and the method has huge potential clinically.
Description
Technical field
The present invention relates to a kind of method of foundation and cultivator induced multi-potent stem cells.
Background technology
Embryonic stem cell is a class totipotency stem cell, the potential polytype cell that is divided into various histoorgans.In the last few years, utilized gene " reprogramming " technical finesse to become somatocyte, and as skin flbroblast or blood cell, can allow them obtain totipotency as embryonic stem cell.This technology is called induced multi-potent stem cells (iPS) technology, and the totipotent cell of generation is called iPS cell.IPS cell has medical use widely and is worth, and has brought new hope particularly to the clinical application in regenerative medicine field.Because iPS technology can be cultivated totipotency stem cell from soma through reprogramming of somatic cells with patient, the derived cell of then applying these totipotency stem cells carries out clinical treatment.So not only avoid using the ethics problem of embryonic stem cell, and obtained the cell with patient self genetic background, evaded immunological rejection problem.
At present, most of iPS clone is all supported at mouse embryo fibroblasts (MEF).Use MEF can ensure stem cell undifferentiated state and increase as feeder layer cells.Use MEF that higher reprogramming efficiency of somatic cells also can be provided.But, use MEF to there is potential problem, comprise that the inhuman xenogenesis factor is polluted and the risk of the propagation of animal pathogen.These problems become significant obstacle can check the cell-derived cell of use iPS in clinical application regulator time.Although the xenogenesis factor pollution problem of iPS cell also do not report now, this situation is present on human embryo stem cell.HESC after long-time cultivation, can detect at stem cell surface the expression of xenogenesis factor sialic acid NeuGc ALPHA2-3Gal (Neu5Gc) on MEF.Whether this discovery has caused clinically can cause immunoreactive concern to embryonal vaccination stem cell or its daughter cell.
Pollute in order to reduce the xenogenesis factor, many laboratories can be using parental cell simultaneously as feeder layer cells in the time of induction iPS cell.Human fibroblast, adipocyte, and amniocyte was all tried out.Although can avoid the xenogenesis factor to pollute as feeder layer parental cell, due to parental cell number deficiency, cannot large-scale application in long term maintenance and the amplification of iPS cell.This will have influence on the application of iPS cell in cell therapy.2010, Christian etc. as feeder layer cultivator induced multi-potent stem cells, can maintain the growth of induced multi-potent stem cells by human skin fibroblast, shorter but it is held time, and cultivated only 7 generations of algebraically.Publication number be CN102161980 Patent Application Publication using human marrow mesenchymal stem cell as feeder layer cultivator induced multi-potent stem cells, can maintain the growth of induced multi-potent stem cells, but it is still shorter to hold time, cultivate only 14 generations of algebraically, and, this feeder layer adopts containing the culture medium culturing of foetal calf serum and obtains, and contains heterologous protein, still has potential risk for clinical application.Application publication number CN103589686A (Shen Qing Publication day 2014.02.19) discloses the method using human umbilical cord mesenchymal stem cells (hUCMSC) as feeder layer cultivator induced multi-potent stem cells.But the ability of the external long-term cultivation of human umbilical cord mesenchymal stem cells is still limited, mean lifetime (cultivation algebraically) was approximately 30 generations, and the frequent producer sudden change of the human umbilical cord mesenchymal stem cells of long-term cultivation, so clinical treatment is conventionally only with the 3rd human umbilical cord mesenchymal stem cells to the 5th generation.
Another kind method is not used feeder layer cells, but use extracellular matrix (ECM)-or carry out the foundation of iPS cell and maintain taking synthetic as basic culture systems.But while using these culture systems, reprogramming efficiency of somatic cells is conventionally very low, cannot produces more iPS population of cells and carry out examination.When cultivating on without feeder layer cells, iPS cell long-period also can run into genomic instability, the problem of chromosome abnormalities.This problem is observed on hESC.
Summary of the invention
In order to overcome above-mentioned technical problem, the present invention is intended to set up a kind of feeder layer cells system of polluting without the xenogenesis factor based on human body cell, the present invention utilizes people's induced multi-potent stem cells directly to cultivate human desmocyte like cell (FLC), then further apply FLC and generate the cell with amplifying human iPS, the present invention specifically adopts following technical scheme to solve above-mentioned technical problem:
A method for foundation and cultivator induced multi-potent stem cells, comprises the following steps:
A. prepare human desmocyte like cell feeder layer: get human desmocyte like cell, be seeded on culture dish by 10 mg/ml ametycin advanced processing 3 hours, remove ametycin;
B. cultivate: reprogrammed somatocyte, generates people's induced multi-potent stem cells, and people's induced multi-potent stem cells is transplanted in the feeder layer of preparing in step a and cultivated.
Preferably, in the method for above-mentioned a kind of foundation and cultivator induced multi-potent stem cells, in described step a, human desmocyte like cell is adopted with the following method and is generated:
A1: end user's human foreskin fibroblasts reprogrammed obtains people's induced multi-potent stem cells 5.9 clones;
A2: maintain 5.9 clone 7 days on matrigel;
A3: the cultivation of going down to posterity after using 1 mg/ml Dispase to digest 5-7 minute under 37 DEG C of environment, nutrient solution is
1, nutrient solution is changed every day;
A4: reach 80% when full when being incubated at 5.9 clones in matrigel, remove
after 1 substratum, clean twice with 1 × PBS, then directly add human desmocyte like cell substratum and make 5.9 clones start directed differentiation, change substratum first four day every day, within every 2 days afterwards, change once;
A5: induce after 6～12 days 0.05% trypsinase for cell covering with-EDTA is digested, and with 1 × 10
6the concentration in individual/hole is seeded in cell on pretreated 6 orifice plates of matrigel; In the time that cell covers with again, by cell dissociation and with 2 × 10
5the concentration in individual/hole is inoculated in pretreated 6 orifice plates of 0.1% gelatin;
A6: processed at gelatin flat board on cultivate cellular form homogeneous gradually after 3-5 days, be human fibroblasts's sample.
Preferably, in the method for above-mentioned a kind of foundation and cultivator induced multi-potent stem cells, the people's induced multi-potent stem cells in described step b is adopted with the following method and is generated:
B1: get 1 × 10
4human foreskin fibroblast, adopt the Zinc finger nuclease of Baculovirus-mediated in first day and twice transfection in the 8th day, ZFN gene and OSKM gene are proceeded to cell simultaneously, make OSKM gene specific and be incorporated into the AAVS1 site of human foreskin fibroblast, cell culture fluid is FibroGRO
tM-LS perfect medium;
B2: use 200 ug/ml G418 medicine sieves after 10 days, the cell of survival is transferred on human desmocyte like cell feeder layer and formed population of cells, obtain people's induced multi-potent stem cells.
Preferably, in the method for above-mentioned a kind of foundation and cultivator induced multi-potent stem cells, in described step b1, the infection multiplicity of expressing the each cell of baculovirus of ZFN gene and OKSM gene is 100.
Preferably, in the method for above-mentioned a kind of foundation and cultivator induced multi-potent stem cells, 5.9 cells that use are the cell of 60 continuous passages.
Compared with prior art, the present invention has following technique effect:
Adopt design of the present invention, the human desmocyte like cell that end user's induced multi-potent stem cells is differentiated to form is cultivated people's induced multi-potent stem cells as feeder layer, exempt the risk of the propagation of inhuman xenogenesis factor pollution and animal pathogen, simultaneously because people's induced multi-potent stem cells is cultivated in vitro indefinitely, therefore the feeder layer cells that uses the solution of the present invention also can indefinite a large amount of preparations, with respect to other feeder layer cells, method of the present invention had both ensured the efficiency that reprogramming of somatic cells generates, having overcome again prior art feeder layer cells, to maintain the life-span short, cultivate the few problem of algebraically, technical scheme of the present invention has tremendous potential in the clinical application of people's induced multi-potent stem cells.
Brief description of the drawings
Fig. 1 is the embodiment of the present invention 1 5.9 cells through different experimental conditions form and contrast under the microscope;
Fig. 2 is checking and the reprogramming efficiency of the embodiment of the present invention 2 induced multi-potent stem cells Almightiness types;
Fig. 3 is the advantages of the embodiment of the present invention 3 human desmocyte like cells as feeder layer amplification myeloid-lymphoid stem cell;
Fig. 4 is that the embodiment of the present invention 3 human desmocyte like cells do not affect myeloid-lymphoid stem cell differentiation capability;
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, but the present invention is not only confined to following examples.
The preparation of embodiment 1 feeder layer
Mankind iPS cell 5.9 cells that obtained by human foreskin fibroblast (HFFs) reprogrammed are prior art, are not described in detail at this, and 5.9 clones are used as cultivating the source of FLC in the present invention.5.9 clones are maintain 7 days on matrigel first, then uses 1 mg/ml Dispase (StemCell Technologies company) in 37 DEG C of digestion cultivation of going down to posterity after 5～7 minutes.Nutrient solution
change 1 (StemCell Technologies company) every day.In this experiment, we have used three kinds of diverse ways that 5.9 clone inductions are divided into FLC.The inducing culture of FLC comprises DMEM (Thermo Scientific company), 10% foetal calf serum (Thermo Scientific company) and 0.1mM non-essential amino acid (NEAA) (Invitrogen company).
Method A: people iPS cell is digested in 37 DEG C and separate 20～30 minutes with the Dispase of 0.1 mg/ml, be then transferred to the upper embryoid body (Embryoid body, EB) that forms of ultralow adherent cell culture plate (Corning company).The substratum of embryoid tire contains 80% DMEM/F12 (Invitrogen company), 20% knockout serum replacement (Invitrogen company), 2mM L-glutaminate (PAN Biotech company), the beta-mercaptoethanol (Sigma-Aldrich company) of 0.1mM, the non-essential amino acid of 0.1mM and penicillin/streptomycin (PAN Biotech company).Embryoid body cultivation was transferred in the pretreated 6 porocyte culture plates of 0.1% gelatin after 5 days, and nutrient solution is replaced by the inducing culture of FLC.The individual cells outwards being grown by embryoid tire is maintained on the pretreated plank of gelatin always, the cultivation of just going down to posterity when cell covers with.Every 2～3 days of FLC substratum is changed once, and goes down to posterity with 0.05% trypsinase-EDTA (Invitrogen company) peptic cell.
Method B: by 1 mg/ml Dispase digestion 5 to 7 minutes for 5.9 clones, manual separation 5.9 clones are also transferred in pretreated 6 orifice plates of 0.1% gelatin at 37 DEG C, and substratum is same as the inducing culture of the FLC in method A.
Method C: in the time being incubated at 5.9 clones of matrigel and reaching 80%, remove
after 1 substratum, clean twice with 1 × PBS, then directly add FLC substratum and make 5.9 clones start directed differentiation.Change substratum first four day every day, within every 2 days afterwards, change once.Induce after 6～12 days 0.05% trypsinase for cell covering with-EDTA is digested, and with 1 × 10
6the concentration in individual/hole is seeded in cell on pretreated 6 orifice plates of matrigel.In the time that covering with again, cell (is generally 5 days), by cell dissociation and with 2 × 10
5the concentration in individual/hole is inoculated in pretreated 6 orifice plates of 0.1% gelatin.Processed at gelatin flat board on cultivate cellular form homogeneous gradually after 3 to 5 days, be inoblast sample form.The FLCs cultivation of can once going down to posterity for every 3～4 days that success is induced, and can be by frozen FLC in liquid nitrogen, frozen storing liquid is that FLC substratum adds 10% dimethyl sulfoxide (DMSO) (Sigma company).
Induction FLC clone 5.9 used is the cells that exceeded 60 continuous passages.In the present invention, first adopted the formation embryoid body method of standard to make to clone 5.9 cells formation embryoid bodies, then embryoid body has been inoculated on pretreated six orifice plates of gelatin, differentiation culture liquid is the FLC substratum (method A) of people's reports such as Chen.Occur postvaccinal second day unicellular beginning of embryoid body, within general 5 days, just can around embryoid body, form afterwards radial monolayer cell.Fibroblast-like cells just can be observed and obtain at the 7th day, but their breeding ratios are slower, conventionally need could increase into for 4～5 weeks the fibroblast-like cells group (Figure 1A) of homogeneous.Clone 5.9 population of cells is directly transferred on pretreated six orifice plates of gelatin, directly induce differentiation (method B) without the formation of embryoid body.IPS population of cells is in transfer just differentiation immediately afterwards, and a few days ago has a serious necrocytosis phenomenon what start differentiation.(Figure 1B) be similar to the cytodifferentiation method A that embryoid body mediates, when using method B, the overall yield of FLC is not only not high, and needs long divergaence time (4～5 weeks) could form the cell mass of form homogeneous.
The 3rd method (method C) be directly by the substratum of clone 5.9 population of cells by
1 replaces with FLC substratum, allows be seeded in population of cells on the pretreated culture plate of matrigel and directly break up.The cell of differentiation is expanded to rapidly and covers whole culture plate, and forms a large amount of cell masses at the 5th day, and a lot of elongated fibroblast-like cellses just can be observed at first week.(Fig. 1 C), by these cell renewed vaccinations to the pretreated culture plate of matrigel, a Zhou Houzai transfers to (the 14th day) on the pretreated culture plate of gelatin.On the pretreated plank of gelatin, the rapid enrichment of fibroblast-like cell becomes the cell mass of form homogeneous, and whole process probably needs 3 to 4 weeks.General introduction Fig. 1 D has summed up method C.As can be seen from the figure, the older cell (more than 60 generations) of iPS cell algebraically is divided into FLC sooner than the iPS cell of algebraically youth (the approximately the 30th generation), and the former is that 3 weeks and the latter are 4 weeks.The FLC of three kinds of different methods induction generations does not have obvious difference in form, but the cell that the third method obtains has good survival ability and multiplication capacity, thereby has higher induction efficiency, and this method is used to following experiment.
Embodiment 2 cultivator induced multi-potent stem cells
Using FLC for feeder layer cells is for before reprogramming of somatic cells, need to be with 10 mg/ml ametycins (Millipore company) advanced processing FLC 3 hours.Somatocyte for reprogrammed is human foreskin fibroblast (HFFs, Millipore company), the present invention uses the Zinc finger nuclease (ZFN) of Baculovirus-mediated by ZFN gene and OSKM gene (Oct4, Sox2, Klf4 and c – Myc) proceed to cell simultaneously, make OSKM gene specific and be incorporated into the AAVS1 site of HFFs.Carry the baculovirus of ZFN gene and OSKM gene the 1st day and twice transfection 1 × 10 in the 8th day
4hFFs, cell culture fluid is FibroGRO
tM-LS perfect medium (Millipore company).The infection multiplicity (MOI) of expressing the each cell of baculovirus of ZFN gene and OSKM gene is respectively 100 and 50.After 200 ug/ml G418 medicines sieve 10 days, the cell of survival is transferred on FLC feeder layer and forms population of cells.Be covered with Tissue Culture Plate that MEF or matrigel processed as control group.
This reprogramming of somatic cells technological method adopts taking baculovirus transfection as basic ZFN technology, and OSKM gene specific is inserted into the AAVS1 site (19q13.3-qter) on No. 19 karyomit(e)s of the mankind.The HFF cell of same lot number will carry out reprogrammed induction on different feeder layers, and what relate to has FLC feeder layer, MEF feeder layer and a matrigel feeder layer.After baculovirus transfection and drug screening, the HFF cell of survival is inoculated into respectively different feeder layers until cell colony forms.No matter use which kind of feeder layer, ESC sample colony will just occur the earliest for about the 20th day after transfection.Cultivate again after one week, just can observe cellular form compactness, sharp-edged colony, and most colony AP coloration result be positive (Fig. 2 A).Number by the positive colony of several AP-calculates the reprogramming efficiency (Fig. 2 B) of HFF cell on different feeder layers.In the time that baculovirus infection plural number is 50, HFF cell is at FLC, and the reprogramming efficiency on MEF and matrigel is respectively 1.25%, 2.58% and 0.13%.But in the time that virus infection plural number is increased to 100, the reprogramming efficiency (14.7%) on FLC is 2.6 times of (5.6%) on MEF.
By genomic dna pcr amplification, the specificity of the iPS cell clone of eight random chooses of detection on AAVS1 site integrated.Two pairs of PCR primers design respectively at the two ends of integrator gene expression cassette.The clone of all detections has the integration specifically (Fig. 2 C) of gene in AAVS1 site.Real-time PCR Analysis result proves that these clones have classical embryonic stem cell marker gene Nanog than initial HFF cell, Oct4, and the high level expression of Sox2 (Fig. 2 D).In the time iPS cell being inoculated in to ultralow lamina affixad and cultivating in embryoid body division culture medium, cell can the spherical embryoid body of spontaneous formation (Fig. 2 E).And the expression of three germinal layer marker gene, ectoderm Pax6, mesoderm α-MHC is for being significantly improved in embryoid body with entoderm AFP, in undifferentiated iPS cell, but almost can't detect in contrast the expression (Fig. 2 E) of these genes, this has illustrated that the iPS cell of setting up on FLC has full differentiation potential.
Embodiment 3 impacts of human desmocyte like cell on people's induced multi-potent stem cells
In order to detect the impact of the cell-derived FLC of people iPS on mankind's totipotency stem cell growth, the present invention has measured diameter and the daily progression rate of newly-generated people iPS cell and human embryo stem cell H1 population of cells.After these populations of cells at least being cultivated on FLC or MEF to 5 generations, just start to measure.When measurement, after postvaccinal the 2nd day and the 5th day, choose ten populations of cells respectively and take pictures, then on picture, measure the diameter of population of cells.Population of cells's growth velocity is calculated according to following formula: (diameter-of population of cells population of cells's diameter of 2 days of the 5th day)/3 days.The expression level analysis of the totipotency stem cell marker gene of population of cells is by quantitative qPCR.Cultivate at matrigel or
population of cells on 1 is by contrast.
In order to detect derivative FLC to the totipotent impact of human stem cells, newly-generated people iPS cell and human embryo stem cell H1 are used to form embryoid body.Population of cells at least will be cultivated for 5 generations on FLC or MEF before this, the embryoid body obtaining will carry out expression analysis with real-time quantitative PCR.
Form teratomatous ability in order to detect newly-generated people iPS cell, Accutase (Millipore company) has been digested 1 × 10
6individual iPS cell and 0.5 × 10
6the pretreated HFF of individual mitomycin mixes and is resuspended in the PBS of 50 microlitres.First matrigel good with the undiluted precooling of 50 μ l resuspended cell liquid (Becton Dickinson company) is mixed, be then just injected into the back leg of non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mouse in 5 week age.Take out after two months teratoma, then fix with 4% paraformaldehyde, paraffin embedding, is cut into 5 microns of sections, and with the dyeing of phenodin and eosin.Laboratory animal nursing and using priciple that all experimentation on animalies all specify according to the consultative council of Animal Experimental Study country of Singapore are carried out.
Can find by above-mentioned experiment, the colony form of the stem cell of cultivating on two kinds of different feeder layers is consistent: flat and compact, and colony edge clear (Fig. 3 A).No matter but be mankind iPS clone or hESC's clone, cultivate all large than on MEF of colony on FLC.In order to quantize concrete difference, choose at random ten cell clones from each group, then within postvaccinal the 5th day, measure cell colony diameter at cell.In the time that iPS cell and H1 hESC clone are cultivated on FLC, cell colony is obviously greater than to be cultivated on MEF (Fig. 3 B).Measure the cell colony size of the 2nd day and by the poor average growth rate of calculating cell colony of cell colony diameter between the 5th day and the 2nd day simultaneously.Experimental result proving again the superior growth-promoting effect (Fig. 3 B) of FLC.Other one group of parallel laboratory test, by iPS cell and H1 hESC clone, at FLC, MEF, and cultivating on cutose for 5 generations, then extracts the expression of RNA Real-time PCR Analysis versatility marker gene.Result shows there is no significant difference between different culture medium system, except cultivating on the expression level of Nanog of iPS cell on FLC and MEF higher than the cell (Fig. 3 C) of cultivating on matrigel.Result of study shows than MEF, and FLC maintains of mankind's totipotency expansion of stem cells better to select.
Further, the present embodiment has detected iPS cell and H1 hESC clone and cultivated the differentiation potential after 10 generations on the FLC of deactivation and MEF.This detects the cell of collecting by use and carrys out the outer embryoid body (Fig. 4 A) of organizer.The expression level of three germinal layer marker gene of embryoid body is measured with real-time quantitative RT-PCR, and six different marker gene that detect comprise NELF, Pax6, α-MHC, PPAR_r, AC133, and AFP.The expression level of most of marker gene does not have significant difference between cultured cells on FLC and MEF, except the expression amount of the upper AFP of H1 embryoid body cultivating of FLC and the Pax6 of iPS cell embryoid body all increases (Fig. 4 B) to some extent than the cell on MEF.
The present embodiment has carried out experimentation on animals to detect iPS clone differentiation capability in vivo simultaneously.The back leg that iPS cell is injected into NOD/SCID mouse, after 6 weeks, can be observed teratomatous formation.After injection the 8th week, teratoma is drawn in histological examination differentiation degree.Fig. 4 C is presented at and in teratoma, has three germinal layer cells simultaneously, thereby has confirmed to set up and in the still totipotency of tool differentiation of iPS cell maintaining on FLC.
Adopt design of the present invention, the human desmocyte like cell that end user's induced multi-potent stem cells is differentiated to form is cultivated people's induced multi-potent stem cells as feeder layer, exempt the risk of the propagation of inhuman xenogenesis factor pollution and animal pathogen, simultaneously because people's induced multi-potent stem cells is cultivated in vitro indefinitely, therefore the feeder layer cells that uses the solution of the present invention also can indefinite a large amount of preparations, with respect to other feeder layer cells, method of the present invention had both ensured the efficiency that reprogramming of somatic cells generates, having overcome again prior art feeder layer cells, to maintain the life-span short, cultivate the few problem of algebraically, technical scheme of the present invention has tremendous potential in the clinical application of people's induced multi-potent stem cells.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. a method for foundation and cultivator induced multi-potent stem cells, is characterized in that, comprises the following steps:
A. prepare human desmocyte like cell feeder layer: get human desmocyte like cell, be seeded on culture dish by 10 mg/ml ametycin advanced processing 3 hours, remove ametycin;
B. cultivate: reprogrammed somatocyte, generates people's induced multi-potent stem cells, and people's induced multi-potent stem cells is transplanted in the feeder layer of preparing in step a and cultivated.
2. the method for a kind of foundation according to claim 1 and cultivator induced multi-potent stem cells, is characterized in that, in described step a, human desmocyte like cell is adopted with the following method and generated:
A1: end user's human foreskin fibroblasts reprogrammed obtains people's induced multi-potent stem cells 5.9 clones;
A2: maintain 5.9 clone 7 days on matrigel;
A3: the cultivation of going down to posterity after using 1 mg/ml Dispase to digest 5-7 minute under 37 DEG C of environment, nutrient solution is
1, nutrient solution is changed every day;
A4: reach 80% when full when being incubated at 5.9 clones in matrigel, remove
after 1 substratum, clean twice with 1 × PBS, then directly add human desmocyte like cell substratum and make 5.9 clones start directed differentiation, change substratum first four day every day, within every 2 days afterwards, change once;
A5: induce after 6～12 days 0.05% trypsinase for cell covering with-EDTA is digested, and with 1 × 10
6the concentration in individual/hole is seeded in cell on pretreated 6 orifice plates of matrigel; In the time that cell covers with again, by cell dissociation and with 2 × 10
5the concentration in individual/hole is inoculated in pretreated 6 orifice plates of 0.1% gelatin;
A6: processed at gelatin flat board on cultivate cellular form homogeneous gradually after 3-5 days, be human fibroblasts's sample.
3. the method for a kind of foundation according to claim 1 and cultivator induced multi-potent stem cells, is characterized in that, described step b specifically comprises the steps:
B1: get 1 × 10
4human foreskin fibroblast, adopt the Zinc finger nuclease of Baculovirus-mediated in first day and twice transfection in the 8th day, ZFN gene and OSKM gene are proceeded to cell simultaneously, make OSKM gene specific and be incorporated into the AAVS1 site of human foreskin fibroblast, cell culture fluid is FibroGRO
tM-LS perfect medium;
B2: use 200 ug/ml G418 medicine sieves after 10 days, the cell of survival is transferred on human desmocyte like cell feeder layer and cultivated and form population of cells, obtain people's induced multi-potent stem cells.
4. the method for a kind of foundation according to claim 3 and cultivator induced multi-potent stem cells, is characterized in that, in described step b1, the infection multiplicity of expressing the each cell of baculovirus of ZFN gene and OKSM gene is 100.
5. the method for a kind of foundation according to claim 2 and cultivator induced multi-potent stem cells, is characterized in that, 5.9 cells that use are the cell of 60 continuous passages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410382321.2A CN104140951B (en) | 2014-08-06 | 2014-08-06 | A kind of method set up and cultivate people induced multi-potent stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410382321.2A CN104140951B (en) | 2014-08-06 | 2014-08-06 | A kind of method set up and cultivate people induced multi-potent stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104140951A true CN104140951A (en) | 2014-11-12 |
| CN104140951B CN104140951B (en) | 2016-08-24 |
Family
ID=51850293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410382321.2A Expired - Fee Related CN104140951B (en) | 2014-08-06 | 2014-08-06 | A kind of method set up and cultivate people induced multi-potent stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104140951B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726395A (en) * | 2015-03-20 | 2015-06-24 | 深圳市人民医院 | Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells |
| CN105670989A (en) * | 2014-11-18 | 2016-06-15 | 朱向情 | Kit and method for effectively inducing somatic cell phenotype reprogramming |
| CN106256900A (en) * | 2016-08-12 | 2016-12-28 | 浙江译美生物科技有限公司 | A kind of stem cell cultivating system of non-animal derived property |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161980A (en) * | 2011-02-12 | 2011-08-24 | 浙江大学 | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast |
| CN103348002A (en) * | 2010-12-31 | 2013-10-09 | 维也纳农业大学 | Method of generating induced pluripotent stem cells and differentiated cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
-
2014
- 2014-08-06 CN CN201410382321.2A patent/CN104140951B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103348002A (en) * | 2010-12-31 | 2013-10-09 | 维也纳农业大学 | Method of generating induced pluripotent stem cells and differentiated cells |
| CN102161980A (en) * | 2011-02-12 | 2011-08-24 | 浙江大学 | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
Non-Patent Citations (2)
| Title |
|---|
| TE LIU ET AL.: "Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression", 《EXPERIMENTAL CELL RESEARCH》 * |
| 刘德伍等: "人诱导多能干细胞定向分化为表皮样干细胞的研究", 《中华医学会整形外科学分会第十一次全国会议、中国人民解放军整形外科学专业委员会学术交流会、中国中西医结合学会医学美容专业委员会全国会议论文集》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105670989A (en) * | 2014-11-18 | 2016-06-15 | 朱向情 | Kit and method for effectively inducing somatic cell phenotype reprogramming |
| CN104726395A (en) * | 2015-03-20 | 2015-06-24 | 深圳市人民医院 | Method for inducing directional differentiation of human induced pluripotent stem cells into pancreatic cells |
| CN106256900A (en) * | 2016-08-12 | 2016-12-28 | 浙江译美生物科技有限公司 | A kind of stem cell cultivating system of non-animal derived property |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104140951B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sugii et al. | Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells | |
| Beers et al. | Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions | |
| Wu et al. | Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts | |
| JP2020115866A (en) | Methods of direct differentiation from pluripotent stem cells to functional heart muscle | |
| Correia et al. | Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes | |
| KR101861171B1 (en) | Cardiomyocyte medium with dialyzed serum | |
| CN101402943B (en) | Method for in vitro induction and cultivation of multi-potentiality stem cell | |
| CN102686724A (en) | Method for producing mesenchymal stem cells from human pluripotent stem cells, and mesenchymal stem cells produced by same | |
| Ghasemi‐Dehkordi et al. | Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder‐and serum‐free system (Matrigel matrix), MEF and HDF feeder cell lines | |
| Slamecka et al. | Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions | |
| Cao et al. | In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes | |
| Lorenzo et al. | Generation of mouse and human induced pluripotent stem cells (iPSC) from primary somatic cells | |
| CN102161980B (en) | Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast | |
| CN103555661A (en) | Culture method free of multipotential stem cell without serum and feeder layer | |
| CN103667349A (en) | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) | |
| CN103589686B (en) | A kind of human umbilical cord mesenchymal stem cells that uses is as the method for feeder layer cultivator induced multi-potent stem cells | |
| CN101984050B (en) | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof | |
| CN107002035A (en) | Stem cell composition and the method for producing the stem cell for treatment use | |
| CN104140951A (en) | Method for developing and cultivating human induced pluripotent stem cells | |
| CN105420193A (en) | Differential medium and application thereof in preparing neural stem cells | |
| Rodríguez Gutiérrez et al. | Pluripotent cell models for gonadal research | |
| Gao et al. | A protocol for the generation of patient-specific iPSC lines from peripheral blood mononuclear cells | |
| Mohamed et al. | Umbilical cord tissue as a source of young cells for the derivation of induced pluripotent stem cells using non-integrating episomal vectors and feeder-free conditions | |
| JP2020517281A (en) | Cryopreserved cells ready for use | |
| TWI280280B (en) | Culture system and method for expansion and undifferentiated growth of human embryonic stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhuo Lang Inventor after: Wang Hui Inventor after: Wang Ke Inventor before: Wang Shuo |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WANG SHUO TO: ZHUO LANG WANG HUI WANG KE |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160824 Termination date: 20170806 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |