WO2008146991A1

WO2008146991A1 - Process for the isolation of placenta-derived trophoblast stem cells

Info

Publication number: WO2008146991A1
Application number: PCT/KR2007/005234
Authority: WO
Inventors: Gi-Jin Kim; Kyung-Sun Shin; Kyu-Hwan Na
Original assignee: Chabiotech Co., Ltd.; College Of Medicine Pochon Cha University Industry-Academic Cooperation Foundation
Priority date: 2007-05-29
Filing date: 2007-10-24
Publication date: 2008-12-04
Also published as: KR20080104844A

Abstract

The present invention provides a method for isolating trophoblast stem cells, the method including: (a) harvesting placental villi from a detached normal placenta; (b) adding an enzyme solution containing trypsin, DMase I, and dispase to the placental villi of step (a) to perform an enzymatic reaction and adding a fetal bovine serum thereto to terminate the enzymatic reaction; (c) centrifuging the reaction solution of step (b) and separating cytotrophoblasts from the recovered cells by a density-gradient separation method; and (d) culturing the cytotrophoblasts of step (c) in a medium containing a fetal bovine serum and an antibiotic.

Description

PROCESS FOR THE ISOLATION OF PLACENTA-DERIVED TROPHOBLAST STEM

CELLS

TECHNICAL FIELD

The present invention relates to a method for isolating placenta-derived trophoblast stem cells at highly purity.

BACKGROUND ART

The placenta is a disk-shaped organ that develops in the uterine wall during pregnancy and has abundant vascular tissues. The nutritive, respiratory, and excretory functions of the fetus are carried by means of the placenta. Based on the scientific evidences that the basic foundation of people's health is determined during pregnancy, the importance of the placenta has been getting higher. Much effort has been made to elucidate mutual relations between various materials related thereto. The placenta is composed of various types of cells according to week of gestation or the position thereof, and its applicability has been extremely limitedly. Studies about classification and functions of trophoblast cells and establishment of the cell lines have been insufficiently carried out in domestic and foreign countries. Some trophoblast cell lines have been established in a mouse model. This provides a new cell culture system required for studying trophoblast development and placentation, but the establishment of trophoblast cell lines in the other mammals has not yet been reported clearly.

The placenta is the most different organ among mammal species, and in particular, is significantly different between humans and mice in trophoblast development. It is very difficult to elucidate the metabolism of female sterility and placental insufficiencies in human, by using trophoblast cell lines established in mice. Blastocyst formation is similar between humans and mice, but post-implantation procedures are significantly different between humans and mice. The trophectodermal cells of mouse blastocysts rapidly proliferate, and after implantation, form extraembryonic ectoderm (ExE). On the other hand, many trophectodermal cells of human blastocysts fuse to form multinucleated syncytiotrophoblasts, and cytotrophoblasts, which are diploid trophoblast cells. The trophoblast cells have different shapes and functions according to week of gestation or the position of the placenta. Thus, trophoblast cell lines have been extremely limitedly utilized in studying placental insufficiencies and female sterility.

In this regard, there is an increasing need to develop a method of isolating human trophoblast cells, in particular, a method of isolating human trophoblast stem cells.

Recently, U.S. Pat. Pub. No. 2006/0211110 discloses a method of isolating human trophoblast stem cells. According to this patent document, trophoblast stem cells are isolated from early-stage trophoblastic villi, i.e., chorionic villi of pregnant women in 4-5 weeks of pregnancy by laparoscopy and enzymatic treatment with trypsin/EDTA.

However, in the isolation method disclosed in the patent document, pregnant women must be subjected to an invasive surgery such as laparoscopic surgery. That is, laparoscopic chorionic villi sampling (CVS) must be essentially performed for prenatal diagnosis of genetic absormalities. For this, a cathether is inserted into the uterus via the cervix or abdomen to take a sample of chorionic villi. This is a hard and very invasive surgery. Moreover, abortion may occur during laparoscopic surgery, which limits the use of laparoscopic surgery in stem cell isolation.

In addition, according to the isolation method disclosed in the patent document, trophoblast stem cells are obtained from the chorionic villi of pregnant women in the early stage of pregnancy, and are Oct-4 positive cells. Considering the fact that while

Oct-4 expression in inner cell mass, which is an origin of embryonic stem cells, is increased, Oct-4 expression in trophoblast cells is reduced, the trophoblast stem cells obtained by the above patent document are different from pure trophoblast stem cells since they exhibit the characteristics of embryonic stem cells as well as those of trophoblast stem cells. DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

While endeavoring to develop a method for isolating trophoblast stem cells from the placenta of a pregnant woman at high purity without using an invasive surgery while avoiding ethical problems associated with stem cells, the present inventors have found that when chorionic villi are harvested from the placenta and treated with enzymes, and cyto-trophoblasts are isolated from the chorionic villi using a density-gradient separation method and cultured, trophoblast stem cells can be isolated at high purity.

Therefore, the present invention provides a method for isolating trophoblast stem cells from placental villi at high purity.

TECHNICAL SOLUTION

According to an aspect of the present invention, there is provided a method for isolating trophoblast stem cells, the method including: (a) harvesting placental villi from a detached normal placenta; (b) adding an enzyme solution containing trypsin, DNase I, and dispase to the placental villi of step (a) to perform an enzymatic reaction and adding a fetal bovine serum thereto to terminate the enzymatic reaction; (c) centrifuging the reaction solution of step (b) and separating cytotrophoblasts from the recovered cells by a density-gradient separation method; and (d) culturing the cytotrophoblasts of step (c) in a medium containing a fetal bovine serum and an antibiotic.

ADVANTAGEOUS EFFECTS

A trophoblast stem cell isolation method according to the present invention uses a placenta discarded after birth. Thus, there is no need to perform an invasive laparoscopic surgery during pregnancy, i.e., a laparoscopic surgery at the early stage of pregnancy, and further, there is no likelihood that abortion occurs. Moreover, trophoblast stem cells obtained by the present inventive method exhibit the characteristics (Nanog and Sox2 positive and Oct-4 negative) of pure trophoblast stem cells, unlike stem cells obtained by the prior art method (U.S. Pat. Pub. No. 2006/0211110). Therefore, the present inventive isolation method enables to isolate pure trophoblast stem cells, and the obtained trophoblast stem cells can be used in the screening of drug candidates, toxicity test, etc. for the treatment of female sterility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 3 respectively show the morphology, karyotype, and cell cycle of trophoblast stem cells obtained according to the present inventive isolation method;

FIG. 4 shows fluorescence activated cell sorting (FACS) analysis results of trophoblast stem cells obtained according to the present inventive isolation method;

FIGS. 5 and 6 show immunocytometrical analysis results of trophoblast stem cells obtained according to the present inventive isolation method; FIG. 7 shows RT-PCR analysis results of genes expressed from trophoblast stem cells obtained according to the present inventive isolation method; and

FIG. 8 shows the degree of invasion of the subcutaneous tissues of nude mice transplanted with trophoblast stem cells obtained according to the present inventive isolation method.

BEST MODE FOR CARRYING OUT THE INVENTION

A trophoblast stem cell isolation method according to the present invention uses a placenta discarded after birth. Thus, there is no need to perform an invasive laparoscopic surgery during pregnancy, i.e., a laparoscopic surgery at the early stage of pregnancy, and further, there is no likelihood that abortion occurs. Moreover, trophoblast stem cells obtained by the present inventive method exhibit the characteristics (Nanog and Sox2 positive and Oct-4 negative) of pure trophoblast stem cells, unlike stem cells obtained by the prior art method (U.S. Pat. Pub. No. 2006/0211110). Therefore, the present inventive isolation method enables to isolate pure trophoblast stem cells, and the obtained trophoblast stem cells can be used in the screening of drug candidates, toxicity test, etc. for the treatment of female sterility. A present inventive isolation method includes harvesting placental villi from a detached normal placenta [step (a)]. The detached normal placenta may be a placenta separated and discarded from a healthy woman after birth. That is, the "detached normal placenta" refers to a placenta separated from the body of a woman after birth. The detached normal placenta may be promptly stored in a sterilized bag placed in an ice bath. The harvesting of the placental villi from the detached normal placenta may be performed by a conventional anatomical method, e.g., by mincing placental villi present in the placenta using sterilized scissors. The placental villi thus obtained are washed twice or more, preferably five times, with an antibiotic (e.g., penicillin, stereptomycin)-containing phosphate buffered saline (PBS), to remove contaminants such as blood present in the tissues.

The harvested placental villi may be directly treated with an enzyme. Preferably, the placental villi may be further minced using sterilized scissors or the like and then treated with an enzyme. More preferably, the placental villi may be minced (e.g., a size of about 1 mm or less to form a turbid state) using sterilized scissors, washed, concentrated by centrifugation at about 1000 rpm for about 5 minutes, and treated with an enzyme. The washing may be performed twice or three times using a buffer such as HBSS (Hank's balanced salt solution), and the centrifugation may be performed at 1000 to 1200 rpm for about 5 to 10 minutes, preferably at about 1 ,000 rpm for about 5 minutes.

The enzyme treatment of step (b) may be performed by adding an enzyme solution containing trypsin, DNase I, and dispase to the placental villi. The concentration of trypsin, DNase I, and dispase is not particularly limited. For example, a solution containing about 20 mg of trypsin, 10 mg/ml of DNase I, and 1.2 U/ml of dispase may be used.

Trophoblast stem cells are positioned mainly in the inner portions of the placental villi. Thus, if necessary, the enzyme treatment and the enzymatic reaction termination may be repeatedly performed to increase the yield of trophoblast stem cells. That is, the enzyme treatment and the enzymatic reaction termination may be performed once or twice. When the enzyme treatment and the enzymatic reaction termination are performed once, the enzyme treatment may be continued for about one hour. When the enzyme treatment and the enzymatic reaction termination are performed twice, each enzyme treatment may be continued for about 30 minutes.

In the present inventive isolation method, the enzyme treatment may be gradually performed at a relatively low temperature, e.g., at about 20 to 30 ^°C , preferably at room temperature, unlike a conventional enzyme treatment at about 37 ^°C . By doing so, cell damage can be significantly reduced.

After the enzymatic reaction is terminated, the obtained solution is centrifuged at about 1 ,000 rpm for about 5 minutes.

The present inventive isolation method includes isolating cytotrophoblasts from the cells obtained in step (b) using a density-gradient separation method [step (c)J. The cells obtained in step (b) are a mixture of cytotrophoblasts, which are a source of trophoblast stem cells, with syncytiotrophoblasts. The cytotrophoblasts can be isolated by a density-gradient separation method based on a cell density difference (specific gravity of cytotrophoblasts: about 1.062-1.048, specific gravity of syncytiotrophoblasts: 1.013-1.039). Preferably, the density-gradient separation method may be a Ficoll density-gradient separation method. The cytotrophoblasts can be obtained by performing a Ficoll density-gradient separation method and isolating cells between 30 and 70% layers. In detail, a mixture of the solution obtained in step (b) and HBSS (Hank's balanced salt solution) is added to Ficoll density-gradient solutions (70%, 30%, 10%) and centrifuged at about 2,000 rpm for about 20 minutes, and cells present between 30 and 70% layers are isolated to thereby obtain cytotrophoblasts.

The present inventive isolation method includes culturing the cytotrophoblasts obtained in step (c) in a stem cell culture medium, e.g., a fetal bovine serum (FBS) and antibiotic-containing medium. The culture medium may be an alpha-MEM supplemented with 10% FBS, 1 % penicillin-streptomycin, 2 mM L-glutamine, 100 uM beta-mercaptoethanol, 1 ug/ml heparin, and 25 ng/ml fibroblast growth factor-4 (FGF-4). The culturing may be performed under conventional culture conditions, e.g., at 37 °C in a CO₂ incubator. Hereinafter, the present invention will be described more specifically with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 : Isolation of trophoblast stem cells

After an informed consent form was signed by a healthy pregnant woman, a normal placenta was obtained from the woman after birth, and promptly stored in a sterilized bag placed in an ice bath. Then, the morphological and structural characteristics of the placenta was visually observed and recorded. The placental villi present in the placenta were cut into pieces using sterilized scissors, and washed five times with an antibiotic (penicillin and streptomycin)-containing PBS to remove blood in the tissues.

After sufficiently removing the blood, the placental villi (pink) were minced into small pieces (about 1 mm or less) using sterilized scissors, washed three times with a sterilized HBSS solution, and centrifuged at 1 ,000 rpm for 5 minutes.

After removing the supernatant, 10 ml of a cocktailed enzyme solution (20 mg trypsin, 10 mg/ml DNase I, and 1.2 U/ml dispase) was added to the resultant residue, and the mixture was incubated at room temperature for 30 minutes while gradually stirring. The obtained enzyme solution was transferred to a 50 ml conical tube, and 1 ml of FBS was added thereto to terminate the enzymatic reaction. 10 ml of a fresh cocktailed enzyme solution was added to the conical tube, and the reaction solution was incubated at room temperature for 30 minutes while gradually stirring, and 1 ml of FBS was added thereto to terminate the enzymatic reaction. Then, the resultant solution was centrifuged at 1 ,000 rpm for 5 minutes.

The cells remained after removing the supernatant were mixed with 1 ml of HBSS. Ficoll density-gradient solutions (70%, 30%, 10%, each 3 ml) were gradually transferred to a 15 ml conical tube. 1 ml of the cell-containing HBSS solution was carefully added to the conical tube, and the reaction solution was centrifuged at about 2,000 rpm for 20 minutes. After removing 10% and 30% Ficoll density-gradient layers, cells present in a white and thin layer between the 70% and 30% Ficoll density-gradient layers were harvested.

The harvested cells were washed three times with a HBSS solution, and added to 3 ml of a culture medium (alpha-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 2 mM L-glutamine, 100 uM bera-mercaptoethanol, 1 ug/ml heparin, and 25 ng/ml fibroblast growth factor-4 (FGF-4)). The resultant culture mixture was placed in a T25 flask and incubated at 37⁰C in a CO2 incubator.

Cell isolation was initiated on January 15, 2007, and the cells were subcultured for 10 passages (once per about five days) considering the growth rate of the cells. The characteristics of cells obtained after the 10 passages were evaluated, and the cells were designated "CHA-TBSC-1".

Example 2: Analysis of morphological characteristics

The cells (CHA-TBSC-1) obtained in Example 1 were observed with a phase contrast microscope, and the result is shown in FIG. 1. The morphology of CHA-TBSC-1 show cuboidal types, which is typical morphology of trophoblast stem cells. In a Mycoplasma test using a Mycoplasma detection kit (iNtRON Biotechnology, Inc.), the cells were found to be negative. The cells were treated with colcemid (Invitrogen) and KCI solution (0.075M KCI), and stained with Trypsin-Giemsa, and the karyotypes of the cells were determined using CytoVision (Applied Imaging). As a result, the karyotypes of the cells were 46 XX (see FIG. 2). In addition, the cells were stained with propidium iodide (Pl), and the cell cycle of the cells was measured using a flow cytometer (FACS, Beckman). As a result, the cells showed a faster than normal cell cycle (see FIG. 3).

The above results show that the placenta-derived cells obtained in Example 1 are new and safe trophoblast cells which are Mycoplasma negative, have normal karyotypes, and show a rapid cell division.

Example 3: Fluorescence activated cell sorting (FACS) and immunocytometrical analyses In order to identify specific antigens present on surfaces of the placenta-derived cells obtained in Example 1 using various antibodies, fluorescence activated cell sorting

(FACS) analysis was performed. That is, when the cells were grown to 80% confluence, 1 ml of a cell dissociation buffer (GIBCO) was added to the cells to dissociate the cells from the culture tube. The cells were incubated with green or blue fluorescent material-labeled human specific antibodies, i.e., anti-CD13, anti-CD71 , anti-CD178, anti-CD44, anti-CD45, anti-CD105, anti-CD90, anti-CD95, anti-HLA-ABC, anti-HLA-DR, anti-HLA-G, anti-cytokeratin 7 and anti-Vimentin at room temperature for one hour and washed three times with PBS. FACS analysis was performed using a flow cytometer, and the results are shown in FIG. 4.

As shown in FIG. 4, the placenta-derived cells obtained in Example 1 according to the present inventive isolation method were determined to be CD13 positive (≥99.71), CD71 negative (<0.01), CD178 negative (<4.58), CD44 positive (>98.54), CD105 negative (≤1.55), CD90 positive (>99.74), CD95 positive (>94.40), HLA-ABC positive (>99.42), HLA-DR negative (≤0.21), and the characteristics of trophoblast cells, i.e., CD45 negative (≤0.32), Vimentin negative (≤4.67), HLA-G positive (≥4.59), cytokeratin 7 positive (>98.25).

Meanwhile, in order to perform an immunocytometrical analysis, the cells obtained in Example 1 were fixed with methanol on a glass slide at 4^°C for 10 minutes, treated with a blocking solution (DAKO) for 10 minutes to prevent nonspecific protein reactions, and incubated with human trophoblast-specific monoclonal antibodies, anti-cytokeratin 7 and anti-HLA-G, as primary antibodies at room temperature for one hour. The cells were washed with PBS and incubated with a green fluorescent material-labeled secondary antibody for 30 minutes. The cell nuclei were stained with propidium iodide (Pl). The results are shown in FIG. 5 (cytokeratin 7) and FIG. 6

(HLA-G).

The above results show that the placenta-derived cells obtained in Example 1 are trophoblast cells.

Example 4: Analysis of RNA expression levels of stem cell-associated genes RT-PCR was performed for genes expressed from the cells (CHA-TBSC-1) obtained in Example 1 according to the present inventive isolation method. That is, when the cells obtained in Example 1 were grown to about 80% confluence in a T25 flask, the cells were recovered and RT-PCR was performed as follows. That is, the recovered cells were lysed with Trizol to extract total RNA. cDNAs were synthesized from the total RNA using reverse transcriptase, and PCR was performed using cDNA-specific primers and Tag DNA polymerase. The PCR products were subjected to electrophoresis on agarose gel to identify the amplified genes. The primer sequences, the composition of a PCR solution, and the conditions of PCR are summarized in Tables 1 to 3 below.

Table 1

Table 2

Table 3

The results of RT-PCR are shown in FIG. 7. The differentiation of trophoblast cells at the blastocyst stage is controlled by the balance of expression of Oct-4 between inner cell mass (ICM) and trophectoderm. Generally, the fate of trophectoderm was determined by decreasing Oct-4 expression in the blastocyst. The trophectoderm gradually differentiates into trophoblasts constituting about 40-50% of placenta. The expression of Nanog and Sox2 genes necessary for cell self-proliferation is a critical marker demonstrating the presence of trophoblast stem cells. The expression of AFP(alpha fetoprotein), cardiac actin, and neuronal factor 68 (NF68) after differentiation into endoderm, mesoderm, and ectoderm indicates that trophoblast stem cells can differentiate into various cell lineages. CDX-2, cytokeratin 7, and HLA-G are genes specific for trophoblast cells, and thus, the expression of these genes demonstrates the presence of highly purified trophoblast cells. The expression of TERT gene associated with cell proliferation can also be used as a marker demonstrating the presence of trophoblast stem cells. FIG. 7 shows that the cells obtained in Example 1 according to the present inventive isolation method are pure trophoblast stem cells.

Example 5: Analysis of invasive function of trophoblast stem cells

One of the characteristic functions of trophoblast stem cells is to invade tissues by secreting various matrix metalloproteinases. The cells (CHA-TBSC-1) obtained in Example 1 were labled with a red fluorescent material, and were then transplanted into the subcutaneous tissues of male nude mice (weight: 40 g, 1 x 10⁶ cells/mouse). The degree of migration and invasion of the cells was analyzed using a fluorescence detector (Xenogen) for 4 weeks, and the result is shown in FIG. 8. As shown in FIG. 8, the cells obtained in Example 1 according to the present inventive isolation method exhibited an invasive property.

Claims

1. A method for isolating trophoblast stem cells, the method comprising: (a) harvesting placental villi from a detached normal placenta;

(b) adding an enzyme solution containing trypsin, DNase I₁ and dispase to the placental villi of step (a) to perform an enzymatic reaction and adding a fetal bovine serum thereto to terminate the enzymatic reaction;

(c) centrifuging the reaction solution of step (b) and separating cytotrophoblasts from the recovered cells by a density-gradient separation method; and

(d) culturing the cytotrophoblasts of step (c) in a medium containing a fetal bovine serum and an antibiotic.

2. The method of claim 1 , wherein in step (b), the placental villi are cut into pieces before adding the enzyme solution.

3. The method of claim 2, wherein in step (b), the placental villi are cut into pieces, washed, and concentrated by centrifugation at 1000 rpm for 5 minutes, before adding the enzyme solution.

4. The method of claim 1 , wherein in step (b), the enzymatic reaction and the enzymatic reaction termination are repeated twice.

5. The method of claim 4, wherein in step (b), each enzymatic reaction is performed for 30 minutes.

6. The method of any one of claims 1 through 5, wherein in step (b), the enzymatic reaction is performed at 20 to 30⁰C .

7. The method of claim 6, wherein in step (b), the enzymatic reaction is performed at room temperature.

8. The method of claim 1 , wherein in step (c), the density-gradient separation method is a Ficoll density-gradient separation method.

9. The method of claim 8, wherein in step (c), after performing the Ficoll density-gradient separation, cells present between 30% and 70% layers are isolated to obtain the cytotrophoblasts.

10. The method of claim 1 , wherein in step (d), the medium is alpha-MEM supplemented with 10% fetal bovine serum, 1 % penicillin-streptomycin, 2 mM L-glutamine, 100 uM beta-mercaptoethanol, 1 ug/ml heparin, and 25 ng/ml fibroblast growth factor-4 (FG F-4).