CN106479956B - A kind of culture medium and method improving reprogramming efficiency of somatic cells - Google Patents
A kind of culture medium and method improving reprogramming efficiency of somatic cells Download PDFInfo
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Abstract
The present invention provides a kind of culture medium for inducing somatic reprogramming, and the culture medium includes the culture medium and phosphatidic acid of conventional inducing somatic reprogramming.The present invention also provides a kind of method of inducing somatic reprogramming, the method includes any periods reprogrammed in inducing cell using culture medium of the invention or in any period of inducing cell reprogramming, adds phosphatidic acid in the medium.The study find that the simplest lipid of phosphatidic acid-, it is added in the culture medium for inducing somatic reprogramming, the reprogramming efficiency of body cell can be significantly improved, and add the Apoptosis reduced during reprogramming, it is hereby achieved that the iPS cell of a large amount of high quality, and lay a good foundation for iPS cell in drug screening and its application on the following translational medicine.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of culture medium and method for improving reprogramming efficiency of somatic cells.
Background technique
Embryonic stem cell (embryonic stem cells, ESCs) is used as a kind of myeloid-lymphoid stem cell, has the complete of development
Energy property, can be divided into all organs, tissue even reproduction cell, this brings for gene and the research of cell therapy and regenerative medicine
New opportunity.But embryonic stem cell is mainly separately cultured by the inner cell mass of blastula stage and is generated, this needs to destroy
Embryo in mesoderm growing early stage, therefore the research of human embryo stem cell has caused dispute in ethics because carrying out source problem.Separately
Outside, immunological rejection caused by heteroplastic transplantation usually also limits the clinical application of ESCs.And inductivity pluripotent stem cell
The appearance of (induced pluripotent stem cells, iPSCs) is expected to break through the bottleneck problem of embryonic stem cell application,
Extensive material and method are provided for therapeutic treatment and biological study.
Can there are Oct4, Sox2, Klf4 and c-Myc using foreign gene with the reprogramming of inducing somatic, the common factor
(OSKM).But there are still many urgent problems to be solved in iPSCs Induction Process, as reprogramming four factors in c-
Itself is a proto-oncogenes by Myc, so that iPSCs has certain oncogenicity；The induced efficiency of iPSCs is low, insufficient
1%；And the iPS cell line obtained exists heterogeneous, quality is inhomogenous, and most second-rate (only about 10% clone can
To generate allophenic mice).It, may be there is several factors that the quality of iPSCs generation be affected, such as foreign gene used through studying
Dosage, difference of condition of culture, and the additive for initial cell etc..
2012, Esteban et al. reported a kind of natural compound, and ascorbic acid (vitamin C, Vc) can
As a kind of additive of culture medium, inhibit the expression of p53 and p21, while slowing down cell ageing, improves reprogramming efficiency.Most
It is close the study found that human embryo stem cell (hESCs) versatility is maintained but to lead to the differentiation of mouse embryo stem cell (mESCs)
Factor FGF2 can hinder the expression of the extracellular matrix of reprogramming of somatic cells, to improve the efficiency of reprogramming by lowering.
Phosphatidic acid (phosphatidic acid, PA) is that the common phosphatide of one kind is as the constituent of cell membrane
The biosynthesis precursor of glycerophosphatide, has received more and more attention in recent years.Phosphatidic acid can be by phospholipase D
(PLD) hydrolysis generates, and phosphorylation can also occur through diacylglycerol kinase and generate.In mammals, PA can pass through tune
The various vital movements of the number of ways participation cell such as section enzymatic activity or membrane structure change, such as vesicular traffic, secretion,
Endocytosis and exocytosis, dynamic construction and cell differentiation of cytoskeleton etc..Nearest result of study shows that PA can pass through
TRIAP1/PREL1 complex regulates and controls the synthesis and accumulation of cuorin (Cardiolipin, CL) on mitochondrial membrane, prevents cell color
The release of plain C (Cytochrome, Cty c) inhibits Apoptosis.
Body cell dedifferentes technology and continuously achieves a series of important breakthroughs in recent years, provides for the application of regenerative medicine
New approach, makes it possible the regeneration of histoorgan.In addition, establishing the cell of disease using the iPS cell in disease source
Model and animal model are also that discussion disease incidence mechanism and drug platform screening open new field.Meanwhile going deep into system
Research dedifferente mechanism, it is not only most important for solving Basic Science Problem, and for being transformed and improving cell fate
The theoretical basis of decision is also held the balance, so be applied to regenerative medicine application also have will have greatly enlightenment use for reference
Effect.And the inhomogenous of extremely low induced efficiency and gained cell seriously limits the application of the technology, such as high-flux sequence and
Drug screening etc..These problems require inventor by furtheing investigate the regulatory mechanism of cell de-differentiation to answer.
However, research reported at present, has focused largely on transcription factor, micromolecular inhibitor etc. during reprogramming
Effect, and Effect study of the lipid in reprogramming is had not been reported.
Summary of the invention
Based on object above, the application the study found that cell membrane normal constituent phosphatidic acid (PA), be added to and rearrange
In journey culture medium, the reprogramming efficiency of body cell can be significantly improved and reduce apoptosis of the cell in reprogramming, so as to
To obtain the iPS cell of a large amount of high quality, and established for iPS cell in drug screening and its application on the following translational medicine
Basis is determined.
It is an advantage of the invention to provide a kind of culture medium for inducing somatic reprogramming, the culture mediums
Containing phosphatidic acid, and provide purposes of the culture medium in inducing somatic reprogramming.
It is a further object of the invention to provide a kind of methods for improving reprogramming efficiency of somatic cells.
The purpose of the present invention is achieved through the following technical solutions.
In one aspect of the invention, special the present invention provides a kind of culture medium for inducing somatic reprogramming
Sign is that the culture medium includes the culture medium and phosphatidic acid of conventional inducing somatic reprogramming.
Preferably, the concentration of phosphatidic acid is 100-800 μM in the culture medium, it is highly preferred that concentration is 200-600 μM,
It is further preferred that concentration is 400 μM.
Preferably, the culture medium of the conventional inducing somatic reprogramming be N2B27 culture medium, KOSR culture medium or
FBS culture medium.
Wherein, the N2B27 culture medium mainly consists of the following compositions: the DMEM/F12 base mixed by the volume ratio of 1:1
Basal culture medium and Neurobasal basal medium and percent by volume be the culture medium 1~2% N2 additive,
Percent by volume is the bovine serum albumin(BSA) (BSA) and 1000 of 1~2% B27 additive of the culture medium, 25ng/mL
The LIF ELISA (LIF) of units per ml.
Wherein, KOSR (serum substitute) culture medium mainly consists of the following compositions: Knock out DMEM is dry thin
Born of the same parents' culture medium, percent by volume are Sodium Pyruvate, the 0.1mM of 20% KOSR, NEAA, Glutmax, 1mM of the culture medium
The LIF ELISA (LIF) of beta -mercaptoethanol and 1000 units per mls
Wherein, the FBS culture medium mainly consists of the following compositions: DMEM/F12 basal medium, percent by volume are
Sodium Pyruvate, 0.1mM beta -mercaptoethanol and the 1000 units/milli of 20%FBS, NEAA, Glutmax, 1mM of the culture medium
The LIF ELISA (LIF) risen.
Wherein, the NEAA is cell culture additive, containing nonessential amino acid glycine, L- needed for 7 kinds of cell culture
Alanine, altheine, L-Aspartic acid, Pidolidone, L-PROLINE, Serine.Generally can be commercially available, such as
The NEAA is purchased from Gibco, article No. 11140.
The Glutmax is the dipeptides containing L-Glutamine, Ala-Gln stable form.Generally may be used
With commercially available, such as the Glutmax is purchased from Gibco, article No. 35050.
Preferably, the body cell can derive from different kinds, the including but not limited to mankind, gorilla, monkey etc..
It is furthermore preferred that the body cell is thin for the skin fibroblasts of primate, mucomembranous epithelial cell, lymph
Born of the same parents, hair follicle cell, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells, mesenchymal stem cell can be from primate
Species are isolated or buy from relevant cell bank.
It is furthermore preferred that the body cell is thin for the skin fibroblasts of people, mucomembranous epithelial cell, lymphocyte, hair follicle
Born of the same parents, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells, mesenchymal stem cell.
Preferably, the culture medium also added fortimicin (Doxcycline, Dox), hepatic glycogen synthesis 3 β of kinases
The selective depressant CHIR99021, non ATP competitiveness mek inhibitor PD0325901 of (GSK3 β) receptor；
Preferably, the concentration of the fortimicin Dox is 1-5ng/ml, more preferably 2ng/ml；
Preferably, the concentration of the CHIR99021 is 1-5 μM, more preferably 3 μM；
Preferably, the concentration of the PD0325901 is 1-5 μM, more preferably 1 μM.
In another aspect of the invention, the present invention provides a kind of method of inducing somatic reprogramming, the method packet
Any period in inducing cell reprogramming is included using culture medium culture body cell of the invention or in inducing cell reprogramming
Any period, add phosphatidic acid in the medium, continue cultivate body cell.
Preferably, early stage cell reprograms and/or mid-term using culture medium culture body cell of the invention or
Phosphatidic acid is added in culture medium, continues to cultivate body cell.
Specifically, it the described method comprises the following steps:
1) versatility related gene is imported into body cell；
2) body cell for having imported versatility related gene is prepared as single cell suspension, is walked using conventional medium culture
Rapid 1) the middle body cell for importing versatility related gene, example of spatial compartmentalizationis, and using the conventional medium culture
During body cell, select one day perhaps several days using culture medium culture body cell of the invention or described in the use
During conventional medium culture body cell, one day or several days addition phosphatidic acid is selected, so that it is dry to obtain induced multi-potent
Cell.
Preferably, the body cell can derive from different kinds, the including but not limited to mankind, gorilla, monkey etc..
It is furthermore preferred that the body cell is thin for the skin fibroblasts of primate, mucomembranous epithelial cell, lymph
Born of the same parents, hair follicle cell, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells, mesenchymal stem cell can be from primate object
Kind is isolated or buys from relevant cell bank.
It is furthermore preferred that the body cell is thin for the skin fibroblasts of people, mucomembranous epithelial cell, lymphocyte, hair follicle
Born of the same parents, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells, mesenchymal stem cell.
Preferably, first day of reprogramming to third day using culture medium culture body cell of the invention or it is described often
Phosphatidic acid is added in rule culture medium；
Preferably, in the third day of reprogramming to the 6th day using culture medium culture body cell of the invention or described normal
Phosphatidic acid is added in rule culture medium；
Preferably, culture medium culture body cell of the invention is used within the 6th day to the 9th day or described normal in reprogramming
Phosphatidic acid is added in rule culture medium；
Preferably, in the third day of reprogramming to the 9th day using culture medium culture body cell of the invention or described normal
Phosphatidic acid is added in rule culture medium；
Preferably, in the third day of reprogramming to fortnight using culture medium culture body cell of the invention or described
Phosphatidic acid is added in conventional medium.
Preferably, the concentration of the phosphatidic acid of the addition is 100-800 μM, it is preferable that concentration is 200-600 μM, into one
Preferably, concentration is 400 μM to step.
Preferably, the culture medium of the conventional inducing somatic reprogramming be N2B27 culture medium, KOSR culture medium or
FBS culture medium.
Wherein, the N2B27 culture medium mainly consists of the following compositions: the DMEM/F12 base mixed by the volume ratio of 1:1
Basal culture medium and Neurobasal basal medium and percent by volume be the culture medium 1~2% N2 additive,
Percent by volume is the bovine serum albumin(BSA) (BSA) and 1000 of 1~2% B27 additive of the culture medium, 25ng/mL
The LIF ELISA (LIF) of units per ml.
Wherein, KOSR (serum substitute) culture medium mainly consists of the following compositions: Knock out DMEM is dry thin
Born of the same parents' culture medium, percent by volume are Sodium Pyruvate, the 0.1mM of 20% KOSR, NEAA, Glutmax, 1mM of the culture medium
The LIF ELISA (LIF) of beta -mercaptoethanol and 1000 units per mls.
Wherein, the FBS culture medium mainly consists of the following compositions: DMEM/F12 basal medium, percent by volume are
Sodium Pyruvate, 0.1mM beta -mercaptoethanol and the 1000 units/milli of 20%FBS, NEAA, Glutmax, 1mM of the culture medium
The LIF ELISA (LIF) risen.
Wherein, the NEAA is cell culture additive, containing nonessential amino acid glycine, L- needed for 7 kinds of cell culture
Alanine, altheine, L-Aspartic acid, Pidolidone, L-PROLINE, Serine.Generally can be commercially available, such as
The NEAA is purchased from Gibco, article No. 11140.
The Glutmax is the dipeptides containing L-Glutamine, Ala-Gln stable form.Generally may be used
With commercially available, such as the Glutmax is purchased from Gibco, article No. 35050.
Meanwhile the inventors of the present application found that endogenous phosphatidic acid be suppressed after, the reprogramming efficiency of body cell significantly drops
Low, this illustrates that endogenous phosphatidic acid participates in reprogramming, and plays indispensable role during reprogramming.Namely
It says, the missing of endogenous PA can significantly reduce reprogramming efficiency, but can be made up by exogenous PA.
The pluripotent stem cell obtained by the method for the invention has with embryonic stem cell (ES) similar characteristic, these are special
Property include: proliferative capacity, express embryonic stem cell marker, formed comprising it is interior, in, the monster of the tissue of outer three germinal layers and cell
Tumor.
The present invention has meaning below:
The study find that the simplest lipid of phosphatidic acid-, is added in the culture medium for inducing somatic reprogramming, it can
To significantly improve the reprogramming efficiency of body cell.Obtained PA-iPS cell is reprogrammed in multipotency sex factor Oct4, Nanog etc.
Expression it is higher, all have in vivo and in vitro to triploblastica break up ability.Also, PA-iPS cell is injected into blastaea
When can form hair color and be fitted into normal chimera of the rate up to 90% or more, and the ability being fitted into system genitale.Meanwhile the addition of PA
Reduce the Apoptosis during reprogramming, it may be possible to which cuorin (Cardiolipin, CL) on mitochondrial membrane is adjusted by PA
Accumulation, to prevent the release of cytochrome c (Cty c, Cytochrome), and then inhibit Apoptosis.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 is the flow chart of the technical solution of the application；
Fig. 2 is influence of the PA of various concentration to reprogramming of somatic cells；Wherein, figure A is to be contaminated using alkaline phosphatase (AP)
Color statisticallys analyze reprogramming efficiency of somatic cells；B is schemed to analyze GFP positive cell ratio using Fluorescein activated cell sorter, wherein
(400 μM) of PA groups 37.30%, control group only 8.77%；Scheming C is representativeness GFP positive colony and statistical analysis；Figure D is representative
Property AP coloration result and its statistical analysis；
Fig. 3 is effect stage of the PA in reprogramming of somatic cells；Wherein, figure A is that AP dyes representative result, and d expression is rearranged
The number of days of journey；Figure B is the AP dyeing statistical analysis for scheming A；
Fig. 4 is that endogenous PA is inhibited to significantly affect reprogramming of somatic cells；Wherein, figure A is representativeness AP coloration result, schemes B
Statistical analysis is dyed to scheme the AP of A；
Fig. 5 shows that PA-iPSCs of the invention has the versatility of development；Wherein figure A shows PA-iPSCs and mES cell
System has similar versatility gene expression dose；C represents different cell clones；Scheming B is Western Blot as a result, display
In the same high expression versatility marker gene of protein level PA-iPS cell line；Figure C is immunofluorescence dyeing, and display versatility turns
The expression of the record factor and surface markers SSEA1 are positive；Scheming D is that immunofluorescence dyeing shows triploblastica marker gene
Expression is positive；Scheme E for differentiation in vivo detection: hematoxylin-eosin (HE) dyeing has triploblastica structure；Scheming F is the chimeric bodily form
At experiment, there is completely black mouse to be born after diagram display F1 generation allophenic mice and the backcrossing of ICR female rat, i.e., this chimeric ability can be into
The transmitting of row system genitale；
Fig. 6 shows that phosphatidic acid significantly reduces the Apoptosis during reprogramming, the PA group in programming process wherein figure A attaches most importance to
Compare representative picture with each period cellular morphology of control group (scale bar is 100 μm)；Scheming B is Annexin-V detection reprogramming
Apoptosis in the process；Figure C is the statistical analysis for scheming the Apoptosis detection of B；Figure D, which shows PA group, makes apoptosis execute albumen
The expression of caspase7 is lowered, and Apoptosis is further suppressed；
Fig. 7 is phosphatidic acid by inhibiting Apoptosis to promote the ideograph of reprogramming.
Specific embodiment
Unless specifically stated otherwise, cell used herein is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Unless specifically stated otherwise, reagent used in following embodiment is cell culture grade reagent, and can be from regular channel
It is commercially available.
The preparation of 1 pluripotent stem cell of embodiment
1. the culture medium inducing somatic using the PA containing various concentration reprograms
1) prepare body cell.Before inducing somatic reprogramming, recovery or passage OG2MEF (Oct4 drive GFP
mouse embryonicBoth Oct4-GFP fibroblast is purchased from Institute of Zoology, Academia Sinica) in six
In orifice plate, every hole inoculation 1~2 × 105Cell, after culture for 24 hours, TetO-FUW-OSKM and FUW- after 1ml concentration is added in 1:1
M2rtTA (is purchased from Addgene, article No. is 20321 and 20342) viral mixed liquor, and supplements 0.5ml MEF cell culture medium
(DMEM culture medium (is purchased from Gibco, article No. 11965), the FBS and NEAA for being 10% containing volume ratio), is placed in 37 DEG C of cultures
Continue to cultivate in case.Virus infection for 24 hours after, abandon virus liquid, PBS washes twice, replaces fresh MEF culture medium and continues culture until thin
Born of the same parents are long to 90% meet, pass in the feeder cells culture plate being ready for.
2) inducing somatic reprograms.0th day (d0): when the MEF cell length of virus infection to 90% is converged, with 0.25%
Pancreatin (be purchased from Gibco) digestion, be centrifuged after terminating digestion, then be resuspended with the N2B27 culture medium of the PA containing 100 μM, with every
Hole 2.5~4 × 104The density of a cell is inoculated in the feeder cells culture plate being ready for, while adding 2ng/ml's
Fortimicin Dox (D9891, be purchased from Sigma), 1 μM of PD0325901 (PD0325901 is purchased from Stemgent) and 3 μM
The inducing expression that CHIR99021 (being purchased from Stemgent) carries out foreign gene, is denoted as d0.D1-d14: example of spatial compartmentalizationis.
According to above method, the culture medium containing 100,200,400,600 and 800 μM of PA is used respectively and is free of
There is the culture medium of PA to prepare pluripotent stem cell.
The iPS cell line that PA group induced synthesis is not added is named as control-iPS, abbreviation C-iPS；It is added to PA group induction shape
At iPS cell line be named as PA-iPS.
3) picking monoclonal, which is built, is.D14 days of reprogramming, clone is long to certain size, chooses under microscope mellow and full
There is relief monoclonal to be marked, expands culture with mouth suction pipe or medium size pipette tips picking monoclonal, construct cell line.And
The d14 of reprogramming counts the reprogramming of different PA concentration using alkaline phosphatase (alkaline phosphatase, AP) dyeing
Efficiency.
2. section is reprogrammed using the culture medium inducing somatic containing PA in different times
To choose the time range that PA is acted on, according to the method for embodiment 1, respectively in the different phase of reprogramming, i.e. d1-
3, d3-6, d6-9, d3-9, d3-14 use the N2B27 culture medium culture medium containing PA, until the d14 of reprogramming utilizes alkaline phosphatase
Influence of enzyme (alkaline phosphatase, AP) the dyeing statistics different phase addition PA to reprogramming efficiency.
The iPS cell line that PA group induced synthesis is not added is named as control-iPS, abbreviation C-iPS；It is added to PA group induction shape
At iPS cell line be named as PA-iPS.
3. examining influence of the endogenous PA to reprogramming of somatic cells
To examine the influence of endogenous PA and the PA of external source addition to reprogramming, inventor is added to respectively inhibits PA to generate
Inhibitor PLD1i (VU0359595, be purchased from Alabaster, AL), PLD2i (VU0285655-1 is purchased from Alabaster,
AL), PLD1/2i (VU0155056 is purchased from Alabaster, AL) and DGKi (R59022 is purchased from La Jolla, CA).Pass through list
Solely effect and analysis of The Combined inhibit endogenous PA to count the influence for reprogramming, and whether this influence can pass through
Restored after external source supplement PA, further verifies effect of the PA in reprogramming.
4.PA-iPS cell line Quality Identification
For the quality for verifying PA-iPS cell, inventor passes through molecular biology method PCR, biochemical method respectively
The expression of the technologies such as Western and immunofluorescence dyeing detection molecules level, protein level multipotency sex factor.And lead to
Crossing vitro differentiation, the experiment of internal teratoma and chimera experiment further proves that the obtained clone of PA reprogramming is high quality
PA-iPS cell.
5.Apoptosis detection
Further to study mechanism of action of the phosphatidic acid in reprogramming.Inventor observes relative to control group, phosphatide
Acid group finds a small amount of incomplete situation of clone.Supposition may there are apparent Apoptosis in control group.Therefore inventor
Apoptosis detection is carried out to cell with Annexin V-PE/7-ADD (BD), is divided using FlowJo 7.6.5 streaming data
Analysis carries out statistical analysis using GraphPad Prism 5.It is withered simultaneously with the detection of the biochemistries means such as Western Blot
Die GAP-associated protein GAP such as Bax (Cell Signaling Technology, 2772), Bcl-2 (Cell Signaling
Technology, 2876) and the expression of Caspase7 (abcam, ab181579).
The identification and interpretation of result for the pluripotent stem cell that embodiment 2 obtains
1. phosphatidic acid (PA) effectively facilitates reprogramming efficiency
For the most suitable activity for selecting phosphatidic acid (PA), inventor adds in the medium respectively when inducing reprogramming
100 μM, 200 μM, 400 μM, 600 μM and 800 μM of PA, d14 carry out alkaline phosphatase (AP) dyeing.Statistical result showed, PA
It is remarkably improved reprogramming efficiency, addition PA group AP positive colony number is shown as and is significantly more than control group, and be in PA concentration
Gradient relies on formula and improves (Fig. 2A).Wherein flow cytometry and 400 μM of PA function and effect are the most significant.With fluidic cell
Art analyzes the GFP positive cell ratio of d14, the results show that the GFP positive cell ratio of 400 μM of PA groups of addition is significant
Higher than control group, PA group is 37.30%, and control group only 8.77% (Fig. 2 B).AP dyeing simultaneously also shows addition PA can be significant
It improves 4-5 times of reprogramming efficiency (Fig. 2 C and D).Synthesis result shows that PA is added into culture medium is remarkably improved body cell weight
The efficiency of programming, and the most suitable activity of PA is 400 μM.
2.PA plays a significant role early stage reprogramming to mid-term
For the time range for choosing PA effect, inventor respectively in the different phase of reprogramming, i.e. d1-3, d3-6, d6-9,
The PA (400uM) of optimum concentration is added in d3-9, d3-14, and equivalent PBS is added in control group, carries out AP dyeing in the d14 of reprogramming
And it statisticallys analyze.AP dyes statistical result showed, is remarkably improved reprogramming efficiency in the PA that any stage is added 400 μM,
Especially d3-9 obtains most clone's numbers, is approximately 4 times of control group, and group difference is smaller (Fig. 3 A and B).As it can be seen that PA master
It to play a role in the early metaphase of reprogramming, therefore, inventor can be by adding 400 μ in the d3-9 external source of reprogramming process
The method of this simplicity of MPA significantly improves reprogramming efficiency, to obtain a large amount of iPS cell clone.
3. endogenous PA is inhibited to seriously affect reprogramming
Since the source of intracellular PA mainly there are two approach, i.e., obtained by PLD (phospholipase D) hydrolysis PC (phosphatidyl choline)
It arrives or DAG (diacylglycerol) is generated by DGK (diacylglycerol kinases) phosphorylation, inventor adds PLD suppression into culture medium simultaneously
Preparation (PLDi) and DGK inhibitor (DGKi) inhibit the generation of endogenous PA.Reprogramming d3 start simultaneously persistently with PLDi and
DGKi handles cell, and the DMSO (two inhibitor are dissolved by DMSO) of equivalent is added in control group, and d14 carries out AP dyeing, counts AP
The quantity of positive colony.(Fig. 4) as the result is shown, after endogenous PA is suppressed, AP positive colony is substantially reduced, i.e. reprogramming efficiency
It significantly reduces, this illustrates that endogenous PA participates in reprogramming and plays extremely important effect during reprogramming；But when same
When adding 400 μM of PA to cultivating system, the quantity of AP positive colony and DMSO+PA group and indifference, this redemption experiment table
Bright external source PA can replace endogenous PA to play a role.Synthesis result explanation, PA plays indispensable during reprogramming
Effect, the missing of endogenous PA can significantly reduce reprogramming efficiency, but can be made up by exogenous PA.
4.PA reprograms to obtain the iPS cell clone (PA-iPSCs) of high quality
Obtained cell clone is reprogrammed, establishes stable iPS cell line by choosing monoclonal, PA-iPS cell line is through more
After secondary passage, still normal expression illustrates that multipotency sex factor Oct4 expression is normal to GFP.Further to identify obtained PA-iPS
The quality of cell line, first by molecular biology and biochemical method analyze versatility marker gene such as Oct4,
The expression of Nanog, Sox2, E-cadherin etc., the expression of these versatility marker gene reaches embryo as the result is shown
Expression quantity (Fig. 5 A and B) in the level of expression in stem cell (mESC) or even some a little higher than mESC.In addition, being immunized glimmering
Light coloration result is also shown, and the expression of the transcription factors such as Oct4, Nanog and surface marker gene SSEA1 are positive (Fig. 5 C).
This illustrates that the obtained PA-iPS cell line of inventor has the versatility similar to mESC.
For the differentiation potential for detecting PA-iPSCs, inventor has carried out vitro differentiation experiment, and digestion iPSCs, which is resuspended in, to be free of
Suspend culture in the mES culture medium (FBS preparation) of Lif, can form within second day embryoid body (embryoid bodies, EBs),
EB ball is inoculated in the plate that Matrigel was coated with by the d3 of differentiation carries out adhere-wall culture, and immunofluorescence dyeing is carried out after 3 days,
The typical marks gene of three germinal layers is the positive, entoderm: α-fetoprotein as the result is shown；Mesoderm: α-smooth
muscle actin；Ectoderm: β III tubulin (Fig. 5 D).This illustrates that the PA-iPS cell that inventor obtains has in vitro
The potential broken up to triploblastica.In addition, being subcutaneously injected PA-iPSCs into NOD-SCID mouse, diameter about 1- can be formed after 4 weeks
The tumor mass of 2cm, at 6 weeks, tumour can be grown big to diameter 3cm.Hematoxylin-eosin is carried out by histotomy
(Hematoxylin-Eosin, HE) dyeing, it is seen that the Various Tissues structure from three germinal layers, such as the adenoid epithelium of entoderm
Organize (left side), entoderm musculature (in) and ectodermic nerve fiber (right side) (Fig. 5 E).To sum up result illustrates inventor
Constructed PA-iPS cell line either still all has multi-lineage potential in vivo in vitro.
In order to further study the potentiality of development of PA-iPS cell, inventor infuses PA-iPS cell line by embryo operation
It is mapped in the ICR white hair mice embryonic of blastula stage, replants the female rat uterus into false pregnancy, the chimeric energy of analysis birth mouse
Power, although chimeric rate is variant, 4 PA-iPS cell lines have a gomphosis mouse birth, and chimeric rate reach as high as 85% with
On.Also, it is returned with the allophenic mice of birth with ICR female rat, has obtained the completely black progeny mice of hair color, illustrated that this is embedding
Conjunction ability can carry out system genitale transmitting (Fig. 5 F).Result of study shows PA-iPS cell line chimeric ability with higher.
5. phosphatidic acid improves reprogramming efficiency by reducing apoptosis
From the early stage (d4~5) of reprogramming, cell growth state is good, and PA group and control (Control) group are in form
Upper no marked difference, and since d6, poly- heap cell start to occur that shading becomes strong, no longer adherent growth has been hiked up to death
Phenomenon carries out at any time, this phenomenon is more and more obvious in control group, until the cell for having very much " clones " is all dead when d10
It dies, clone completely disappears；In contrast, the clone of PA group death is then considerably less than control group, and more clones are but increasingly tighter
Close, it is clear that edge increasingly comes, and the existing clone morphologically reprogrammed completely of d10 occurs, when d12, most clone cells foots
Reach close, edge clear, at stereo structure (Fig. 6 A).Therefore, inventor withers to the cell of (d4-d12) during entire reprogramming
It dies situation to be analyzed, as the result is shown (Fig. 6 B and C), d4-d6PA group and control group do not have notable difference, but since d8,
The apoptosis rate of PA group maintains always a lower maintenance level (about 10~15%)；Control without adding external source PA
The apoptosis rate of group but persistently increases, and d14 is unexpectedly more than 40%.This illustrates that external source is added PA and the cell during reprogramming is inhibited to wither
It dies.By the detection of biochemical method, during finding PA-iPS reprogramming, Bcl-2 expression up-regulation, and Bax expression is constant,
Promotion is formed Bcl-2/Bax heterodimer by this, and increases the amount of Bcl-2 homodimer, to inhibit cell death.Simultaneously
It was found that the expression that PA group makes apoptosis execute albumen caspase7 is lowered, Apoptosis (Fig. 6 D) is further suppressed.It has been reported
Claim, PA can by prevent mitochondria in Cyt c release inhibit Apoptosis, therefore inventor speculate, a large amount of external source PA into
Enter after cell accumulation on mitochondrial membrane again and form TRIAP1/PRELI complex, this complex can maintain cuorin
The accumulation of (Cardiolipin, CL) on mitochondrial membrane, so that the release of cytochrome c (Cty c, Cytochrome) is prevented,
And then Apoptosis is inhibited, it finally shows as PA group reprogramming efficiency and significantly improves (Fig. 7).
Claims (22)
1. a kind of culture medium for inducing somatic reprogramming, which is characterized in that the culture medium includes conventional inductor
The culture medium and phosphatidic acid of cell reprogramming；
Wherein, the culture medium of the conventional inducing somatic reprogramming is N2B27 culture medium, KOSR culture medium or FBS culture
Base, the concentration of phosphatidic acid is 100-800 μM in the culture medium.
2. culture medium according to claim 1, which is characterized in that the concentration of phosphatidic acid is 200-600 μ in the culture medium
M。
3. culture medium according to claim 1, which is characterized in that the concentration of phosphatidic acid is 400 μM in the culture medium.
4. culture medium according to claim 1, which is characterized in that the culture medium also added fortimicin Dox, glycogen
Selective depressant CHIR99021, the non ATP competitiveness mek inhibitor PD0325901 of original synthesis 3 beta receptor of kinases.
5. culture medium according to claim 4, which is characterized in that the concentration of the fortimicin Dox is 1-5ng/ml.
6. culture medium according to claim 4, which is characterized in that the concentration of the fortimicin Dox is 2ng/ml.
7. culture medium according to claim 4, which is characterized in that the concentration of the CHIR99021 is 1-5 μM.
8. culture medium according to claim 4, which is characterized in that the concentration of the CHIR99021 is 3 μM.
9. culture medium according to claim 4, which is characterized in that the concentration of the PD0325901 is 1-5 μM.
10. culture medium according to claim 4, which is characterized in that the concentration of the PD0325901 is 1 μM.
11. a kind of method of inducing somatic reprogramming, the method includes any periods reprogrammed in inducing somatic to make
With culture medium culture body cell such as of any of claims 1-10, or inducing cell reprogramming it is any when
Phase adds phosphatidic acid in the medium, continues to cultivate body cell；
Wherein, the concentration of the phosphatidic acid of the addition is 100-800 μM；The culture medium of the conventional inducing somatic reprogramming
For N2B27 culture medium, KOSR culture medium or FBS culture medium.
12. according to the method for claim 11, which is characterized in that early stage cell reprogramming and/or mid-term is using such as
Culture medium culture body cell of any of claims 1-10 adds phosphatidic acid in the medium, continues to cultivate body
Cell.
13. according to the method for claim 11, which is characterized in that the described method comprises the following steps:
1) versatility related gene is imported into body cell；
2) body cell for having imported versatility related gene is prepared as single cell suspension, uses conventional medium incubation step 1)
The middle body cell for importing versatility related gene, example of spatial compartmentalizationis, and thin using the conventional medium culture body
During born of the same parents, select use culture medium culture body cell as claimed in any one of claims 1-3 within one day or several days,
Perhaps one day or several days addition phosphatidic acid is selected during using the conventional medium culture body cell, thus
It is induced multi-potent stem cell.
14. according to the method for claim 13, which is characterized in that used such as right at first day of reprogramming to third day
It is required that culture medium culture body cell described in any one of 1-10 or adding phosphatidic acid in the conventional medium.
15. according to the method for claim 13, which is characterized in that in the third day of reprogramming to the 6th day using such as right
It is required that culture medium culture body cell described in any one of 1-10 or adding phosphatidic acid in the conventional medium.
16. according to the method for claim 13, which is characterized in that use such as right within the 6th day to the 9th day in reprogramming
It is required that culture medium culture body cell described in any one of 1-10 or adding phosphatidic acid in the conventional medium.
17. according to the method for claim 13, which is characterized in that in the third day of reprogramming to the 9th day using such as right
It is required that culture medium culture body cell described in any one of 1-10 or adding phosphatidic acid in the conventional medium.
18. according to the method for claim 13, which is characterized in that use in the third day of reprogramming to fortnight as weighed
Benefit requires culture medium culture body cell described in any one of 1-10 or adds phosphatidic acid in the conventional medium.
19. according to the method for claim 11, which is characterized in that the body cell is the skin of primate into fiber
Cell, mucomembranous epithelial cell, lymphocyte, hair follicle cell, fat mesenchymal stem cell, between umbilical cord mesenchymal stem cells or marrow
Mesenchymal stem cells.
20. according to the method for claim 11, which is characterized in that the body cell is the skin fibroblasts of people, glues
Film epithelial cell, lymphocyte, hair follicle cell, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells or medulla mesenchyma are dry
Cell.
21. according to the method for claim 11, which is characterized in that the concentration of the phosphatidic acid of the addition is 200-600 μM.
22. according to the method for claim 11, which is characterized in that the concentration of the phosphatidic acid of the addition is 400 μM.
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| CN102093981A (en) * | 2010-12-17 | 2011-06-15 | 深圳市北科生物科技有限公司 | Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells |
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| CN102093981A (en) * | 2010-12-17 | 2011-06-15 | 深圳市北科生物科技有限公司 | Method for efficiently inducing reprogramming of human body cells into pluripotent stem cells |
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