CN108998410B - Use of protein kinase inhibitors to inhibit haploid cell doubling - Google Patents
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Abstract
The invention provides the use of a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor in inhibiting haploid cell doubling. The screened CDK1 protein kinase and Rock protein kinase inhibitor can be widely used for culturing and differentiating parthenogenetic and parthenogenetic haploid embryonic stem cells. The technical scheme of the invention provides a basis for research and application based on haploid genomes.
Description
Technical Field
The present invention relates to the field of cell culture, more specifically, the present invention relates to methods of haplotype maintenance in vitro culture or differentiation of haploid cells.
Background
For diploid organisms, haploids have a single copy genome and are of great advantage in genetic studies. However, the haploid embryonic stem cells can be spontaneously doubled in the in vitro culture or differentiation process, and continuous flow enrichment is needed to obtain or maintain the haploid embryonic stem cells, which greatly increases the sample arrangement cost of the haploid, thereby hindering the application of the haploid embryonic stem cells in gene screening and modification (Elling et al, 2011; Leeb and Wutz, 2011; Yang et al, 2012; Zhou et al, 2016); meanwhile, the ploidy maintenance mechanism of haploid stem cells is also an interesting problem, and the mechanism may contain the unique cell cycle regulation mechanism of embryonic stem cells.
In recent years, studies have shown that parthenogenetic haploid embryonic stem cells maintain haploid ploidy in the presence of MEK kinase inhibitors and GSK3 β kinase inhibitors more readily than in serum systems (Leeb et al, 2012). However, although there are reports that the in vitro culture of parthenogenetic haploid embryonic stem cells can be effectively slowed down with tyrosine kinase inhibitors, this approach has not been broadly effective in inhibiting haploid embryonic stem cell doubling, nor has rodents such as mouse haploid somatic cells been obtained (Takahashi et al, 2014). Sagi et al reported that human parthenogenetic haploid embryonic stem cells could differentiate to obtain a blastoderm somatic cell (Sagi et al, 2016). In conclusion, there is still no effective control over the diploid embryonic stem cell doubling during the culture and differentiation process (especially for rodent haploid somatic cells) and no effective haploid cell acquisition for genetic screening.
Disclosure of Invention
In order to solve the technical problems, the inventor of the invention carries out small molecule screening on some important regulatory factors and check points of the cell cycle based on the dynamic change of chromosomes in the cell division process of the haploid embryonic stem cells, and finds that some protease inhibitors can effectively inhibit haploid doubling.
It is therefore an object of the present invention to provide the use of protease inhibitors to inhibit haploid cell doubling.
Based on this new finding, it is another object of the present invention to provide compositions (e.g., culture media) and methods for culturing and/or differentiating haploid cells.
It is a further object of the present invention to provide a method for obtaining haploid cells and haploid somatic cells obtained.
It is also an object of the present invention to provide the use of the obtained haploid somatic cells.
The technical scheme of the invention is as follows.
In one aspect, the invention provides the use of a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor in inhibiting haploid cell doubling.
Preferably, the CDK1 protein kinase inhibitor is Ro 3306; the Rock protein kinase inhibitor is Y-27632;
preferably, the haploid cell is a mammalian, preferably rodent, haploid cell, more preferably a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte, or a haploid endoderm progenitor cell;
preferably, said inhibition is inhibition of diploid haploid cells during culture and/or differentiation of said haploid cells.
During the culture process of the haploid cells and/or the differentiation process of the haploid cells to other types of cells, the protein kinase inhibitor provided by the invention can inhibit the haploid cell from being doubled, maintain the haploid property and simultaneously do not influence the normal growth and differentiation of the cells.
In another aspect, the invention provides a composition for culturing and/or differentiating haploid cells, said composition comprising a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor.
Preferably, the CDK1 protein kinase inhibitor is Ro 3306; the Rock protein kinase inhibitor is Y-27632;
preferably, the composition is a cell culture medium;
preferably, the CDK1 protein kinase inhibitor is present in the composition at a concentration of 1.5-5 μ Μ, preferably 3-5 μ Μ, more preferably 4.5 μ Μ; the concentration of the Rock protein kinase inhibitor in the composition is 5-40 μ M, preferably 10-40 μ M, more preferably 40 μ M;
preferably, the haploid cell is a mammalian, preferably rodent, haploid cell, more preferably a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte or a haploid endoderm progenitor cell.
The composition provided by the invention can be used for culturing or maintaining haploid cells and can also be used for differentiating the haploid cells. The protein kinase inhibitor in the composition provided by the invention can inhibit haploid cell doubling and maintain the monoploidy without influencing the normal growth and differentiation of the cells during the culture process of the haploid cells and/or during the differentiation process of the haploid cells to other types of cells.
In a further aspect, the invention provides a method for culturing and/or differentiating haploid cells, said method comprising culturing and/or differentiating haploid cells in the presence of a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor.
Preferably, the CDK1 protein kinase inhibitor is Ro 3306; the Rock protein kinase inhibitor is Y-27632;
preferably, the haploid cell is a mammalian, preferably rodent, haploid cell, more preferably a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte, or a haploid endoderm progenitor cell;
preferably, the method comprises culturing and/or differentiating the haploid cells with the compositions provided herein.
In the above methods provided by the present invention, the CDK1 protein kinase inhibitor and/or Rock protein kinase inhibitor does not affect the normal culture and/or differentiation of haploid cells, but rather inhibits haploid cell doubling during the process, maintaining the haploid property, while not affecting the normal growth and differentiation of cells.
In yet another aspect, the invention provides a method of obtaining haploid cells, said method comprising inhibiting haploid cell doubling with a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor.
Preferably, the CDK1 protein kinase inhibitor is Ro 3306; the Rock protein kinase inhibitor is Y-27632;
preferably, said inhibition is inhibition of haploid cell doubling during culture and/or differentiation of said haploid cells;
preferably, the haploid cell is a mammalian, preferably rodent, haploid cell, more preferably a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte, or a haploid endoderm progenitor cell;
preferably, the method comprises culturing and/or differentiating the haploid cells with the compositions provided herein.
For example, the invention provides a method of obtaining a haploid neural stem cell, a haploid neural cell, a haploid cardiomyocyte or a haploid endoderm progenitor cell, comprising culturing and/or differentiating a haploid embryonic stem cell or its precursor cell with a composition provided by the invention.
In a further aspect, the present invention also provides various haploid cells obtained by the above method and the use of any of them in genetic studies.
For example, the present invention provides haploid neural cells obtained by the above method and their use in genetic studies such as neurotoxin and neurophysiological functions.
Compared with the prior art, the invention provides a new discovery that the protein kinase inhibitor has the function of inhibiting haploid cell doubling.
The inventor starts from the perspective of cell cycle, observes the dynamic change of chromosome in the process of haploid embryonic stem cell division by using H2B-GFP marked haploid embryonic stem cell, and finds that chromosome is doubled due to abnormality in the process of haploid embryonic stem cell division. Based on the fact, the inventor of the invention screens some important regulatory factors and check points of the cell cycle by small molecules, and screens out small molecule compounds capable of promoting maintenance of the haploid. In particular, inhibitors of CDK1 protein kinase and Rock protein kinase have been found to effectively inhibit haploid doubling, while inhibitors of cell cycle checkpoint protein Chk1 significantly reduce the haploid proportion of haploid stem cells.
The screened CDK1 protein kinase and Rock protein kinase inhibitor, especially Rock protein kinase inhibitor, can be widely used for culturing androgenesis and androgenesis haploid embryonic stem cells. The method is beneficial to the large-scale amplification of the haploid embryonic stem cells, greatly reduces the process of continuously purifying the haploid, simplifies the culture scheme, reduces the cost and promotes the application of the haploid embryonic stem cells.
The screened CDK1 protein kinase and Rock protein kinase inhibitor can also be widely used for differentiating parthenogenetic and parthenogenetic haploid embryonic stem cells. The invention obtains haploid somatic cells such as haploid neural stem cells, nerve cells, cardiac muscle cells, islet progenitor cells and the like, and provides an effective method for obtaining haploid somatic cells from a three germ layer. Due to the characteristics of haploid single copy genotypes, the method and the obtained haploid cells provide a good and efficient genetic model for researching specific life processes such as neurogenesis and neurogenic diseases, myocardial development and cardiovascular diseases and the like. For example, the invention screens genes for improving the resistance of nerve cells to neurotoxic manganese ions by utilizing haploid nerve cells, and provides a new vector for a specific toxicity screening experiment.
In conclusion, the new discovery and the new technical scheme based on the invention provide a basis for research and application based on haploid genomes.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
figure 1 shows the dynamic changes of chromosomes during the division of diploid (control) and haploid embryonic stem cells.
FIG. 2 shows the inhibitory effect of CDK1 and Rock protein kinase inhibitors and their concentrations on the doubling of haploid embryonic stem cells.
FIG. 3 shows inhibition of doubling by Rock protein kinase inhibitors during differentiation of haploid embryonic stem cells into haploid neural stem cells.
Fig. 4 shows the inhibition of doubling by Rock kinase inhibitors during differentiation of haploid neural stem cells into haploid neurons.
FIG. 5 shows inhibition of doubling by Rock kinase inhibitors during differentiation of haploid embryonic stem cells into haploid cardiomyocytes.
FIG. 6 shows inhibition of diploidy by Rock kinase inhibitors during differentiation of haploid embryonic stem cells into haploid endoderm progenitor cells.
FIG. 7 shows the application of the obtained haploid neural stem cells in genetic screening for neurotoxic substance resistance.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
Example 1Dynamic changes of chromosomes during haploid embryonic stem cell division
Diploid embryonic stem cell lines and parthenogenetic (parthenogenetic) haploid embryonic stem cell lines derived from fertilized eggs are respectively established from the same strain of H2B-GFP transgenic mice (introduced from Beijing Wintolite laboratory animal technology Co., Ltd.).
Culturing diploid embryonic stem cell lines and androgenesis haploid embryonic stem cells in 12-well plates. First, feeder layer cells of mice (mitomycin C (sigma, M0503) -treated embryonic fibroblasts of mice E13.5 days) were seeded on a culture well plate, and fibroblast culture fluid (high-sugar DMEM (Gibco, C12430500BT), 1 XP (100 x, Gibco, 11360- Base (DMEM/F12(Gibco, 10565018) was mixed with Neurobasal (Gibco, 21103-049)1 to 1, 1 XN 2 additive (100X, Gibco, 17502048), 1 XB 27 additive (50X, Gibco, 17504044), 2% bovine serum albumin (1000X, sigma, A8022), 1 Xbeta-mercaptoethanol (1000X, Gibco, 21985023), 1 XGlutamax (200X, Gibco, 35050-clan 061), 1. mu.g/ml insulin (Roche, 11376497001)), 10% serum replacement (Gibco, 10828028) and 1 XPenicilin-Streptomycin (Gibco, 15140163, 100;) were added, and 1 micromole of PD 5901 (LI5901, 04-0006-10), 993 micromole of steent (021, Chimilli-04-00010) and 1 micromole of inhibitory factor for leukemia (1000F 2207/ml). Flow purified haploid H2B-GFP labeled embryonic stem cells were observed for entry into M-phase divisions using a live cell workstation, with diploid H2B-GFP labeled embryonic stem cells of diploid origin as controls. FIG. 1A shows (top row) the division of diploid embryonic stem cells into metaphase chromosome condensation (left to right 1), which lines to the metaphase plate (left to right 2), which is pulled to the bipolar and is accompanied by chromosome deaggregation (left to right 4) after approximately 16 minutes into the anaphase (left to right 3). FIG. 1A also shows (bottom row) that haploid embryonic stem cells divide into chromosome condensation during the division phase, line metaphase to metaphase (left to right 1 panel), do not enter anaphase within 1 hour, i.e., chromosomes are pulled apart to the bipolar (left to right 2-3 panels), no late entry is observed for chromosomes within 3 hours after the M phase block occurs, and with chromosome deagglomeration into the terminal phase or interval (left to right 4 panels), a slippage in M phase (slippage) occurs resulting in chromosome doubling. Figure 1B uses ZEN 2011 software to quantify GFP fluorescence intensity (reflecting chromosome content) of diploid and haploid chromosome segments, respectively. FIG. 1C shows the normal M-phase and abnormal M-phase ratios at mitosis for diploid and haploid embryonic stem cells.
A number of observations indicate that at least 152 mitotic phases (chromosome agglutination into M phase) are observed for diploid cells, 100% of the mitotic phases enter the late phase (chromosomes are pulled apart and towards the two poles) within half an hour after the mid-entry phase, and chromosome agglutination occurs after the late phase. At least 160 division phases (chromosome agglutination in M phase) are observed for haploid cells, approximately 10% of the division phases have a block of more than 2 hours after entering the middle phase and do not enter the later phase (chromosomes are pulled apart and pulled to the two poles), and chromosome agglutination directly occurs.
Example 2Screening of protein kinase inhibitor for inhibiting haploid cell doubling and concentration thereof
And (I) screening protein kinase inhibitors and concentrations thereof in 12-well plates.
First, feeder cells of mice (mitomycin C (sigma, M0503) -treated mouse MEF) were inoculated into a culture well plate, and cultured with fibroblast culture solution (same as in example 1) plus 10% fetal bovine serum (Gibco, 16000-. Parthenogenetic (parthenogenetic) haploid embryonic stem cells were stained with a viable cell dye 2 μ g/ml Hoechst 33342(Invitrogen, H3570) for 15-20 minutes at 37 ℃, 1n peak was collected by flow sorting, and diploid ES was used as a control to determine the 2n peak position. The enriched haploid embryonic stem cells were evenly seeded in 12-well plates plated with feeder cells. A specific concentration of a specific type of protein kinase inhibitor was added to a culture solution of mouse embryonic stem cells (same as in example 1). DMSO at the same dose served as a control. At least 3 replicates of each protein kinase inhibitor and DMSO were set up. The results are shown in Table 1 below.
TABLE 1 Effect of different inhibitors on haploid doubling
(II) experiments are carried out by using CDK1 protein kinase inhibitor and Rock protein kinase inhibitor.
After adding the protein kinase inhibitor to the mouse ES culture solution for 12 to 15 days, 10. mu.g/ml Hoechst 33342 stained the haploid ratio of the flow analysis inhibitor group and the DMSO group, using the same procedure as in the first part above.
Figure 2A shows that 4.5 μ M CDK1 inhibitor Ro3306(BioVision, 2039-1) and any Rock inhibitor such as 20-40 μ M Y-27632 (select, S1049) significantly inhibited haploid embryonic stem cell doubling: the pure haploid control group treated with DMSO for 12 days had a haploid rate of 10%, while the experimental group treated with Ro3306 for 12 days had a haploid rate of 80%; the control group treated with DMSO had a haploid rate of 16%, while Y-27632(Y-27) treated for 12 days had a haploid rate of 92%. FIG. 2B shows the classical clonal morphology of mouse ES cells following drug treatment. And as shown in fig. 2C (immunofluorescence staining imaging was performed on a confocal laser microscopy Zeiss LSM780 or Leica two-photon confocal laser microscopy TCS Sp8 instrument), immunofluorescence staining did not affect expression of pluripotency factors OCT4(santa cruz, sc-8628), Nanog (abcam, AB80892), Sox2 (mileore, AB5603), SSEA1 (milepore, MAB4301), and the like.
Screening experiments for their concentrations were performed as described above with 1.5, 3 and 4.5 μ M Ro3306, respectively. Figure 2D shows that concentration of Ro3306 greater than 3 μ M is significant in haploid doubling inhibition, while 4.5 μ M is the most effective, at the optimal concentration, greater than 5 μ M severely affects cell survival, with at least three biological replicates. Subsequent experiments a number of more stringent independent replicates (at least 6 parthenogenetic haploid embryonic stem cells and 3 parthenogenetic haploid embryonic stem cells) were performed with Ro3306 at an optimal concentration of 4.5 μ M, verifying that Ro3306 significantly inhibited haploid embryonic stem cell culture doubling in vitro, as shown in fig. 2F.
Screening experiments for their concentrations were performed as described above with Y-27632 at 5, 10, 20 and 40. mu.M, respectively. Figure 2E shows that Y-27632 at concentrations greater than 20 μ M is significantly inhibited from haploid doubling, while at 40 μ M is the most effective, at the optimal concentration, greater than 40 μ M severely affects ES cell morphology, with at least three biological replicates. Subsequent experiments carried out a number of more stringent independent repeat experiments (at least 3 parthenogenetic haploid embryonic stem cells and 2 parthenogenetic haploid embryonic stem cells) using Y-27632 at an optimal concentration of 40 μ M, confirmed that Y-27632 significantly inhibited haploid embryonic stem cell culture doubling in vitro, as shown in fig. 2G. "Before" in fig. 2D to 2G indicates Before the experiment.
Example 3Haploid neural stem cells obtained from haploid embryonic stem cells using Rock kinase inhibitors
Flow-purified androgenesis (parthenogenetic) haploid embryonic stem cells are obtained, 5 ten thousand cells are planted in a culture dish of 3.5cm with feeder cells, clone grows out after 3-5 days, 0.25% trypsin (Gibco, 25200-072) is digested for 3-5min, 10% FBS is used for stopping digestion, 1 XPBS is used for washing once, mouse embryonic stem cell culture solution (same as example 1) without PD0325901, Chir99021 and LIF is used for suspension culture of haploid ES, and after suspension culture is carried out for 2 days, 500 embryoid balls are planted in a culture dish of 6cm paved with polylysine of 1mg/ml (sigma, P6407) and 5ug/ml laminin (invitrogen, 23017-015). After 16 hours, the medium was changed to neural stem cell culture (N2B27 basic medium (same as example 1), 20ng/ml bFGF (R & D, 233-FB-001MG) and 20ng/ml EGF (R & D, 2028-EG-200)) was added. Any Rock inhibitor, such as 20 μ M Y-27632, was added, with the same dose of DMSO as a control. After adherent differentiation for 3 days, digestion was carried out, and the cells were seeded at 1:10 passages on a 6cm petri dish plated with polylysine at 1mg/ml (sigma, P6407) and 5ug/ml laminin (invitrogen, 23017-. Further differentiation, at day 10 of differentiation, haploids were flow analyzed and sorted for purification. Fig. 3A shows the differentiation process starting with haploid ES (day 0) and differentiating for 30 days. FIG. 3B shows the results of 10 days of differentiation, with almost complete doubling of the DMSO control group haploid (haploid ratio of 1.5%) and the Y-27632 group maintaining a high haploid ratio (haploid ratio greater than 90%). FIGS. 3C-3G are graphs showing the results of measurements after 30 days of differentiation, wherein FIG. 3C shows that the Y-27632 group shows a distinct neural stem cell morphology upon further differentiation; FIG. 3D shows that karyotyping of group Y-27632 revealed that the majority of differentiated cells were 20 chromosomes; FIGS. 3E and 3F show that immunofluorescence staining identifies classical neural stem cell marker proteins, with cells from the Y-27632 group co-expressing Nestin (anti-Nestin, millipore, MAB353) at 95% or more, Sox2(anti-Sox2, millipore, AB5603), Pax6(anti-Pax6, abcam, AB5790), CD133(anti-CD133, milli biotec, 130-; FIG. 3G shows the identification of mRNA upregulation of neural stem cell marker proteins Nestin, Sox2, N-cadherin and Zfp521 by real-time quantitative PCR.
Example 4Haploid neurons from haploid neural stem cells using Rock kinase inhibitors
Haploid neural stem cells were obtained by differentiation (same as example 3).
Differentiation culture was carried out using a differentiation medium (N2B27 basal medium (same as example 1) supplemented with 1% fetal bovine serum, and with Rock inhibitor 20. mu. M Y-27632). At day 7 of stochastic differentiation, the marker protein Tuj1 for neurons was immunofluorescent-stained, FIG. 4A, left panel 1, showing a high neuronal morphology, protruding long axons and expressing Tuj1 protein (anti-Tuj1, millipore, MAB 163); FIG. 4A, right 3, shows the flow analysis Tuj1-FITC positive cell rate of 9%, Tuj1 positive cells are mostly haploid, and undifferentiated diploid neural stem cells are negative control.
Differentiation culture was performed using a differentiation medium (N2B27 basal medium (same as example 1) supplemented with 3% fetal bovine serum, and with Rock inhibitor 20. mu. M Y-27632). At day 14 of stochastic differentiation, labeled protein GFAP of immunofluorescent-stained glial cells, FIG. 4B, left panel 1, shows that there is a large amount of glial morphology, and GFAP is expressed (anti-GFAP, DAKO, Z033429); FIG. 4B, right 3, shows the ratio of GFAP-FITC positive cells in an effluent analysis of 19%, GFAP positive cells being mostly haploid, and undifferentiated diploid neural stems being negative controls.
Neurons that differentiated for 7 days were further purified and the medium was changed to neuronal maturation medium (N2B27 basal medium (same as example 1) with the addition of 100. mu.M cAMP (sigma, D0627), 20ng/ml brain-derived neurotrophic factor (NT-3, peprotech, 450-02), 20ng/ml NT-3(peprotech, 450-03) and 20ng/ml glial-derived neurotrophic factor (peprotech, 450-10)), and the addition of Rock inhibitor 20. mu. M Y-27632, maturation for 14 days, marker proteins Tuj1 and Map-2 for immunofluorescent-stained neurons, FIG. 4C shows that nearly 100% of neurons express Tuj1 and Map-2 (anti-Map-ab-2, abcam, 32454). FIG. 4D shows that the flow analysis Map-2 positive cellular haploid rate was greater than 65%. FIG. 4E shows that mature neurons co-express Tuj1 and neurosynaptic protein Syn1(anti-Syn1, millipore, AB 1543P). FIG. 4F shows the results of patch-clamp measurements of differentiated haploid neuronal electrophysiological functions with sodium ion current, potassium ion current and a certain action potential.
Example 5Haploid cardiomyocytes obtained from haploid embryonic stem cells using Rock kinase inhibitors
Flow-purified androgenesis (parthenogenetic) haploid embryonic stem cells are obtained, 5 ten thousand cells are planted in a culture dish with 3.5cm of feeder cells, clone is grown out after 3-5 days, 0.25% trypsin (Gibco, 25200-072) is digested for 3-5min, 10% FBS is used for stopping digestion, 1 XPBS is used for washing once, mouse embryonic stem cell culture solution without PD0325901, Chir99021 and LIF (same as example 1) is used for suspension culture of haploid ES, and after 2 days of suspension culture, 500 embryoid balls are planted in a culture dish with 6cm of matrix matrigel matrix (BD, 354277). After 16 hours, the cells were replaced with myocardial induction medium (DF12, 1 XB 27 additive (50X, Gibco, 17504044), 10% serum replacement (1000X, sigma, A8022), 1 XB-mercaptoethanol (1000X, Gibco, 21985023), 1 XSGlutamax (200X, Gibco, 35050-. On the fifth day, after 2 μ M IWR1 replaced GSK3 β inhibitor Chir99021 and induced for another 2 days, BMP4 and Activin A were withdrawn on the seventh day. The Rock inhibitor 20 mu M Y-27632 is added during the culture process when the culture medium is changed into the myocardial culture medium. FIG. 5A shows the differentiation process, myocardial differentiation day 12, appearance of beating myocardium, and FIG. 5B shows immunofluorescence staining to identify differentiation appearance of cardiomyocyte-specific cardiac troponin T (anti-cTnT, abcam, ab 8295). Undifferentiated mouse embryonic stem cells were used as a negative control, fig. 5C shows that haploid embryonic stem cells differentiated to obtain cTnT-positive cardiomyocytes, and fig. 5D shows that the majority of cTnT-positive cardiomyocytes were haploid.
Example 6Obtaining haploid inner embryo layer group cells from haploid embryonic stem cells by using Rock kinase inhibitor
Flow-purified androgenesis (hermaphrodite) haploid embryonic stem cells are obtained, 5 ten thousand cells are planted in a culture dish with 3.5cm of feeder cells, clone grows out after 3-5 days, 0.25% trypsin (Gibco, 25200-072) is digested for 3-5min, 10% FBS is stopped from digesting, 1 XPBS is used for washing once, mouse embryonic stem cell culture solution without PD0325901, Chir99021 and LIF (same as example 1) is used for suspension culture of haploid ES, and after 2 days of suspension culture, 500 embryoid balls are planted in a culture dish paved with fibronectin (fibronectin, millipore, fc010) for 6 cm. Endoderm induction medium (DF12, 1 XB 27 additive (50X, Gibco, 17504044), 10% serum replacement (1000X, sigma, A8022), 1 XB-mercaptoethanol (1000X, Gibco, 21985023), 1 XSGlutamax (200X, Gibco, 35050 one 061)), 1 XPenicillin-Streptomyces (Gibco, 15140163, 100X) and 100ng/ml Activin A (R & D, 338-AC-050/CF), 3 μ M GSK3 β inhibitor Chir99021 (mgsteent, 04-0004-10) were exchanged after 16 hours. And the culture is carried out by replacing endoderm induction culture solution, and a Rock inhibitor 20 mu M Y-27632 is added all the time during the culture. Fig. 6A shows the differentiation process. Specific protein PDX1 staining pancreatic islet progenitor cells after 16 days of culture. FIG. 6B shows immunofluorescence staining to identify the specific protein Pdx1(anti-Pdx1, abcam, ab47383) that differentiated into pancreatic islet progenitor cells. Undifferentiated mouse embryonic stem cells were used as a negative control, fig. 6C shows that haploid embryonic stem cells differentiated to give pdx1 positive islet cells, and fig. 6D shows that the majority of islet cells positive for pdx1 are haploid.
Example 7Application of haploid neural stem cells in genetic research and toxic genetic screening
Genetic screening for neurotoxic substance resistance was performed using haploid neural stem cells (from example 3).
The experimental procedure is shown in fig. 7A, with 1 mmol of manganese chloride treatment as a control. After 4 days of screening, the wild-type haploid neural stem cells of the control group were almost all dead, while a small number of cells in the gene trap pool survived as shown in fig. 7B (arrows indicate viable cells) (independent 3 times).
Amplifying living cells, namely the manganese chloride-resistant neural stem cells, and fig. 7C shows the results of at least 2 experiments, wherein the manganese chloride-resistant cells are subjected to high-throughput sequencing to verify that gene capture site genes are enriched in genes in biological processes such as synaptic transmission, membrane potential, neuron delivery and the like, wherein the capture rate of Park2 gene is higher in at least two experiments.
Specifically knocking out Park2 in the primary diploid neural stem cell by using Crisper-Cas9 so as to improve the manganese ion resistance of the primary diploid neural stem cell. FIG. 7D shows wild-type diploid and Park knockout primary diploid neural stem cells treated with 500 μ M MnCl2Cell morphology after 3 days of treatment, respectively, and fig. 7E shows viable cell count statistics after 3 days of treatment with 500 micromolar manganese chloride, respectively, for wild type and Park knockout primary diploid neural stem cells.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
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Claims (11)
- Use of a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor for inhibiting haploid cell doubling, wherein said CDK1 protein kinase inhibitor is Ro3306, used at a concentration of 3-5 μ Μ; the Rock protein kinase inhibitor is Y-27632, and the use concentration is 20-40 mu M; and the haploid cells are murine haploid cells.
- 2. The use of claim 1, wherein said haploid cell is a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte, or a haploid endoderm progenitor cell.
- 3. Use according to claim 1 or 2, wherein said inhibition is the inhibition of the doubling of haploid cells during their culture and/or differentiation.
- 4. A method of obtaining haploid cells, said method comprising inhibiting haploid cell doubling with a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor, wherein said CDK1 protein kinase inhibitor is Ro3306 used at a concentration of 3-5 μ Μ; the Rock protein kinase inhibitor is Y-27632, and the use concentration is 20-40 mu M; and the haploid cells are murine haploid cells.
- 5. The method of claim 4, wherein the haploid cell is a haploid embryonic stem cell, a haploid neural cell, a haploid cardiomyocyte, or a haploid endoderm progenitor cell.
- 6. The method according to claim 4 or 5, comprising culturing and/or differentiating said haploid cells with a composition comprising said Ro3306 at a concentration of 3-5 μ M used.
- 7. The method of claim 6, wherein the composition is a cell culture medium.
- 8. The method of claim 6, wherein the composition comprises Ro3306 at a concentration of 4.5 μ Μ.
- 9. The method of claim 6, wherein said composition further comprises Y-27632 at a concentration of 20-40 μ Μ.
- 10. The method of claim 9, wherein the composition comprises Y-27632 at a concentration of 40 μ Μ.
- 11. The method of claim 6, wherein said inhibiting inhibits diploid haploid cells during culture and/or differentiation of said haploid cells.
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| Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes;Hui Yang等;《Cell Research》;20130716;第23卷;第1197页右栏第4段 * |
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