CN106867961A - A kind of induced multi-potent stem cell forms mesoblastemic inducing culture and abductive approach - Google Patents
A kind of induced multi-potent stem cell forms mesoblastemic inducing culture and abductive approach Download PDFInfo
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- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Abstract
Mesoblastemic inducing culture is formed the invention provides a kind of induced multi-potent stem cell, the inducing culture includes each component of following concentration：The 15mM of LiCl 5, glutamine 0.5 2%, nonessential amino acid 0.5 2%, 5 15 μM of CHIR99021, the 0.2mmol/l of 8 15ng/ml, β mercaptoethanols of bFGF 0.05, the μ g/ml of hydrocortisone 0.2 0.6, the μ g/ml of insulin 4 10, the μ g/ml of transferrins 80 120, the 12ng/ml of sodium selenite 8, the 0.2mg/ml of penicillin 0.05, the 0.2mg/ml of streptomysin 0.05.Inducing culture of the present invention is to improve the probability that induced multi-potent stem cell (ips) is broken up to mesoblastema by adding some specific cell factors and supplement on the basis of DMEM/F12 culture mediums.In addition, the present invention provides a kind of induced multi-potent stem cell forms mesoblastemic abductive approach, methods described is that the simple embryoid body of induced multi-potent stem cell is inoculated in inducing culture to carry out Fiber differentiation, so as to efficiently be divided into mesoblastema.
Description
Technical field
The present invention relates to a kind of inducing culture and abductive approach, and in particular to a kind of induced multi-potent stem cell forms middle embryo
The inducing culture and abductive approach of confluent monolayer cells.
Background technology
Skin tissue engineering is study hotspot in recent years, but the relevant cell of skin belongs to terminally differentiated cells,
It is difficult to carry out large-scale culture amplification in vitro again, seed cell comes the development that source problem hinders organizational project, autologous kind
There is rejection in daughter cell limited source, the seed cell of allosome, stem cell has ethics problem again, therefore is received from urine
Collection urine cell, by urine cell induction turn into IPS cells, ips cells induce again as mesoblastema, fibroblast,
Sweat gland cells, mesoblastema etc. can solve the problems, such as the seed source of skin tissue engineering, and cell is obtained from urine, convenient
Fast, obtained without by wound, wide material sources.
Each cell induction scheme that multi-functional stem cell is induced to differentiate into skin at present mainly has the simple cell factor
Induction, co-culturing, inducing, embryoid body induction etc..But simple cell factor induced efficiency is low, there is immunogene in co-culturing, inducing
Property, embryoid body exist non-directional induction feature, ultimately form three germinal layers, it is impossible to efficiently obtain some germinal layer, therefore, this hair
A kind of bright efficient inducing pluripotent stem cells of offer are divided into mesoblastemic method.
The content of the invention
A kind of induced multi-potent stem cell is provided it is an object of the invention to the weak point for overcoming prior art to exist
Form mesoblastemic inducing culture and abductive approach.
To achieve the above object, the technical solution used in the present invention is：A kind of induced multi-potent stem cell forms mesoderm
The inducing culture of cell, the inducing culture includes each component of following concentration：
Wherein LiCl is lithium chloride, CHIR99021 is GSK-3 (glycogen synthase kinase 3) inhibitor, and b FGF are alkalescence
Fibroblast growth factor.
Induced multi-potent stem cell of the present invention forms mesoblastemic inducing culture and abductive approach can be with height
Effect inducing pluripotent stem cells are divided into mesoblastema, while whole process does not use serum, effectively avoid animal
The risk that borne causal agent brings, improves the security of clinical practice.
Preferably, induced multi-potent stem cell described above forms mesoblastemic inducing culture includes following concentration
Each component：
Using induced multi-potent stem cell described above to form mesoblastemic inducing culture and abductive approach can be with
At utmost, most efficiently inducing pluripotent stem cells are divided into mesoblastema, the base of mesoblastema marker gene Flk-1
Because of expression highest.
Preferably, the inducing culture also includes basic culture solution, and the basic culture solution is DMEM/F12E.
Inducing culture of the present invention is by adding some specific cell factors on the basis of DMEM/F12 culture mediums
With supplement so as to improve the probability that induced multi-potent stem cell (ips) is broken up to mesoblastema, for skin tissue engineering is provided
Seed cell is originated.
In addition, forming mesoblastemic abductive approach, methods described the invention provides a kind of induced multi-potent stem cell
It is that the simple embryoid body of induced multi-potent stem cell is inoculated in inducing culture described above to carry out Fiber differentiation.
Preferably, the Fiber differentiation temperature is 25~28 DEG C.
Preferably, the preparation method of the embryoid body is to be inoculated in the induced multi-potent stem cell of single recovery to contain embryoid
Body is formed in culture medium, is put into after centrifugation after cultivating 48h in incubator, that is, form embryoid body.
Preferably, the inoculum concentration of the induced multi-potent stem cell of the single recovery is 1 × 106Individual cells/well.
Preferably, the Parameter Conditions of the culture plate low-speed centrifugal are：700r/min, 5min.
Preferably, the temperature of the incubator be 37 DEG C, carbon dioxide content be 5%CO2。
Specifically, the preparation method of embryoid body is：(1), recovery cell：The IPS cells of P30 are selected to be recovered, with nothing
The human embryonic stem cell medium (mTeSR1) of matrix carries out cell culture, when cell reaches 80% degree of converging, uses Accutase
Cell dissociation buffer digestion is unicellular, is counted；(2) and then by each AggreWellTM1x10 is added in 800 cultivation plate holes6It is individual
Cell, adds EB to form culture medium, operation manual is used by product, by AggreWellTM800 culture plates are with 700r/min low speed
Centrifugation 5min, is then placed in containing 5%CO2, cultivate in 37 DEG C of incubators, form embryoid body after culture 48h.
On the other hand, the present invention provides a kind of induced multi-potent stem cell and forms mesoblastemic inducing culture in induction
Multipotential stem cell forms application during mesoblastema.
The beneficial effects of the present invention are：Mesoblastemic luring is formed the invention provides a kind of induced multi-potent stem cell
Culture medium and abductive approach are led, induced multi-potent stem cell can be efficiently divided into using the inducing culture and abductive approach for middle embryo
Confluent monolayer cells.
Brief description of the drawings
Fig. 1 breaks up simple to be induced in inducing culture of the present invention and control medium in the embodiment of the present invention 1
The expression comparison diagram of the Flk-1 in cell is formed after embryoid body.
Specific embodiment
It is right below in conjunction with the accompanying drawings and the specific embodiments to better illustrate the object, technical solutions and advantages of the present invention
The present invention is described further.
Embodiment 1
A kind of induced multi-potent stem cell described in the present embodiment forms mesoblastemic inducing culture includes following concentration
Each component：
Embodiment 2
A kind of induced multi-potent stem cell described in the present embodiment forms mesoblastemic inducing culture includes following concentration
Each component：
Embodiment 3
A kind of induced multi-potent stem cell described in the present embodiment forms mesoblastemic inducing culture includes following concentration
Each component：
The external evoked ips of embodiment 4 breaks up to mesoblastema
First, the preparation of experimental group culture medium
According to addition Insulin 3 0mg, hydrocortisone 0.2mg, transferrins in 500ml DMEM/F12 culture mediums
50mg, sodium selenite 5 μ g, β mercaptoethanols 0.05mmol, bFGF 6 10 μM of μ g, CHIR99021, LiCl 10mM, nonessential ammonia
The proportions induced multi-potent stem cell of the present invention of base acid 5ml, glutamine 5ml, penicillin 50mg, streptomysin 50mg
Mesoblastemic inducing culture is formed, and as the experimental group culture medium in following experiment.
2nd, the preparation of control group culture medium
By 500ml EB culture mediums culture medium as a control group.
3rd, recovery cell
Select the IPS cells of P30 to be recovered, cell is carried out with the human embryonic stem cell medium (mTeSR1) without matrix
Culture, when cell reaches 80% degree of converging, it is unicellular, counting to be digested with Accutase cell dissociation buffers.
4th, the formation of embryoid body：By addition 1 × 10 in each AggreWellTM800 cultivation plate hole6Individual cell, adds EB
Culture medium is formed, operation manual is used by product, by AggreWellTM800 culture plates with 700r/min low-speed centrifugal 5min, so
After be put into containing 5%CO2, cultivate in 37 DEG C of incubators, form embryoid body after culture 48h.
5th, induction differentiation
Simple embryoid body is transferred in 24 orifice plates of low absorption, 24 orifice plates are divided into two groups, respectively experimental group and control group,
Every group of 12 holes, experimental group experimental group medium culture continues to use EB medium cultures, and control group is trained with control group culture medium
Support, liquid is changed every other day, cultivate to after 7 days, the gene expression dose to mesoblastemic marker gene Flk-1 is detected.
Wherein, the specific method that mesodermal precursor cells are identified is：By control group and experimental group cell respectively with containing
0.25% trypsase and 0.02%EDTANa2Digestive juice process 1~3 minute, make cell detachment culture matrix and turn into singly
Cell, adds the culture medium containing 10% hyclone to carry out neutralization digestion, and 1200rpm is centrifuged 5 minutes, is moistened with cold PBS
Wash cell, cell precipitation be collected in 1.5mlEP pipes, using cell total rna extracts kit (RNeasyMiniKit,
QIAGEN74104), by its operation instruction cell lysis and intracellular total serum IgE is extracted, RNA is determined using ultraviolet specrophotometer
After concentration and purity, using reverse transcription reagent box (ReverTraAceqPCRRTMasterMix, TOYOBOFSQ-201) by 1 μ g
Total serum IgE reverse transcription is cDNA.By cDNA, primer with qPCR reaction premix reagents
(THUNDERBIRDSYBRqPCRMix, TOYOBOQPS-201) mixes, by the opticon2 of Bio-Rad companies
Fluorescence real-time quantitative PCR instrument is expanded to genes of interest, and with people's GAPDH gene expression amounts as reference, the reaction of each sample is only
Stand in triplicate to eliminate systematic error.
6th, result
As shown in figure 1, compared when forming mesoblastema using experimental group culture medium induction ips using control group culture medium
When induction ips forms mesoblastema, the gene expression dose of the mesoblastema marker gene Flk-1 of experimental group is far above right
According to the gene expression dose of the mesoblastema marker gene Flk-1 of group, show the inducing culture and abductive approach of experimental group
The efficiency broken up to mesoblastema is significantly higher than control group, illustrates that inducing culture of the present invention and abductive approach can be with height
Effect inducing pluripotent stem cells are divided into mesoblastema.Whole process does not use serum simultaneously, effectively avoids animal
The risk that borne causal agent brings, improves the security of clinical practice.
It is last to should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (10)
1. a kind of induced multi-potent stem cell forms mesoblastemic inducing culture, it is characterised in that the inducing culture
Each component including following concentration：
2. induced multi-potent stem cell as claimed in claim 1 forms mesoblastemic inducing culture, it is characterised in that institute
State each component of the inducing culture including following concentration：
3. induced multi-potent stem cell as claimed in claim 1 or 2 forms mesoblastemic inducing culture, and its feature exists
In the inducing culture also includes basic culture solution, and the basic culture solution is DMEM/F12.
4. a kind of induced multi-potent stem cell forms mesoblastemic abductive approach, it is characterised in that methods described is to induce
The embryoid body of multipotential stem cell carries out Fiber differentiation in being inoculated in inducing culture as claimed in claim 1.
5. abductive approach according to claim 3, it is characterised in that the temperature of the Fiber differentiation is 25~28 DEG C.
6. abductive approach according to claim 3, it is characterised in that the preparation method of the embryoid body is by single recovery
Induced multi-potent stem cell be inoculated in and form culture medium containing embryoid body, be put into after centrifugation after cultivating 48h in incubator, i.e.,
Form embryoid body.
7. abductive approach according to claim 3, it is characterised in that the induced multi-potent stem cell of the single recovery connects
The amount of kind is 1 × 106Individual cells/well.
8. abductive approach according to claim 3, it is characterised in that the Parameter Conditions being centrifuged are：700r/min,
5min。
9. abductive approach according to claim 3, it is characterised in that the temperature of the incubator is 37 DEG C, carbon dioxide
Content is 5%CO2。
10. the induced multi-potent stem cell as described in claims 1 to 3 forms mesoblastemic inducing culture in induced multi-potent
Stem cell forms application during mesoblastema.
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| CN111304157A (en) * | 2020-03-16 | 2020-06-19 | 吉林大学 | Method for obtaining bovine initial state induced pluripotent stem cells |
| CN111321110A (en) * | 2020-02-28 | 2020-06-23 | 上海市东方医院（同济大学附属东方医院） | Method for differentiating human pluripotent stem cells into mesoderm |
| CN111647552A (en) * | 2020-06-18 | 2020-09-11 | 中国人民解放军联勤保障部队第九二〇医院 | Method for rapidly and efficiently preparing embryoid bodies by inducing pluripotent stem cells |
| CN116445408A (en) * | 2023-05-22 | 2023-07-18 | 呈诺再生医学科技(北京)有限公司 | Use of LSD1 inhibitors to promote iPSC differentiation to HSCs and maintenance of HSC dryness |
| CN117305241A (en) * | 2023-11-28 | 2023-12-29 | 上海兴瑞一达生物科技有限公司 | Method for inducing and differentiating hiPSCs into NK cells |
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