JP2002176973A - Mammalian embryonic stem cell and method for establishing the same and subculture method for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide multifunctional embryonic stem cells, to provide a method for establishing the stem cells, and to provide a subculture method for the embryonic stem cells. SOLUTION: The objective mammalian embryonic stem cells are characterized by having all of the following cell biological characteristics: (1) derived from blastodermic embryos, (2) continuing the a proliferation in an undifferentiated condition, (3) expressing a sugar chain SSEA-1 antigen, (4) alkali phosphatase activity is positive, (5) expressing transcription factor Oct 3/4, and (6) having multidifferentiation potency. The method for establishing the embryonic stem cells comprises culturing cells obtained from the inner cell mass of mammalian blastodermic embryos on a feeder of umbilical cord endothelial cells isolated from an umbilical cord by the use of fetal bovine serum-containing MEMαas the medium to form a colony. The subculture method for the embryonic stem cells is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an embryonic stem cell, a method for establishing the same, and a method for subculturing the embryonic stem cells. More specifically, the present invention is used as research material for elucidating the cell biology and molecular biology control and conversion functions of differentiating embryonic stem cells into organs and organs into individuals, or producing organs for xenotransplantation. Embryonic stem cells, which have the potential for high frequency of homologous recombination with foreign genes, can be useful as donor cells for producing recombinant cloned animals that serve as bioreactors for useful drugs. The present invention relates to a sex stem cell and a method for establishing the same, and a method for subculturing the embryonic stem cells.

[0002]

BACKGROUND OF THE INVENTION Since the discovery of mouse embryonic stem cells as cultured cells showing the ability to differentiate into living organisms, the regeneration of tissues and organs using these embryonic stem cells, the pursuit of embryonic development mechanisms, and cloning Embryology, which aims to create individuals, has been highlighted, and is expected to be one of the directions to be pursued in the next generation of medicine and biotechnology.

[0003] Since a group of researchers in the United States first reported the establishment of embryonic stem cells in humans in 1998 (Thompson TA, et al., Science).
ce 282, 1145-1147, 1998; Sha
mblott MJ, et al. Proc. Nat
l. Acad. Sci. USA. 1995, 13726
Embryonic stem cells have suddenly come into the limelight as universal cells that are paving the way for regenerative medicine and gene therapy of the human nervous system, blood cell system, organs, and the like.
However, in animal species other than mice, the production of germline chimeras using embryonic stem cells (or embryonic stem-like cells) has not been successful yet (SticeS, et al., Bi).
ol. Reprod. 48, 715-719, 199
6; Strelchenko N, Therio-ge
No., 37, 111-126, 1996), genetic manipulation of embryonic stem cells in vitro, and the production of transgenic individuals via transformed stem cells is currently limited to mice (Bradley). A, et
al. , Nature, 399, 255, 198.
4).

[0004] Stellechenko reported in the above-mentioned article that he established bovine embryonic stem-like cells, performed nuclear transfer using these cells as donor nuclei, and transplanted cloned embryos into recipient cattle. are doing. But,
The expression of alkaline phosphatase, which the present inventors have listed as a feature of the stem cells, is negative, and it seems that other conditions have not been cleared. Also, Campbell et al. Succeeded in producing cloned offspring from cultured cells derived from embronic discs in sheep (Nature, 38).
0, 64-66, 1996), the expression of alkaline phosphatase and the sugar chain SSEA-1 which are characteristics of embryonic stem cells.
No description is given regarding the expression of the antibody.
The cells from the nic disc are morphologically like epithelial-like cells.

[0005] Stellechenko, Campbel
have used mouse STO, a fetal fibroblast cell line, or primary fibroblasts derived from a mouse fetus in order to culture embryonic stem-like cells. Primary fibroblasts used for the maintenance of embryonic stem cells can be used as cell lines if used after the secondary culture of primordial germ cells, but cannot be subcultured when used in primary cultures (Stewart, CL, et al.). al., Dev.
iol. 161, 626-628, 1994), it is highly likely that the use of the primary fibroblasts for the establishment of embryonic stem cells of livestock species such as cattle and sheep prevented the establishment of stem cells. It is also known that embryonic stem cells are maintained in an undifferentiated state in the presence of a leukemia inhibitory factor, and promote cell proliferation. However, even with the addition of leukemia inhibitory factors, rodents other than mice and livestock embryonic stem cells have not been successfully established, and an unknown growth factor may be present.

As described above, no animal species in which embryonic stem cells have been established have been confirmed other than mice and humans, and the expression of sugar chains SSEA-1 essential for identifying embryonic stem cells, There are no reports that the culture system has pluripotency.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has as its object to provide embryonic stem cells other than mice and humans. Another object of the present invention is to provide a method for establishing those cells and a method for continuously culturing those cells.

[0008]

The present invention firstly solves the above-mentioned problems by providing the following cell biological features:
(1) that it is derived from the blastocyst stage embryo, (2) that it continues to grow in an undifferentiated state, (3) that the sugar chain SSEA-1 antigen is expressed, and (4) that its alkaline phosphatase activity is positive. A mammalian embryonic stem cell, characterized in that it has all of the following: (5) expression of a transcription factor Oct3 / 4; and (6) multipotency.

The present invention also relates to a method for culturing cells obtained from the inner cell mass of a mammalian embryo on a feeder of umbilical cord endothelial cells separated from the umbilical cord using MEMα containing fetal bovine serum as a medium to form colonies. Or, this colony was tested for the presence or absence of alkaline phosphatase activity, sugar chain SSEA.
The present invention provides a method for establishing embryonic stem cells, characterized in that screening is performed using the expression of -1 antigen, the expression of Oct3 / 4, the presence or absence of pluripotency, and the proliferation in an undifferentiated state as indices.

Further, the present invention provides a method for primary culturing cells obtained from the inner cell mass of a mammalian embryonic stem cell stage embryo on a feeder of umbilical cord endothelial cells separated from the umbilical cord using MEMα containing fetal bovine serum as a medium. To form colonies,
The colony cells were detached from the medium with a 0.25% trypsin 0.1% EDTA solution, and the detached cells were washed with PBS.
A method for subculturing embryonic stem cells, comprising washing the cells by centrifugation together with a solution, dispersing the cells into individual cells, and reculturing the cells in the medium used for the primary culture.

In the above-mentioned method for establishing embryonic stem cells and the subculture method, cells obtained from the inner cell mass of a mammalian blastocyst-stage embryo are subjected to the treatment of fetal bovine serum, epidermal growth factor, In a preferred embodiment, the cells are cultured in a MEMα medium containing a leukemia inhibitory factor.

[0012]

First, the method for establishing embryonic stem cells of the present invention will be described in more detail. Usually, in order to establish embryonic stem cells, mitomycin treatment or γ
Mouse STO feeder cells that have been irradiated to stop cell division are required. In the present invention, umbilical cord endothelial cells are used as feeders. The culture medium contains fetal calf serum (FC
S), contains leukemia inhibitory factor (LIF). Epidermal growth factor (EGF) is not essential for the formation of embryonic stem cell colonies, but has a remarkable effect on cell lineage of embryonic stem cells, and it is important to add it to the culture medium as a growth factor for cells. It is. Using umbilical cord cells as feeders,
The addition of EGF to the culture medium acts synergistically on the growth of embryonic stem cells, allowing the establishment of cell lines.

The amounts of these components added to the medium are, for example, 5-10% for FCS and 10-50 ng / m for LIF.
l, EGF can be about 10 to 50 ng / ml. The cultivation is performed at a temperature of about 39 ° C. under 5% CO ₂ conditions. By the culture as described above, a colony of embryonic stem cells is obtained. Furthermore, cells that form these colonies must, for example, express the sugar chain SSEA-1 antigen, be positive for alkaline phosphatase, express the transcription factor Oct3 / 4, and have pluripotency. The expression of the function as an embryonic stem cell can be confirmed by screening using, as an index.

For example, the expression of the SSEA-1 antigen can be confirmed by staining the cells with the SSEA-1 antibody and separating the cells by flow cytometry. Having pluripotency is to differentiate into neurons in a culture system,
al fibrillary acidic prot
The pluripotency can be confirmed by staining with ein (GFAP) and a Nestin antibody marker. Furthermore, nuclear transfer to unfertilized eggs derived from in vitro culture in which the embryonic stem cells were enucleated,
Further, it can be confirmed by developing the blastocyst by in vitro culture and transferring the blastocyst to the uterus of a recipient female to make it pregnant.

Next, the method for subculturing embryonic stem cells according to the present invention will be described. In this method, a colony of embryonic stem cells (primary cultured cells) obtained by the above-described establishment method is detached from an incubator, recultured in another incubator, and expanded. When detaching the colony, after removing the culture medium from the incubator, add about 0.1 to 0.25% trypsin EDTA solution and treat at about 39 ° C. for about 6 minutes to remove the colony together with the feeder cells. Can be peeled off. By culturing this in a medium similar to that used for primary culture derived from umbilical cord endothelial cells in which cell division has been suppressed in advance (that is, a medium containing FCS, LIF, and EGF), the colonies are adhered to the feeder, and Can be multiplied. Other than the cells used for subculture, they can be cryopreserved in liquid nitrogen according to a standard method.

As described above, the method of the present invention can be applied to the establishment of embryonic stem cells of all mammals including humans, and can be used to establish embryonic stem cells from various animal species, Sex stem cells can be cultured continuously. For example, embryonic stem cells of pigs, cows, and horses can be used to create organs for xenotransplantation,
It can be used for the production of recombinant cloned animals as a bioreactor for useful drugs, and is expected to contribute greatly to the development of regenerative medicine and biotechnology.

Hereinafter, the present invention will be described in more detail and concretely with reference to examples, and the characteristics of the embryonic stem cells obtained by this method will be explained by showing test results. However, the present invention is not limited by the following examples.

[0018]

Example 1 Embryonic stem cells of the present invention were obtained by the following method, and the cells were tested for biological properties.

(1) A method for separating and culturing umbilical cord endothelial cells and preparation as feeder cells will be described. The bovine neonatal umbilical cord was cut to approximately 1 cm ² with sterile surgical scissors,
% Ethanol, spray-sterilized, and washed several times with PBS (-) (without Dulbecco, Ca, Mg) containing antibiotics (2000 units / ml of penicillin;
Meiji Seika, Streptomycin 100 μg / ml; Meiji Seika, Fangisone 25 μg / ml; GIBCO BR
L). Further, the above sample was cut into about 1 mm ² on a sterile petri dish, and then 3 ml of 0.25% trypsin 0.1% E
Treated with DTA (GIBCO BRL) for 10 minutes. Trypsin solution containing this sample in 5 ml of PBS
After dilution with (-), centrifugation (1000 rpm, 5 minutes)
The precipitate was suspended in 5 ml of PBS (-) and centrifuged again under the same conditions. The obtained precipitate was subjected to 10% FCS (GIB
CO BRL) and MEMα (GIB) containing antibiotics
CO BRL) 3 ml culture dish (primary,
Falcon, 6cm diameter), 38.6 ℃, 5% CO
Culture was performed under the conditions of ₂ . After 3 to 4 days of culture, endothelial cells appeared as colonies around the minced umbilical cord sample. After further culturing for 7 to 10 days until reaching confluence, the umbilical cord sample mass was removed and subculture was performed. The medium is replaced with a new one every two days. Passage the cells to PB
After washing twice with S (-), the cells were treated with trypsin EDTA for several minutes, and the cells were plated at a concentration of about 1/5 in a 6 cm culture dish. Almost confluent after 4 days. Two
After repeating the passage three times, the cells were cryopreserved according to the conventional method. When used as a feeder, the frozen cells were thawed and a new culture was started. Procedure for creating feeder cells. a Mitomycin (Sigma) was added to umbilical cord endothelial cells near a confluence at a concentration of 10 μg / ml and cultured for 3 to 4 hours. b medium was removed and the cells were washed three times with PBS (-), treated with trypsin to detach cells, and then centrifuged (10
00 rpm, 5 minutes). The cells were suspended at a concentration of 2 × 10 ⁶ / ml. 0.5 ml of the suspension was added dropwise to each well of a c4 well culture dish (Nunc). The uniform feeder cells, 2 days or more after preparation, were used for the establishment of embryonic stem cells.

(2) Establishment and Culture of Embryonic Stem Cells Embryos that had been cryopreserved in advance were thawed according to a conventional method and then subjected to a test. In rats, blastocyst stage embryos 4 days after copulation was confirmed, and in cows and horses, blastocyst stage embryos 7 days after artificial or natural mating were used, respectively. An inner cell mass (ICM) portion was cut out from all blastocysts by microsurgery, and cultured on feeder cells. 20 ng / ml EGF (S
cultivation was continued in MEMα (containing 10% FCS, penicillin and streptomycin) supplemented with 20 ng / ml LIF (Sigma) at 38.6 ° C. and 5% CO ₂ . The medium was changed once every two days. The ICM on the feeder adhered to the feeder in 1-2 days and continued to grow. After 4 to 5 days of culture, surrounding cell colonies have developed. Further culturing was continued until the colony diameter reached 3000.
When the thickness reached ４4000 μm, the cells were detached from the feeder using a pipette, treated with a trypsin solution for about 6 to 7 minutes, and then the cells were dissociated using a glass pipette. After dissociation, the cells were diluted with PBS (-), centrifuged twice and washed, and the resulting precipitate was plated on feeder cells at a 1/3 concentration under the same culture conditions as described above (primary culture).

(3) Test Method A morphology that is one of the indicators of embryonic stem cells was observed under a phase-contrast microscope, and proliferation in an undifferentiated state was examined. Alkaline phosphatase activity, which is a characteristic of undifferentiated cells, is characterized by fast red TR salt (Si
gma) containing Naphthol AS MX Pho
Detected by staining with sphate (Sigma). Expression of the undifferentiated marker SSEA-1 antigen in embryonic stem cells was determined by flow cytometry (fluorescence activation) by anti-SSEA-1 antibody staining.
SS using ed cell sorter (FACS)
An attempt was made to separate a population of stem cells that bind to the EA-1 antibody. That is, 2 × 10 ⁵ rat embryonic stem cells were used as anti-rat IgG
And after reaction with anti-mouse SSEA-1 antibody, FITC (f
fluorescein isothio-cyanat
e) Secondary staining with labeled rabbit anti-mouse IgG was performed and analyzed by FACS. After reacting 2 × 10 ⁵ bovine embryonic stem cells with anti-bovine IgG and anti-mouse SSEA-1 antibody, FIT
Secondary staining with C-labeled rabbit anti-mouse IgG was performed and analyzed by FACS. Furthermore, in horses, 2 × 10 ⁵ embryonic stem cells were reacted with anti-horse IgG and anti-mouse SSEA-1 antibodies, then secondary-stained in the same manner as rats and cows, and analyzed by FACS. The transcription factor Oct3 / 4 used to identify mouse undifferentiated cells was detected by PCR.
In order to examine the pluripotency of embryonic stem cells, the obtained stem cells are differentiated into nervous cells, and they are detected immunohistochemically using GFAP, a nestin antibody, a cell marker antibody for astrocytes and neural stem cells. Tried. That is, horse and rat embryonic stem cells were subcultured three times in MEMα medium containing EGF, fibroblast growth factor (FGF) 2 and FGF9, and when glial cells were observed, mouse anti-Nes
After the reaction with the tin antibody, a secondary reaction was performed with an alkaline phosphatase-conjugated anti-mouse IgG antibody, and staining was performed with Fast red. Similarly, after subculture three times in a place containing EGF, FGF2 and FGF9, these cytokines are removed,
When the cells were further cultured for 7 days, the cells were reacted with a rabbit anti-GFAP antibody, then subjected to a secondary reaction with an alkaline phosphatase-conjugated anti-rabbit IgG antibody, and stained in the same manner. Further, in order to examine the pluripotency of bovine embryonic stem cells, the stem cells were used as donor nuclei, nuclear transfer was performed, cloned embryos were produced, and then transplanted into recipient females. EGF and LIF, which are additional factors to the medium, are used, respectively, and one or three types of horse embryonic stem cells are cultured in a control medium excluding both, and the effect of the additional factors on cell growth is examined. Examined. In addition, to examine the effect of feeder cells on stem cell establishment, bovine ICM was cultured without feeder cells and compared with a control.

(4) Test Results As a result of culturing under the conditions of (2) above, embryonic stem cells in which cells very similar to mouse embryonic stem cells were closely aggregated as shown in the phase contrast micrographs of FIGS. Colonies were obtained. Primary cultures required 7-10 days to reach confluence. Alkaline phosphatase activity was positive (FIGS. 4, 5, and 6). The percentage of anti-mouse SSEA-1 antibody-positive cells in embryonic stem cells is shown in FIG.
It is indicated by 8 and 9. 24.5% rats, 1 cow
The ratio was 0.2% and the proportion of horses was 38.4%, suggesting that there was a difference between animal species.

[0023]

[Table 1] As is clear from Table 1, Oct3 / 4 was positive in embryonic stem cells of all animal species.

Next, as evidence of the pluripotency of embryonic stem cells, equine astrocyte marker GPAP antibody-positive cells (FIG. 10) and neural stem cell marker Nest
In antibody positive cells (FIG. 11) were generated by an in vitro culture system. This result proved that the equine embryonic stem cells had pluripotency. Similar results were obtained with rat embryonic stem cells.

[0025]

[Table 2]

Table 2 shows the pregnancy rates of nuclear transfer embryos using bovine embryonic stem cells as donor nuclei. This also confirmed the pluripotency of the cell nucleus.

As shown in FIGS. 1 to 11 and Tables 1 and 2, it was confirmed that the cell lines established in the present invention completely exhibited the characteristics as embryonic stem cells having pluripotency.

As apparent from FIG. 12, EGF, LIF
In the control excluding both, the cells did not reach confluence even if the culture was continued for 1 week, whereas in the system added with EGF alone, 4 days, the combined use of EGF and LIF resulted in an additional 3 days.
At day, there was a noticeable proliferative effect reaching confluence.

[0029]

[Table 4]

As is clear from Table 4, it was impossible to establish bovine embryonic stem cells without the presence of feeder cells, but by using feeder cells, 5 out of 9 ICMs (56%) were established as stem cells. It was suggested that the use of feeder cells is essential for the establishment of embryonic stem cells.

Example 2 The embryonic stem cells obtained in Example 1 were subcultured by the following method. After removing the medium from the culture dish of the bovine embryonic stem cells that reached confluence on the feeder cells, 0.25% trypsin 0.1% EDTA
For 6 to 10 minutes to disperse embryonic stem cells and feeder cells in the solution. This suspension was centrifuged twice (1000 rpm, 5 minutes), and the obtained precipitate was plated on a medium having the same composition as that used in the subculture at a concentration of 2 × 10 ⁵ cells / ml. Active division and proliferation of the cells on the culture dish were observed (FIG. 13), and the cells reached confluence in about 4 to 5 days.

[0032]

As described above in detail, the present invention provides an embryonic stem cell, a method for establishing the cell, and a method for continuously culturing the embryonic stem cell. This will enable stem cell regeneration, elucidation of differentiation mechanisms, creation of organs for xenotransplantation, and production of recombinant cloned animals that will serve as bioreactors for useful drugs, contributing to the development of regenerative medicine and biotechnology. Can be expected.

[Brief description of the drawings]

FIG. 1: Micrograph 200 of rat embryonic stem cell colony microscopy
Times.

FIG. 2 is a photomicrograph of a bovine embryonic stem cell colony at a magnification of 200 times.

FIG. 3 is a photomicrograph of a horse embryonic stem cell colony at a magnification of 100 times.

FIG. 4 is a photomicrograph of a rat embryonic stem cell colony alkaline phosphatase staining microscope at a magnification of × 200.

FIG. 5 is a photomicrograph of a bovine embryonic stem cell colony alkaline phosphatase staining microscope at a magnification of 200 times.

FIG. 6 is a photomicrograph 200 times of equine embryonic stem cell colony alkaline phosphatase staining microscopy.

FIG. 7 shows the results of FACS of rat embryonic stem cells by anti-SSEA-1 antibody. The area shaded in black is the percentage of positive cells.

FIG. 8. F of bovine embryonic stem cells with anti-SSEA-1 antibody
ACS results. The area shaded in black is the percentage of positive cells.

FIG. 9: F of horse embryonic stem cells by anti-SSEA-1 antibody
ACS results. The area shaded in black is the percentage of positive cells.

FIG. 10 is a photomicrograph of GFAP antibody-positive cells derived from equine embryonic stem cells at 100 ×.

FIG. 11 is a 200 × photograph of a Nestin antibody-positive cell micrograph derived from a horse embryonic stem cell.

FIG. 12 is a graph showing the effect of cell growth factors on horse embryonic stem cell proliferation.

FIG. 13: Photomicrograph 20 of bovine embryonic stem cells (passage number 5)
0 times.

Claims (9)

[Claims] 1. The following cell biological characteristics (1) derived from a blastocyst stage embryo, (2) continued growth in an undifferentiated state, and (3) expression of a sugar chain SSEA-1 antigen To do,
(4) that the alkaline phosphatase activity is positive, (5) that the transcription factor Oct3 / 4 is expressed,
(6) Mammalian embryonic stem cells having all of pluripotency. 2. The embryonic stem cell according to claim 1, wherein the mammal is a rat, cow or horse. 3. The cells obtained from the inner cell mass of a mammalian blastocyst stage embryo are cultured on a feeder of umbilical cord endothelial cells separated from the umbilical cord using MEMα containing fetal bovine serum as a medium to form a colony. A method for establishing embryonic stem cells, comprising: 4. The cells obtained from the inner cell mass of a mammalian blastocyst stage embryo are cultured on a feeder of umbilical cord endothelial cells separated from the umbilical cord using MEMα containing fetal bovine serum as a medium to form colonies. And screening the colonies using as indicators the proliferation ability in an undifferentiated state, the sugar chain SSEA-1 antigen expression, the presence or absence of alkaline phosphatase activity, the expression of Oct3 / 4, and the presence or absence of pluripotency. Method for establishing embryonic stem cells. 5. The cell obtained from the inner cell mass of a mammalian blastocyst stage embryo is cultured in a MEMα medium containing fetal calf serum, epidermal growth factor, and leukemia inhibitory factor.
Method for establishing embryonic stem cells. 6. The method for establishing embryonic stem cells according to claim 3, wherein the mammal is a rat, cow, or horse. 7. A colony obtained by primary culture of cells obtained from the inner cell mass of a mammalian blastocyst stage embryo on a feeder of umbilical cord endothelial cells separated from the umbilical cord using MEMα containing fetal bovine serum as a medium. Once formed, the colony cells are detached from the medium with a trypsin EDTA solution, the detached cells are washed with a PBS solution by centrifugation, washed and dispersed into individual cells, and then re-dispersed in the medium used for the primary culture. A method for subculturing embryonic stem cells, which comprises culturing. 8. The embryonic stem cell according to claim 7, wherein cells obtained from the inner cell mass of the mammalian blastocyst stage are primarily cultured in a MEMα medium containing fetal bovine serum, epidermal growth factor, and leukemia inhibitory factor. Subculture method. 9. The method for subculturing embryonic stem cells according to claim 7, wherein the mammal is a rat, cow or horse.