CN104946581A - Special culture medium and method for culturing porcine trophoderm stem cells - Google Patents

Abstract

The invention discloses a special culture medium and method for cultivating porcine trophoblast stem cells. The special culture medium comprises bFGF, activin A, Y27632, Knockout SR and β-mercaptoethanol. The culture method is to pre-coat the stem cell culture vessel with extracellular matrix; then digest the porcine blastocyst with protease to remove the zona pellucida, transfer it to the stem cell culture vessel, and add the special medium of the present invention; cultivate until the total number of cells is 1000 ~2000 trophoblast stem cell clones were digested into single cells, and transferred to new culture vessels coated with extracellular matrix for subculture of cells. The composition of the special culture medium of the invention is determined, the use safety is high, and the contamination of heterologous cells is avoided. The culture method is simple and efficient, and has the advantages of high clone formation rate, low cell apoptosis rate, strong proliferation and passage ability, and safety and stability for porcine trophoblast stem cell culture.

Description

A special medium and method for cultivating porcine trophoblast stem cells

technical field

The invention belongs to the field of stem cell technology research, in particular to a special culture medium and method for cultivating porcine trophoblast stem cells.

Background technique

Stem cells are a kind of primitive undifferentiated cells with multi-directional differentiation potential and self-replication ability, and are the original cells that form various tissues and organs of mammals. The medical profession calls it "universal cell".

The first cell differentiation occurs when the animal embryo develops to the blastocyst in the early stage, forming two kinds of inner cell mass and trophoblast with different characteristics and functions. Trophoblast stem cells are the progenitor cells of placental tissue cells, and gradually differentiate into trophoblast precursor cells, chorionic trophoblast, syncytiotrophoblast and multinucleated giant cells. During the apparent reprogramming process in early embryonic development, DNMTs, TETs, HDACs and other related enzymes work together to maintain the overall low methylation level of the trophoblast cell genome and the chromatin condensation of the chorionic trophoblast, and participate in the regulation of trophoblast differentiation, migration and Ability to invade the endometrium (Logan et al., 2013). In order to reveal such a complex molecular mechanism in trophoblast differentiation and development, it is necessary to isolate and purify trophoblast stem cells from early embryos and establish their in vitro culture model. The earliest successful isolation of trophoblast stem cells from mouse blastocyst trophectoderm, they can maintain self-renewal in the environment of adding basic fibroblast growth factor bFGF and human leukemia inhibitory factor hLIF, and can chimerize after injection into mouse blastocysts into various parts of the mouse placenta, but not in the germ layers of the mouse fetus (Tanaka et al., 1998; Takahashi and Yamanaka., 2006; Rielland et al., 2008). The trophoblast cell lines of some other animals have also been established, including cattle, sheep and pigs, etc. These cells can be continuously subcultured and exhibit the characteristics of epithelial cells. However, there are many unknown differences in the key signaling pathways related to cell proliferation and renewal of trophoblast stem cells in ungulates and mice, and this classic mouse trophoblast stem cell culture system is not suitable for livestock and primates Isolation and culture of trophoblast stem cells from other large animals. The growth factors and regulatory pathways required for the isolation and culture of stem cells from different species of animals are quite different. At present, there are no reports on the successful establishment of trophoblast stem cells derived from early blastocysts of pigs and the identification of stem cell characteristics at home and abroad. Due to the difficulty in obtaining early embryos in primates such as humans and rhesus monkeys, it will take a longer time to explore a suitable trophoblast stem cell culture system. This is similar to the fact that there is still no successful lineage of embryonic stem cells from domestic animals and primates.

Trophoblast stem cells have been isolated from both pre-implantation and post-implantation embryos in mice. However, there is no report at home and abroad that similar trophoblast stem cells can be obtained in ungulates such as pigs, cattle, and sheep. This kind of trophoblast stem cells that can proliferate and renew has multipotency. Porcine placental-derived trophoblast cell lines have been established that can be serially subcultured and exhibit epithelial characteristics, expressing the differentiation markers lamin A/C, but not pluripotent stem cell characteristics (Flechon et al., 1995; La Bonnardiere et al., 2002). Song Zhiqiang et al. (2013) used mouse fetal fibroblasts as feeder cells, added fibroblast growth factor FGF4 and human leukemia inhibitory factor LIF to the basal culture medium, and isolated pig trophoblasts from day 6 parthenogenetic blastocysts. layer of stem cell-like cells. In the experiment, the culture efficiency was low, and about 30 blastocysts could obtain one cell clone group, which reflected the characteristics of trophoblast stem cells in morphology, but failed to prove its stem cell characteristics, that is to say, no real porcine trophoblast had been obtained. stem cell. Li Rongfeng et al. (2013) invented a method for establishing bovine trophoblast stem cells. Bovine blastocysts were inoculated into the feeder layer of mouse fetal fibroblasts and subcutaneous connective tissue cells of Wnt-3A mice. Small molecule inhibitors PD0325901 and CHIR99021 were used to subculture the cells. This culture system is a classic embryonic stem cell culture method. The trophoblast stem cells thus obtained are a type of cells derived and transformed from embryonic stem cells under specific conditions, and may have some essential and potential characteristics with stem cells derived from trophectoderm. the difference.

Contents of the invention

In order to overcome the above-mentioned problems in the prior art, the present invention provides a special medium for cultivating porcine trophoblast stem cells.

The special medium for cultivating porcine trophoblast stem cells provided by the invention, the components include bFGF, activin A, Y27632, Knockout SR and β-mercaptoethanol. The above ingredients have been commercialized and can be directly purchased from commercially available products, for example, bFGF, activin A, Knockout SR and β-mercaptoethanol can be purchased from Gibco brand, and Y27632 can be purchased from Sigma brand.

bFGF: basic fibroblast growth factor, a heparin-binding protein that promotes cell division, activates the FGF signaling pathway during multiple biological reactions in embryonic development and differentiation, and is a biological agent widely used in cell culture active protein.

activate A: Activin A is a member of the TGFβ superfamily of cytokines. The signaling pathways mediated by the TGFβ superfamily have different biological effects in maintaining the self-renewal and specific differentiation of embryonic stem cells. The specific cell fate mainly depends on the cell Phases of signal reception, as well as cytokine levels and interactions with other cytokines. Long-term effects of bFGF and activin A alone can induce stem cell differentiation, but they can maintain the pluripotency of stem cells when they act together, suggesting that there is a mechanism of mutual regulation between them.

Y27632: chemical formula C ₁₄ H ₂₁ N ₃ O, is a Rho protein kinase (ROCK) specific inhibitor, the structural formula is as follows:

Studies have shown that Y27632 can cause changes in the morphology of human embryonic stem cells, from compact cell colonies to loose and flat, and Y27632 When applied to the passage of human embryonic stem cells, the passage efficiency can be significantly improved. In terms of maintaining the characteristics of embryonic stem cells, the role of ROCK inhibitors has not yet been determined. However, in the present invention, it is proved that Y27632 It will not adversely affect the maintenance of pluripotency of stem cells, but can inhibit the apoptosis effect in the process of cell subculture, indicating that it can be safely used for the cultivation of porcine trophoblast stem cells.

Knockout SR: Knockout Serum Replacement, is a defined serum-free preparation used to replace fetal bovine serum to promote the growth of undifferentiated pluripotent stem cells. Most traditional embryonic stem cell culture medium formulations require fetal bovine serum, which contains many unknown components and some factors that induce cell differentiation, and will cause contamination of foreign serum proteins. Therefore, in order to solve this problem, scientists proposed a serum-free culture system and developed Knockout SR can effectively maintain the undifferentiated state of embryonic stem cells and maintain stem cell proliferation.

β-mercaptoethanol: β-mercaptoethanol can induce cell proliferation, promote DNA synthesis, and at the same time avoid peroxide damage to in vitro cultured cells and hinder the differentiation trend of pluripotent stem cells. It is often used to culture embryonic stem cells, induced pluripotent stem cells and neural stem cells.

Experiments found that the combination of bFGF, activin A, Y27632, Knockout SR and β-mercaptoethanol can effectively maintain the rapid proliferation and renewal of porcine trophoblast stem cells. Among them, the removal of bFGF, activin A or Knockout SR in the culture system resulted in a significant decrease in cell proliferation, and the absence of Y27632 resulted in most of the cells undergoing apoptosis after passage, and only about 30% of the cells continued to grow. Without the addition of β-mercaptoethanol, the edges of stem cell clones became loose, and the cells tended to differentiate.

Preferably, in the special medium for cultivating porcine trophoblast stem cells, the concentration of bFGF is 12.5-50 ng/mL, the concentration of activin A is 5-20 ng/mL, the concentration of Y27632 is 2-10 nmol/mL, and the volume of Knockout SR The concentration is 2%~5%, and the concentration of β-mercaptoethanol is 20~55 nmol/mL. The concentrations of bFGF, activin A, Y27632, Knockout SR and β-mercaptoethanol in this range can more effectively maintain the pluripotency and proliferation renewal of stem cells.

Preferably, the above-mentioned special medium for cultivating porcine trophoblast stem cells also includes DMEM/F12 medium, Neurobasal medium, N-2 supplement, B-27 supplement, MEM NEAA and L-Glutamine. The above ingredients have been commercialized and can be purchased directly, for example, these ingredients can be purchased from Gibco brand.

DMEM/F12 medium has complex components and contains a variety of trace elements, which is suitable for the culture of mammalian cells under the condition of low serum content.

Neurobasal medium is a basal medium used to meet the special needs of prenatal and fetal neuronal cells, and is suitable for the growth of epithelial cells such as nerve cells.

N-2 Supplement is a chemically defined, serum-free supplement mainly used in Neurobasal medium. B-27 supplement is a serum-free supplement that can be used in combination with Neurobasal medium. In the culture system of the present invention, the combined use of N-2 supplement and B-27 supplement can promote the adherent growth of trophoblast-like epithelial cells.

MEM NEAA contains glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine. These 7 kinds of non-essential amino acids required for cell culture can effectively improve the ratio of cell culture medium and reduce the side effects of the cells themselves producing non-essential amino acids during cell culture. L-glutamine is an essential amino acid for cell growth. After removing the amino group, it provides an important source of energy for cultured cells and participates in protein synthesis and nucleic acid metabolism. Add MEM in the culture system of the present invention NEAA and L-glutamine are beneficial to maintain long-term culture of cells in vitro.

Preferably, in the above-mentioned special medium for cultivating porcine trophoblast stem cells, DMEM/F12 medium and Neurobasal medium are mixed according to a volume ratio of 1:1 as the basal culture medium; every 20mL of basal culture medium is added with N-2 supplement 50~200μL, B-27 supplement 100~400μL, MEM NEAA50~200μL, L-glutamine 100~200μL.

The present invention also provides a method for cultivating porcine trophoblast stem cells, comprising the following steps:

(1) The stem cell culture vessel is pre-coated with extracellular matrix;

(2) The porcine blastocyst is digested with protease to remove the zona pellucida, and transferred to step (1) the stem cell culture vessel coated with extracellular matrix, and cultured with any of the above-mentioned special medium for cultivating porcine trophoblast stem cells;

(3) After culturing to form a clonal group with a total number of 1000-2000 cells, digest them into single cells and transfer to a new step (1) subculture the cells on culture vessels coated with extracellular matrix.

Preferably, in the above method for culturing porcine trophoblast stem cells, the blastocyst is a blastocyst derived from artificial insemination, a blastocyst derived from somatic cell nuclear transfer, a blastocyst derived from in vitro fertilization or a parthenogenetic blastocyst.

Preferably, in the above method for culturing porcine trophoblast stem cells, the extracellular matrix in step (1) is rat type I collagen, Matrigel or fibronectin. The above ingredients are all commercialized and can be purchased directly, for example, these ingredients can be purchased from Gibco brand.

Rat Type I Collagen is a common fibrous collagen found in skin, bones, tendons, and other connective tissues. Gibco brand Rat Type I Collagen can be diluted flexibly to produce hardened gelatin through a gelation step.

Gibco brand Matrigel is a basement membrane matrix extracted from EHS mouse tumors rich in extracellular matrix proteins. Its main components are laminin, type IV collagen, nestin, heparan sulfate glycoprotein, and growth factors and matrix metalloproteinases. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix.

Fibronectin is an extracellular glycoprotein that exists in body fluids in a soluble form or in the extracellular matrix in an insoluble form, respectively. It can connect with cell fibers and proteoglycans and play a bonding role.

Experiments in the present invention verify that the use of rat type I collagen, Matrigel or fibronectin-coated culture vessels can greatly increase the adhesion at the bottom of the cell culture vessel, impel pig blastocysts to adhere to the wall, and isolate trophoblast stem cells , and promote the attachment to the culture dish during cell division and proliferation, and maintain the growth of cell clones attached to the wall.

Preferably, in the above-mentioned method for culturing porcine trophoblast stem cells, the protease in step (2) is pronase protease; blastocysts are washed with pig embryo culture medium PZM-3 before digestion, and then transferred to the culture medium of porcine trophoblast stem cells. Wash in a special medium; the ambient temperature for cultivation is 39°C, the volume concentration of CO ₂ is 5%, and the saturation humidity.

Preferably, in the above method for culturing porcine trophoblast stem cells, the subculture method in step (3) is: after rinsing with DPBS, add an appropriate amount of TRYPLE select trypsin replacement enzyme (Gibco brand) to digest them into single cells, and then add pig trophoblast The special culture medium for porcine trophoblast stem cells was used to stop digestion, the cells were suspended by pipetting, collected and centrifuged, diluted with the special medium for cultivating porcine trophoblast stem cells, and passaged to stem cell culture vessels coated with extracellular matrix at a ratio of 1:3 .

The present invention also provides porcine trophoblast stem cells cultured by the method for culturing porcine trophoblast stem cells.

Compared with the prior art, the present invention has the following beneficial effects:

The use of feeder cells in traditional stem cell culture experiments can secrete some key growth factors, promote the proliferation of pluripotent cells, remove toxins in the in vitro culture environment and inhibit their differentiation. However, the interference of a large number of unknown components on the identification of stem cells and the contamination of heterologous cells will affect researchers' research on stem cell culture systems, so establishing a feeder-free culture system for trophoblast stem cells is more advantageous than a feeder-layer system .

The present invention successfully develops a feeder-free culture system for trophoblast stem cells of livestock large animals for the first time, and replaces traditional heterogeneous feeder layer cell support by screening growth factor combination culture fluid with clear chemical composition. The composition of the special medium is determined, the use safety is high, and the contamination of heterologous cells is avoided. It is of great significance for the study of the biological mechanism of this type of stem cells, as well as the early development of embryos and placental differentiation, and will further promote the development of trophoblast stem cells in somatic cells. Applications in cloning and large animal transgenic research.

The method for cultivating porcine trophoblast stem cells provided by the invention is simple and efficient, and has the advantages of high clone formation rate, low cell apoptosis rate, strong proliferation and passage ability, and safety and stability for culturing porcine trophoblast stem cells. Among them, the efficiency of artificial insemination-derived blastocysts to form stem cell clones was 38.79%, and the efficiency of somatic cell nuclear transfer-derived blastocysts to form stem cell clones was 6.34% (statistical results are shown in Table 1), and they can maintain the characteristics of stem cells stably for a long time.

Description of drawings

Figure 1 shows the morphology of porcine blastocysts (A: blastocysts derived from artificial insemination, B: blastocysts derived from somatic cell nuclear transfer).

Figure 2 is the morphology of porcine blastocysts after pronase digestion of the zona pellucida (A: blastocysts derived from artificial insemination, B: blastocysts derived from somatic cell nuclear transfer).

Figure 3 is the morphology of porcine blastocysts inoculated in a special medium for trophoblast stem cells and growing on the wall (A: blastocysts derived from artificial insemination on the second day after inoculation, B: somatic cell nuclear transfer derived from somatic cell nuclear transfer on the second day after inoculation Blastocysts, C: the artificial insemination-derived blastocysts grew adherently on the third day after inoculation, D: the somatic cell nuclear transfer-derived blastocysts grew adherently on the third day after inoculation).

Figure 4 shows the growth morphology of porcine trophoblast stem cells of F0 and F3 generations (A: porcine trophoblast stem cells isolated from artificial insemination blastocysts in F0 generation, B: porcine trophoblast stem cells isolated from artificial insemination blastocysts in F3 generation, C : F0 somatic cell nuclear transfer derived porcine trophoblast stem cells isolated from blastocysts, D: F3 generation somatic cell nuclear transfer derived porcine trophoblast stem cells isolated from blastocysts.

Figure 5 shows the expression of related marker genes detected by agarose gel electrophoresis in porcine trophoblast stem cells. AI-TSCs-cDNA is the cDNA of porcine trophoblast stem cells isolated from blastocysts derived from artificial insemination, SCNT-TSCs-cDNA is the cDNA of porcine trophoblast stem cells isolated from blastocysts derived from somatic cell nuclear transfer, Trophoblasts-cDNA is the differentiated trophoblast cell line cDNA, Fibroblasts-cDNA is the cDNA of a fetal fibroblast cell line.

Figure 6 is the immunofluorescence staining of cells to detect the specific expression genes CDX2 and Eomes of porcine trophoblast stem cells (column A shows the cell morphology under white light, column B shows the staining of nuclei by hoest33342 under ultraviolet excitation light, and column C shows incubation with CDX2 or Eomes antibody The color was developed after adding fluorescent secondary antibodies, and column D is the effect after the overlapping of columns B and C. AI-TSCs on the right represent porcine trophoblast stem cells isolated from blastocysts derived from artificial insemination, and SCNT-TSCs represent blastocysts derived from somatic cell nuclear transfer Porcine trophoblast stem cells isolated from embryos.

Figure 7 is the cell morphology of porcine trophoblast stem cells after induction and differentiation (A: trophoblast stem cells isolated from artificial insemination blastocysts differentiate to form multinucleated giant cells, B: trophoblast stem cells isolated from artificial insemination blastocysts differentiate to form trophoblasts Cells, C: Trophoblast stem cells isolated from blastocysts derived from somatic cell nuclear transfer differentiated to form multinucleated giant cells, D: Trophoblast stem cells isolated from blastocysts derived from somatic cell nuclear transfer differentiated to form trophoblast cells.

Detailed ways

The present invention will be further described below in conjunction with the examples, but they are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1: Isolation, culture and identification of porcine trophoblast stem cells from blastocysts derived from artificial insemination (AI)

1. Establishment of the culture system of porcine trophoblast stem cells derived from AI-derived blastocysts

1.1 Obtaining method of blastocyst:

The common estrous sows were bred by AI method, and the blastocysts were collected by the method of in vitro punching after the bridging. The specific operation is: select sows with high gestational age to be slaughtered on the sixth or seventh day after mating, take out the uterus, fix the front end of the uterine horn with a hemostatic forceps, inject egg flushing solution (DPBS+PVA) from the umbrella of the oviduct, and use The catheter was collected at the uterine horn, filtered and washed with egg washing solution, and the blastocysts were collected under a stereomicroscope, and transported to the laboratory at a constant temperature of 39°C (the shape of blastocysts derived from artificial insemination is shown in Figure 1A).

1.2 Preparation of special medium for porcine trophoblast stem cells:

DMEM/F12 and Neurobasal medium are mixed according to the volume ratio of 1:1 as the basic culture medium;

For every 20mL of basal culture medium, add N-2 Supplement 50~200μL, B-27 supplement 100~400μL, MEM NEAA 50~200μL, L-Glutamine 100~200μL. And the concentration is 12.5~50ng/mL bFGF, 5~20ng/mL activin A, 2~10nmol/mL Y27632, 20~55nmol/mL β-mercaptoethanol and Knockout SR at a volume concentration of 2% to 5%.

1.3 Isolation and culture of primary trophoblast stem cells

1.3.1 Extracellular matrix-coated cell culture vessels

You can choose any one of the following methods A, B and C, and the effects are similar.

A: Rat type I collagen-coated petri dish: the specific experimental procedure is to dilute rat type I collagen to 50-200 μg/mL with 0.02 molar acetic acid, and then add it to an ordinary petri dish (Corning brand) at 5-20 μg/ ^cm2 , incubated at room temperature for 1 hour, discarded carefully, washed three times with DPBS, and dried for later use.

B: Matrigel-coated culture dish: The specific experimental steps are to dilute the concentration of Matrigel to 50-200 μg/mL with DMEM/F12, and then add it to a common culture dish (Corning brand) at 5-20 μg/cm ² , at 37 After incubating at ℃ for 1 hour, carefully aspirate and discard, and dry in the air for later use.

C: Fibronectin-coated culture dish: the specific experimental procedure is to dilute fibronectin to 50-200 μg/mL with DMEM/F12, then add it to a common culture dish (Corning brand) at 5-20 μg/ ^cm2 , and keep it at room temperature After incubation for 1 hour, carefully aspirate and discard, and dry for later use.

1.3.2 Inoculation of blastocysts and formation of primary stem cell clones: pig blastocysts developed to the sixth or seventh day were washed twice with embryo culture medium PZM-3, and then digested with pronase to remove the zona pellucida (remove the embryo pellucida The shape of the embryo after the zone is shown in Figure 2A), transferred to the stem cell medium and washed twice, and then the blastocyst with the zona pellucida completely lysed and disappeared was sucked into the special medium of the present invention. Transfer to an incubator at 39°C, 5% CO ₂ and saturated humidity for culture (observe the shape of the blastocyst after inoculation on the second day as shown in Figure 3A), and observe the adherence of the blastocyst and the growth shape of cell clones on the third day (stem cell The clonal morphology formed by adherence is shown in Fig. 3C). The final efficiency of trophoblast stem cell lines isolated from blastocysts derived from artificial insemination was 38.79%. The specific experimental data are shown in Table 1 below.

Table 1

1.4 Subculture and cryopreservation of porcine trophoblast stem cells

The subculture method used is enzymatic digestion, that is, when the stem cell clones are dense and the cells are contact-inhibited, rinse twice with DPBS, add an appropriate amount of TRYPLE select trypsin replacement enzyme (Gibco brand) to digest into single cells, and add porcine trophoblast stem cells The digestion medium was terminated, the cells were suspended by pipetting with a pipette tip, collected and centrifuged, and passaged to a new extracellular matrix-coated culture dish at a ratio of 1:3 (the morphology of F0 and F3 stem cells are shown in Figure 4A and Figure 4B). The cryopreservation method of porcine trophoblast stem cells is to digest the cells, centrifuge them, add commercial cryopreservation medium (Gibco brand), pipette the tip of the pipette to suspend the cells evenly, transfer them to cryopreservation tubes (Corning brand), and place Store at 4°C for 30 minutes, then store at minus 20°C for 30 minutes, then store at minus 80°C for 16-48 hours, and then transfer to liquid nitrogen for long-term storage.

2. Identification of marker gene expression in porcine trophoblast stem cells

2.1 Detection of gene expression by RT-PCR technique

The detection includes trophoblast stem cell-specific expression genes Eomes, Cdx2, and Tead4; pluripotency genes Oct4, Nanog, and Sox2; trophoblast-specific expression genes Cyp17a1, Plet, Krt8, and Hand1; fibroblast growth factor receptor 2 gene Fgfr2, X chromosome Inactivation of related gene Xist; and internal reference gene Gapdh.

Specific experimental steps: the cultured cells in step 1 were digested and washed with PBS, RNA was extracted with the RNA extraction kit from Qiagen, and reverse-transcribed into cDNA with the reverse transcription kit from Fermentas for storage, targeting the above genes of pigs Sequence-specific primers were designed for amplification, and agarose gel electrophoresis was used to detect whether the gene was expressed. In the experiment, cDNA amplification of differentiated trophoblast cell lines and fetal fibroblast cell lines were used as controls. The results showed that the trophoblast derived from artificial insemination Layer stem cells highly expressed Eomes, Cdx2, Tead4, Cyp17a1, Plet, Krt8, Hand1, and Fgfr2, and weakly expressed Oct4, Nanog, and Sox2 (the gene expression of stem cells detected by electrophoresis is shown in Figure 5).

The PCR primer sequences used to detect the expression of all genes are shown in Table 2:

Table 2

2.2 Detection of CDX2 and Eomes specifically expressed in trophoblast stem cells by immunofluorescent staining

When the pig trophoblast stem cells of the F3 generation cultivated in the special medium of the present invention reach a confluence of about 80%, they are washed twice with DPBS, fixed with 4% paraformaldehyde for 5 minutes, and then the cell membrane is perforated by TritonX-100. Then add goat serum to block for 30 minutes, then add 1:200 diluted CDX2 or Eomes primary antibody to incubate overnight, wash with DPBS more than three times, then add 1:1000 diluted Alexa Fluor ^® 568 fluorescently labeled secondary antibody in the dark, Shows red fluorescence. Afterwards, DAPI nuclear staining, mounting, microscopic examination, and photographing under an inverted fluorescent microscope showed that CDX2 protein was abundantly expressed in the cytoplasm and nucleus, and Eomes protein was abundantly expressed in the nucleus (the expression of CDX2 and Eomes is shown in Figure 6) .

3. Functional identification of multilineage differentiation of porcine trophoblast stem cells

3.1 In the experiment, the induction medium designed to differentiate into multinucleated giant cells was 90% DMEM/F12 and 10% fetal bovine serum (Gibco brand). The specific experimental procedure is to remove the stem cell culture medium after culturing the F3 trophoblast stem cells for 24 hours, wash it twice with DPBS, and replace it with the differentiation medium to continue the culture. Change the medium every two to three days.

In the experiment, it was found that after three days of culture in the differentiation medium, about 5% of the cells had begun to differentiate into multinucleated giant cells, and after 15 days of continuous induction of differentiation, about 50% of the cells had differentiated into typical multinucleated giant cells (multinucleated giant cell morphology as shown in Figure 7A).

3.2 In the experiment, the induction medium designed to differentiate into trophoblast cell line was 95% DMEM/F12, 5% fetal bovine serum FBS and 0.1Unit/mL bovine insulin. The specific experimental procedure is to remove the stem cell culture medium after the F3 trophoblast stem cells are cultured for 24 hours, wash with DPBS twice, and replace with the above induction medium to continue the culture. Change the medium every two to three days. In the experiment, it was found that after one week of culture in the induction medium, the cell clones were loose, the growth rate was slow, and the growth was in sheets, showing the characteristics of flat epithelial cells (the morphology of differentiated trophoblast cells is shown in Figure 7 B).

Example 2: Isolation, culture and identification of porcine trophoblast stem cells derived from somatic cell nuclear transfer (SCNT) blastocysts

1. Establishment of culture system for porcine trophoblast stem cells isolated from SCNT blastocysts

1.1 Acquisition of SCNT blastocysts: using a micromanipulator to perform nuclear transfer of porcine fibroblasts to obtain cloned embryos, acquisition and maturation of porcine oocytes, cultivation of donor fibroblasts, enucleation and injection of oocytes, Fusion and activation of oocytes and somatic cells were performed according to existing literature standards (Li et al. al., 2013, PMID: 24033142) The embryo culture medium PZM-3 was used to culture the reconstituted embryos to the sixth or seventh day, and the blastocysts were collected. Compared with AI-derived blastocysts, its volume is smaller, the total number of cells in blastocysts is less, and the quality of embryos is poor (SCNT blastocyst morphology is shown in Figure 1B).

1.2 The rest of the establishment method of the culture system of pig trophoblast stem cells from SCNT-derived blastocysts is consistent with the establishment method of the culture system of pig trophoblast stem cells from AI-derived blastocysts described in Example 1. SCNT blastocysts are separated from trophoblast stem cells. The final efficiency is 6.34%. (The morphology of the embryo after removing the zona pellucida is shown in Figure 2B. The morphology of the blastocyst observed after inoculation the next day is shown in Figure 3B. The morphology of the SCNT-derived stem cells attached to the wall is shown in Figure 3D. The morphology of the SCNT-derived stem cells of the F0 and F3 generations As shown in Figure 4C and Figure 4D, respectively, the specific experimental data of the efficiency of SCNT blastocyst isolation trophoblast stem cell lines are shown in Table 1).

2. Identification of related marker genes of SCNT-derived trophoblast stem cells

2.1 The identification method of related marker genes of SCNT-derived trophoblast stem cells is consistent with the method described in Example 1 (the gene expression of SCNT-derived stem cells is shown in Figure 5).

2.2 Detection of trophoblast stem cell-specific expression genes CDX2 and Eomes by immunofluorescent staining: the basic method is the same as in Example 1, and the secondary antibody used is changed to Alexa Fluor 488-labeled fluorescent antibody, which shows green fluorescence (CDX2 and Eomes) The expression is shown in Figure 6).

3. The multi-lineage differentiation function identification method of SCNT-derived trophoblast stem cells is consistent with that described in Example 1 (the morphology of multinucleated giant cells after differentiation of SCNT-derived trophoblast stem cells is shown in Figure 7C, and the morphology of differentiated trophoblast cells is shown in Figure 7D shown).

The present invention is the first time at home and abroad to successfully develop a feeder-free culture system for large animal trophoblast stem cells. The growth factor combination culture solution with a clear chemical composition is selected to replace the traditional heterogeneous feeder layer support, avoiding heterogeneity. Cell contamination is of great significance for the study of the biological mechanism of this type of stem cells, early embryonic development and placental differentiation, and will further promote the application of trophoblast stem cells in somatic cell cloning and large animal transgenic research.

Claims (10)

1. A special culture medium for cultivating porcine trophoblast stem cells, characterized in that the components include bFGF, activin A, Y27632, Knockout SR and β-mercaptoethanol. 2. The special medium for cultivating porcine trophoblast stem cells according to claim 1, wherein the concentration of bFGF is 12.5~50ng/mL, the concentration of activin A is 5~20ng/mL, and the concentration of Y27632 is 2~50ng/mL. 10nmol/mL, the volume concentration of Knockout SR is 2%~5%, and the concentration of β-mercaptoethanol is 20~55 nmol/mL. 3. The special medium for cultivating porcine trophoblast stem cells according to claim 1 or 2, further comprising DMEM/F12 medium, Neurobasal medium, N-2 supplement, B-27 supplement, MEM NEAA and L-Glutamine. 4. the special medium for cultivating porcine trophoblast stem cells according to claim 3, is characterized in that, Mix DMEM/F12 medium and Neurobasal medium according to the volume ratio of 1:1 as the basic culture medium; Add N-2 supplement 50~200μL, B-27 supplement 100~400μL, MEM NEAA to every 20mL of basal culture medium 50~200μL, L-glutamine 100~200μL. 5. A method for cultivating porcine trophoblast stem cells, comprising the following steps: (1) The stem cell culture vessel is pre-coated with extracellular matrix; (2) The porcine blastocyst is digested with protease to remove the zona pellucida, and transferred to step (1) using the special medium for cultivating porcine trophoblast stem cells described in any one of claims 1 to 4 on the stem cell culture vessel coated with extracellular matrix to cultivate; (3) After culturing to form a clonal group with a total number of 1000-2000 cells, digest them into single cells and transfer to a new step (1) subculture the cells on culture vessels coated with extracellular matrix. 6. The method for cultivating pig trophoblast stem cells according to claim 5, wherein the blastocyst is a blastocyst derived from artificial insemination, a blastocyst derived from somatic cell nuclear transfer, a blastocyst derived from in vitro fertilization, or a parthenogenetic Blastocyst. 7. The method for culturing porcine trophoblast stem cells according to claim 5, wherein the extracellular matrix in step (1) is rat type I collagen, Matrigel or fibronectin. 8. The method for cultivating porcine trophoblast stem cells according to claim 5, wherein the protease in step (2) is pronase protease; blastocysts are cleaned with pig embryo culture medium PZM-3 before digestion, and then Transfer to the special medium for cultivating porcine trophoblast stem cells for washing; the ambient temperature for culturing is 39°C, the volume concentration of CO ₂ is 5%, and the saturation humidity. 9. The method for culturing porcine trophoblast stem cells according to claim 5, characterized in that the subculture method in step (3) is: rinse with DPBS, add an appropriate amount of TRYPLE select trypsin replacement enzyme to digest into single cells, Add a special medium for porcine trophoblast stem cells to stop digestion, pipette the cells to suspend, collect and centrifuge, dilute with a special medium for cultivating porcine trophoblast stem cells, and pass them to stem cell culture vessels coated with extracellular matrix at a ratio of 1:3 middle. 10. The method for cultivating porcine trophoblast stem cells described in claims 5 to 9 cultivates the porcine trophoblast stem cells obtained.