CN109628383B - The cultural method that reprogramming culture medium and reprogramming induce multi-potent stem cell - Google Patents
The cultural method that reprogramming culture medium and reprogramming induce multi-potent stem cell Download PDFInfo
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Abstract
The present invention relates to the cultural methods that a kind of reprogramming culture medium and reprogramming induce multi-potent stem cell, the reprogramming culture medium is made of DMEM-F12 basal medium and adding ingredient, the adding ingredient includes the L-AA of 60 μ g/mL-180 μ g/mL, the hydrocortisone of 5.3 μm of ol/L-74 μm of ol/L, the sodium selenite of 3ng/mL-89ng/mL, the Optiferrin of 8 μm of ol/L-23 μm of ol/L, the retinyl acetate of 0.5 μm of ol/L-7.4 μm of ol/L, the plant source recombination human basic growth factor of 40ng/mL-60ng/mL, the IGF of 8 μ g/mL-12 μ g/mL, 0.2 μm of ol/L-0.6 μ g/m The sodium butyrate of CHIR99021 and 100 μm of ol/L-450 μm of ol/L of the A-83 of L, 2 μm of ol/L-6 μm of ol/L.The safety that clinical use not only can be improved using the reprogramming culture medium, can also improve adult cell reprogramming is the efficiency induced multi-potent stem cell.
Description
Technical field
The present invention relates to the trainings that biological field more particularly to a kind of reprogramming culture medium and reprogramming induce multi-potent stem cell
The method of supporting.
Background technique
2006, one kind was invented by tetra- kinds of transcription factor structures of OCT4, SOX2, KLF4 and c-Myc by the team stretched in mountain more
At " cocktail " method, successfully the reprogramming of the skin fibroblasts of terminal differentiation can be become with the dry of differentiation versatility
Cell, this stem cell, which is referred to as, induces multi-potent stem cell (induced pl μ ripotentstem cells, abbreviation
"iPSC").These stem cells have the differentiation potential similar with embryonic stem cell (embryonic stem cells), being capable of shape
Adult body develops three most basic germinal layers, and ultimately forms a variety of adult cells.This method breaches medically user
The ethics of embryonic stem cell limits, and can solve the immune rejection problems in cellular transplantation therapy, has expanded stem cell skill significantly
Application potential of the art on clinical medicine.
It, can be with according to reprogramming carrier classification currently, a variety of reprogramming methods have been developed in multiple laboratories in the world
It is roughly divided into following four classes:
(1) viral method: which includes genome, insert type viral vectors (e.g. slow virus) and non genome are inserted immediately
Entering carrier (e.g. sendai virus), the shortcomings that such method, is potential risk brought by the insertion immediately of genome, or
Interminable viral dilution process bring is constant.
(2) DNA method: including a variety of plasmids including Piggy Bac, Sleeping Bea μ ty and Episomal
Type, the feature of such methods are that reprogramming efficiency is low.
(3) RNA method and protein act: such methods operating process is complicated, poor in industrial application practicability.
(4) compound small molecule method: this is to apply a kind of more and more extensive reprogramming method at present, but its reprogramming is imitated
There are biggish individual differences for rate.
However, preceding 3 kinds of methods use the reprogramming culture medium containing serum composition in Induction Process at present, such as
Add in the presence of feeder layer using knockout serum or using containing N2 or B27 in the case where no feeder layer
Add the culture medium of agent, and all also containing the haemocyanin or other animal sources including transferrins in these culture mediums
Sexual element.Due to the presence of serum protein composition, the inconvenience induced multi-potent stem cell in process of clinical application is caused, it is unfavorable
In the industrialization induction of reprogramming and the formulation of standardization process.And compound small molecule method is big there is still a need for individual difference is solved
The problem of, i.e., how various kinds of cell type realize Approximate Equivalent reprogramming efficiency be still regenerative medicine field a disaster
Topic.
It reprograms from the noble cells of differentiation terminal to inducing multi-potent stem cell, it is the widest in industry and clinical application at present
There are two types of general reprogramming cultural methods:
(1) it nourishes revulsion: adult cell being reprogrammed in the case where there is trophoblastic situation and obtains inducing multi-potent stem cell simultaneously
It is expanded.The shortcomings that this method be to induce multi-potent stem cell be required to during follow-up cultivation trophocyte be subject to it is auxiliary
Culture is helped, trophocyte is mostly source of mouse embryo fibroblast, and in subsequent directed differentiation and organizational project application, purpose is thin
Born of the same parents are easy the pollution by trophocyte, this becomes the big obstacle for hindering to induce multi-potent stem cell application.
(2) panoistic method culture: what this method obtained induces multi-potent stem cell the auxiliary for not needing trophocyte, Neng Gou
By continued propagation under the support of macromolecular matrices glue or Laminin ELISA.
Currently, non-trophoblast method is the industry mainstream induced multi-potent stem cell.But non-trophoblast method needs cell culture
Base provides complicated nutritional ingredient, the especially substitute of serum, and a variety of composite parts is needed to be adjusted by accurately concentration
Match, so that sufficient nutrition can be provided for the growth of multipotential stem cell by reaching, while guaranteeing to maintain multipotential stem cell again
Various performances.
But the cultivating system that both cultural methods use uses animal blood serum composition living as maintenance cell
The important source material of power, this makes containing a large amount of plant and animal material albumen in such culture medium, and the chemical component of additive is also inadequate
It is clear, subsequent processing application can be adversely affected, such as target protein isolates and purifies difficulty etc., while culture medium at
This is also higher.
Summary of the invention
The present invention for the technical problems in the prior art, provides both without using serum composition or without containing serum
The reprogramming culture medium of transferrins and the cultural method induced multi-potent stem cell using reprogramming culture reprogramming, not only may be used
To improve the safety of clinical use, can also improve adult cell reprogramming is the efficiency induced multi-potent stem cell.
The technical scheme to solve the above technical problems is that
A kind of reprogramming culture medium, the reprogramming culture medium are made of DMEM-F12 basal medium and adding ingredient,
The adding ingredient include the L-AA of 60 μ g/mL-180 μ g/mL, 5.3 μm of ol/L-74 μm of ol/L hydrocortisone,
The view of the sodium selenite of 3ng/mL-89ng/mL, the Optiferrin of 8 μm of ol/L-23 μm of ol/L, 0.5 μm of ol/L-7.4 μm of ol/L
Flavol acetic acid esters, the plant source recombination human basic growth factor of 40ng/mL-60ng/mL, 8 μ g/mL-12 μ g/mL IGF, 0.2 μ
The butyric acid of CHIR99021 and 100 μm of ol/L-450 μm of ol/L of the A-83 of mol/L-0.6 μ g/mL, 2 μm of ol/L-6 μm of ol/L
Sodium.
The adding ingredient includes L-AA, 40 μ of 70 μ g/mL-90 μ g/mL in one of the embodiments,
The hydrocortisone of mol/L-60 μm of ol/L, the sodium selenite of 10ng/mL-30ng/mL, 8 μm of ol/L-12 μm of ol/L
Optiferrin, retinyl acetate, the plant source recombination human basic of 45ng/mL-55ng/mL of 3 μm of ol/L-6 μm of ol/L are raw
The long factor, the IGF of 8 μ g/mL-12 μ g/mL, the A-83 of 0.3 μm of ol/L-0.5 μ g/mL, 3 μm of ol/L-5 μm of ol/L
The sodium butyrate of CHIR99021 and 280 μm of ol/L-410 μm of ol/L.
The adding ingredient includes the hydrogenation of the L-AA of 80 μ g/mL, 50 μm of ol/L in one of the embodiments,
Cortisone, the sodium selenite of 20ng/mL, the Optiferrin of 10 μm of ol/L, the retinyl acetate of 4 μm of ol/L, 50ng/mL
Plant source recombination human basic growth factor, the IGF of 10 μ g/mL, the A-83 of 0.4 μ g/mL, 4 μm of ol/L CHIR99021 and
The sodium butyrate of 400 μm of ol/L.
It is a kind of to reprogram the cultural method induced multi-potent stem cell, using adult cell using described in any of the above embodiments heavy
Programming culture medium carries out reprogramming culture.
In one of the embodiments, the adult cell be umbilical cord source human mesenchymal cells, people CD34+ cell and/or
Human skin fibroblasts.
The cultural method that the reprogramming induces multi-potent stem cell in one of the embodiments, includes the following steps:
Reprogramming uses Epi5 Reprogramming Kit, and adult cell is resuspended in and is provided in electrotransformation kit
In Resuspension Buffer, electricity turns, the cell after obtaining electric turn；
Cell after electricity is turned is squeezed into the hole of Matrigel coating culture plate, and reprogramming described in any of the above embodiments is utilized
Culture medium is placed in 37 ± 0.5 DEG C of temperature, is cultivated in 5% carbon dioxide environment, in incubation, partly changes liquid to electricity daily and turns
It afterwards the tenth day, is every other day changed the liquid once since the tenth day, until the 28th day.
The cultural method that the reprogramming induces multi-potent stem cell in one of the embodiments, includes the following steps:
Reprogramming uses CytoTuneTM2.0 Sendai Reprogramming Kit of-iPS, according to 2 × 106Cell,
The ratio of MOI=5 carries out viral transduction, after virus is added, is placed in 37 ± 0.5 DEG C of temperature, is incubated in 5% carbon dioxide environment
12-16h starts for culture medium to be changed to reprogramming culture medium described in any of the above embodiments for second day, cultivates to the 28th day.
The cultural method that the reprogramming induces multi-potent stem cell in one of the embodiments, includes the following steps:
Reprogramming uses SimpliconTMRNA Reprogramming Kit recycles described in any of the above embodiments rearrange
Journey culture medium, by cell culture to the 28th day.
The cultural method that the reprogramming induces multi-potent stem cell in one of the embodiments, further includes making to reprogram
The step of inducing multi-potent stem cell differentiating into nerve cells.
The beneficial effects of the present invention are:
Reprogramming culture medium of the invention is added by adding particular types into DMEM-F12 basal medium with what is matched
Addition point, the mode combined using the growth factor of the pure chemistry factor and non-animal are free of serum composition, also not contained
Serum transferrin can not only overcome the problems, such as animal derived components and trophocyte pollution during conventional reprogram, mention
The safety of high clinical use can also overcome the problems, such as that the conventional process efficiency that reprograms is low, to significantly improve adult cell
Reprogramming is the efficiency induced multi-potent stem cell.
In addition, still may be used using the cultural method that the reprogramming culture medium is induced multi-potent stem cell by lasting passage
Karyotypic stability, cells pluripotency and differentiation capability is set all to maintain higher level.
Detailed description of the invention
Fig. 1 is that adult cell reprogramming is the detection figure induced multi-potent stem cell；Wherein, figure a is CD34+ cell induction the
1 day (being denoted as origin) detection figure；Figure b is the inspection that CD34+ cell reprograms that the iPSC to be formed clones (being denoted as Codonies)
Mapping；Figure c is that CD34+ cell reprograms the iPSC alkaline phosphatase staining to be formed (being denoted as AP positive) figure；Scheming d is
CD34+ cell reprograms the iPSC to be formed clone OCT3/4 identified by immunofluorescence figure (being denoted as Oct4/DAPI)；Scheming e is that CD34+ is thin
Born of the same parents reprogram the iPSC to be formed clone's caryogram detection；Figure f is the detection figure that fibroblast (HDF) induces the 1st day；Scheming g is that HDF is thin
Born of the same parents reprogram the detection figure of the iPSC to be formed clone；Figure h is that HDF cell reprograms the iPSC alkaline phosphatase staining figure to be formed；
Figure i is that HDF cell reprograms the iPSC to be formed clone's OCT3/4 identified by immunofluorescence figure；Figure j is that HDF cell reprograms to be formed
IPSC clones caryogram detection；Figure k is the detection figure that interstitial cell (MSC) induces the 1st day；Figure l is that MSC cell reprograms to be formed
The detection figure of iPSC clone；Figure m is that MSC cell reprograms the iPSC alkaline phosphatase staining figure to be formed；Figure n is MSC cell weight
The iPSC that programming is formed clones OCT3/4 identified by immunofluorescence figure；Figure o is that MSC cell reprograms the iPSC to be formed clone's caryogram inspection
It surveys.
Fig. 2 induces multi-potent stem cell the quantity variance figure of clone for the generation of embodiment 2 and comparative example 1；Wherein, figure a is
AP stained positive is induced multi-potent stem cell using the source of people that traditional N2B27+bFGF reprogramming culture medium induces to clone；Scheme b
To use the serum-free of embodiment 1 to reprogram culture medium without transferrins, the source of people induced induces multi-potent stem cell AP dye
Color positive colony.
Fig. 3 induces multi-potent stem cell the quantity variance figure of clone for the generation of embodiment 3 and comparative example 2；Wherein, figure a is
AP stained positive is induced multi-potent stem cell using the source of people that traditional N2B27+bFGF reprogramming culture medium induces to clone；Scheme b
AP dyeing is induced multi-potent stem cell to use the serum-free of embodiment 1 to reprogram the source of people that culture medium induces without transferrins
Positive colony.
Fig. 4 induces multi-potent stem cell the quantity variance figure of clone for the generation of embodiment 4 and comparative example 3；Wherein, figure a is
AP stained positive is induced multi-potent stem cell using the source of people that traditional N2B27+bFGF reprogramming culture medium induces to clone；Scheme b
AP dyeing is induced multi-potent stem cell to use the serum-free of embodiment 1 to reprogram the source of people that culture medium induces without transferrins
Positive colony.
Fig. 5 carries out Endoderm differentiation for inducing multi-potent stem cell for the preparation of embodiment 2 respectively, Mesoderm breaks up,
The detection figure of Ectoderm differentiation.
Fig. 6 is the expression knot for inducing multi-potent stem cell the neural stem cell marker in atomization prepared by embodiment 2
Fruit statistical chart.
Fig. 7 induces multi-potent stem cell rear institute to carry out adult cell reprogramming behaviour using each group culture medium in comparative example 4
The AP stained positive clone obtained.
Specific embodiment
Citing description is carried out to the present invention below, the given examples are served only to explain the present invention, is not intended to limit the present invention
Range.Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Embodiment 1
The present embodiment provides a kind of serum-frees to reprogram culture medium without transferrins, by DMEM-F12 basal medium and
Adding ingredient composition, adding ingredient are as follows: the Asia of the L-AA of 80 μ g/mL, the hydrocortisone of 50 μm of ol/L, 20ng/mL
Sodium selenate, Optiferrin, retinyl acetate, the plant source recombination human basic of 50ng/mL of 4 μm of ol/L of 10 μm of ol/L are raw
The long factor (OsrbFGF), the IGF of 10 μ g/mL, the A-83 of 0.4 μ g/mL, 4 μm of ol/L CHIR99021 and 400 μm of ol/L
Sodium butyrate.
Embodiment 2
The present embodiment provides the cultural methods that a kind of reprogramming behaviour of adult cell induces multi-potent stem cell, including walk as follows
It is rapid:
6 well culture plates are coated with using Matrigel (STEMCELL Technologies), bed board is placed on 37 DEG C of insulating boxs
It is middle that it is more than hour to be incubated for one.
Reprogramming uses Epi5 Reprogramming Kit (specific steps can be found in kit specification), uses Neon
Electrotransformation kit (Thermo Fisher) carries out the operation of a variety of people's adult cells respectively, and operating procedure is as follows: respectively by navel
Band source human mesenchymal cells, people CD34+ cell, human skin fibroblasts people's adult cell are resuspended in after centrifuge washing
In the Resuspension Buffer provided in electrotransformation kit, electricity is carried out according to following procedure and is turned: PulseVoltage:
1650V, PulseWidth:10ms, PulseNumber:3, electricity turn to terminate, the cell after obtaining electric turn.
Cell after electricity is turned is driven into the pipe of reprogramming culture medium of embodiment containing 6mL 1, after mixing uniformly, then is divided
It is not added in each hole in 6 well culture plates, carries out cell culture.The condition of cell culture: 37 DEG C of temperature, 5% carbon dioxide.This
Afterwards, it carries out changing after liquid turns to electricity the tenth day by the way of partly changing liquid daily, since the tenth day, every other day change the liquid once straight
To the 28th day, occurs a large amount of people in culture plate and induce multi-potent stem cell clone.
Secondary culture: the hole in every kind of cell type selection 6 orifice plates is incubated for 5 at 37 DEG C using Accutase digestive juice
Minute, to be individual cells by the cell dissociation in culture plate, new training is used respectively after postdigestive cell centrifuge washing
It is outstanding to support base weight, is passaged to same size culture plate according to 1:1 ratio, is trained at TeSRTM-e8 (STEMCELL Technologies)
It supports and is cultivated in base, until there is sizeable clone in plate.Use the Matrigel (STEMCELL of 1 μ g/mL
Technologies 48 well culture plates) are coated with, are sucked out after under a dissecting microscope moving the clone reprogrammed in plate with pipette tips pinch,
It is placed in 48 orifice plates, a clone, commodity in use TeSRTM-e8 (STEMCELL Technologies) is placed in every hole
Culture medium is cultivated in 37 DEG C, 5% CO2.When cell growth reaches 70% coverage rate, 0.05% is used
Trypsin/EDTA is digested in 37 DEG C of incubation 5min, uses new culture medium after postdigestive cell centrifuge washing respectively
It is resuspended, and culture medium passage is added into T25 culture bottle be coated with, cultivated in 37 DEG C, 5% CO2 to next biography
Generation.Cell after subculture 10 times, is carried out core in TeSRTM-e8 (STEMCELL Technologies) culture medium by cell
Type compares.Cell culture carries out Molecular Identification to 10 generations or so, to cell, and verifying induces in above-mentioned reprogramming culture medium to be obtained
People induce multi-potent stem cell and have complete molecular labeling.Molecular Identification is combined by immunofluorescence and Q-PCR and is carried out.
Induce multi-potent stem cell using alkaline phosphatase staining (Cat#SCR004, Millipore) counting of clone
And versatility identification: absorbing the cell culture fluid in culture dish, be added after being cleaned using PBS 4% paraformaldehyde fix 1~
2min, TBST is cleaned 3 times later, and the AP dyeing working fluids of enough Fresh is added, and (by taking 24 orifice plates as an example, every hole is added
0.5mL), 15~20min of room temperature avoid light place；Dyeing liquor is siphoned away after dyeing, is cleaned 2-3 times with PBS, microscopically observation
Coloration result.
Inducing multi-potent stem cell for preparation is subjected to entoderm (Endoderm) differentiation: under a dissecting microscope will reprogramming
Clone in plate is sucked out after being moved with pipette tips pinch, is placed in 48 orifice plates, and a clone, commodity in use are placed in every hole
TeSRTM-e8 (STEMCELL Technologies) culture medium is cultivated in 37 DEG C, 5% CO2.When induced multi-potent is dry
When cell reaches 70% coverage rate, is handled 5 minutes under the conditions of 37 DEG C with 0.05%trypsin/EDTA, terminated using DMEM
Cell dissociation.After cell washing centrifugation, according to 1 × 105The ratio in every hole is reinoculated in 24 orifice plates, is placed in 37 DEG C, 5%
It is cultivated 48 hours in CO2 environment.Then Endoderm differentiation is carried out using following culture medium: in DMEM/F12 basal medium
It adds 1xN-2 supplement (Invitrogen), 1xB-27 supplement (Invitrogen), non-essential
Amino acids is 0.1mM, and GlutaMAX 1mM, β-mercaptoethanol are 0.1mM, and Activin is that 10 μ g/ml (match
Come from bibliography D'Amour, (2005) et al.Nat Biotechnol 23 (12): 1534-1541 in side).Differentiation culture item
Part are as follows: 37 DEG C, 5% CO2In.Culture is identified after 4 days.
Inducing multi-potent stem cell for preparation is subjected to mesoderm (Mesoderm) differentiation: being reached when inducing multi-potent stem cell
When 70% coverage rate, is handled 5 minutes with 0.05%trypsin/EDTA at 37 DEG C, terminate cell dissociation using DMEM.Cell
According to 1 × 10 after washing centrifugation5The ratio in every hole is reinoculated in 24 orifice plates, is placed in the CO of 37 DEG C, 5%248 are cultivated in environment
Hour.Mesoderm differentiation is carried out using following culture medium: 1xB-27 supplement is added in RPMI basal medium
(Invitrogen), CHIR99021 is that (formula comes from bibliography Lian et al., ProcNatlAcadSci to 5 μ g/ml
USA,2012.).Break up condition of culture are as follows: 37 DEG C, 5% CO2In.Culture is identified after 4 days.
Inducing multi-potent stem cell for preparation is subjected to ectoderm (Ectoderm) differentiation: being reached when inducing multi-potent stem cell
When 70% coverage rate, is handled 5 minutes with 0.05%trypsin/EDTA at 37 DEG C, terminate cell dissociation using DMEM.Cell
According to 5 × 10 after washing centrifugation5The ratio in every hole is reinoculated in 24 orifice plates, is placed in the CO of 37 DEG C, 5%248 are cultivated in environment
Hour.Endoderm differentiation is carried out using following culture medium: adding 1xN-2 in DMEM/F12 basal medium
supplement(Invitrogen),1xB-27 supplement(Invitrogen),non-essential amino
Acids is 0.1mM, and GlutaMAX 1mM, β-mercaptoethanol are 0.1mM, 10uM SB431542 and 2uM JW55.
Break up condition of culture are as follows: 37 DEG C, 5% CO2In.Culture 21 days is carried out, changes the liquid once, is identified later daily.Identification knot
Fruit sees Fig. 5.
Transcription variation using Q-PCR detection from from embryonic stem cell to neural stem cell transition process.Nerve is taken respectively
It is expanded 2 weeks after the completion of the how tender stem cell of induction (iPSC), induction before stem cell induction using neural stem cell amplification culture medium
Neural stem cell (NSC) afterwards, is analyzed as two kinds of materials.It is detected by Q-PCR thin from embryonic stem cell to nerve cord
The transcription situation of change of SOX1, PAX6, OCT3/4, NANOG during dysuria with lower abdominal colic becomes.The total serum IgE of two kinds of cells uses RNeasy
Mini or Micro Kit (QIAGEN) is stripped, 1mg RNA SuperScript III First-Strand
Synthesis System (Invitrogen) synthesizes cDNA.With SYBR Premix Ex Taq (TaKaRa) and Thermal
The label of Cycler Dice Real Time System (TaKaRa) Lai Jinhang Quan-titative PCR and reaction, beta-
Actin is used as internal reference.All data are analyzed with delta-Ct method.For identifying neural stem cell marker
The primer sequence of the encoding gene of SOX1, PAX6, OCT3/4, NANOG is as shown in table 1 below.
Primer sequence table of the table 1 for the encoding gene of identification of cell marker
It is identified using immunofluorescence experiment from the protein labeling induced multi-potent stem cell into neural stem cell transition process
(immunofluorescence dyeings of a variety of difference cell type markers): iPSC, induction before taking neural stem cell to induce respectively are completed
Neural stem cell after being expanded 2 weeks using neural stem cell amplification culture medium afterwards carries out following immunofluorescence as two kinds of materials
Dyeing identification: fixed cell 40 minutes using 4% paraformaldehyde room temperature, with twice of DPBS buffer solution for cleaning；Then with 0.1%
Triton X-100 is permeabilized 5 minutes, with twice of DPBS buffer solution for cleaning；Then with containing 10% horse serum and 0.1%
4 DEG C of cell are incubated overnight by the DPBS buffer of Triton X-100；Then it is added and uses the diluted antibody of DPBS buffer, 37 DEG C
2h is incubated for take pictures after DPBS buffer solution for cleaning three times.Antibody details of use are as shown in table 2.
The antibody that the immunofluorescence dyeing of 2 neural stem cell marker of table uses
| Antibody | It is purchased from | Extension rate |
| Sox1 | Abcam | 1:150 |
| NESTIN | Abcam | 1:500 |
| GATA4 | Abcam | 1:500 |
| BRACHYURY | Abcam | 1:250 |
| OCT3/4 | Abcam | 1:150 |
The expression quantity of iPSC marker before being induced using neural stem cell is calculated induction and uses nerve cord after the completion as 1
The relative fold expression of neural stem cell marker and iPSC marker after cell amplification culture medium amplification 2 weeks.Q-PCR detection
As a result as shown in Figure 6.
Karyotype detection: chromosome karyotype analysis process is according to Chromosome Preparation From
Cultured Cells (Howe etc., 2014, DOI:doi:10.3791/50203) is carried out.Human chromosome is prepared using pendency method
Slide handles 2h in 70 DEG C of ovens.Solution with normal saline containing 0.01%EDTA, 0.05% trypsase is adjusted
PH is 7.0.Water-bath after chromosome slide is handled 5-20s in the above solution, is reshuffled twice to after 37 DEG C with physiological saline.
Then slide is immersed in Giemsa solution and is dyed 5-10 minutes until colour developing is clear.Rear microscopy is dried into slide flushing.
Testing result is detailed in Fig. 1, Fig. 5 and Fig. 6.
As seen from Figure 1, this reprogramming culture can be such that a variety of different adult cells formation induce multi-potent stem cell, and
And what is formed induces multi-potent stem cell the label that multipotential stem cell is expressed with normal caryogram.
As seen from Figure 5, the people induces multi-potent stem cell three kinds of germinal layers for being divided into allelotaxis's needs: (1) breaking up
Obtain the positive endoderm cell of GATA4 expression；(2) differentiation obtains the positive mesoblastema of Brachyury expression；(3) divide
Change and obtains the positive ectoderm cell of SOX1 and NESTIN expression.To demonstrate the reprogramming cultural method using the present embodiment
What is obtained induces multi-potent stem cell with the ability for being divided into three kinds of different germinal layers, to demonstrate its complete differentiation potential.
As seen from Figure 6, this method induces multi-potent stem cell the source of people induced nerve stem cells of culture using people, significantly
The expression of the neural stem cell marker (SOX1, PAX6) of cell has been raised, meanwhile, iPSC marker (OCT3/4,
NANOG expression quantity) is remarkably decreased.What the present embodiment reprogramming culture obtained induces multi-potent stem cell high in the expression of transcription level
Horizontal multipotential stem cell marker gene, and can successfully be divided into the neural precursor of expression neural markers object.
Embodiment 3
The present embodiment provides the cultural methods that a kind of reprogramming behaviour of adult cell induces multi-potent stem cell, including walk as follows
It is rapid:
6 well culture plates are coated with using Matrigel (STEMCELL Technologies), bed board is placed on 37 DEG C of insulating boxs
It is middle that it is more than hour to be incubated for one.Reprogramming uses CytoTuneTM2.0 Sendai Reprogramming Kit of-iPS is (specific
Step can be found in kit specification), according to 2 × 106The ratio progress viral transduction of cell, MOI=5, after virus is added, by
Cell is placed in 37 DEG C, the environment Hu of 5% carbon dioxide is always incubated for 12-16 hours.Start within second day for culture medium to be changed to implementation
The reprogramming culture medium of example 1 is cultivated to the 28th day, occurs a large amount of people in culture plate and induce multi-potent stem cell clone.
It is subsequent that inducing multi-potent stem cell in the step of clone breaks up, identifies and embodiment 4 for preparation is broken up, is identified
The step of it is identical.
Embodiment 4
The present embodiment provides the cultural methods that a kind of reprogramming behaviour of adult cell induces multi-potent stem cell, including walk as follows
It is rapid:
6 well culture plates are coated with using Matrigel (STEMCELL Technologies), bed board is placed on 37 DEG C of insulating boxs
It is middle that it is more than hour to be incubated for one.According to SimpliconTMRNA Reprogramming Kit (OKSG) (Millipore) specification
It is operated, using the reprogramming culture medium culture of embodiment 1 to the 28th day, until there are a large amount of people's induced multi-potents in culture plate
Stem cell clone.
It is subsequent that inducing multi-potent stem cell in the step of clone breaks up, identifies and embodiment 4 for preparation is broken up, is identified
The step of it is identical.
Comparative example 1
This comparative example provides a kind of cultural method that adult cell reprogramming behaviour induces multi-potent stem cell, using Epi5
The step of Reprogramming Kit, operating procedure is with embodiment 4, is essentially identical, and difference is: the training that this comparative example uses
Supporting base is that tradition N2B27+bFGF reprograms culture medium, i.e., 1xN-2 supplement is added in DMEM/F12 basal medium
(Invitrogen), 1xB-27 supplement (Invitrogen), non-essential amino acids are 0.1mM,
GlutaMAX is 1mM, and β-mercaptoethanol is 0.1mM, and bFGF is that (formula comes from bibliography to 100ng/ml: Human
induced pluripotent stem cells free of vector and transgene sequences.Science
324(5928):797-801)。
The people that this comparative example is obtained induce multi-potent stem cell using alkaline phosphatase staining (Cat#SCR004,
Millipore) carry out inducing multi-potent stem cell the counting of clone, statistical result is shown in Fig. 2 a.
As seen from Figure 2, it compared with traditional N2B27+bFGF reprograms culture medium, is using
In the case of Epi5Reprogramming Kit, it is dry thin that more source of people multipotencys can be obtained using the reprogramming culture of embodiment 1
Born of the same parents clone.
Comparative example 2
This comparative example provides a kind of cultural method that adult cell reprogramming behaviour induces multi-potent stem cell, and uses
CytoTuneTM- iPS 2.0Sendai Reprogramming Kit (Thermo Fisher), operating procedure and embodiment 2
The step of it is essentially identical, difference is: the culture medium that this comparative example uses also for traditional N2B27+bFGF reprogram culture medium, i.e.,
1xN-2supplement (Invitrogen) is added in DMEM/F12 basal medium, 1xB-27 supplement
(Invitrogen), non-essential amino acids is 0.1mM, GlutaMAX 1mM, β-mercaptoethanol
It is that (formula comes from bibliography to 100ng/ml: Human induced pluripotent stem cells for 0.1mM, bFGF
free of vector and transgene sequences.Science 324(5928):797-801)。
The people that this comparative example is obtained induce multi-potent stem cell using alkaline phosphatase staining (Cat#SCR004,
Millipore) carry out inducing multi-potent stem cell the counting of clone, statistical result is shown in Fig. 3.
As seen from Figure 3, compared with traditional N2B27+bFGF reprograms culture medium, CytoTune is being usedTM-iPS
In the case of 2.0Sendai Reprogramming Kit, it is more that more source of people can be obtained using the reprogramming culture of embodiment 1
It can stem cell clone.
Comparative example 3
This comparative example provides a kind of cultural method that adult cell reprogramming behaviour induces multi-potent stem cell, and uses
SimpliconTMRNA Reprogramming Kit (OKSG) (Millipore), the step base of operating procedure and embodiment 2
This is identical, and difference is: the culture medium that this comparative example uses also reprograms culture medium for traditional N2B27+bFGF, i.e., in DMEM/
1xN-2supplement (Invitrogen) is added in F12 basal medium, 1xB-27 supplement (Invitrogen),
Non-essential amino acids is 0.1mM, and GlutaMAX 1mM, β-mercaptoethanol are 0.1mM, bFGF
For 100ng/ml, (formula comes from bibliography: Human induced pluripotent stem cells free of
vector and transgene sequences.Science 324(5928):797-801)。
The people that this comparative example is obtained induce multi-potent stem cell using alkaline phosphatase staining (Cat#SCR004,
Millipore) carry out inducing multi-potent stem cell the counting of clone, statistical result is shown in Fig. 4.
As seen from Figure 4, compared with traditional N2B27+bFGF reprograms culture medium, Simplicon is being usedTMRNA
In the case of Reprogramming Kit (OKSG) (Millipore), it can be obtained more using the reprogramming culture of embodiment 1
Source of people multipotential stem cell clone.
Comparative example 4
This comparative example probe into reprogramming culture medium in constituent part concentration to reprogramming culture influence, using with implementation
The cultural method that the adult cell reprogramming behaviour of 4 same steps of example induces multi-potent stem cell, specific test are grouped as follows 3 institute of table
Show:
Table 3 probes into the grouping sheet of influence of the concentration of constituent part in reprogramming culture medium to reprogramming culture
Cell clone is induced multi-potent stem cell using gained in each group test in alkaline phosphatase staining method detection comparative example 1
Form, statistical result is shown in Fig. 7.As seen from Figure 7, after mentioned component goes beyond the scope in culture medium, mentioned component is utilized
Off-limits reprogramming culture medium cannot smoothly maintain the stability induced multi-potent stem cell, and alkaline phosphatase staining is shown carefully
There is dead or abnormal colony morphology in born of the same parents.
In addition, applicant is largely probed into, in reprogramming culture medium of the invention, when L-AA is 60-180 μ
G/mL, hydrocortisone are 5.3-74 μm of ol/L, sodium selenite 3-89ng/mL, Optiferrin are 8-23 μm of ol/L, view is yellow
Alcohol acetic ester is 0.5-7.4 μm of ol/L, plant source recombination human basic growth factor be 40-60ng/mL, IGF be 8-12 μ g/mL,
A-83 is that 0.2-0.6 μ g/mL, CHIR99021 are 2-6 μm of ol/L, when sodium butyrate is 100-450 μm of ol/L, on the whole can be suitable
Benefit maintains the stability induced multi-potent stem cell.
Wherein, when L-AA is 70 μ g/mL-90 μ g/mL, L hydrocortisone is 40 μm of ol/L-60 μm of ol/, sub- selenium
Sour sodium be 10ng/mL-30ng/mL, Optiferrin be 8 μm of ol/L-12 μm of ol/L, retinyl acetate is 3 μm of ol/L-6 μ
Mol/L, plant source recombination human basic growth factor are that 45ng/mL-55ng/mL, IGF are 8 μ g/mL-12 μ g/mL, 0.3 A-83
When μm ol/L-0.5 μ g/mL, CHIR99021 are 3 μm of ol/L-5 μm of ol/L and sodium butyrate is 280 μm of ol/L-410 μm of ol/L,
The stability induced multi-potent stem cell for reprogramming preparation is best.
Each technical characteristic of above embodiments can be combined arbitrarily, for simplicity of description, not to above-described embodiment
In each technical characteristic it is all possible combination be all described, as long as however, the combination of these technical characteristics be not present lance
Shield all should be considered as described in this specification.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
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Claims (8)
1. a kind of reprogramming culture medium, which is characterized in that the reprogramming culture medium is by DMEM-F12 basal medium and addition
At being grouped as, the adding ingredient includes the hydrogen of the L-AA of 60 μ g/mL-180 μ g/mL, 5.3 μm of ol/L-74 μm of ol/L
Change Optiferrin, the 0.5 μm of ol/L-7.4 μ of cortisone, the sodium selenite of 3ng/mL-89ng/mL, 8 μm of ol/L-23 μm of ol/L
The retinyl acetate of mol/L, the plant source recombination human basic growth factor of 40ng/mL-60ng/mL, 8 μ g/mL-12 μ g/mL
IGF, the A-83 of 0.2 μm of ol/L-0.6 μ g/mL, 2 μm of ol/L-6 μm of ol/L CHIR99021 and 100 μm of ol/L-450 μ
The sodium butyrate of mol/L.
2. reprogramming culture medium according to claim 1, which is characterized in that the adding ingredient includes 70 μ g/mL-90 μ
The L-AA of g/mL, the hydrocortisone of 40 μm of ol/L-60 μm of ol/L, 10ng/mL-30ng/mL sodium selenite, 8 μ
The plant of the retinyl acetate, 45ng/mL-55ng/mL of the Optiferrin of mol/L-12 μm of ol/L, 3 μm of ol/L-6 μm of ol/L
A-83, the 3 μm of ol/L-5 of material resource recombination human basic growth factor, the IGF of 8 μ g/mL-12 μ g/mL, 0.3 μm of ol/L-0.5 μ g/mL
The sodium butyrate of CHIR99021 and 280 μm of ol/L-410 μm of ol/L of μm ol/L.
3. reprogramming culture medium according to claim 2, which is characterized in that the adding ingredient includes the L- of 80 μ g/mL
Ascorbic acid, the hydrocortisone of 50 μm of ol/L, the sodium selenite of 20ng/mL, 10 μm of ol/L Optiferrin, 4 μm of ol/L
Retinyl acetate, the plant source recombination human basic growth factor of 50ng/mL, the IGF of 10 μ g/mL, 0.4 μ g/mL A-83,
The sodium butyrate of CHIR99021 and 400 μm of ol/L of 4 μm of ol/L.
4. a kind of reprogram the cultural method induced multi-potent stem cell, which is characterized in that use claim 1 using adult cell
Reprogramming culture is carried out to 3 described in any item reprogramming culture mediums.
5. according to claim 4 reprogram the cultural method induced multi-potent stem cell, which is characterized in that the adult is thin
Born of the same parents are umbilical cord source human mesenchymal cells, people CD34+ cell or human skin fibroblasts.
6. according to claim 5 reprogram the cultural method induced multi-potent stem cell, which is characterized in that including walking as follows
It is rapid:
Reprogramming uses Epi5 Reprogramming Kit, and adult cell is resuspended in and is provided in electrotransformation kit
In Resuspension Buffer, electricity turns, the cell after obtaining electric turn；
Cell after electricity is turned is squeezed into the hole of Matrigel coating culture plate, described in any item heavy using claims 1 to 33
Programming culture medium is placed in 37 ± 0.5 DEG C of temperature, is cultivated in 5% carbon dioxide environment, in incubation, partly changes liquid extremely daily
It the tenth day after electricity turn, is every other day changed the liquid once since the tenth day, until the 28th day.
7. according to claim 5 reprogram the cultural method induced multi-potent stem cell, which is characterized in that including walking as follows
It is rapid:
Reprogramming uses CytoTuneTM2.0 Sendai Reprogramming Kit of-iPS, according to 2 × 106Cell, MOI=
5 ratio carries out viral transduction, after virus is added, is placed in 37 ± 0.5 DEG C of temperature, is incubated for 12-16h in 5% carbon dioxide environment,
Start within second day for culture medium to be changed to the described in any item reprogramming culture mediums of claims 1 to 3, cultivate to the 28th day.
8. according to claim 5 reprogram the cultural method induced multi-potent stem cell, which is characterized in that including walking as follows
It is rapid:
Reprogramming uses SimpliconTMRNA Reprogramming Kit recycles claims 1 to 3 described in any item
Culture medium is reprogrammed, by cell culture to the 28th day.
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| 人诱导性多潜能干细胞系的建立;师迎旭等;《中国组织工程研究》;20150205;第19卷(第6期);第868-875页 * |
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