CN115927166A - A method for the isolation and establishment of a rat primitive endoderm stem cell - Google Patents

Abstract

The invention discloses a method for separating and establishing a rat primitive endoderm stem cell line, which comprises the steps of separating a rat implantation embryo, culturing a system and identifying a cell line. The embryo 8.5 days after implantation of SD rat is taken as a research object, and the embryo 8.5 days after fertilization of the SD rat is separated to separate the primary endoderm stem cell after implantation, so that the method can be used for the model of early embryo development in vitro research and the exploration of regenerative medicine (stem cell treatment), and makes up the defect of the separation source of the prior rat primary endoderm stem cell.

Description

A method for the isolation and establishment of rat primitive endoderm stem cells

technical field

The invention belongs to the field of methods for isolating and establishing lines of rodent primitive endoderm stem cells, and relates to a method for isolating and establishing lines of primitive endoderm stem cells of rats, in particular to a method for isolating and establishing lines of primitive endoderm stem cells of rat embryos after implantation .

Background technique

Primitive endoderm stem cells (Extraembryonicendodermstemcell, XEN) are derived from the primitive endoderm (primaryendoderm, PrE) produced by the inner cell mass (ICM) in mammalian blastocysts, and have the ability of self-renewal and multilineage differentiation of stem cells. Changes gradually with embryonic development. In the early stage of mammalian development, the fertilized egg undergoes multiple cleavages to form a blastocyst, and the ICM in the blastocyst expresses the characteristic markers of PrE cells and EPI (epiblast) cells at the same time. At E4.5, the two lineages of PrE-specific marker Gata6 ⁺ cells and EPI marker Nanog ⁺ cells were spatially separated (Nakai-FutatsugiYetal., 2015). PrE cells migrate to the blastocoel side and further differentiate into visceral endoderm (visceral endoderm, VE) and parietal endoderm (parietal endoderm, PE) (NiakanKKetal., 2013). VE cells cover the proximal end of the extraembryonic region and interact with the embryonic gut endoderm at the distal end of the embryonic side, while PE cells secrete a large amount of basement membrane protein - laminin - which together with the trophoblast giant cell layer form the "Reichert membrane" ( HoganBL et al., 1984). VE becomes the main component of visceral yolk sac, and VE is the earliest cell involved in hematopoiesis (TolesJFetal., 1989; McGrathKE, PalisJ2005), which forms blood islands and endothelial cells by expressing Indianhedgehog and vascular endothelial growth factor (VEGF) (Byrd et al., 2002; Damer et al., 2002). PE is involved in the formation of the cavity wall yolk sac (ZhangYetal., 2018). VE and PE act as "early placenta", especially before the establishment of the mother-infant placental cycle, participate in the protection and nourishment of the embryo, and are responsible for the exchange of nutrients and waste (Cross et al., 1994, Igarashi He et al., 2018).

In 2005, Kunath et al. took the lead in isolating XEN cells from mouse blastocyst embryos (Kunath Tetal., 2005), which opened the way for the research of XEN cells. In 2009, Debeb et al. isolated extraembryonic endoderm precursor stem cells (extraembryonic endoderm precursor, XEN-P) from rat blastocysts. The XEN-P cell line highly expresses Oct4, SSEA, Gata6 and Gata4, so it is named extraembryonic endoderm precursor (XEN-P) (Debebetal., 2009). In 2018, Zhong et al. reported the isolation of mouse primitive endoderm stem cell (pXEN) lines from mouse blastocysts, which expressed Oct4 and had characteristics similar to rat XEN-P cells. pXEN cells are highly similar to XEN cells in terms of morphology, gene expression profiles and lineage contributions. pXEN cells can be transformed into XEN-like cells, and pXEN cells are more representative and typical than XEN cells of blastocyst PrE (Zhong et al., 2018). Transcription factors (transcription factors TFs) mediate CR to obtain iXEN cells, overexpress exogenous TFs (Gata3, Eomes, Tfap2c, Myc and Esrrb (GETME) to reprogram MEFs into iXEN cells, and XEN-like cells highly express Oct4 (BenchetritHetal.,2019 ). XEN cell blastocyst injection is mainly involved in the chimerism of extraembryonic lineage PE, and rarely involved in the chimerism of VE (KunathTetal., 2005). At the same developmental stage, XENP cells expressing Oct4 have more Great plasticity (GrabarekJBetal., 2012).

Factors maintaining biological properties of XEN cells

The biological properties of endogenous XEN cells are mainly regulated by transcription factors (TFs) and the epigenetic state of cells. TFs (Gata4, Gata6, and Sox17) played an important role in regulating the formation of XEN cells during the formation of XEN precursor cells at the blastocyst stage or the induction of XEN cells by PSCs in vitro. Studies have shown that Gata6 is not only located downstream of FGF4/MAPK signaling pathway effector Grb2, but also upstream of secondary XEN genes (Gata4, PDGFRa and Sox17) during PrE development. Gata6 can quickly and directly inhibit the pluripotency gene regulatory network and concomitantly activate the XEN gene (WamaithaSE etal., 2015; SchrodeNetal., 2014). There is also an important stemness factor Sall4 in XEN cells, and Sall4 is located upstream of Gata4 and Gata6. Sall4 functions as an important lineage-specific gene activator in XEN cells (LimCYetal., 2008; EllingUetal., 2006). After the establishment of an expression network of lineage-specific TFs, the transcriptional memory of each precursor cell is maintained through epigenetic modifications (DNA methylation, histone modifications, and microRNAs). DNA methylation is generally considered to be a stable epigenetic modification that can compact chromatin and repress genes (Rugg-GunnPJ et al., 2010; SennerCE etal., 2012; MoroLNetal., 2018; ZengZLetal ., 2018).

The ERK signaling pathway plays an important role in maintaining the development of XEN progenitor cells in blastocysts (NicholsJ et al., 2009). In blastocysts, the effect of ERK signaling on the development of XEN progenitor cells relies on fibroblast growth factor 4 (fibroblast growth factor 4, FGF4) (Azami Te et al., 2019). At the same time, the proliferation of XEN cells requires the RTK family member platelet-derived growth factor (platelet-derived growth factor, PDGF), and the proliferation ability of XEN is reduced in the absence of PDGF (RalstonA., 2018). XEN cells induced by Nodal and BMP4 can differentiate to VE both in vivo and in vitro (ArtusJetal., 2012; PacaAetal., 2012; Kruithof-deJulioMetal., 2012).

In summary, the close relationship between XEN cells and EPI in development, and the compatibility of gene expression between XEN cells and PSCs, XEN cells become a transitional state bridging the interconversion between somatic cells and pluripotent cells. XEN cells play a vital role in directing the differentiation of embryonic cell types, so XEN cells can be used to simulate the development process in vitro, using XEN cells or XEN cell culture conditions to influence and guide the differentiation of PSCs, studies have shown that XEN cells can be used Promote the differentiation of hematopoietic cells and cardiomyocytes (Paca Aetal., 2012; BrownKetal., 2010). Through the induction of ciXEN cells, functional liver cells and neurons can be formed (Lietal., 2017), which provides a large source of functional cells for cell therapy after tissue and organ lesions. With the deepening of stem cell therapy and organoid research, the isolation and establishment of XEN cells, molecular characteristics and functional research are urgent problems to be solved.

At present, the sources of primitive endoderm stem cells that have been reported in mice mainly include prebed embryos (blastocysts), postimplantation embryos, small molecule compounds induce somatic cell reprogramming to generate ciXEN cells, and transdifferentiation of pluripotent stem cells. However, there are only reports on the isolation of primitive endoderm stem cells from blastocysts in rats, and it is not clear whether there are other sources of primitive endoderm stem cells in rats.

Contents of the invention

In order to make up for the deficiency of the existing isolation source of rat primitive endoderm stem cells, the present invention provides a method for efficiently separating and establishing a line from rat postimplantation embryos to obtain postimplantation rat primitive endoderm stem cells. The specific method is as follows:

A method for isolating and establishing a line of rat primitive endoderm stem cells, the steps comprising:

Step 1: Obtain the post-implantation embryo, dissect the female rat 8.5 days after fertilization and cut the uterine implantation point to obtain the post-implantation embryo;

Step 2: Isolate the extraembryonic tissue corresponding to the primitive endoderm lineage from the implanted embryo, culture it in the primitive endoderm stem cell culture system and place it in a carbon dioxide cell incubator, and obtain rat primitive endoderm stem cells after subculture cell line.

Further, it also includes: Step 3: identification of rat primitive endoderm stem cell lines.

Further, in the second step, primitive endoderm stem cells are isolated from post-implantation embryos of rats to establish a line.

Further, the primitive endoderm stem cell culture system in the second step is obtained by culturing under the conditions of fetal bovine serum (FBS), leukemia inhibitory factor (LIF) and an activator of Wnt signaling pathway (CHIR99021).

Further, the incubator in the second step is a carbon dioxide cell incubator with a constant temperature of 37°C and 5% CO ₂ .

Further, in the second step, the interval between changing the medium is 2 days, and the cells are subcultured when the density reaches 80%.

Further, the method takes 25-30 days to obtain a stable rat primitive endoderm stem cell line.

Further, the identification method in the third step is a combination of two biological methods, immunofluorescence in molecular biology and RNA seq in omics.

Compared with prior art, the beneficial effect of the present invention is:

(1) To explore and optimize the culture system, add the small molecule inhibitor 3uM CHIR99021 (an activator of Wnt signaling pathway) to the reported culture system, which can efficiently isolate the primordial endoderm stem cell line from post-implantation rat embryos;

(2) Established a method for isolating primitive endoderm stem cells from rat post-implantation embryos for the first time, and successfully obtained 3 primitive endoderm stem cell lines, which were identified and analyzed by immunofluorescence and transcriptome sequencing. The gene expression profiles of the primitive endoderm stem cells after implantation are basically the same as those of the primitive endoderm stem cell lines derived from preimplantation embryos (blastocysts), and highly express the specific marker genes of the primitive endoderm lineage;

(3) Make up for the deficiency of the existing source of isolated rat primitive endoderm stem cells.

Description of drawings

Figure 1: XEN cells isolated from pre-implantation embryos (E4.5) (A: bright field image of the isolation process, B: immunofluorescence experiment).

Figure 2: XEN cells isolated from post-implantation embryos (E8.5) (A: bright field image of the isolation process, B: immunofluorescence experiment).

Figure 3: RNAseq gene expression heat map analysis.

Figure 4: PCA analysis of RNAseq.

Figure 5: Heatmap analysis of three cell lineage-specific marker genes by RNAseq.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention. Also, in the following invention, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concept of the present invention.

1. Obtaining embryos before and after implantation

1. Acquisition of pre-implantation embryos (blastocysts)

Select healthy 4-6 week female SD rats for superovulation, inject pregnant horse serum gonadotropin (PMSG), inject human chorionic gonadotropin (HCG) 48 hours later, and mate with male mice of the same strain , see the plug on the next day and mark it as E0.5, kill the female mice by neck breaking on the next day after seeing the plug, cut the oviducts of both female mice with sterile surgical instruments, and draw the egg mass in the embryo operation solution (HCZB), Digested in hyaluronidase. The fertilized eggs washed in the embryo operation solution (HCZB) were transferred into the rat embryo culture medium mR1ECM-2C, and placed in a 5% CO2 incubator at 37° C. for further cultivation.

2. Acquisition of embryos after implantation (E8.5)

Select healthy 8-10-week-old female SD rats in estrus, and mate them with male mice of the same strain. The next day when the plug is seen, it is recorded as E0.5. At 8.5 days after seeing the plug, the female rats are killed by neck dissection, and sterile surgical instruments are used. The uterus was cut, and the post-implantation embryos were separated using a stereo microscope in the embryo operating solution, and the collected rat post-implantation embryos were used for future use.

2. Isolation of primordial endoderm stem cells from rat embryos before and after implantation

1. Isolation of primitive endoderm stem cells from rat preimplantation embryos (blastocysts)

Remove the blastocyst zona pellucida with a benchtop solution under a stereo microscope, and transfer it into the rat primitive endoderm stem cell culture medium with mouse feeder cells added in advance, and put it in a 5% CO2 incubator at 37°C for 3 days. The inner cell mass grows adherently, forming clonal protrusions. Cell clones were picked at day 14 and transplanted into new 24-well plates for growth. Thereafter, the culture medium of rat primitive endoderm stem cells was replaced with a new one at intervals, and the primitive endoderm stem cells were passaged when the cell density reached 80%.

2. Isolation of primordial endoderm stem cells from post-implantation rat embryos

The collected 8.5-day post-implantation embryos were separated from the tissues corresponding to the primitive endoderm lineage under a stereomicroscope, and transferred into the culture medium of rat primitive endoderm stem cells that had been added with mouse feeder cells in advance. Put it in a 5% CO2 incubator at 37°C for 5-7 days, the embryos will grow adherently, replace the medium, and continue to culture for about 14 days. Primitive endoderm stem cell clones will form. Use mechanical methods to transfer the cell clones to fresh Cultured in the culture medium of rat primitive endoderm stem cells, and then replaced with new rat primitive endoderm stem cell culture medium every other day, and when the cells reached 80% density, the primitive endoderm stem cells were passaged.

Rat Implantation Primitive Endoderm Stem Cell Medium, DMEM (GibcoC1199500BT), 15% FBS (Gibco#10099-141C), 2mM GlutaMAXSupplement (Gibco#35050-061), 0.1mMnonessentialaminoacids (Gibco#11140-035), 1mMsodiumpyruvate (Gibco co #11360-070), 0.1mMβ-mercaptoethanol (Gibco#21985-023), 1% penicillinstreptomycin (BI#QDZ200), 1000IU/mlleukemiainhibitoryfactor (LIF) (Millipore#ESG1107) leukemia inhibitory factor, 3μM CHIR99021 (Axon# 1386) A Wnt Activator of signaling pathways.

3. Identification of primordial endoderm stem cells in rat embryos before and after implantation

1. Immunofluorescence staining

After the stable establishment of primitive endoderm stem cell lines derived from embryos in rats before and after implantation, immunofluorescence identification of different germ layer-specific markers was performed on them (Figure 1), so as to evaluate the relationship between primitive endoderm stem cell lines derived from embryos after implantation and Characterization of preimplantation embryo-derived stem cell lines. The primordial endoderm stem cells of the stably cultured rats before and after implantation were washed 3 times with DPBS, fixed with 4% paraformaldehyde for 30 minutes, and permeabilized with 0.5% TritonX-100-PBS at room temperature for 30 minutes , 2% BSA was blocked at room temperature for 1 hour, the primary antibody was diluted 1:200 with 2% BSA, and incubated overnight. Wash 3 times with 0.5% TritonX-100-PBS, dilute the secondary antibody at 1:500 with 0.5% TritonX-100-PBS, and incubate at room temperature for 2 hours. Wash 3 times with 0.5% TritonX-100-PBS, dilute DAPI at 1:3000 with 0.5% TritonX-100-PBS, stain nuclei in the dark for 5 minutes, wash 3 times with 0.5% TritonX-100-PBS, each A piece of anti-fluorescence extinction agent, nail polish to seal the piece. The staining results were observed with a confocal laser microscope. As shown in Figure 1 and Figure 2, we found that both post-implantation embryo-derived primitive endoderm stem cell lines and pre-implantation embryo (blastocyst)-derived stem cell lines highly expressed primitive endoderm-specific markers GATA6, Sox17, and PDGFRa . At the same time, like the primitive endoderm stem cells derived from rat blastocysts, some cells express the epiblast-specific marker OCT4, but the epiblast-specific marker pluripotency gene Nanog and the trophectoderm-specific marker CDX2 do not express, This is consistent with the results of primitive endoderm stem cells derived from rat blastocysts studied by others (Debebetal., 2009).

2. Transcriptome sequencing analysis (RNA-seq)

(1) Extraction of RNA

Digest the primitive endoderm stem cells of the stably cultured rats before and after implantation with 0.05% trypsin for 5 minutes, collect ^5X106 cells by centrifugation, wash 3 times with DPBS, and use the cell samples in Trizol to make them completely thaw . Add 1/5 volume of chloroform, shake vigorously for 15s, let stand at room temperature for 5min, and centrifuge at 16000×g4°C for 15min. Transfer 400-500 μL of the upper aqueous phase to a new RNase-free 1.5 mL centrifuge tube. Add an equal volume of isopropanol, invert and mix well, place at -20°C for 1 hour, and centrifuge at 16000×g 4°C for 10 minutes. Carefully discard the supernatant, and add 1 mL of 75% ethanol prepared with DEPC water to wash the pellet. Centrifuge at 16000×g for 3 minutes at 4°C, discard the supernatant, repeat twice, and be careful not to discard the RNA pellet. Place at room temperature for 2-3 minutes, dry the RNA precipitate, add 30 μL RNasefreewater, after the precipitate is completely dissolved, take a small amount of RNA for detection, and store the rest at -70°C for later use. Use agarose gel electrophoresis to preliminarily analyze the integrity of the RNA sample and whether there is DNA contamination, select the complete and non-contaminated RNA, use the NanoDrop2000 spectrophotometer to detect the concentration and purity of the RNA, and finally use the Agilent2100Bioanalyzer to accurately detect the integrity of the RNA.

(2) Sequencing and Analysis

The Nanodrop spectrophotometer detects the RNA concentration and purity, and the concentration and purity are qualified. The company (Novogene) conducts library construction and machine sequencing, and obtains seq whole gene expression heat map analysis, RNAseq PCA analysis and RNAseq three Heatmap analysis of cell lineage-specific marker genes.

As shown in Figure 3, RNAseq whole gene expression heat map analysis, the whole gene expression profiles of primitive endoderm stem cell lines of rat postimplantation embryos and preimplantation embryos (blastocysts) that we isolated were compared with those of The gene expression profiles of the reported primitive endoderm stem cell lines from rat preimplantation embryos (blastocysts) were similar but distinct from those of the mouse primitive endoderm stem cell lines (XEN and pXEN), suggesting that our isolated Primitive endoderm stem cells after implantation in rats are similar to blastocyst-derived primitive endoderm stem cells that have been reported in rats.

As shown in Figure 4, PCA analysis of RNAseq, we isolated primitive endoderm stem cell lines from rat post-implantation embryos (blastocysts) and compared with reported rat implantation The clustering analysis of the primitive endoderm stem cell lines of the pre-embryo (blastocyst) was similar, but could not cluster together with the reported mouse primitive endoderm stem cell lines (XEN and pXEN). It shows that the primitive endoderm stem cells isolated from rats after implantation are consistent with the blastocyst-derived primitive endoderm stem cells that have been reported in rats.

As shown in Figure 5, RNAseq heat map analysis of three cell lineage-specific marker genes, primitive endoderm stem cell lines from rat post-implantation embryos versus pre-implantation embryos (blastocysts) that we isolated Consistent with the reported gene expression of three cell lineage-specific markers in primitive endoderm stem cell lines of rat and mouse preimplantation embryos (blastocysts), high expression of primitive endoderm lineage-specific markers such as GATA6, GATA4 , Sox17, Sox7, PDGFRa, Apoe, Krt8, etc., but do not express epiblast and trophectoderm lineage-specific marker genes, such as epiblast pluripotency markers Nanog, Sox2, Pou5f1, Etv4 and trophectoderm specificity Sex markers CDX2, GATA3, Tfap2c, etc. It shows that the primitive endoderm stem cell lines isolated from rat post-implantation embryos are similar to the isolated and reported primitive endoderm stem cell lines of rats and mice.

The above results show that we can also successfully isolate and establish a rat primitive endoderm stem cell line from rat post-implantation embryos by exploring the optimized culture system. The origin of primitive endoderm stem cells has consistent molecular characteristics.

It should be understood that the above-mentioned specific embodiments of the present invention are only used to illustrate the invention or explain the principle of the present invention, but not to limit the present invention. Therefore, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention shall fall within the protection scope of the present invention. Furthermore, it is intended that the appended claims of the present invention embrace all changes and modifications that come within the scope and metesques of the appended claims, or equivalents of such scope and metes and bounds.

Claims (8)

1. A method for separating and establishing a lineage of a rat primitive endoderm stem cell is characterized by comprising the following steps:

the method comprises the following steps: obtaining implanted embryos, dissecting female rats 8.5 days after fertilization, and shearing uterus implanted points to obtain implanted embryos;

step two: extraembryonic tissues corresponding to the original endoderm lineage are separated from the nidation embryo, cultured in an original endoderm stem cell culture system and placed in a carbon dioxide cell incubator, and the rat original endoderm stem cell lineage is obtained after liquid change and passage.

2. The method for isolation and lineage establishment of rat primitive endoderm stem cells of claim 1, further comprising:

step three: identifying rat primitive endoderm stem cell lines.

3. The method of claim 1, wherein in step two the primitive endoderm stem cells are lineage isolated from rat post-implantation embryos.

4. The method for separating and lineage establishing rat primitive endoderm stem cells in claim 1, wherein the culture systems of the primitive endoderm stem cells in step two are cultured under the conditions of Fetal Bovine Serum (FBS), leukemia Inhibitory Factor (LIF) and activator of Wnt signaling pathway (CHIR 99021).

5. The method of claim 1, wherein in step two the incubator is thermostatted at 37 ℃ and contains 5% CO ₂ The carbon dioxide cell incubator of (1).

6. The method for separating and establishing the lineage of rat primitive endoderm stem cells in claim 1, wherein in the second step, the liquid change interval is 2 days, and the cells are passaged when reaching 80% density.

7. The method for separating and establishing the lineage of rat primitive endoderm stem cells according to any one of claims 1 to 6, wherein the time for obtaining stable rat primitive endoderm stem cell lineage is 25 to 30 days.

8. The method for separating and establishing the lineage of rat primitive endoderm stem cells according to claim 2, wherein the identification method in step three is a combination of molecular biological immunofluorescence and omic RNAseq biological method.

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