JP2002360243A - Hepatocyte derived from human cord blood nucleated cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell supply source slight in burden on a relevant donor, easily differentiatedly inducible into hepatocytes (or hepatic precursor cells) usable in hepatocyte transplantation therapy or the like, to provide a method for differentiatedly inducing the cell supply source into hepatocytes (or hepatic precursor cells), and to provide the hepatocytes (or hepatic precursor cells) obtained by the method. SOLUTION: This method for producing hepatocytes and/or hepatic precursor cells is characterized in comprising the step of differentiatedly inducing the nucleated cells in human cord blood. The 2nd objective hepatocytes and/or hepatic precursor cells obtained by the method are provided. The other objective hepatocytes and/or hepatic precursor cells to be used in hepatocyte transplantation therapy are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for inducing nucleated cells in human umbilical cord blood into hepatocytes or hepatic progenitor cells, and to a hepatocyte or hepatic progenitor cell obtained by the method.

[0002]

2. Description of the Related Art At present, there is no effective treatment other than liver transplantation for patients with severe liver disease such as fulminant hepatitis, cirrhosis, and liver cancer. In Japan, approximately 40,000 deaths from liver disease are counted annually in Japan. ing. In the liver, which is the central organ of metabolism, there is a strong demand for organ transplantation. There is also the problem of rejection due to immunity. It has also been pointed out that the development of an artificial liver is difficult.
Although hepatocyte transplantation has been attempted as an alternative treatment, human liver is required as a cell source, and at present it is difficult to expand the application. In 1988, two groups in the United States reported on human pluripotent embryonic stem / germline cells (ES / EG) (Thomson, JA, et al. (1998) Science, 28
2 , 1145-1147; Shamblotte, MJ, et al. (1998) Proc.
Natl. Acad. Sci. USA, 95 , 13726-13731), human (E
The possibility that various cells differentiated from S / EG) cells can be applied to transplantation therapy has increased. However, research on the induction of hepatocyte differentiation from mouse ES cells has just begun, and at present, there are many restrictions on human ES (like) cell research itself at public institutions. In experimental animal model systems, rat donor-derived hepatocytes are found in the liver of rats that have undergone bone marrow transplantation [Petersen et al., (1999) Science, 284 ,
1168-1170] that rat bone marrow cells can be differentiated into liver (like) cells in a culture system (Oh, S., et al. (2000) Biochem. Bi)
ophys. Res. Commun., 279, 500-504). Hematopoietic stem cells derived from donors differentiated into hepatocytes in mice [Lagasse et al., (2000) Nature Med. 6 , 1229-1234.
In addition, human-derived hepatocytes are also found in the liver of bone marrow transplant patients in humans [Theise, NO, et al., (200)
0) Hepatology, 32 , 11-16] suggests that bone marrow hematopoietic stem cells could be used as a source of human hepatocytes, but it places a considerable burden on donors. On the other hand, there have been no reports on studies using human cord blood.

[0003]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned situation, and provides a cell supply capable of easily inducing differentiation into hepatocytes (or hepatic progenitor cells) which can be used for hepatocyte transplantation therapy. It is an object of the present invention to provide a cell source which is a source and has a small burden on a donor, a method for inducing the differentiation into hepatocytes (or hepatic progenitor cells), and a hepatocyte (or hepatic progenitor cell) obtained by this method. Aim.

[0004]

Means for Solving the Problems In the course of intensive research to achieve the above object, the present inventors focused on human umbilical cord blood and cultured human umbilical cord blood nucleated cells by adding various growth and differentiation inducing factors. Cells expressed the human albumin gene,
Production of albumin was observed in the culture solution. Also, SCID
When nucleated cells in cord blood were transplanted into the spleen of a mouse,
After 4 and 6 weeks, expression of the human albumin gene and human albumin were observed in hepatocytes. Albumin is a protein that is biosynthesized by hepatocytes.Human albumin gene and human albumin were detected in cultured cells and transplanted animals, indicating that cord blood nucleated cells were induced to differentiate into hepatocytes. . The present inventors have conducted studies based on the above facts and findings, and have reached the present invention.

That is, the present invention relates to hepatocytes and / or hepatic progenitor cells obtained by inducing differentiation of nucleated cells in human cord blood.

[0006] The present invention also relates to the above hepatocytes and / or hepatic progenitor cells used for hepatocyte transplantation therapy.

Further, the present invention relates to a method for producing hepatocytes or / and hepatic progenitor cells, which comprises inducing differentiation of nucleated cells in human cord blood.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, as a method for inducing differentiation of nucleated cells in human umbilical cord blood, a method for differentiating and growing directly or in vitro is exemplified. Examples of the case of differentiation and proliferation in direct culture include, for example, a method of inducing differentiation into hepatocytes or / and hepatic progenitor cells by transplanting nucleated cells separated and prepared from human umbilical cord blood into immunodeficient animals. No. Here, examples of the immunodeficient animal include animals exhibiting the same pathology as human severe combined immunodeficiency disease (SCID), such as SCID mice. Differentiation into human hepatocytes can be confirmed by human albumin gene expression (RT-PCR method) and albumin protein expression (immunohistological staining and Western blotting method).

[0009] As a method of differentiating and proliferating by in vitro culture, a nucleated cell separated and prepared from human umbilical cord blood is cultured by adding a growth / differentiation inducing factor to hepatocytes or / and
And a method for inducing differentiation into hepatic progenitor cells.
Here, as the proliferation / differentiation inducing factor, for example, LIF
(Human leukemia inhibitory factor), SCF (human stem cell factor),
Examples include FGF (fibroblast growth factor), HGF (human hepatocyte growth factor), OSM (Oncostatin M), dexamethasone, etc., and a combination of FGF / HGF / LIF / SCF is particularly preferred. Expression of hepatocyte-related genes (such as albumin) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined by RT-PCR.
It can be confirmed by law. Albumin-producing cells can also be confirmed by immunohistological staining.

The hepatocytes (or hepatic progenitor cells) of the present invention
Is considered to be applicable to the treatment of various liver diseases. In addition, when this is used for hepatocyte transplantation treatment, the shortage of donors, which is a problem in conventional hepatocyte transplantation treatment, is solved, the burden on the donor is eliminated, and the problem of immune rejection is also solved. Can greatly reduce costs.

[0011]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Example 1 Separation of nucleated cells from human umbilical cord blood Umbilical cord blood from normal healthy labor of 15 healthy adult women who obtained informed consent was subjected to a 10 ml vacuum test containing ACD-A solution as an anticoagulant. Dispensed into tubes (manufactured by Nipro) and used within 8 hours after storage at room temperature. Equivalent amount of this cord blood to 6
% Hydroxyethyl starch (HES) and physiological saline (Nipro ampoule), and allowed to stand for 90 minutes. After that, the upper layer was collected and separated from most erythrocytes in the lower layer. An equal volume of saline with phosphate buffer was added, the cells were collected by centrifugation and washed once with saline with phosphate buffer. Although many erythrocytes contaminated in addition to nucleated cells are observed in this cell preparation, the number gradually decreases after the medium is exchanged (see FIG. 1).

Example 2 Induction of Human Cord Blood Nucleated Cells into Hepatocytes in a Culture System Culture of nucleated cells isolated from human cord blood was performed using Dulbecco's modified minimal basal medium containing high glucose and 15% fetal bovine serum (FBS). (DMEM; Gibco-BRL) and various growth / differentiation-inducing factors were added thereto and cultured in a tissue culture plate for 3 weeks. The plates were coated with 0.1% gelatin and were usually seeded at a cell density of 2 × 10 ⁶ / cm ² .
The added factors and concentrations are shown below. LIF (R and D Sy
stems, 10 ng / ml), SCF (R and D Systems
10 ng / ml), FGF [fibroblast growth factor 1
(Gibco-BRL, 20 ng / ml) + human fibroblast growth factor 2 (Gibco-BRL, 10 ng / ml)], HGF (Mochida Pharmaceutical Co., Ltd., 10 ng / ml), OSM (DIACLONE Re
search company, 10 ng / ml). About half of the medium was replaced with a fresh medium containing a growth and differentiation inducing factor every 3 to 5 days to maintain the concentration of the growth and differentiation inducing factor which is liable to deteriorate. FGF /
When HGF / LIF / SCF was added and cultured, round cells were observed from about day 7 and the number of cells significantly increased on day 21. The results are shown in FIG.

[0014] Cells cultured under the same conditions are added 3 times a week.
Harvested until week, mRNA was isolated from all cells (Roche
After synthesizing complementary strand DNA (cDNA) using reverse transcriptase (RT), albumin and glyceraldehyde-3-phosphate dehydrogenase (GA)
PDH) and albumin and GAPDH mRNA by polymerase chain reaction (PCR) using primers.
Was detected. The results are shown in FIG. GAPDHmRN used as a control because it is always expressed regardless of cell type
Compared with A, some expression of albumin mRNA was observed on the 7th day, and the expression increased on the 14th and 21st days. Quantitative PC
As quantified by R (Roche), the relative expression of albumin mRNA on days 14 and 21 was approximately 10-fold on day 7. In addition, albumin mRNA
The size of the product of RT-PCR of the GAPDH mRNA of and the control is 350 bp and 400 bp, respectively.

On the other hand, FGF / LIF / SC excluding HGF
Circular cells were hardly observed on the 21st day of the culture with the addition of F (upper right of FIG. 3), and the frequency of appearance of the round cells was lower in the culture with the addition of FGF / HGF / LIF or FGF / HGF / SCF (lower left of FIG. Lower right). FGF without HGF
In the cultures supplemented with / LIF / SCF, the expression of albumin mRNA was considerably low. When quantified by quantitative PCR, an approximately 5-fold difference was observed. The results of this experiment show that four types of cytokines, FGF / H, are required for the effective differentiation and proliferation induction of hepatocyte-like round cells expressing the albumin gene.
It indicates that GF / LIF / SCF is essential. RT-PCR of albumin and GAPDH mRNA
Product size is 400bp and 500bp respectively
It is. Since glucose 6-phosphatase (G6Pase) mRNA cannot be confirmed by the RT-PCR method, it can be assumed that the induced hepatocytes are like liver stem cells before reaching the maturation stage. OSM, known as hepatocyte maturation factor, was added in combination with dexamethasone, but had no maturation effect.

Next, in order to examine the expression of albumin protein in the small round cells, immunohistological staining was performed. The cells were subjected to normal acetone fixation, incubated with a rabbit anti-human albumin antibody (DAKO), and reacted with an anti-rabbit IgG antibody (Sigma, F1262) conjugated with FITC, which is a fluorescent dye, to obtain a fluorescence microscope. And by a phase contrast microscope. FGF / H
Approximately half of the cells 21 days after the addition of GF / LIF / SCF were fluorescently stained intensely, confirming that albumin was expressed in the cells (FIG. 4, left; 40-fold). The right figure in FIG. 4, which was magnified 400 times, showed that the cytoplasm of the cells was mainly stained, reflecting the localization of albumin in the cytoplasm. In addition, the upper part of FIG. 4 is by a phase contrast microscope, and the lower part is by a fluorescence microscope.

Example 3 Experiment on Transplantation of Human Cord Blood Nucleated Cells into Severe Combined Immunodeficiency (SCID) Mice 0.4 m per SCID mouse (6-7 weeks old)
g of 2-acetylaminofluorene (2-AAF) on day 7 after subcutaneous administration with 10% partial hepatectomy.
^Seven nucleated cells (0.1 ml phosphate buffered saline) were injected into the spleen, sacrificed 4-6 weeks later, and serum separation and liver extraction were performed. Human albumin in mouse serum was detected by Western blotting. After mouse serum was subjected to SDS-polyacrylamide electrophoresis / Western blot, a mouse monoclonal antibody against human albumin (CORTEX BIOCHEM, CR2116M) was used.
Horseradish peroxidase-conjugated goat anti-mouse IgG antibody was used as a secondary antibody. The peroxidase reaction was detected by chemiluminescence using an ECL kit (Amersham). This specific monoclonal antibody against human albumin also crosses mouse albumin with an efficiency of about 1 / 1,000, but significantly expresses human albumin compared to control mouse serum not injected with human umbilical cord blood nucleated cells. It has been shown. FIG. 5 shows the results. In FIG. 5, lane 1 is human serum (1 μl of 1000-fold dilution is used), and lanes 3 and 4 are SCID mice transplanted with human cord blood nucleated cells (#
1, # 2) serum (use 1 μl of 20-fold dilution)
Lane 2 shows the results for the serum of a control mouse (this specific antibody cross-reacts with mouse albumin at about 1 / 1,000 of the efficiency, so that a weak signal is also observed in the serum of the control mouse). This result suggests that human cord blood nucleated cells produce albumin in immunodeficient mice and have differentiated into functional hepatic parenchymal cells that secrete into the blood.

[0018]

According to the present invention, if liver cells, which play a central role in metabolism, can be differentiated and proliferated in large amounts from cord blood in vitro and used, it will be possible to treat various liver diseases. The ability to induce liver (stem) cells from human umbilical cord blood also makes it easy to prepare a repertoire of liver (stem) cells that does not cause rejection without relying on cell nuclear transfer. The shortage of donors,
It can be expected to reduce the burden on donors, resolve immune rejection reactions, and significantly reduce costs. Furthermore, there is no concern about human clones due to nuclear transfer, etc., and there are few ethical problems.

[Brief description of the drawings]

FIG. 1 shows that human umbilical cord blood nucleated cells were FGF / HGF /
Cultured in a medium containing LIF / SCF for 3 weeks,
It is the figure which image | photographed the cell by the phase-contrast microscope on the 7,11,15,21 days (400 times magnification).

FIG. 2 shows that human umbilical cord blood nucleated cells were FGF / HGF /
The cells were cultured in a medium containing LIF / SCF for 3 weeks, cells were collected every 7 days, and human albumin mRNA expression was measured by RT-PC.
It is the figure analyzed by the R method.

FIG. 3 shows that human umbilical cord blood nucleated cells were expressed by FGF, respectively.
/ HGF / LIF / SCF (upper left), FGF / LIF / SC
F (upper right), FGF / HGF / LIF (lower left), FGF /
After culturing for 3 weeks under the conditions of HGF / SCF (lower right), after cell recovery, human albumin mRNA and control GA
FIG. 3 is a diagram in which PDH mRNA expression was analyzed by an RT-PCR method.

FIG. 4 shows human cord blood nucleated cells cultured in a medium containing various cytokines for 3 weeks, and human albumin was detected by immunostaining (left: magnification of 40; right:
Magnification 400).

FIG. 5 shows SCs 4 weeks after human cord blood nucleated cell transplantation.
FIG. 3 is a diagram showing detection of human albumin in ID mouse serum by an immunoblotting method.

 ────────────────────────────────────────────────── ─── Continued on front page (72) Inventor Yujiro Tanaka 2-6-8 Oyamadai, Setagaya-ku, Tokyo F-term (reference) 4B065 AA93X BA24 BB19 BB34 BD39 CA44 4C087 BB52 BB59 CA04 MA01 NA14 ZA75 ZB21

Claims (9)

[Claims] 1. Hepatocytes and / or hepatic progenitor cells obtained by inducing differentiation of nucleated cells in human cord blood. 2. The method according to claim 1, wherein the nucleated cells separated and prepared from human cord blood are differentiated and expanded directly or in vitro.
2. The hepatocyte or / and hepatic progenitor cell according to 1. 3. The hepatocyte or / and liver according to claim 1, wherein nucleated cells separated and prepared from human umbilical cord blood are induced to differentiate into hepatocytes and / or hepatic progenitor cells by transplantation into immunodeficient animals. Progenitor cells. 4. The hepatocyte according to claim 1, wherein nucleated cells separated and prepared from human umbilical cord blood are induced to differentiate into hepatocytes and / or hepatic progenitor cells by culturing by adding a growth / differentiation-inducing factor. / And hepatic progenitor cells. 5. The hepatocyte or / and hepatic progenitor cell according to claim 1, which is used for hepatocyte transplantation treatment. 6. A method for producing hepatocytes and / or hepatic progenitor cells, which comprises inducing differentiation of nucleated cells in human cord blood. 7. The production method according to claim 6, wherein the nucleated cells separated and prepared from human umbilical cord blood are differentiated and expanded directly or in vitro. 8. The production method according to claim 6, wherein nucleated cells separated and prepared from human umbilical cord blood are transplanted into an immunodeficient animal to induce differentiation into hepatocytes and / or hepatic progenitor cells. 9. The production method according to claim 6, wherein the nucleated cells separated and prepared from human umbilical cord blood are induced to differentiate into hepatocytes and / or hepatic progenitor cells by culturing with a growth and differentiation inducing factor.