CN118064355A - A method for rapidly inducing mouse totipotent stem cells in vitro and preparing blastocyst-like cells based on the induction - Google Patents
A method for rapidly inducing mouse totipotent stem cells in vitro and preparing blastocyst-like cells based on the induction Download PDFInfo
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Abstract
The invention discloses a method for preparing blastocysts based on the method for rapidly inducing totipotent sample stem cells of mice in vitro. A method for rapidly preparing totipotent sample stem cells of a mouse, comprising: the mouse embryonic stem cells are treated with a spliceosome inhibitor Pladienolide B with the concentration of 10-20nM for 3-12 hours to obtain the instant totipotent stem cells. A method for preparing blastocysts in vitro rapidly comprises the steps of using a blastocyst induction medium to resuspend totipotent sample stem cells obtained by the method of the invention, uniformly mixing, and transferring the mixture to a microporous culture plate treated by an anti-adhesion flushing fluid; the microwell plates were placed in a37℃cell incubator for 5 days. The invention provides a method for inducing the totipotent sample stem cells of mice and preparing the blastocysts based on the totipotent sample stem cells, which is faster and more convenient than the prior art, and greatly shortens the time for preparing the blastocysts in vitro.
Description
Technical Field
The invention belongs to the field of in-vitro culture of cell tissues, and relates to a method for rapidly inducing totipotent stem cells in vitro and preparing blastocysts based on the totipotent stem cells.
Background
Mouse embryos are often used to explore the dynamic process of early embryo development in mammals, and although the use of mouse embryos in animal experiments meets animal ethical requirements, it does not meet the 3R guidelines of animal research-substitution, reduction and improvement. Therefore, the mouse embryo stem cells (Embryonic STEM CELLS, mESCs) which are easy to obtain are used for constructing the blastocysts with the shape, structure and gene expression profile similar to those of the natural blastocysts of the mice in vitro, and have great application value.
However, mESCs are derived from epiblast cells of mouse blasts and can only differentiate into intrablasts, and thus, they cannot form a complete blastlike structure by aggregation and differentiation of mESCs alone. In contrast, mouse totipotent-like stem cells have the potential to differentiate bi-directionally both intra-and extra-embryonally, and should theoretically be able to serve as starting cells for the construction of blastocysts. In 2012, a mouse totipotent-like stem cell called 2-cell-like cell (2 CLC) was found to exist in culture conditions of serum+lif-based mESCs (abbreviated as mESM medium). Over the last decade, numerous researchers have focused on exploring how to obtain mouse totipotent-like stem cells in vitro that can be stably cultured for a long period of time. Until 2021, the Du Peng team at Beijing university first found that treatment of mESCs cultured in mESM medium with 2.5nM of spliceosome inhibitor Pladienolide B (PlaB) for a prolonged period of time (6-7 passages, 3-5 days per passage, at least 18 days required) successfully transformed mESCs into mouse totipotent stem cells. In 2023, the Guangzhou laboratory is full of groups, which find that the totipotent sample stem cells can be used for successfully constructing the mouse blastocysts in vitro, but the time for obtaining the mouse totipotent sample stem cells is not shortened because the induction of the mouse totipotent sample stem cells is not improved, and the construction of the mouse blastocysts is at least more than 23 days.
However, the above-mentioned mice must be subjected to long-term subculture, and the low dose PlaB still has cytotoxicity, and long-term continuous treatment can result in prolonged doubling time and reduced viability of the cells, which ultimately limits the possibility of mass production of the cells. On the other hand, the time period required for preparing totipotent stem cells is much longer than the formation period of blastocysts, resulting in a longer preparation time and lower efficiency for preparing blastocysts in this manner. These disadvantages lead to the difficulty in popularizing and applying the technology for preparing blastocysts from mouse totipotent stem cells.
Disclosure of Invention
The invention aims at providing a method for rapidly inducing totipotent sample stem cells of mice in vitro aiming at the defects of the prior art.
The invention also aims to provide a method for constructing an artificial mouse blastula in vitro based on the mouse totipotent stem cells, which provides a substitute model for researching early embryogenesis, implantation research and embryo toxicology screening of mice.
The inventors of the present invention found in the study that the short-time and high-dose treatment of the spliceosome inhibitor does not impair the survival and proliferation ability of the cells, and that both the real-time quantitative PCR and RNA sequencing results show that the cells treated in a short time will up-regulate the expression of the totipotent gene and reduce the expression of part of the pluripotent gene. These results show that cells treated with high concentrations of spliceosome inhibitors exhibit totipotent characteristics and that methods of blastocyst-like construction based on the derivation of such totipotent-like stem cells have been developed.
A method for rapidly preparing totipotent sample stem cells of a mouse, comprising: the mouse embryonic stem cells are treated by adopting a spliceosome inhibitor with the concentration of 10-20nM for 3-12 hours to obtain totipotent stem cells.
As a preferred aspect of the invention, the mouse embryonic stem cells are dissociated into single cells and then distributed on mouse feeder cells, and medium is replaced daily for no more than 48 hours using mESM medium.
As a preferred embodiment of the present invention, the mouse feeder cells are mouse fibroblasts treated with 10. Mu.g/ml mitomycin or commercially available mouse fibroblasts are used as they are.
As a further preferred aspect of the invention, the density of mouse embryonic stem cells distributed on mouse feeder cells is 1-2 tens of thousands of cells/cm 2; the mESM medium was used for 44-48 hours with fresh mESM medium changed daily.
As a preferred embodiment of the present invention, the spliceosome inhibitor is Pladienolide B, abbreviated PlaB hereinafter.
Preferably, the embryonic stem cells of the mouse are established isolated from the epiblast lineage of the mouse blasts, and are believed to have no ability to differentiate towards extraembryonic, i.e., trophoblast cells. However, after being treated by PlaB at high concentration, the mouse totipotent-like stem cells can differentiate into trophoblast-like cells and primitive endoderm-like cells in a 2D differentiation system. The present invention demonstrates that the treatment system for spliceosome inhibitors can be applied to a number of mouse embryonic stem cell lines of different origin, such as embryonic stem cell lines isolated from the C57BL/6 mouse blastula epiblast lineage (B6-mESCs) and commercially available TT2-mESCs.
The mESM medium mentioned in the present invention can be obtained by searching in any article concerning the culture of mouse embryonic stem cells, and the required components can be obtained from the market, and the specific configuration system is as follows:
A method for rapidly preparing blastocysts in vitro comprises
S1, preparing totipotent sample stem cells according to the method;
S2, re-suspending the totipotent sample stem cells obtained in the step S1 by using a blastocyst-like induction culture medium, uniformly mixing, and transferring the totipotent sample stem cells into a micropore culture plate treated by using an anti-adhesion flushing fluid;
S3, placing the micro-pore culture plate in a cell culture box at 37 ℃ for culturing for 4-6 days.
As a preferable method for treating the micro-porous culture plate by using the anti-adhesion flushing liquid, AGGREWELL TM 400 micro-porous culture plate is treated by using the anti-adhesion flushing liquid for more than 30 minutes, the flushing liquid is discarded, and PBS is used for flushing.
Different initial cell inoculum sizes in S2 affect the subsequent blastocyst-like formation, including blastocyst-like diameter and blastocyst-like formation efficiency. In a preferred embodiment of the present invention, as a preferred embodiment of the present invention, the initial totipotent-like stem cells are seeded in an amount of 6 to 25 cells per microwell. Further preferably, when the cell inoculum size is 6-15 cells per microwell, the diameter of the formed blastocyst is more similar to that of a mouse natural blastocyst. More preferably, the cell inoculum size is 8-12 cells per microwell, for example 8, 10 or 12 cells per microwell.
As a preferable mode of the invention, the blastocyst-like induction medium is prepared by the following steps:
Constant volume according to 50 ml;
wherein the N2B27 basal medium is prepared according to a final volume of 50ml and comprises the following components:
wherein the TSC basal medium is formulated in a final volume of 50ml and has the composition:
the method for constructing the blastocyst-like comprises the following steps:
(1) Based on the culture condition of the mouse embryo stem cells, the totipotent stem cells can be induced to generate by short treatment of the high-concentration spliceosome inhibitor PlaB, and the totipotent stem cells are determined as the cell sources for constructing the blastocysts.
(2) And (3) collecting the cells in the step (1), and performing in-vitro assembly by using AGGREWELL microporous culture plates, wherein the specific culture medium is induced for 5 days, so that a model similar to a mouse blastula structure can be obtained.
(3) According to the blastocyst-like structural model obtained by the method, the evaluation is carried out in terms of size, morphology, molecular characteristics, gene expression and the like, and the difference between the blastocyst-like structural model and a mouse natural embryo (E3.5-E4.5) is compared, on the other hand, an embryo in vitro culture system is used for in vitro culture, the development after early embryo implantation is simulated, and a research model is provided for early embryo development research.
In the invention, in order to verify that the mouse embryo stem cells treated by the high-concentration spliceosome inhibitor PlaB have totipotency, a 2D differentiation experiment can be carried out by using a blastocyst-like induction culture medium, and three cell lineages of OCT4 protein, CDX2 protein and GATA6 protein corresponding to the blastocyst stage of the mouse can be detected by utilizing an immunofluorescence technology. The invention also includes verifying totipotency of totipotent-like stem cells under a 2D differentiation system, for example, using the following method:
(1) Treating mouse embryonic stem cells cultured in mESM medium with a high concentration of a spliceosome inhibitor PlaB;
(2) Using 0.05% pancreatin to digest totipotent stem cells into single cells, re-inoculating onto laminin coated pore plates, and culturing for 5 days using blastocyst-like induction medium;
(3) OCT4 protein, CDX2 protein and GATA6 protein are detected by immunofluorescence staining technique. Preferably, conventional mouse embryonic stem cells are cultured to 44-48 hours or at about 80% confluence with the splice inhibitor PlaB, the time to begin treatment being dependent on the growth state of the mouse embryonic stem cells.
Preferably, the present invention contemplates two sources of embryonic stem cell lines, respectively TT2mESCs from the commercial market and B6-mESCs isolated from the C57BL/6 mouse blastula epiblast lineage. The embryonic stem cell lines of different origins and different cell densities may respond differently to the spliceosome inhibitor, the specific treatment time being dependent on the cell state. In a preferred embodiment of the invention, the spliceosome inhibitor is treated for 3 to 9 hours, more preferably 6 to 8 hours.
Preferably, the treatment time of the spliceosome inhibitor is such that it does not affect the status of the mouse embryonic stem cells. In a preferred embodiment of the invention, the test concentration of spliceosome inhibitor PlaB is 10-20nM.
Preferably, the AGGREWELL microwell culture plates are from STEMCEL company, cat: #34415.
Preferably, anti-ADHERENCE RINSING Solution (Anti-adhesion rinse) is used, manufacturer STEMCEL, cat# 07010, which is treated for more than 30 minutes at room temperature.
In the present invention, cells to be cultured are usually placed in a cell culture tank for culture. For example, the temperature of the cell culture is 37 ℃,5% co 2.
The innovation point of the invention is that:
1. The mouse totipotent stem cell is induced fast by means of high concentration of spliceosome inhibitor PlaB, has the characteristics of mouse totipotent cell and capacity of differentiating into blastula to three lineages, and provides one blastula-like constructing method based on the method.
2. The method for constructing the mouse blastula takes the mouse totipotent sample stem cells generated by the rapid induction as initial cells, shortens the induction time of the totipotent sample cells to a great extent, and can be used for a blastula-like formation experiment after 6-9 hours of induction.
3. The blastocyst-like embryo derived from the totipotent sample stem cells generated by rapid induction well simulates the morphological structure and transcriptome characteristics of the embryo before implantation of the mouse, and provides a more convenient substitution model for researching early embryogenesis, implantation research and embryo toxicology screening of the mouse.
The invention has the beneficial effects that:
The invention takes mESCs cultured in mESM culture medium as initial cells, proposes to treat mESCs in a dosage ten times that of PlaB in the prior art in a short time, and the mice totipotent sample stem cells are obtained and the blastocyst-like cells are prepared while the totipotent of the mice is endowed to the maximum extent and the survival and proliferation capacities of the mice are not damaged. Compared with the prior art, the method greatly shortens the culture period of preparing the blastocysts based on the totipotent stem cells, reduces the culture cost and the technical difficulty, and is easier to popularize and apply. In addition, the blastocysts prepared by the method are closer to the natural blastocysts of the mice in transcriptome level than the blastocysts derived by the prior art, and a research model closer to the natural blastocysts of the mice is provided for researching early embryogenesis, implantation research and embryo toxicology screening of the mice.
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In order to more clearly illustrate the technical solutions of the present application, the drawings that are required to be used in the embodiments will be briefly described below, and it is apparent that each of the drawings in the following description is directed to some embodiments of the present application.
FIG. 1 shows the effect of different PlaB treatment concentrations and treatment times on TT 2-mESCs. Wherein, A represents the bright field state diagram of TT2-mESCs at PlaB different doses and different treatment durations. Since TT2-mESCs appeared to be significantly apoptotic after both 9 and 12 hours, the subsequent experiments excluded the 9 and 12 hour groups. The B diagram corresponds to the expression condition of the totipotent genes MERVL and ZFP352 of TT2-mESCs in the (A) after different concentrations and different treatment time periods.
FIG. 2 shows the gene expression of the mouse totipotent stem cells induced by the above method. Wherein, the A graph shows the expression condition of typical totipotent genes and multipotent genes of TT2-mESCs treated by different doses PlaB for corresponding durations. Panel B shows the change in the expression level of TT2-mESCs after treatment for 6 hours at 20nM PlaB compared to the transcriptome of untreated cells.
FIG. 3 shows the formation of B6-mESCs induced totipotent-like stem cells and derived blastocysts of mice in the present invention. FIG. A shows a schematic representation of the formation of totipotent stem cells from mouse embryonic stem cells after treatment for 6 hours with 20nM PlaB and the preparation of blastocysts based thereon. Panel B shows the bright field image of cells before and after treatment of B6-mESCs for 6 hours with 20nM PlaB. Panel C shows the formation of blastocysts in AGGREWELL well plates after treatment of B6-mESCs for 6 hours at 20nM PlaB. Panel D shows the case in the AGGREWELL well plate of (B), including blastula, solid cell mass and blank wells.
FIG. 4 shows TT2-mESCs induced formation of totipotent stem cells and derived blastocysts in mice in accordance with the present invention. Panel A shows the bright field image of cells of TT2-mESCs before and after 20nM PlaB treatment. Panel B shows the formation of blasts from TT2-mESCs in AGGREWELL well plates after PlaB induction. Panel C shows bright field pictures of blastocyst-like structures prepared with TT2-mESCs and totipotent-like stem cells induced by TT2-mESCs as starting cells, respectively, wherein only loosely connected cell masses can be produced with TT2-mESCs as starting cells. Panel D shows the conditions in AGGREWELL well plates, including blasts, solid cell clusters and blank wells, when blasts were prepared with TT2 mESCs and totipotent stem cells induced with TT2 mESCs as starter cells, respectively.
FIG. 5 shows the identification of the presence of cell lineages in TT 2-like blasts. Panels a-B show immunofluorescent staining of CDX2 and OCT4 or OCT4 and GATA6 of the mouse natural blastula and TT 2-like blastula, and the right bar graph shows the ratio of different types of natural blastula or TT 2-like blastula in all samples tested. Panel C shows the proportion of cells of each lineage present in the mouse natural blastula and TT 2-like blastula.
FIG. 6 shows key biological events that closely mimic the development of a mouse embryo prior to implantation during blastocyst-like development. Panels a-B show localization of zona-1 in the cell mass formed during blastocyst-like formation of mouse totipotent-like stem cells and statistics of the proportion of ZO-1 positive cell mass. The mouse natural mulberry embryo is used as a positive control. Panels C-D show localization of polarized protein Ezrin in cell clusters formed during blastocyst-like formation of mouse totipotent-like stem cells and statistics of the proportion of Ezrin positive cell clusters. The mouse natural mulberry embryo is used as a positive control. Panels E-F show localization of activated YAP1 protein within the cell mass formed during blastocyst-like formation of mouse totipotent-like stem cells and statistics of the proportion of cell mass positive for the protein.
FIG. 7 shows the results of RNA sequencing and analysis of mouse embryonic stem cells using blastocysts formed after 20nM PlaB hours of treatment as samples. The figure shows that the blastocyst-like cell derived from totipotent stem cells in the invention is very similar to the natural blastocyst of a mouse. Among them, panel a shows the expression levels of typical genes of epiblast (Epiblast, EPI), trophoblast (Trophectoderm, TE) and primitive endoderm (PRIMITIVE ENDODERM, prE) during blastocyst-like formation. Panel B shows the Principal Component Analysis (PCA) of the transcriptome of mouse 16-cell embryos, E3.5 and E4.5 blasts, TBLC-and EPS-like blasts, and totipotent stem cell-derived blasts of the invention (i.e., "blasts" in the figures (this study) "). Panel C shows a comparison of differential genes of blastocysts of the present invention with mouse E3.5 blastocysts or E4.5 blastocysts. Panels D-E show the results of GO analysis between the blastocysts of the present invention and either the E3.5 blastocysts or the E4.5 blastocysts. Panel F shows the expression of marker genes in each lineage of blastocysts in blastocysts of the present invention and E3.5 and E4.5 blastocysts.
Detailed Description
The invention mainly relates to a novel method for constructing a mouse blastula in vitro by utilizing a mouse totipotent stem cell, which comprises the following steps:
1. obtaining mouse embryo stem cells cultured in vitro;
2. treating the mouse embryonic stem cells under mESM medium culture conditions with a high concentration of a spliceosome inhibitor PlaB, and marking the mouse embryonic stem cells as a starting point day 0;
3. collecting the totipotent stem cells treated by the spliceosome inhibitor PlaB for 6-12 hours;
4. Treating AGGREWELLTM400,400 microporous culture plates with anti-adhesion flushing liquid for more than 30 minutes, discarding the flushing liquid, and flushing with PBS for later use;
5. Re-suspending the cells obtained in the step S3 by using a blastocyst-like induction medium, uniformly mixing, and transferring the cells to a micropore culture plate treated in the step S4;
6. The microwell plates were placed in a 37℃cell incubator for 5 days.
Example 1: the concentration and treatment time of the spliceosome inhibitor PlaB treatment were explored.
The purpose is as follows: obtaining the totipotent sample stem cell with high proliferation speed and survival capacity and high expression totipotent gene.
The method comprises the following steps:
1. Mouse embryonic stem cells cultured in mESM medium were taken and digested into single cells, which were inoculated onto mitomycin-treated mouse fibroblasts. Here, the mouse embryonic stem cells are commercially available TT2-mESCs.
2. After 48 hours, the fresh mESM medium was changed. The time was again recorded as 0h and PlaB treatments of 10nM and 20nM, respectively, were added to the medium and the cell status was noted and cell pellet collected every 3 hours.
3. The totipotent marker genes were evaluated on 0h, 3h and 6h cells untreated with PlaB and 10nM and 20nM nM PlaB using real-time quantitative RT-PCR technique.
4. Further gene expression detection and transcriptome level analysis were performed on 6h of cells not treated with PlaB and 20: 20nM PlaB using real-time quantitative RT-PCR and RNA sequencing techniques.
Results and discussion:
Because PlaB has strong cytotoxicity, the treatment time of PlaB must be controlled within a proper range. The bright field image (panel A of FIG. 1) shows that the state of mouse embryonic stem cells does not change significantly when they are treated with PlaB at 10nM and 20nM for 3, 6 hours, whereas apoptotic cells appear around the clones when the treatment time is prolonged to 9 hours, and a large number of cells die by 12 hours. Real-time quantitative PCR showed that the expression levels of the totipotent marker genes MERVL and ZFP352 were higher in cells treated for 6 hours with 20nM PlaB than in cells treated for 10nM PlaB for the same time (fig. 1, panel B). Subsequently, we focused on the effect of 20nM PlaB treatments on the pluripotency and totipotency gene expression of mouse embryonic stem cells. The 20nM PlaB treatment resulted in cells that were highly expressing totipotency genes from 3 hours and exhibited more significant elevation at 6 hours compared to untreated cells (panel a of fig. 2); whereas the expression of the pluripotency gene is not significantly affected. This is further demonstrated by the results of RNA sequencing techniques (panel B of FIG. 2).
In order to achieve both good cell status and higher expression of the totipotent gene, 20nM PlaB hours of treatment was considered to be the most suitable treatment regimen.
Example 2: obtaining the totipotent sample stem cells of the mice and preparing the blastocyst-like based on the totipotent sample stem cells.
The purpose is as follows: obtaining the totipotent sample stem cells of the mice and deriving the totipotent sample stem cells to form the blastula structures of the mice based on the totipotent sample stem cells.
This embodiment is divided into two aspects, namely:
first aspect: a method of inducing totipotent sample stem cells in a mouse using an optimized PlaB treatment regimen, the method comprising:
1. Mouse embryonic stem cells cultured in mESM medium were taken and digested into single cells, which were inoculated onto mitomycin-treated mouse fibroblasts. Here, the mouse embryonic stem cells are commercially available TT2-mESCs, respectively, and embryonic stem cell lines (B6-mESCs) isolated from the C57BL/6 mouse blastula epiblast lineage.
2. When 48 hours after inoculation (cell confluence reached 80%), fresh mESM medium was changed and treated with 20nM splice inhibitor PlaB in medium for 6 hours.
Results and discussion:
The invention provides a novel method for rapidly deriving totipotent stem cells from conventional mouse embryonic stem cells (A diagram of FIG. 3), wherein the treatment scheme of PlaB is optimized, and the method has little influence on the state of the mouse embryonic stem cells whether the B6-mESCs (B diagram of FIG. 3) obtained by separation or the commercially available TT2-mESCs (A diagram of FIG. 4) are acted. The proposal greatly shortens the induction time of the mice totipotent sample stem cells and provides a new idea for the establishment of the mice totipotent sample stem cells.
Second aspect: the method for constructing the blastocyst-like cell in vitro by utilizing the totipotent sample stem cell comprises the following steps:
(1) Treatment with 20nM spliceosome inhibitor PlaB for 6 hours successfully induced totipotent-like stem cells, which were digested into single cells using 0.05% pancreatin, and cell pellet was collected by centrifugation as starting cells for in vitro construction of mouse blasts.
(2) Preparing a blastocyst-like induction medium:
Constant volume according to 50 ml;
Wherein the N2B27 basal culture is prepared according to a final volume of 50ml, and comprises the following components:
wherein the TSC basal culture is prepared according to a final volume of 50ml and comprises the following components:
(3) AGGREWELL TM 400 microwell plates were treated with anti-adhesion rinse for 40 minutes, the liquid was discarded, and washed once with PBS.
(4) The totipotent stem cells were resuspended and counted using blastocyst-like induction medium, transferred to AGGREWELL TM 400,400 microwell plates, and the microwell plates were placed in 37℃incubator to be cultured as day0.
(5) Half of the blastocyst-like induction medium was changed daily, culture was continued for 4 days, and blastocysts were harvested on the fifth day.
Results and discussion:
The TT 2-derived totipotent stem cells obtained in PlaB treatment protocol spontaneously aggregated to form a tight cell mass on the first day after inoculation into the microwell culture plates, the volume of which increased continuously with the time of incubation (panel B of FIG. 4), and blastocysts of the typical mouse blastocyst morphology were collected on the fifth day, in contrast to the untreated TT2 cell aggregation to form only a loose cell mass (panel C of FIG. 4). By counting the total microwell plates, we found that cells after PlaB induction formed blastlike structures with an efficiency of 5.53% ± 0.31% of total microwells, whereas only 0.14% ± 0.04% of microwells inoculated with embryonic stem cells not induced by PlaB formed blastlike structures (panel D of fig. 4).
Likewise, the method is equally applicable to B6-mESCs. B6-derived totipotent stem cells can also aggregate and differentiate in microwell plates after PlaB treatment and form blastocysts on the fifth day (panel C of FIG. 3). By counting the microplates, the B6-derived blastocysts after PlaB treatment accounted for 4.14% + -0.26% of all microplates (FIG. 3, panel D).
The results show that the method can successfully convert the mouse embryo stem cells into totipotent stem cells, and based on the totipotent stem cells, the blastocyst-like structure is prepared, and the method which is more convenient and has shorter preparation period is provided for the construction of novel blastocysts.
Example 3: identification and analysis of blastocysts derived from totipotent stem cells.
The purpose is as follows: and evaluating the different points of the blastocyst derived from the totipotent stem cells and the natural blastocyst of the mice, and analyzing the advantages of the novel mouse blastocyst.
This embodiment is divided into three aspects, namely:
First aspect: and (3) carrying out statistical analysis on the positioning and the existing proportion of the cell lineage marker proteins of the blastocysts by utilizing an immunofluorescence technology, and comparing the cell lineage marker proteins with the natural blastocysts of the mice.
The method comprises the following steps:
1. collecting TT 2-blastocysts derived from totipotent-like stem cells induced by TT 2;
2. Identifying the existence of marker proteins corresponding to three cell lineages in the natural blastula period of the mice in blastula-like by using immunofluorescence staining technology;
3. The localization of each positive cell was recorded under a fluorescence microscope and the proportion of different positive cells present in the blastocysts was calculated.
Results and discussion:
The invention confirms that CDX2, OCT4 and GATA6 positive cells exist in TT 2-blastocysts through immunofluorescence technology, and the positioning is similar to that of a natural blastocyst of a mouse, but part of TT 2-blastocysts still have no CDX2 positive cells or OCT4 positive cells (A-B diagram of figure 5). The corresponding cell lineages were indicated by positive cells, and the results showed that they also closely resemble the lineage constitution of the mouse natural blastula, although some cells in TT 2-like blasts did not belong to these three cell lineages, accounting for 4.4% + -0.7% of the total cells (panel C of FIG. 5).
Second aspect: using immunofluorescence techniques, it was assessed whether key biological events of embryo development prior to implantation in mice could be reproduced during blastocyst-like formation, which consisted mainly of densification, polarization and nuclear entry of YAP1 proteins.
The method comprises the following steps:
1. Cell aggregates collected in AGGREWELL TM 400 well plates for 24, 48 and 72 hours, respectively;
2. Staining the compact junction protein ZO-1 indicating densification, the polar proteins Ezrin and YAP1 indicating polarization phenomenon by immunofluorescence staining technology;
3. the presence and localization of positive cells was recorded under a fluorescence microscope.
Results and discussion:
The immunofluorescence technology proves that all cell aggregates are densified in the development process of the blastocyst-like, and the tight junction protein ZO-1 is highly expressed between cells (A-B diagram of FIG. 6); polar protein Ezrin begins to localize to the periphery of a portion of the cell aggregates 48 hours after cell aggregation, with the occurrence rate rising to about 80% from about 50% at 48 hours to about 72 hours (C-D plot of fig. 6); YAP1 protein appeared in the nuclei of peripheral cells 48 hours after cell aggregation, and the proportion of appearance increased to about 40% with the time of about 30% at 48 hours to about 72 hours (E-F diagram of fig. 6). These phenomena all demonstrate that the novel blastocysts of the present invention can reproduce key biological events of pre-implantation embryos of mice during formation, including densification, polarization and nuclear entry of YAP1 proteins.
Third aspect: the blastocysts were evaluated at the transcriptome level using RNA sequencing techniques.
The method comprises the following steps:
1. Collecting cell aggregates produced by totipotent-like stem cells daily for 5 days; .
2. Performing transcriptome level evaluation on blastocysts at each time point by using an RNA sequencing technology;
3. Transcriptome level analysis was performed on the finally obtained blastocysts using RNA sequencing techniques and compared with the natural blastocysts of mice.
Results and discussion:
The present invention compares the expression levels of marker genes defining EPI, TE and PrE cell lines during blastocyst-like development, and the results show that the expression of the genes associated with TE cell lines (Tfap c, gata2 and Gata 3) was significantly increased during blastocyst-like formation, whereas the expression of the genes associated with EPI and PrE cell lines (Esrrb, dppa3 and Nr5a2 at EPI, gata6, col4a1 and Sox17 at PrE) was not significantly increased (FIG. 7A). PCA analysis of blastocysts with mouse natural 16-cell stage embryos and E3.5, E4.5, and TBLC-and EPS-type blastocysts revealed that the blastocysts formed by the present invention tended to lie more between E3.5 and E4.5, while TBLC-type blastocysts were closer to E4.5 (panel B of FIG. 7). ; the differential genes of the blastoid and E3.5 and E4.5 respectively show that E3.5 has 1604 up genes and 3226 down genes compared with the blastoid; e4.5 has 979 up genes and 2869 down genes compared to blastocysts (panel C of FIG. 7). GO analysis between blastocysts and E3.5 blastocysts showed that these up-regulated genes are mainly enriched in "ribonucleoprotein complex biosynthesis", "phospholipid metabolic processes" and "purine ribonucleotide metabolic processes", which have been shown to contribute to differentiation of the TE lineage (panel D of fig. 7). In addition, down-regulated gene enrichment is in processes associated with cell cycle and down-regulating organelle tissue. GO analysis between blastocysts and mouse E4.5 blastocysts showed that up-regulated genes are mainly enriched in "ribosomal subunits", "mitochondrial translation" and "cellular respiration", which are also involved in differentiation of trophoblast cell lines, whereas down-regulated genes are enriched in down-regulated phosphometabolic and catabolic processes (E panel of fig. 7). By comparing the representative genes of all cell lineages in the blastula period of the mice, the results show that the blastula-like has no lower expression level than the natural blastula of the mice, whether Polar TE or Mural TE (panel F of FIG. 7). These results all show that the totipotent stem cells have strong potential for differentiation of trophoblast cells, and the derived blastocysts well simulate the development of the natural blastocysts of mice to a certain extent, which becomes a powerful cell tool for understanding the development events before implantation of mice.
Reference to the literature
(1)Deng,Q.,D.,Reinius,B.,&Sandberg,R.(2014).Single-cell RNA-seq reveals dynamic,random monoallelic gene expression in mammalian cells.Science 343,193-196.
(2)Hu,Y.,Yang,Y.,Tan,P.,Zhang,Y.,Han,M.,Yu,J.,Zhang,X.,Jia,Z.,Wang,D.,&Yao,K.(2023).Induction of mouse totipotent stem cells by a defined chemical cocktail.nature 617,792-797.
(3)Li,R.,Zhong,C.,Yu,Y.,Liu,H.,Sakurai,M.,Yu,L.,Min,Z.,Shi,L.,Wei,Y.,&Takahashi,Y.(2019).Generation of blastocyst-like structures from mouse embryonic and adult cell cultures.Cell 179,687-702.e618.
(4)Mohammed,H.,Hernando-Herraez,I.,Savino,A.,Scialdone,A.,Macaulay,I.,Mulas,C.,Chandra,T.,Voet,T.,Dean,W.,&Nichols,J.(2017).Single-cell landscape of transcriptional heterogeneity and cell fate decisions during mouse early gastrulation.Cell reports 20,1215-1228.
(5)Shen,H.,Yang,M.,Li,S.,Zhang,J.,Peng,B.,Wang,C.,Chang,Z.,Ong,J.,&Du,P.(2021).Mouse totipotent stem cells captured and maintained through spliceosomal repression.Cell 184,2843-2859.e2820.
(6)Xu,Y.,Zhao,J.,Ren,Y.,Wang,X.,Lyu,Y.,Xie,B.,Sun,Y.,Yuan,X.,Liu,H.,&Yang,W.(2022).Derivation of totipotent-like stem cells with blastocyst-like structure forming potential.Cell Research 32,513-529.
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Claims (9)
1. A method for rapidly preparing totipotent sample stem cells of a mouse, comprising: the mouse embryonic stem cells are treated by adopting a spliceosome inhibitor with the concentration of 10-20nM for 3-12 hours to obtain the totipotent sample stem cells of the mice.
2. The method of claim 1, wherein the mouse embryonic stem cells are dissociated into single cells and then distributed on mouse feeder cells using mESM medium for no more than 48 hours.
3. The method of claim 2, wherein the mouse feeder cells are mitomycin treated mouse fibroblasts, or commercially available mouse fibroblasts.
4. The method of claim 1, wherein the spliceosome inhibitor is Pladienolide B.
5. A method for rapidly preparing blastocysts in vitro, comprising the steps of:
S1, preparing totipotent sample stem cells according to the method of any one of claims 1-4;
S2, re-suspending the totipotent sample stem cells obtained in the step S1 by using a blastocyst-like induction culture medium, uniformly mixing, and transferring the totipotent sample stem cells into a micropore culture plate treated by using an anti-adhesion flushing fluid;
S3, placing the micro-pore culture plate in a cell culture box at 37 ℃ for culturing for 4-6 days.
6. The method of claim 5, wherein the anti-adhesion rinse is applied to the microplate by treating AGGREWELL TM 400,400 microplates with the anti-adhesion rinse for more than 30 minutes, discarding the rinse, and rinsing with PBS.
7. The method of claim 5, wherein the average number of totipotent-like stem cells seeded per microwell in S2 is 8-12.
8. The method of claim 5, wherein the blastocyst-like induction medium is configured as follows:
Constant volume according to 50 ml;
wherein the N2B27 basal medium is prepared according to a final volume of 50ml and comprises the following components:
wherein the TSC basal medium is formulated in a final volume of 50ml and has the composition:
9. the method of claim 5, wherein half of the blastocyst-like induction medium is replaced daily in S3.
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