CN108998410A - Kinases inhibitor is inhibiting the purposes in haploid cell diplodization - Google Patents
Kinases inhibitor is inhibiting the purposes in haploid cell diplodization Download PDFInfo
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Abstract
The present invention provides CDK1 kinases inhibitor and/or Rock kinases inhibitor and is inhibiting the purposes in haploid cell diplodization.Using the CDK1 protein kinase and Rock kinases inhibitor of screening, the culture and differentiation of lonely male, lonely female monoploid embryo stem cell can be carried out extensively.The technical scheme is that being provided the foundation based on the research of haploid genome and application.
Description
Technical field
The present invention relates to field of cell culture, more particularly it relates in vitro culture or differentiation haploid cell
In haplotype maintain method.
Background technique
For diplont, monoploid has single copy gene group, has big advantage in genetics research.So
And monoploid embryo stem cell is cultivated in vitro or atomization in spontaneous diplodization can occur, to obtain or keep haploid embryo
Tire stem cell just needs constantly to carry out streaming enrichment, this significantly increases haploid stock layout cost, thus hinder its
Genescreen and modification in application (Elling et al., 2011；Leeb and Wutz, 2011；Yang et al., 2012；Zhou etc.
People, 2016)；Meanwhile the ploidy support mechanism of monoploid stem cell is also an interesting problem, which may contain
The unique cell cycle of embryonic stem cell.
In recent years, researches show that lonely female monoploid embryo stem cells deposits in MEK kinase inhibitor and GSK3 beta kinase inhibitor
Compared to more easily maintenance monoploid ploidy (Leeb et al., 2012) under Serum System under.But it although it has been reported that can be with
Effectively slow down the diplodization of lonely female monoploid embryo Stem cells cultured in vitro with tyrosine kinase inhibitor, the method still can not
It is widely and effectively and inhibits the diplodization of monoploid embryo stem cell, can not also obtain rodent such as mouse monoploid body cell
(Takahashi et al., 2014).The female monoploid embryo stem cell of orphan of the report people such as Sagi can break up that obtain triploblastica body thin
Born of the same parents (Sagi et al., 2016).In short, still failing to efficiently control monoploid embryo stem cell at present in culture and atomization
Middle diplodization (especially for rodent monoploid body cell), while haploid cell can not be obtained effectively also to carry out
Genetic screening.
Summary of the invention
In order to solve the above-mentioned technical problem, the present inventor is based on the cell division to monoploid embryo stem cell
The dynamic change of chromosome in journey, some important regulating and controlling factors and check point of cell cycle carry out small molecule screening, discovery
Some protease inhibitors can effectively inhibit Haploid diploidization.
Therefore, it is an object of the present invention to provide protease inhibitors to inhibit the use in haploid cell diplodization
On the way.
Based on the new discovery, it is a further object to provide the compositions for cultivating and/or breaking up haploid cell
(such as culture medium) and method.
A further object of the present invention is to provide the monoploid body cell of the method and acquisition that obtain haploid cell.
Of the invention a further object is provides the purposes of the monoploid body cell obtained.
Technical scheme is as follows.
On the one hand, the present invention provides CDK1 kinases inhibitor and/or Rock kinases inhibitor at single times of inhibition
Purposes in body cell diplodization.
Preferably, the CDK1 kinases inhibitor is Ro3306；The Rock kinases inhibitor is Y-
27632；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably singly
Times body embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral
Cell；
Preferably, described to inhibit to be to inhibit the haploid cell in the culture of haploid cell and/or atomization
Diplodization.
In the incubation of the haploid cell, and/or in the haploid cell to other types cell differentiation
During, kinases inhibitor provided by the invention is able to suppress haploid cell diplodization, maintains haploidy, while not
Influence normal growth and the differentiation of cell.
On the other hand, the present invention provides a kind of for cultivating and/or breaking up the composition of haploid cell, the composition
Include CDK1 kinases inhibitor and/or Rock kinases inhibitor.
Preferably, the CDK1 kinases inhibitor is Ro3306；The Rock kinases inhibitor is Y-
27632；
Preferably, the composition is cell culture medium；
Preferably, the concentration of the CDK1 kinases inhibitor in the composition be 1.5-5 μM, preferably 3-5 μM,
More preferable 4.5 μM；The concentration of the Rock kinases inhibitor in the composition be 5-40 μM, preferably 10-40 μM, more
It is preferred that 40 μM；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably singly
Times body embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral
Cell.
Composition provided by the invention cannot be only used for the culture or maintenance of haploid cell, and it is thin to can be also used for monoploid
The differentiation of born of the same parents.In the incubation of the haploid cell, and/or in the haploid cell to other types cell differentiation
During, the kinases inhibitor in composition provided by the invention is able to suppress haploid cell diplodization, remains single
Ploidy, while normal growth and the differentiation of cell are not influenced.
Another aspect, the present invention provide a kind of method for cultivating and/or breaking up haploid cell, the method packet
It includes, in the presence of CDK1 kinases inhibitor and/or Rock kinases inhibitor, culture and/or differentiation monoploid are thin
Born of the same parents.
Preferably, the CDK1 kinases inhibitor is Ro3306；The Rock kinases inhibitor is Y-
27632；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably singly
Times body embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral
Cell；
Preferably, the method includes thin using composition culture provided by the invention and/or the differentiation monoploid
Born of the same parents.
In the above method provided by the invention, CDK1 kinases inhibitor and/or Rock kinases inhibitor are not
The normal culture and/or differentiation of haploid cell are influenced, but inhibits haploid cell diplodization during its, maintains single times
Property, while normal growth and the differentiation of cell are not influenced.
Also on the one hand, the present invention provides a kind of method for obtaining haploid cell, and the method includes using CDK1 albumen
Kinase inhibitor and/or Rock kinases inhibitor inhibit haploid cell diplodization.
Preferably, the CDK1 kinases inhibitor is Ro3306；The Rock kinases inhibitor is Y-
27632；
Preferably, described to inhibit to be to inhibit the haploid cell in the culture of haploid cell and/or atomization
Diplodization；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably singly
Times body embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral
Cell；
Preferably, the method includes thin using composition culture provided by the invention and/or the differentiation monoploid
Born of the same parents.
For example, the present invention provides a kind of acquisition monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell
Or the method for monoploid entoderm progenitor cells, the method includes single using composition culture provided by the invention and/or differentiation
Times body embryonic stem cell or its precursor.
In another aspect, the present invention also provides any in the various haploid cells obtained by the above method and they
Purposes in genetics research.
For example, the present invention provides through the monoploid nerve cell of above method acquisition and its in neurotoxin and nerve life
Manage the purposes in the genetics research such as function.
Compared with prior art, the present invention provides kinases inhibitor have inhibit haploid cell diplodization this
One new discovery.
The present inventor starts with from cell cycle angle, and the monoploid embryo stem cell marked using H2B-GFP is seen
The dynamic change of chromosome during the monoploid embryo stem cell division is examined, discovery monoploid embryo stem cells divided
It is abnormal in journey, lead to chromosome doubling.Based on this, some important regulating and controlling factors of the present inventor's cell cycle and inspection
A progress molecular sieve choosing is tested, the small molecule compound that monoploid can be promoted to maintain is filtered out.It especially has found, CDK1 egg
White kinases and Rock kinases inhibitor can effectively inhibit Haploid diploidization, and cell cycle checkpoint PROTEIN C hk1 presses down
Preparation then significantly reduces the monoploid ratio of monoploid stem cell.
Using the CDK1 protein kinase and Rock kinases inhibitor, particularly Rock kinases inhibitor of screening,
The culture of lonely male, lonely female monoploid embryo stem cell can be carried out extensively.This facilitates a large amount of expansions of monoploid embryo stem cell
Increase, substantially reduces and constantly purify haploid process, simplified culture scheme reduces cost, has pushed monoploid embryo stem cell
Application.
Using the CDK1 protein kinase and Rock kinases inhibitor of screening, lonely male, lonely female list can also be carried out extensively
The differentiation of times body embryonic stem cell.Present invention obtains monoploid neural stem cell, nerve cell, cardiac muscle cell, pancreas islet ancestral are thin
The monoploid body cell such as born of the same parents, and provide a kind of effective ways of monoploid body cell for obtaining triploblastica source.Due to single times
The characteristics of body single copy gene type, the haploid cell of the method and acquisition are research such as nerve to occur and nerve disease
Disease, cardiac development and cardiovascular disease etc. specific life process provide good Efficient Genetic model.For example, of the invention
It has been screened using monoploid nerve cell and has improved nerve cell to the gene of neurotoxicity manganese ion resistance, sieved for specific toxicity
Choosing experiment provides new carrier.
To sum up, new discovery of the invention and the new technical solution based on it are research based on haploid genome and answer
With providing the foundation.
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 shows the chromosome during diploid embryonic stem cell (control) and monoploid embryo stem cell division
Dynamic change.
Fig. 2 shows the suppression of CDK1 and Rock kinases inhibitor and its concentration to monoploid embryo stem cell diplodization
Production is used.
Fig. 3 shows that Rock kinases inhibitor is divided into monoploid neural stem cell mistake in monoploid embryo stem cell
To the inhibiting effect of diplodization in journey.
Fig. 4 show Rock kinase inhibitor monoploid it is neural stem cell differentiating in monoploid neuronal process to two
The inhibiting effect changed again.
Fig. 5 shows that Rock kinase inhibitor is right during monoploid embryo stem cell is divided into monoploid cardiac muscle cell
The inhibiting effect of diplodization.
Fig. 6 shows that Rock kinase inhibitor is divided into monoploid entoderm progenitor cells process in monoploid embryo stem cell
In to the inhibiting effect of diplodization.
Fig. 7 shows application of the monoploid neural stem cell of acquisition in the screening of neurotoxic substances genetics of resistance.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.
Embodiment 1The dynamic change of chromosome during monoploid embryo stem cell division
From same strain H2B-GFP transgenic mice (being introduced from Beijing Vital River Experimental Animals Technology Co., Ltd.) point
Male (lonely female) the monoploid embryo stem cell line of diploid embryonic stem cell line and the orphan for not establishing fertilized eggs source.
The culture of diploid embryonic stem cell line and lonely male monoploid embryo stem cell is carried out in 12 orifice plates.Firstly, will
Feeder cells (mouse E13.5 days embryo fibroblasts of mitomycin C (sigma, M0503) processing) inoculation of mouse
In culture orifice plate, with Fibroblast culture solution (DMEM in high glucose (Gibco, C12430500BT), 1 × Sodium Pyruvate (100 ×,
Gibco, 11360-070), 1 × nonessential amino acid (100 ×, Gibco, 11140-050), 1 × Penicillin-
Streptomycin (Gibco, 15140163,100 ×) plus 10% fetal calf serum (Gibco, 16000-044) culture are spare.It will
Lonely male (lonely female) monoploid embryo stem cell using 2 μ g/ml Hoechst 33342 of live cell dye (Invitrogen,
H3570) in 37 degrees Celsius dyeing 15-20 minutes, airflow classification (airflow classification instrument be MoFlo XDP high speed polychrome streaming it is thin
Born of the same parents' sorter) peak 1n is collected, diploid ES compares the determining peak position 2n.By the monoploid embryo stem cell of enrichment averagely plant in
Complete 12 orifice plates of feeder cells.Utilize Mouse Embryonic Stem Cell Culture liquid: N2B27 basal medium (DMEM/F12
(Gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1 to 1 are mixed, addition 1 × N2 additive (100 ×,
Gibco, 17502048), 1 × B27 additive (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma,
A8022), 1 × beta -mercaptoethanol (1000 ×, Gibco, 21985023), 1 × Glutamax (200 ×, Gibco, 35050-
061), 1 μ g/ml insulin (Roche, 11376497001)), be added 10% serum substitute (Gibco, 10828028) and 1 ×
Penicillin-Streptomycin (Gibco, 15140163,100 ×), then plus 1 micromolar PD0325901
The white blood of (stemgent, 04-0006-10), 3 micromolar Chir99021 (stemgent, 04-0004-10) and 1000U/ml
Sick inhibiting factor LIF (millipore, ESG2207).The embryonic stem cell of the monoploid H2B-GFP label of streaming purifying utilizes
The case where living cells work station observation cell enters the M phase and divides, the diploid H2B-GFP Embryonic Stem Cell in diplodization source
As control.Figure 1A shows (upper row) diploid embryo's stem cell division and enters division stage chromosome condensation (from left to right
1st figure), metaphase chromosome agglutination is aligned to metaphase plate (from left to right the 2nd figure), enter after general 16 minutes the later period (from
3rd figure from left to right), chromosome, which is opened, moves towards the two poles of the earth, and with generation chromosome deagglomeration (the 4th figure from left to right).
Figure 1A is also shown (following a line) monoploid embryo stem cell division and enters division stage chromosome condensation, arrangement mid-term to metaphase plate
(from left to right the 1st figure) in 1 hour does not all enter into later period i.e. chromosome and is opened and move towards the two poles of the earth (2-3 from left to right
Open figure), block the entrance that the later period is not observed in after stain colour solid in 3 hours in the generation M phase, and with generation chromosome deagglomeration
Into latter stage or interphase (the 4th figure from left to right), the sliding (slippage) that the M phase has occurred leads to chromosome doubling.Figure 1B
It quantifies diploid respectively using 2011 software of ZEN and the GFP fluorescence intensity of haploid chromosomes part (reflects containing for chromosome
Amount).When Fig. 1 C shows diploid and monoploid embryo stem cell mitosis, normal M phase and exception M phase ratio.
A large amount of observation is the results show that (it is solidifying to enter M phase chromosome for diploid cell observation at least 152 split coil methods
Collection), 100% split coil method enters later period (chromosome is opened, and pulls to the two poles of the earth) within half an hour after entering mid-term, after
Chromosome deagglomeration occurs after phase.For haploid cell observation at least 160 split coil methods (entering M phase chromosome condensation), connect
Nearly 10% split coil method has after entering mid-term not to be entered into the later period up to retardance in 2 hours or more (chromosome is opened, and pulls to two
Pole), chromosome deagglomeration directly occurs.
Embodiment 2Inhibit the kinases inhibitor of haploid cell diplodization and its screening of concentration
(1) screening of kinases inhibitor and its concentration is carried out in 12 orifice plates.
Firstly, the feeder cells (the mouse MEF of mitomycin C (sigma, M0503) processing) of mouse are inoculated in training
Orifice plate is supported, it is spare with Fibroblast culture solution (with embodiment 1) plus 10% fetal calf serum (Gibco, 16000-044) culture.
By lonely male (lonely female) monoploid embryo stem cell using 2 μ g/ml Hoechst 33342 of live cell dye (Invitrogen,
H3570) in 37 degrees Celsius dyeing 15-20 minutes, airflow classification collects the peak 1n, and diploid ES compares the determining peak position 2n.It will
The monoploid embryo stem cell of enrichment is averagely planted in 12 orifice plates for completing feeder cells.Utilize Mouse Embryonic Stem Cell Culture liquid
Certain concentration, particular kind of kinases inhibitor is added in (with embodiment 1).The DMSO of same dose is as control.Every kind
3 Duplicate Samples are at least arranged in kinases inhibitor and DMSO.As a result it see the table below 1.
The different inhibitor of table 1 influence Haploid diploidization
(2) it is tested with CDK1 kinases inhibitor, Rock kinases inhibitor.
Using operation identical with (one) part above, mouse ES culture solution culture is added in kinases inhibitor
After 12-15 days, 10 μ g/ml Hoechst, 33342 staining for flow analyzes the monoploid ratio of inhibitor group and DMSO group.
The CDK1 inhibitor Ro3306 (BioVision, 2039-1) and any Rock inhibitor that Fig. 2A shows 4.5 μM are such as
20-40 μM of Y-27632 (selleck, S1049) inhibits monoploid embryo stem cell diplodization significantly: pure monoploid control
It is 10% that group DMSO, which handles 12 days monoploid ratios, and it is 80% that experimental group Ro3306, which handles 12 days monoploid ratios,；Control group
It is 16% that DMSO, which handles monoploid ratio, and it is 92% that Y-27632 (Y-27), which handles 12 days monoploid ratios,.Fig. 2 B shows drug
ES cells are at classical colony morphology after processing.And as (it is micro- that immunofluorescence dyeing is imaged on laser co-focusing to Fig. 2 C
Completed on mirror Zeiss LSM780 or Leica two-photon laser Laser Scanning Confocal Microscope TCS Sp8 instrument) shown in, immunofluorescence dye
Color does not influence multipotency sex factor OCT4 (santa cruz, sc-8628), Nanog (abcam, ab80892), Sox2
The expression such as (milipore, AB5603) and SSEA1 (millpore, MAB4301).
Carry out the screening experiment of its concentration with 1.5,3 and 4.5 μM of Ro3306 respectively as described above.Fig. 2 D shows Ro3306
Concentration significant is inhibited to Haploid diploidization when being greater than 3 μM, and effect is best at 4.5 μM, is optium concentration, be greater than 5 μM it is tight
Ghost image rings cell survival, and as a result at least biology repeats three times.Subsequent experimental is carried out big using 4.5 μM of optium concentration of Ro3306
The tightened up independent repetition experiment (at least 6 plants lonely male monoploid embryo stem cells and the female monoploid embryo stem cell of 3 plants of orphans) of amount,
Verifying Ro3306 inhibits monoploid embryo Stem cells cultured in vitro diplodization significantly, as shown in Figure 2 F.
Carry out the screening experiment of its concentration with 5,10,20 and 40 μM of Y-27632 respectively as described above.Fig. 2 E shows Y-
Significant is inhibited to Haploid diploidization when 27632 concentration is greater than 20 μM, and effect is best at 40 μM, is optium concentration, is greater than
40 μM seriously affect ES cellular morphology, and as a result at least biology repeats three times.Subsequent experimental uses 40 μM of optium concentration of Y-
27632 carry out a large amount of tightened up independent repetition experiment (at least 3 plants lonely male monoploid embryo stem cells and 2 plants of female monoploid of orphan
Embryonic stem cell), verifying Y-27632 inhibits monoploid embryo Stem cells cultured in vitro diplodization significantly, as shown in Figure 2 G.Figure
Before " Before " in 2D to Fig. 2 G indicates experiment.
Embodiment 3Using Rock kinase inhibitor, monoploid neural stem cell is obtained by monoploid embryo stem cell
Male (lonely female) the monoploid embryo stem cell of orphan for obtaining streaming purifying, by 50,000 cell kinds in thin with feeder layer
The culture dish of the 3.5cm of born of the same parents grows clone for 3-5 days, and 0.25% trypsase (Gibco, 25200-072) digests 3-5min,
10%FBS terminates digestion, and 1 × PBS is washed one time, is trained with no PD0325901, the mouse embryo stem cell without Chir99021 and LIF
Nutrient solution (with embodiment 1) suspend culture monoploid ES, suspend culture 2 days after, by 500 embryoid body ball kinds in be covered with poly rely ammonia
The culture dish of sour 1mg/ml (sigma, P6407) and 5ug/ml laminin (invitrogen, 23017-015) 6cm.16 is small
When after change Culture of neural stem cells liquid into (N2B27 basic culture solution (with embodiment 1) add 20ng/ml bFGF (R&D, 233-
FB-001MG) and 20ng/ml EGF (R&D, 2028-EG-200)).Any Rock inhibitor such as 20 μM of Y-27632, phase is added
DMSO with dosage is as control.After adherent differentiation 3 days, digestion, 1:10 passage kind is in being covered with poly-D-lysine 1mg/ml
The culture dish of (sigma, P6407) and 5ug/ml laminin (invitrogen, 23017-015) 6cm.Further differentiation,
In differentiation the 10th day, flow cytometer showed simultaneously sorted purifying monoploid.Fig. 3 A is shown since monoploid ES differentiation in (the 0th day) 30 days
Atomization.Fig. 3 B show differentiation 10 days as a result, diplodization (monoploid ratio almost occurs for DMSO control group monoploid
For 1.5%), Y-27632 group maintains high monoploid ratio (monoploid ratio is greater than 90%).After Fig. 3 C-3G is differentiation 30 days
Testing result, wherein Fig. 3 C shows Y-27632 group further apparent neural stem cell form occurs in differentiation；Fig. 3 D shows Y-
The cell of 27632 groups of karyotyping discovery differentiation is largely 20 chromosomes；Fig. 3 E and 3F show immunofluorescence dyeing mirror
Fixed classical neural stem cell marker albumen, Y-27632 group 95% or more cell coexpression Nestin (anti-Nestin,
Millipore, MAB353), Sox2 (anti-Sox2, milipore, AB5603), Pax6 (anti-Pax6, abcam,
Ab5790), CD133 (anti-CD133, miltenyi biotec, 130-105-226) etc.；Fig. 3 G shows real-time quantitative PCR mirror
Determine the mRNA up-regulation of neural stem cell marker albumen Nestin, Sox2, N-cadherin and Zfp521.
Embodiment 4Using Rock kinase inhibitor, monoploid neuron is obtained by monoploid neural stem cell
Differentiation obtains monoploid neural stem cell (with embodiment 3).
Using differential medium, (N2B27 basal medium (with embodiment 1) adds 1% fetal calf serum, and adds Rock inhibition
20 μM of Y-27632 of agent) carry out differentiation culture.At random differentiation the 7th day, the labelled protein Tuj1 of immunofluorescence dyeing neuron,
The left side Fig. 4 A 1 has illustrated a large amount of neuron morphology, stretch out long aixs cylinder and expression Tuj1 albumen (anti-Tuj1,
Millipore, MAB163)；The cells ratio that the right side Fig. 4 A 3 illustrates the flow cytometer showed Tuj1-FITC positive is 9%, Tuj1 positive
Cell is largely monoploid, and undifferentiated diploid neural stem cell is negative control.
Using differential medium, (N2B27 basal medium (with embodiment 1) adds 3% fetal calf serum, and adds Rock inhibition
20 μM of Y-27632 of agent) carry out differentiation culture.At random differentiation the 14th day, the label egg of immunofluorescence dyeing Deiter's cells
The left side white GFAP, Fig. 4 B 1 has illustrated a large amount of neuroglia form, and expresses GFAP (anti-GFAP, DAKO, Z033429)；
It is largely single times that the right side Fig. 4 B 3, which illustrates the cells ratio of the flow cytometer showed GFAP-FITC positive as 19%, GFAP positive cell,
Body, undifferentiated diploid nerve cord are negative control.
The neuron broken up 7 days is further purified, culture medium changes the (basis the N2B27 culture of neuronal maturation culture medium into
Base (with embodiment 1) 100 μM of cAMP (sigma, D0627) of addition, 20ng/ml brain derived neurotrophic factor (NT-3,
Peprotech, 450-02), 20ng/ml NT-3 (peprotech, 450-03) and 20ng/ml glia cell line-derived neurotrophy
The factor (peprotech, 450-10)), and 20 μM of Y-27632 of Rock inhibitor are added, maturation culture 14 days, immunofluorescence dye
The labelled protein Tuj1 and Map-2 of color neuron, Fig. 4 C show neuron expression Tuj1 and the Map-2 (anti-close to 100%
Map-2, abcam, ab32454).The cell monoploid ratio that Fig. 4 D shows the flow cytometer showed Map-2 positive is greater than 65%.Fig. 4 E shows
Mature neuron co-expresses Tuj1 and nerve synapse Protein S yn1 (anti-syn1, millipore, AB1543P) out.Fig. 4 F shows
The monoploid neuron electrophysiological function of patch-clamp detection differentiation out, the result is that having sodium ion electric current, potassium current and certain
Action potential.
Embodiment 5Using Rock kinase inhibitor, monoploid cardiac muscle cell is obtained by monoploid embryo stem cell
Male (lonely female) the monoploid embryo stem cell of orphan for obtaining streaming purifying, by 50,000 cell kinds in thin with feeder layer
The culture dish of the 3.5cm of born of the same parents grows clone for 3-5 days, and 0.25% trypsase (Gibco, 25200-072) digests 3-5min,
10%FBS terminates digestion, and 1 × PBS is washed one time, is trained with no PD0325901, the mouse embryo stem cell without Chir99021 and LIF
Nutrient solution (with embodiment 1) suspends culture monoploid ES, after suspending culture 2 days, by 500 embryoid body ball kinds in being covered with matrigel
The culture dish of matrigel matrix (BD, 354277) 6cm.Change myocardium induction broth (DF12,1 × B27 after 16 hours into
Additive (50 ×, Gibco, 17504044), 10% serum substitute (1000 ×, sigma, A8022), 1 × beta -mercaptoethanol
(1000 ×, Gibco, 21985023), 1 × Glutamax (200 ×, Gibco, 35050-061)), addition 1 ×
Penicillin-Streptomycin (Gibco, 15140163,100 ×) and 10ng/ml bone morphogenetic protein 4 (BMP4, R&
D, 5020-BP-010), 10ng/ml Activin A (R&D, 338-AC-050/CF), 6 μM of GSK3 beta inhibitor Chir99021
(stemgent, 04-0004-10).5th day, after 2 μM of IWR1 replacement GSK3 beta inhibitor Chir99021 induce 2 days again, the
Remove BMP4 and Activin A within seven days.Cardiomyocyte culture liquid is changed into carry out that 20 μM of Y- of Rock inhibitor are added always in incubation
27632.Fig. 5 A shows atomization, Myocardium Differentiation the 12nd day, the cardiac muscle beated occurs, Fig. 5 B shows immunofluorescence dyeing identification
There is the special serum cardiac troponin T of cardiac muscle cell (anti-cTnT, abcam, ab8295) in differentiation.Undifferentiated mice embryonic
Stem cell is as negative control, and Fig. 5 C shows the differentiation of monoploid embryo stem cell and obtains cTnT positive cardiomyocytes, and Fig. 5 D is shown
The cTnT positive cardiomyocytes overwhelming majority is monoploid.
Embodiment 6Using Rock kinase inhibitor, monoploid entoderm group cell is obtained by monoploid embryo stem cell
Male (lonely female) the monoploid embryo stem cell of orphan for obtaining streaming purifying, by 50,000 cell kinds in thin with feeder layer
The culture dish of the 3.5cm of born of the same parents grows clone for 3-5 days, and 0.25% trypsase (Gibco, 25200-072) digests 3-5min,
10%FBS terminates digestion, and 1 × PBS is washed one time, is trained with no PD0325901, the mouse embryo stem cell without Chir99021 and LIF
Nutrient solution (with embodiment 1) suspend culture monoploid ES, suspend culture 2 days after, by 500 embryoid body ball kinds in be covered with fiber connection
The culture dish of albumen (fibronectin, millipore, fc010) 6cm.Change entoderm induction broth after 16 hours into
(DF12,1 × B27 additive (50 ×, Gibco, 17504044), 10% serum substitute (1000 ×, sigma, A8022), 1
× beta -mercaptoethanol (1000 ×, Gibco, 21985023), 1 × Glutamax (200 ×, Gibco, 35050-061)), it is added 1
× Penicillin-Streptomycin (Gibco, 15140163,100 ×) and 100ng/ml Activin A (R&D, 338-
AC-050/CF), 3 μM of GSK3 beta inhibitor Chir99021 (stemgent, 04-0004-10).Change entoderm induction broth into
It carries out that 20 μM of Y-27632 of Rock inhibitor are added always in incubation.Fig. 6 A shows atomization.Cultivate 16 days poststainings
The differential protein PDX1 of islet progenitor cells.Fig. 6 B shows immunofluorescence dyeing identification differentiation and the special egg of heart islet progenitor cells occurs
White Pdx1 (anti-Pdx1, abcam, ab47383).Undifferentiated mouse embryo stem cell shows list as negative control, Fig. 6 C
Times body ES cell differentiation obtains pdx1 positive islet cells, Fig. 6 D show pdx1 positive islet cells be largely it is single again
Body.
Embodiment 7Application of the monoploid neural stem cell in genetics research and the screening of toxicity science of heredity
The science of heredity screening of neurotoxic substances resistance is carried out using monoploid neural stem cell (coming from embodiment 3).
1 mM of manganese chloride is used to handle as control, experimentation is as shown in Figure 7 A.After screening 4 days, control group is wild
Type monoploid neural stem cell almost all is dead, and has a small amount of cell survival in gene trap library, and (arrow shows as shown in Figure 7 B
Living cells out) (being independently repeated 3 times).
Living cells, the i.e. neural stem cell of manganese chloride resistance are expanded, Fig. 7 C shows at least 2 experimental results, and manganese chloride is anti-
Property cell high-flux sequence verifying gene trap locus gene be enriched in cynapse transmitting, film potential, neuron deliver etc. biologies
Gene during, wherein Park2 gene is higher in experiment capture rate at least twice.
Using the Park2 in the primary diploid neural stem cell of Crisper-Cas9 specific knockdown, to improve primary two
The resistance of times somatic nerves stem cell manganese ion.Fig. 7 D shows wild type diploid and the primary diploid nerve cord of Park knockout is thin
Born of the same parents are with 500 μM of MnCl2Cellular morphology figure after handling 3 days respectively, the primary diploid mind that Fig. 7 E shows wild type and Park is knocked out
Viable count counts after stem cell handles 3 days with 500 micromole's manganese chlorides respectively.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
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Claims (10)
1.CDK1 kinases inhibitor and/or Rock kinases inhibitor are inhibiting the use in haploid cell diplodization
On the way.
2. purposes according to claim 1, which is characterized in that the CDK1 kinases inhibitor is Ro3306；It is described
Rock kinases inhibitor is Y-27632；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably monoploid
Embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral are thin
Born of the same parents；
Preferably, described to inhibit to be to inhibit two times of the haploid cell in the culture of haploid cell and/or atomization
Change.
3. a kind of for cultivating and/or breaking up the composition of haploid cell, the composition inhibits comprising CDK1 protein kinase
Agent and/or Rock kinases inhibitor.
4. composition according to claim 3, which is characterized in that the CDK1 kinases inhibitor is Ro3306；Institute
Stating Rock kinases inhibitor is Y-27632；
Preferably, the composition is cell culture medium；
Preferably, the concentration of the CDK1 kinases inhibitor in the composition is 1.5-5 μM, preferably 3-5 μM, more excellent
Select 4.5 μM；The concentration of the Rock kinases inhibitor in the composition be 5-40 μM, preferably 10-40 μM, more preferably
40μM；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably monoploid
Embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral are thin
Born of the same parents.
5. a kind of method for cultivating and/or breaking up haploid cell, the method includes in CDK1 kinases inhibitor
And/or in the presence of Rock kinases inhibitor, culture and/or differentiation haploid cell.
6. according to the method described in claim 5, it is characterized in that, the CDK1 kinases inhibitor is Ro3306；It is described
Rock kinases inhibitor is Y-27632；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably monoploid
Embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral are thin
Born of the same parents；
Preferably, the method includes thin using composition culture described in claim 3 or 4 and/or the differentiation monoploid
Born of the same parents.
7. a kind of method for obtaining haploid cell, the method includes using CDK1 kinases inhibitor and/or Rock egg
White kinase inhibitor inhibits haploid cell diplodization.
8. the method according to the description of claim 7 is characterized in that the CDK1 kinases inhibitor is Ro3306；It is described
Rock kinases inhibitor is Y-27632；
Preferably, described to inhibit to be to inhibit two times of the haploid cell in the culture of haploid cell and/or atomization
Change；
Preferably, the haploid cell is the haploid cell of mammal, preferably rodent, more preferably monoploid
Embryonic stem cell, monoploid neural stem cell, monoploid nerve cell, monoploid cardiac muscle cell or monoploid entoderm ancestral are thin
Born of the same parents；
Preferably, the method includes thin using composition culture described in claim 3 or 4 and/or the differentiation monoploid
Born of the same parents.
9. the monoploid body cell obtained by method described in claim 7 or 8.
10. purposes of the monoploid body cell as claimed in claim 9 in genetics research.
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| CN114457113A (en) * | 2022-03-07 | 2022-05-10 | 南开大学 | Method for inhibiting haploid embryonic stem cell from being doubled |
| WO2022228481A1 (en) * | 2021-04-28 | 2022-11-03 | Shandong University | Methods for improving early embryo development |
| WO2023116649A1 (en) * | 2021-12-24 | 2023-06-29 | 北京仁基源医学研究院有限公司 | Composite reagent for preparing ultra-high-resolution chromosomes and use method thereof |
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| WO2022228481A1 (en) * | 2021-04-28 | 2022-11-03 | Shandong University | Methods for improving early embryo development |
| CN116457457A (en) * | 2021-04-28 | 2023-07-18 | 山东大学 | Method for improving early embryo development |
| CN116457457B (en) * | 2021-04-28 | 2024-03-01 | 山东大学 | Method for improving early embryo development |
| WO2023116649A1 (en) * | 2021-12-24 | 2023-06-29 | 北京仁基源医学研究院有限公司 | Composite reagent for preparing ultra-high-resolution chromosomes and use method thereof |
| CN114457113A (en) * | 2022-03-07 | 2022-05-10 | 南开大学 | Method for inhibiting haploid embryonic stem cell from being doubled |
| CN114457113B (en) * | 2022-03-07 | 2023-11-17 | 南开大学 | Method for inhibiting haploid embryonic stem cell doubling |
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