JP2002153281A - Bovine ES-like cell establishment method and bovine ES-like cell line - Google Patents

Abstract

(57) [Summary] [Problem] To provide a bovine ES cell having pluripotency and capable of long-term subculture in an undifferentiated state, and a method for establishing the same. SOLUTION: After the inner cell mass of bovine blastocyst is cultured on a mouse fetal fibroblast feeder layer to isolate primary ES-like cells, the primary ES-like cells are coated with extracellular matrix protein. For establishing bovine ES-like cells, characterized by subculturing on a prepared plate, and bovine ES-like cells established by this method

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The invention of this application relates to a bovine ES.
And a bovine ES-like cell line. More specifically, the invention of this application relates to a method for establishing bovine ES-like cells useful for producing chimeric bovines and cloned bovines, producing bovine organs in a test, and the like, and a bovine ES-like cell line established by this method. is there.

[0002]

BACKGROUND ART As embryonic stem cells (ES cells), a plurality of cell lines have been established in mice, which can be cultured in cells differentiated in vitro, and chimeric mice can be produced by transplanting them into early mouse embryos ( Forrester L. M et al., Proc.
Natl. Acad. Sci. USA, 88: 7514-7577, 1991); Palac
ios R et al., Dev. Biol, 92: 7530-7534, 1995). Recently, an ES-like cell line having pluripotency from porcine escaped blastocysts (Chen L. R et al., Theriogenology. 52: 195-212, 1
999) and ES cell lines with pluripotency from human blastocysts have been established (Thomson J. A et al.,
Science, 282: 1145-1147, 1998). However, no bovine ES cell line has been reported so far.

[0003] Saito et al (Ropux's Arch. Dev. Biol,
201: 134-141, 1992) report that bovine ES-like cells were subcultured several times. Recently, it was reported that bovine ES-like cells were established, chimeric embryos were produced, embryos were transplanted, and that the fetus was a chimeric bovine (Cibelli: J.
B et al., Nature Biotechnology, 16: 642-646, 199
8). In this report, the inner cell mass of bovine blastocysts (Inner c
ell mass: ICM). The characteristics of ES-like cells are morphologically that the ratio of the nucleus area to the cytoplasm is high and that the cytoplasm contains abundant lipids in mouse ES.
Similar to cells. It also shows that there is no production of vimentin and cytokeratin, which are markers for differentiated cells. However, in the case of bovine ES-like cells that have been reported so far, it has been confirmed that the alkaline phosphatase activity found in mouse ES cells is negative and that the expression of other undifferentiated marker genes has not been examined. It is not proof of the cell.

A recent report using stem cells (Prowse KR, a
nd Greider CW., Proc Natl Acad Sci USA., 92: 4818-4
822 (1995), Chadeneau RS, et al., Oncogene., 11: 893.
-898 (1995), Wright WE, et al., Dev Genet., 18: 173-
179 (1996), Broccoli D, etal., Proc Natl Acad Sci U
SA., 92: 9082-9086 (1995), Counter CM, et al., Bloo
d., 85: 2315-2320 (1995), Pain B, et al., Developme
nt., 122: 2339-2348 (1996), Thomson JA, et al., Scie
According to nce., 282: 1145-1147 (1998)), in germ cells (including ES cells) and somatic stem cells that continue to proliferate actively in an undifferentiated state, they are enzymes involved in the synthesis and elongation of partial telomeres at the chromosome end. The activity of a certain telomerase has been shown to be extremely high, and is considered to be one of the marker molecules that can characterize cells in an undifferentiated state. In recent studies using mouse ES cells, Oct-3 / 4 molecule (Okamo), a gene transcription factor, has been identified as a molecule involved in maintaining the undifferentiated state.
toK, et al., Cell., 60: 461-472 (1990), Scholer HR.,
Trends Genet., 7: 323-9 (1991), Shimazaki T, et a
l., EMBO J., 12: 4489-4498 (1993), Rosfjord E, eta
l., Biochem Biophys Res Commun., 203: 1795-1802 (19
94)) is attracting attention. Oct-3 / 4 is a multipotent undifferentiated cell during development (8-cell embryo-blastocyst stage IC
It is known that it is specifically expressed in M, ES cells and primordial germ cells (Okamoto K, et al., Cell., 60: 461).
-472 (1990), Scholer HR., Trends Genet., 7: 323-9 (19
91), Yoshimizu T, et al., Dev Growth Differ., 41:
675-684 (1999)).

[0005]

As described above, in cattle, ES cells such as mouse ES cells that can be cultured in an undifferentiated state even after multipotency or long-term subculture have not been established. If it is possible to establish bovine ES cells that have pluripotency and have no cell life, they can be used for the production of cloned cattle from nuclear transplantation using ES cells as donor cells. Mass production of individuals becomes possible. Furthermore, by creating transgenic individuals by introducing and modifying genes into ES cells, it is expected to contribute to the medical field, including application to production of useful proteins. For that purpose, it is necessary to establish ES cells that have pluripotency and can be subcultured for a long period of time in an undifferentiated state, and to establish a culture method.

The invention of this application has been made in view of the circumstances described above, and a bovine ES cell having pluripotency and capable of long-term subculture in an undifferentiated state, and the establishment thereof. The task is to provide a method.

[0007]

Means for Solving the Problems According to the invention of this application, the present invention solves the above-mentioned problems by culturing an inner cell mass of a bovine blastocyst on a mouse fetal fibroblast feeder layer to obtain primary ES-like cells. And then subculturing the primary ES-like cells on a plate coated with extracellular matrix protein, to provide a method for establishing bovine ES-like cells.

[0008] The invention of this application also relates to a bovine ES-like cell line established by the method of the present invention, wherein the bovine ES-like cell line is characterized in that chromosomal telomeres are not shortened and have normal telomerase activity. And a bovine ES-like cell line characterized by expressing the Oct-4 gene.

[0009]

DETAILED DESCRIPTION OF THE INVENTION According to the method of the present invention, an internal cell mass (ICM) is isolated from a bovine blastocyst isolated by a conventional method, and the ICM is isolated from a mouse fetal fibroblast. Primary ES-like cells are isolated by culturing on the feeder layer for 1-2 weeks. Then, the bovine ES-like cells are established by subculturing the primary cells on a plate coated with an extracellular matrix protein. As extracellular matrix proteins,
Type I collagen, fibronectin and the like used as a culture substrate for animal cells can be used. In addition, as a medium for primary culture and subculture, a basal medium (basal medium Eagk) usually used as an animal cell medium is used.
e: BME), minimum essential medium:
MEM), Dulbecco's modified Eagl
e medium: DMEM) or the like can be appropriately selected and used.

The bovine E established by the method of the present invention
One of the features of the S-like cell line is that the chromosomal telomeres are not shortened and have normal telomerase activity. The measurement of the telomere size and its activity can be confirmed, for example, by the methods described in the following Examples.

The bovine ES-like cell line established by the method of the present invention shows, as another characteristic, dominant expression of the Oct-4 gene specific to ES cells. This gene expression can be confirmed by measurement of mRNA by RT-PCR as shown in the Examples below, or by Western blot using an antibody against the transcript.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

[0013]

[Examples] Example 1: Establishment of bovine ES-like cells and long-term subculture (1) Method ES-like cells were obtained from Yamashita et al. (Cytotechnology, 31: 121-12).
9 (1999)), and established from bovine blastocyst stage embryos (8-9 days after insemination) created by in vitro culture. Specifically, immature eggs collected from slaughtered bovine ovaries contained 25
Transfer each of the 30 cells to a Repro C-1 culture plate containing 230 μl of serum-free medium (IVMD101; manufactured by Functional Peptide Research Laboratories).
In vitro maturation was carried out by culturing for 20 to 22 hours under conditions of 3% C ₂ /95% air, 38.5 ° C. and saturated humidity. Next, the matured ova and sperm were combined in an in vitro fertilization medium (IVF100; manufactured by Functional Peptide Research Institute) under the same culture gas phase conditions, and cultured for 6 hours to perform in vitro fertilization. After in vitro fertilization, put 25-30 eggs into one well of Repro C-1 culture plate containing 230 μl of IVMD101 medium,
The cells were cultured for 8-9 days under the conditions of 5% CO ₂ /95% air, 38.5 ° C., and saturated humidity to produce blastocysts.

For the establishment of bovine ES-like cells, blastocysts on day 8-9 of developmental culture were used. The isolation and culture of ICM is basically performed by Cibelli et al. (Nature Biotechnology, 16: 642-646 (1.
998)). The ICM from which trophoblast cells had been surgically and mechanically removed was first cultured on the feeder layer of mouse fetal fibroblasts for 7 to 10 days to establish primary ES-like cells. Here, bovine ES-like cells generally tended to grow densely in sheet form.

As a medium for growing ES-like cells, Dulbecco's modified Eagle MEM (DMEM; High Glucose: GIBCO) is 10%.
Fetal bovine serum (FBS, CELLect GOLD; ICN Biomedicals), 1
00 μM mercaptoethanol (Hansui Science), 1 mM MEM
Using a medium supplemented with a non-essential amino acid solution (x 1: GIBCO) and 1 mM sodium pyruvate (x 1: GIBCO), 5% CO2 / 9
The test was performed under the conditions of 5% air, 37 ° C. and saturation humidity.

Conventionally, feeder cells of fibroblasts derived from mouse embryos have been used for long-term subculturing of ES-like cells. In addition to the time-consuming operation of preparing feeder cells each time, feeder cells are While factors and the like that contribute to the maintenance of the undifferentiated state of ES-like cells exist, it is considered that there are factors and the like that induce differentiation. Therefore, it is not necessarily appropriate for performing long-term subculture. In the method of the present invention, long-term subculture of ES-like cells was performed without using feeder cells and using a culture system in which a type I collagen, which is a kind of extracellular matrix component, was coated in a thin layer on a culture dish.

Bovine ES-like cells have the property of losing their proliferative properties when completely dispersed in single cells by trypsin treatment or the like. Therefore, the subculture was performed using 100 to 200 cell populations as one unit. In the passage operation, primary ES-like cells growing on the mouse fetal fibroblast feeder layer are:
Under a stereomicroscope, cut out as a 1-1.5 mm square cell subpopulation with a syringe needle (27G) and preliminarily 6-well plate (BECTN D
ICKINSON) 0.5 ml of 0.15% type I collagen (CELLG) per well
(EN, I-AC; Koken) and transferred to a 6-well plate equilibrated with a growth medium (3 ml). The cell population was cultured by gently pressing it onto a collagen thin layer with a syringe needle (27G) under a stereoscopic microscope, and culturing the cells, followed by subculture at intervals of 10 to 12 days. (2) Results ICM was isolated from bovine blastocyst stage embryos, and ES-like cells grown and cultured on feeder cells of mouse fetal fibroblasts for about 10 days were treated with a 0.15% type I collagen-coated plate. Subcultured above. The ES-like cells cultured on the type I collagen thin layer had the same cell morphology as in the case of using the mouse fetal fibroblast feeder layer, and good and uniform proliferation was observed (FIG. 1-A). On the other hand, when ES-like cells are subcultured on a mouse fetal fibroblast feeder layer,
Uneven growth and differentiation of ES-like cells occur and cell clumps (Fig. 1-
B, arrow) was observed, and growth of compliant cells was inhibited.

Bovine ES cultured on a type I collagen thin layer
Cell-like morphology (Fig. 2)
(A, -B) showed morphology of epithelial cells as observed in mouse ES cells, and favorable cell growth with extensibility was observed. Example 2: Analysis of undifferentiated marker of bovine ES-like cells Properties of bovine ES-like cells established in Example 2 (undifferentiated)
In addition to the characterization of ES-like cells previously shown, telomerase activity and Oct-3 / 4 molecule expression, which are characteristically expressed in mouse ES cells and the like, were recently examined. a) Measurement of telomerase activity The telomerase activity was measured using a TeloChaser telomerase activity measurement kit (Toyobo). The amount of the ES-like cell extracted protein used in one reaction was 20 μg / sample. Telomerase relative activity was determined by electrophoresis and SYBR Gre
en (Takara Shuzo) Take a photograph of the gel after staining, and use the positive control HeLa cell extract (2.5 μg / sample: 1.25 x 10 ⁴ cel) based on the staining intensity of the amplified PCR product band.
(equivalent to ls) was set to 100 and calculated as relative activity. b) Analysis of telomere length Genomic DNA prepared from cells was digested with the restriction enzyme Hinf I (Takara Shuzo)
After complete digestion with RasI and Takara Shuzo, 1.1% agarose gel pulse field electrophoresis (BIORADSHEF-DRII)
After electrophoresis in I), the gel was dried under suction. After drying, the gel was treated with 0.5 M NaOH / 1.5 M NaCl solution for 30 minutes, 0.5 M Tri
s, pH 8 / 1.5 M NaCl solution for 30 minutes, ³² P-label (TTA
GGG) Hybridization using ₃ telomere detection probe (5X SSCPE, 5X Denhardt's sol., 42 ℃, 8-
12 hr). After hybridization, run the gel
The excess probe was removed by washing three times at 0.1 X SSC at 25 ° C, and the average TRF (Terminal) was measured with BAS5000 (Fuji Photo Film).
Restriction Fragment) was measured. c) Expression analysis of bovine Oct-4 gene Expression analysis of Oct-4 gene in bovine ES cells was performed using bovine (van E
ijk, MJT, et al., Biol. Reprod., 60: 1385-1391 (199
Primers (SEQ ID NOS: 1 and 2) were synthesized based on the Oct-4 cDNA base sequence reported in 9)), and the synthesis was performed by RT-PCR. The PCR product amplified by RT-PCR using bovine Oct-4 analysis primers was obtained from TA-Cloning Kit (Invitor
gen), and the nucleotide sequence of the PCR product was confirmed. For nucleotide sequencing, use the Dye Terminator cycle Seq
This was performed using an ABI373A sequencer (ABI) using uencing kit (PERKIN ELMER). (2) Results The established bovine ES-like cells showed relatively high telomerase activity, indicating that the cells can be cultured in a state where they are maintained in an undifferentiated state. The established two ES-like cell lines (ES-A, ES-B) were continuously passaged, and changes in telomerase activity were examined at each passage number (Fig. 3-A,-
B). Although there was some difference in telomerase activity at each passage number, it was confirmed that the telomerase activity of ES-like cells was retained even after passage of 20-30 times or more (around 1 year of continuous culture). (Fig. 3). Since the telomerase activity did not disappear even when the established ES-like cells were subcultured for a long period of time, it was expected that the chromosome telomere length of the subcultured ES-like cells was maintained or extended. To confirm this, the chromosome telomere length of ES-like cells was measured. As shown in FIG. 4, the ES-like cell line was subcultured (culturing days: about 200 days at P18, about 360 days at P32)
However, the size of the chromosome telomere was not shortened even when the reaction was continued, and the long level of 24 kb was maintained. On the other hand, fibroblasts without telomerase activity (data not published)
Means long-term subculture (culture days: P2 about 4 days, P32 about 110 days)
As a result, the telomere length was significantly shortened from 18 kb to 11 kb.

Recently cloned POU5F1 (bovine Oct-4
Structural gene corresponding to van Eijk, MJT, et al., Bio
l. Reprod., 60: 1385-1391 (1999)), synthesize a primer for bovine Oct-4 analysis based on the gene sequence of bovine Oct-4
Was analyzed. Bovine Oct-4 gene expression is expressed in blastocysts and ES-like cell lines -A, established from blastocysts, and
In -B, it was confirmed at a relatively high level, but hardly found in adult bovine fibroblasts (somatic cell line) (Fig. 5-A, -B). The expression of the Oct-4 gene in bovine ES-like cells was maintained even after repeated passages (FIG. 5-C).

From the above results, it was confirmed that the cells established by the method of Example 1 were bovine ES-like cells.

[0021]

As described above in detail, according to the invention of this application, a method for simply and reliably establishing bovine ES-like cells having molecular biological characteristics as ES cells,
Bovine ES-like cells established by this method are provided. This makes it possible to perform various genetic manipulations and the like on bovine individuals.

[0022]

[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Corporation et al. <120> Bovine ES cells and a Method for obtaining the cells <130> NP00494-YS <160> 2 <210> 1 <211> 25 <212 > DNA <213> Artificial Sequence <220> <213> Synthesized oligonucleotide <400> 1 caggccgatg tggggctcac cctgg 25 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <213> Synthesized oligonucleotide <400 > 2 cagtttgaat gcaagggaga gcccag 26

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a micrograph image of a bovine ES-like cell passaged (second time) on day 8 of culture. A shows cells on a plate coated with type I collagen, and B shows cells on a mouse fibroblast feeder layer.

FIG. 2 is a micrograph image showing the cell growth morphology of bovine ES-like cells having different passage numbers. A is the second passage, B is the passage
This is the 16th time.

FIG. 3 shows changes in telomerase activity of subcultured bovine ES-like cells. A is a gel electrophoresis image, and B is a graph of a relative activity value with respect to a control.

FIG. 4 is a gel electrophoresis image showing measurement results of chromosome telomere lengths of bovine ES-like cells and bovine adult muscle tissue-derived fibroblasts having different passage numbers.

FIG. 5 shows measurement results of Oct-4 gene expression in bovine blastocysts, ES-like cells, and cultured somatic cells (fibroblasts). A is a gel electrophoresis image of each RT-PCR product, B is a graph of expression intensity relative to control, C is Oct-4 gene expression product at each passage number of subcultured ES-like cells. It is a gel electrophoresis image of (RT-PCR product).

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Claims (3)

[Claims] 1. An inner cell mass of a bovine blastocyst is cultured on a mouse fetal fibroblast feeder layer to isolate primary ES-like cells, and then the primary ES-like cells are treated with extracellular matrix proteins. A method for establishing bovine ES-like cells, comprising subculturing on a coated plate. 2. A bovine ES-like cell established by the method of claim 1, wherein the bovine ES has a normal telomerase activity without shortening of chromosomal telomeres.
Cell line. 3. A bovine ES-like cell line established by the method of claim 1, wherein the Bovine ES-like cell line expresses the Oct-4 gene.