JP2001037471A - Production of transplantable hematopoietic cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing hematopoietic mast cells by transforming mammalian animal cells such as embryonic type hematopoietic cells, etc., to the hematopoietic mast cells. SOLUTION: This method for producing hematopoietic mast cells comprises co-culturing mammalian animal cells capable of being transformed to the hematopoietic mast cells by a co-culture of a cell line separated from the AGM zone of a mammalian animal fetus, made as the cell line and capable of sustaining the propagation or living of the hematopoietic mast cells and hematopoietic precursor cells e.g. umbilical blood cells, embryonic mast cells, embryonic germ cells, primordial germ cells, cells derived from para-aorta splanchnic mesoderm or umbilical vesicle, neural mast cells or endothelial cells, with the above cell line for transforming the mammalian animal cells to the hematopoietic mast cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing hematopoietic stem cells, and more particularly, to a method for producing hematopoietic stem cells by converting mammalian cells into hematopoietic stem cells.
INDUSTRIAL APPLICABILITY The present invention can be applied to medical fields such as an operation for expanding hematopoietic stem cells in vitro and bone marrow transplantation.

[0002]

2. Description of the Related Art The life span of mature blood cells flowing through a living body is short (about 120 days for red blood cells and about 7 days for platelets in humans), and new mature blood cells are constantly supplied by differentiation from immature hematopoietic cells. Have been. It is believed that these hematopoietic stem cells, which are considered to be the most upstream of the differentiation of hematopoietic cells, produce cells downstream and slowly self-renew to replenish hematopoietic cells throughout their lives. Unlike differentiated hematopoietic cells, hematopoietic stem cells can survive for life. In bone marrow transplantation, peripheral blood stem cell transplantation, and umbilical cord blood transplantation, it is significant to transplant viable hematopoietic stem cells. In other words, once transplantation is successful, the benefits of transplantation can be expected for a lifetime.

[0003] By the way, it is considered that blood cells are generated by almost the same mechanism in mammals regardless of species, and the mode of generation of hematopoietic cells has been studied using a mouse as a model animal.

[0004] Hematopoietic cells originate from two different sites (Dzierzak, E., Immunology Today, 19: 228-236,
1998). One of them is the extraembryonic region (extraembryonic sit
e) Yolk sac, which appears earliest on day 7 of embryo. Hematopoietic cells proliferate in the yolk sac while forming cell clumps called blood islands. However, hematopoietic cells present in the yolk sac do not survive for a long time even when transplanted into adult mice, and do not have the properties of hematopoietic stem cells.

[0005] Apart from the yolk sac, the embryo (em)
bryo proper) para-aortic mesoderm (PAS: paraaort)
ic splanchnopleural mesoderm) confirms the appearance of hematopoietic cells. At 8.5 days of fetal life, a vasculature is established and blood flow is started between the yolk sac and the embryo. Experiments separating and culturing the yolk sac and PAS before the establishment of the vasculature showed that PAS also contained cells that differentiated into hematopoietic cells.
It is thought that hematopoietic cells appear independently in two places of S. In other words, from embryos 7.5 to 8.5 days old,
When PAS was isolated and cultured under conditions that could differentiate myeloid hematopoietic cells and lymphocyte cells in vitro, the appearance of myeloid hematopoietic cells and lymphoid hematopoietic cells was confirmed (Cuma
no, A., Cell, 86: 907-916, 1996). Thereafter, the embryos 8.
On the 5th, CFU-S (spleen-
clony forming unit) has been confirmed and its frequency is higher than that in the yolk sac.
There are many opinions that S originated.

[0006] When fetal hematopoietic cells generated by PAS progress until organ formation progresses and is recognized as aorta-gonad-mesonephros (AGM) (embryo 10.5 days), they acquire the characteristics as hematopoietic stem cells. Have the ability to survive in adults (Muller, AM, Immunity, 1: 291-301, 1994, Medvi
nsky, A., Cell, 86; 897-906, 1996). Thereafter, the hematopoietic cells present in the AGM migrate to the liver, which also functions as a hematopoietic organ right after birth. Around the 16th day of fetal life, hematopoietic stem cells migrate to the bone marrow, which is an adult hematopoietic organ, and the bone marrow functions as a hematopoietic organ for life. As described above, cells generated by PAS gradually change into hematopoietic stem cells by AGM, proliferate,
It is thought that an adult type hematopoietic system will be constructed.

As described above, studies on the development and differentiation of hematopoietic stem cells are steadily progressing, and a method of expanding hematopoietic stem cells in vitro has been proposed. For example, it is known that adult hematopoietic stem cells present in AGM can be expanded by tissue culture of the AGM region (WO98).
/ 12304). Also, separated from the AGM region of the mammalian fetus,
A method for supporting proliferation or survival of hematopoietic stem cells or hematopoietic progenitor cells by culturing hematopoietic stem cells or hematopoietic progenitor cells together with a cell line that is established and can support the proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells Has been reported (WO99 / 03980).

However, a method for converting mammalian cells such as fetal hematopoietic cells into hematopoietic stem cells has not yet been reported. Yoder et al. Have shown that cells of the yolk sac on the 9th day of embryo transfer to newborn mice (Yoder, M. et al.
C., Immunity 7: 335-344, 1997), the result after the vasculature was established in the fetus.
The possibility that hematopoietic stem cells have migrated to the yolk sac cannot be ruled out. It is also transplanted into newborn mice,
No mention is made of adult transplantability.

[0009]

An object of the present invention is to provide a method for producing hematopoietic stem cells by converting mammalian cells such as fetal hematopoietic cells into hematopoietic stem cells.

[0010]

Means for Solving the Problems The present inventors have isolated and established mammalian cells, such as fetal hematopoietic cells, from the AGM region of mammalian fetuses, and have grown or survived hematopoietic stem cells and hematopoietic progenitor cells. It has been found that the above-mentioned mammalian cells can be converted into hematopoietic stem cells by co-culturing with a cell line capable of supporting E. coli, and the present invention has been completed.

That is, the present invention relates to a mammalian fetal AGM.
Co-culturing mammalian cells that can be converted into hematopoietic stem cells by co-culturing with a cell line that can support proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells, which is isolated from the region and established, and that supports hematopoietic stem cells and hematopoietic progenitor cells. And converting the mammalian cells into hematopoietic stem cells.

[0012] The present invention also relates to the above method, wherein the mammalian cells are cord blood cells, embryonic stem cells.
m cell; ES cell), embryonic germ cell
l; EG cell), primordial germ cell
Provided is a method selected from cells derived from para-aortic mesoderm or yolk sac, neural stem cells, vascular endothelial cells, or embryonic stem cells.

Further, the present invention provides the above method, wherein the mammalian cell is a cell derived from para-aortic mesoderm or yolk sac. The present invention also provides the method, wherein the cell line is derived from a mouse or a human.

[0014] The present invention further provides a method according to the above method, wherein the method is accompanied by expansion of hematopoietic stem cells converted from the mammalian cells. Further, the present invention provides the method, wherein the cell line is adheredly grown on an inner wall of an incubator to form a layer, and a mammalian cell is overlaid on the cell line layer and cultured. I will provide a.

Further, the present invention provides the above method, wherein the hematopoietic stem cell has an ability to transplant into an adult mammal. In this specification, hematopoietic stem cells refer to cells that show long-term engraftment in adult individuals and have the ability to differentiate into various hematopoietic cell lineages. In addition, hematopoietic cells derived from hematopoietic stem cells capable of engraftment are referred to as adult hematopoietic cells, and hematopoietic cells in the early development that are not derived from hematopoietic stem cells are referred to as fetal hematopoietic cells. Also, embryo (embryo proper)
Is the part that grows as an individual in the future.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, hematopoietic stem cells are produced by converting mammalian cells into hematopoietic stem cells.

The mammalian cell used in the present invention is a cell line (hereinafter referred to as "AGM-derived stromal cell") which is isolated and established from the AGM region of a mammalian fetus and which can support the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells. A cell that can be converted into a hematopoietic stem cell by co-culture with a cell line. Hereinafter, a mammalian cell having such a potential property may be simply referred to as a “mammalian cell”. Mammalian cells can be obtained from various tissues and organs described below. Specifically, cord blood cells, cells derived from PAS, or embryonic stem cells, embryonic germ cells, primordial germ cells, cells derived from yolk sac And neural stem cells, vascular endothelial cells, and the like, but are not limited thereto as long as they can be converted into hematopoietic stem cells by co-culture with an AGM-derived stromal cell line. The mammalian cell does not need to be completely separated from other cells, and may be a mixture of cells present in the tissue containing the target cell.

As shown in the Examples, cells in the yolk sac or PAS region can be differentiated into viable hematopoietic cells. Hematopoietic cells present in adult bone marrow produce fetal hemoglobin when transplanted into embryos (Geige
r, H., Cell, 93: 1055-1065, 1998). This indicates that the environment surrounding the cells can direct the cells toward dedifferentiation. It is presumed that bone marrow, peripheral blood, and umbilical cord blood that have been subjected to hematopoietic stem cell transplantation, in addition to hematopoietic stem cells, have cells capable of engraftment by culturing with an AGM-derived stromal cell line. It is thought that by culturing the tissue with an AGM-derived stromal cell line, hematopoietic cells capable of engraftment can be produced.

Hematopoietic cells have been shown to originate from vascular endothelial cells (Nishikawa, SI., Immunit
y, 8: 761-769, 1998), it is also presumed that among vascular endothelial cells, there are cells that can be differentiated into hematopoietic stem cells.

[0020] Embryonic stem cells (ES cells)
l) has pluripotency, and mouse ES cells can differentiate into all somatic cells when transplanted into blastula. Recently, it has become possible to differentiate and produce hematopoietic cells from ES cells in vitro via hematopoietic cell precursor cells having characteristics of vascular endothelial cells (Nishikawa, SI., Develop
ment 125: 1747-1757, 1998, Choi, K., Development 12
5: 725-732, 1998). That is, by culturing ES cells on an incubator coated with type IV collagen to differentiate into Flk-1 positive cells, and further culturing Flk-1 positive cells under the above culture conditions, Flk-1 VE-cadherin-positive cells that are positive and are markers for vascular endothelial cells can be obtained. From VE-cadherin-positive cells exhibiting the characteristics of vascular endothelial cells, hematopoietic cells expressing CD45, which is a blood cell marker, can appear. However, the viability of these hematopoietic cells in transplanted individuals has not been confirmed. last year,
ES cells even in humans (Thomson, JA, Science, 282: 1145-1
147, 1998), EG cells (Shamblott, MJ, Proc. Natl.
Acad. Sci. USA, 95: 13762-13731, 1998) has been established, and it has become feasible to differentiate such cells into various cell lineages and use them clinically in the future. Using an AGM-derived stromal cell line, it would be possible to convert ES cells into hematopoietic cells capable of engraftment in adults.

The use of the cloning technique makes it possible to reproduce the embryo development of a specific individual. Using the present invention, developing cells could be intentionally differentiated toward hematopoietic cells.

Recently, it has been shown that transplantation of neural stem cells present in the adult mouse brain differentiates into hematopoietic cells (Bj
ornson, CRR, Science, 283: 534-537, 1999). It shows that stem cells present in adults can be converted to stem cells of other cell lineages. Neural stem cells are EGF or bFG
Can be grown on F (Reynolds, BA, De
v. Biol., 175; 1-13, 1996; Gritti, A., J. Neurosc
i., 16: 1091-1100, 1996). It is presumed that after proliferating neural stem cells, it is possible to differentiate them into hematopoietic stem cells using an AGM-derived stromal cell line. Similarly, it is presumed that stem cells of other organs can be differentiated into hematopoietic stem cells.

As described above, if it becomes possible to use stem cells, ES cells, and the like of tissues that can be easily collected by humans, other than blood, hematopoietic cells can be collected from blood cells of the patient,
It can produce hematopoietic stem cells or various blood cells to promote blood cell recovery in patients with impaired or abnormal blood cell production.

The AGM-derived stromal cell line used in the present invention is isolated and established from the AGM region of a mammalian fetus.
In addition, a cell line capable of supporting the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells, and a method for separating and establishing the cell line is described in WO99 / 03980. Specifically, for example, they can be separated and established as follows.

The male and female mice were converted to SPF (specific pathogen).
-free), females are placed in the same cage with males overnight, and the following morning, female mice in which the presence of a vaginal plug has been confirmed are transferred to new cages and raised. The day when the vaginal plug was confirmed,
9. 0.5 days of gestation, 8 to 13 days of gestation, preferably 10.
The fetus is removed from the mouse on day 5. A method for separating the AGM region from a fetus is described in Godin et al. (Godin, I., Proc. Natl. A
cad.Sci. USA, 92: 773-777, 1995), Medvinsky et al. (Me
dvinsky, AL, Blood, 87: 557-566, 1996). That is, the fetus was placed in a culture dish containing phosphate-buffered saline to such an extent that the fetus was immersed, and AG was placed under a stereoscopic microscope.
The M region is excised without any other regions and transferred to a new culture dish.

In the AGM region obtained as described above,
One drop of a medium, for example, a MEM medium containing 10% FCS (fetal bovine serum) is added, and the cells are cultured overnight at 37 ° C., 5% CO ₂ and 100% humidity. When the cells in the AGM region adhered to the culture dish, the culture was further continued by adding a medium,
The stromal cells appear around the region tissue piece. After further culturing for about one week, the adherent cells are detached by trypsin treatment, washed with a medium, and seeded on a culture dish. The next day, cells that have not adhered to the culture dish are removed together with the medium, and fresh medium is added. Two weeks after the start of the culture after the trypsin treatment, gamma rays of about 900 rads are irradiated to remove endogenous hematopoietic cells. Two weeks later,
The culture system is trypsinized to suspend the cells,
000 cells / well, preferably 50-100 cells
/ Well in a 24-well culture dish. At this time, if the number of cells to be seeded is reduced and cloning by the direct limiting dilution method is attempted, the cells do not proliferate. After acclimating the cells to
It is preferable to carry out cloning by the limiting dilution method.
Subsequently, after culturing is continued for about 3 weeks, the cells are seeded by a limiting dilution method in a 96-well culture dish at 0.05 to 1 cell / well, preferably 0.3 cells / well. The cells that grow from the wells in which only the cells are seeded are expanded and cultured.

From the cells obtained as described above,
By selecting cells capable of supporting the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells, the stromal cell line used in the present invention can be obtained. Such cell selection can be performed by co-culturing the candidate strain with hematopoietic stem cells or hematopoietic progenitor cells, and then evaluating the degree of proliferation of hematopoietic stem cells or hematopoietic progenitor cells. The degree of proliferation of hematopoietic stem cells or hematopoietic progenitor cells is determined by culturing the cells at the start of coculture and after coculture in the presence of cytokines, respectively.
This is done by comparing the number of colonies of blood cells.

In the above description, the method of establishing a stromal cell line from the mouse AGM region has been specifically described. However, the AGM region used for establishing the stromal cell line is not limited to mouse-derived ones. AGM regions of mammals such as pigs, sheep, and humans may be used. AG
The appearance of hematopoietic stem cells in the M region has been observed in humans (Tavian, M., Blo
od, 87: 67-72, 1996), mouse (Sanchez, MJ., Immun
ity, 5: 513-525, 1996), suggesting a common mechanism during mammalian development. still,
Hematopoietic stem cells derived from human umbilical cord blood can be expanded using a stromal cell line derived from the mouse AGM region (WO99 / 03980). It is strongly supported that stromal cells equivalent to the stromal cell lines shown can be isolated and used in the present invention.

When mammalian cells are co-cultured with an AGM-derived stromal cell line, the mammalian cells are converted to hematopoietic stem cells. On the other hand, even when mammalian cells are cultured alone, conversion to hematopoietic stem cells does not occur. The mammalian cell and the AGM-derived stromal cell line may be derived from a mammal of the same species or may be derived from a different mammal.

Co-culture of AGM-derived stromal cell lines and mammalian cells can be performed by various known methods (Xu, MJ, Blood, 9
2: 6 2032-40, 1998). Preferably, the AGM-derived stromal cell line is cultured in an incubator in advance, and is grown adheringly on the inner wall of the incubator to form a cell layer. Then, mammalian cells are overlaid on the layer and cultured.

Hematopoietic stem cells converted from mammalian cells by co-culture with an AGM-derived stromal cell line are supported for growth or survival by co-culture with an AGM-derived stromal cell line. That is, when the stromal cell line and the hematopoietic stem cells converted by the method of the present invention are co-cultured,
Hematopoietic stem cells rotate through the cell cycle, partly self-replicate as stem cells, partly proliferate while differentiating into hematopoietic progenitor cells, and hematopoietic stem cells are maintained in this culture system. In addition, hematopoietic progenitor cells differentiated by the above-described co-culture proliferate while differentiating into cells of a single or multiple differentiation lineages (WO99
/ 03980). In the present invention, it is considered that the conversion of mammalian cells to hematopoietic stem cells and the proliferation of hematopoietic stem cells partially partially progress.

The hematopoietic stem cells produced as described above are:
It preferably has the ability to transplant into adult mammals. As described above, in a medical field, the therapeutic effect that can be expected by transplanting hematopoietic stem cells is large. If hematopoietic stem cells capable of engraftment could be produced and expanded from various tissues and cells, transplant medical treatment would be greatly advanced.

It has been clarified that the proliferation and differentiation of hematopoietic cells are regulated by various growth factors and cytokines. The differentiation of hematopoietic progenitor cells into neutrophils, erythrocytes, macrophages, etc.
It is regulated by cytokines highly specific to the cell lineage such as CSF, EPO and M-CSF. On the other hand, in vitro differentiation of fetal hematopoietic cells into adult hematopoietic cells has not been successful even in a system using various cytokines or cells, but according to the present invention, it is the first time that hematopoietic stem cells are generated in vitro. Successful.

[0034]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

[0035]

[Example 1] Establishment of AGM-derived stromal cell line capable of supporting proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells <1> Establishment of AGM-derived stromal cell line (1) Isolation of AGM region of mouse embryo C3H / HeNSLc mouse Male and female (purchased from Japan SLC, Inc.) were bred under SPF (specific pathogen-free) environment. One or two females with one male overnight
The female mice in the same cage, the presence of a vaginal plug was confirmed the next morning, were transferred to a new cage and bred. The day on which the vaginal plug was confirmed was defined as gestation 0.5 day. The mouse at 10.5 days of gestation was killed by cervical dislocation, and the fetus was removed. AGM separation is described by Godin et al. (Godin, I., Proc. Natl. A
acd.Sci.USA, 92: 773-777, 1995), Medvinsky et al. (Me
dvinsky, AL, Blood, 87: 557-565, 1996). Put the fetus in a culture dish containing PBS (-) (phosphate buffered saline) (manufactured by Nissui Pharmaceutical Co., Ltd.) to the extent that the fetus is immersed, and take care that the AGM region does not include other regions under a stereoscopic microscope. And a new 24-well culture dish (Nu
nc # 143982).

(2) Establishment of AGM-Derived Cell Line A MEM medium (Sigma) containing 10% FCS (Hyclone) in the AGM region transferred to a 24-well culture dish (Nunc # 143982)
Was added and cultured overnight in an incubator. Unless otherwise stated, the cultures in the examples were performed under the conditions of a MEM medium (Sigma) containing 10% FCS (Hyclone), 37 ° C., 5% CO ₂ , and 100% humidity. In overnight culture, cells in the AGM region
When it adhered to the culture dish, 2 ml of MEM medium containing 10% FCS was further added. Thereafter, as the culture was continued, stromal cells gradually appeared around the AGM region tissue piece. After further culturing for another week, the adherent cells were treated with trypsin (0.05% trypsin, 0.53 mM EDTA).
(Gibco BRL) in PBS containing 37 ° C., 3 to 5 minutes), washed twice with medium, and cultured in a 6-well culture dish (Nunc
# 152795). The next day, cells that did not adhere to the culture dish were removed together with the medium, and fresh medium was newly added. Two weeks after the transfer to the 6-well culture dish, 900 Rad gamma rays were irradiated to remove endogenous hematopoietic cells. The cells were directly cloned from this culture system by the limiting dilution method, but no cell growth was observed, and the cells could not be cloned. Therefore, the number of cells seeded in one well was increased, and cells were adapted to withstand proliferation from a small number of cells before cloning by the limiting dilution method.

That is, in the same manner as described above, the AGM was excised and cultured, and the culture system, which had been exposed for 2 weeks after irradiation with γ-rays, was treated with trypsin (0.05% trypsin, 0.53 mM EDT).
A in PBS containing A at 37 ° C for 3-5 minutes) to suspend the cells.
The cells were seeded in 24-well culture dishes at 0-100 cells / well. After culturing for 3 weeks, the cells are seeded in a 96-well culture dish (Nunc # 167008) by limiting dilution at 0.3 cells / well, and grown from a well in which only one cell has been seeded. The grown cells were expanded and cultured. As a result, fibroblast-like cells and pavement-like cells were obtained, and cloning was successful.

The human cord blood-derived CD34-positive cell fraction was co-cultured with fibroblast-like cells for two weeks and examined for the presence of colony-forming cells in the culture system. No colony forming cells were found in the culture system. Therefore, the same examination was carried out for 7 clones showing the cobblestone-like morphology, and three clones having the activity of supporting the growth of human hematopoietic stem cells were obtained. These are AGM-s
1, named AGM-s2 and AGM-s3.

[0039]

[Example 2] Acquisition of viability of embryo-derived 8.5-day embryo-derived cells (1) Production of C57BL6-Ly5.2 pregnant mouse C57BL6-Ly5.2 mouse (about 8 weeks old) (purchased from Japan SLC, Inc.) Male and female are SPF (specific pathogen-f
ree). One or two females were placed in the same cage with one male overnight, and the following morning, the female mice confirmed to have a vaginal plug were transferred to a new cage and reared. The day on which the vaginal plug was confirmed was 0.5 days of gestation (or embryo).

(2) Preparation of Cells Derived from PAS and Yolk Sac A pregnant mouse at 8.5 days of gestation was killed by dislocation of the cervical vertebra, the embryo was excised, and the yolk sac and the PAS region were mixed with other regions. Great care was taken not to separate exactly.
These regions were treated with collagenase (purchased from Sigma, 0.04% collagenase, 10% FCS, PBS solution at 37 ° C, 1%).
(Time treatment) to disperse the cells into single cells.

(3) Transplantation of Cells Derived from Yolk Sac and PAS Region into Mice Cells derived from yolk sac and PAS region of three embryos were irradiated with 9.5 Gy γ-rays at 10 weeks of age for C57BL6-Ly5.1pep mice. Was implanted from the tail vein.

On the other hand, cells derived from the yolk sac and the PAS region for the three embryos prepared in the same manner as above were respectively added onto AGM-S3 cells previously cultured in a 24-well culture dish. After co-culturing for 2, 4 and 6 days, the cells were trypsinized and all cells were collected and irradiated with 9.5 Gy of γ-rays.
One week old C57BL6-Ly5.1pep mice were transplanted from the tail vein.

(4) Confirmation of engraftment of transplanted cells to recipient individuals Ten to twelve weeks after transplantation, blood was collected from the orbital vein of each mouse, erythrocytes were lysed, and the cells were subjected to FITC-labeled anti-Ly5.2. Antibody, P
E-labeled anti-Mac-1 antibody, PE-labeled anti-Gr-1 antibody, APC-labeled anti-B220
Stained with an antibody or APC-labeled anti-Thy-1 antibody, analyzed by flow cytometry, and analyzed for myeloid (Mac-1 positive or Gr-
Individuals with more than 1% of Ly5.2-positive donor cells in all (1 positive) cells and lymphoid (B220-positive or Thy-1 positive) cells were regarded as individuals in which transplanted cells survived. Table 1 shows the results.

[0044]

[Table 1] Table 1 数 Number of surviving mice / Number of transplanted mice ─ ────────────────────── Co-culture period 0 days 2 days 4 days 6 days ───────────────── ──────────── 8.5-day embryo PAS 0/9 0/4 8/9 4/4 8.5-day embryo yolk sac 0/9 0/4 4/8 4/4 ───── ────────────────────────

The cells themselves derived from the 8.5-day embryo do not have the ability to survive into adults. However, it was found that co-culture with AGM-S3 gained the ability to survive into adults as the number of days of culture increased. These results indicate that AG
It is thought that M-S3 stromal cells stimulate cells existing in the yolk sac and PAS to acquire the ability to survive into adults. It is also presumed that cells converted from fetal hematopoietic cells to hematopoietic stem cells due to the activity of AGM-derived stromal cells to expand hematopoietic stem cells or hematopoietic progenitor cells are amplified over time in this culture system.

[0046]

[Example 3] Acquisition of viability of cells derived from embryonic day 8 embryos (1) Creation of C57BL6-Ly5.2 mouse pregnant mice Pregnant mice were prepared in the same manner as in Example 2, and yolk sac, PA
S cells were fractionated.

(2) Preparation of Cells Derived from PAS and Yolk Sac After a pregnant mouse on day 8 of gestation was killed by dislocation of the cervical vertebra, the embryo was excised and the yolk sac and the PAS region were not mixed with other regions. Pay close attention to the exact separation. These regions were treated with collagenase (purchased from Sigma, 0.04%
Collagenase, 10% FCS, PBS solution for 1 hour) to disperse the cells into single cells.

(3) Transplantation of Cells Derived from Yolk Sac and PAS Region into Mice Cells derived from yolk sac and PAS region equivalent to 2 or 5 embryos were irradiated with 9.5 Gy γ-rays for 10-week-old C57BL6- Ly5.1pep
The mice were transplanted from the tail vein.

On the other hand, 2 or 5 prepared in the same manner as above
Cells derived from the yolk sac and the PAS region equivalent to the amount of the embryo were added onto AGM-S3 cells previously cultured in a 24-well culture dish. After culturing for 4 days, the cells were trypsinized to collect all cells, and 10-week-old C57 cells were irradiated with 9.5 Gy of γ-rays.
BL6-Ly5.1pep mice were transplanted from the tail vein.

(4) Confirmation of Engraftment of Transplanted Cells to Recipient Individuals Ten to twelve weeks after transplantation, peripheral blood of the recipient individual was analyzed by flow cytometry in the same manner as in Example 2, and myeloid cells were analyzed. (Mac-1 positive or Gr-1 positive) cells and lymphoid (B
In all of the 220 positive or Thy-1 positive) cells, individuals in which the Ly5.2 positive donor cells exceeded 1% were regarded as individuals in which transplantation was established. Table 2 shows the results.

[0051]

[Table 2] Table 2 数 Number of surviving mice / Number of transferred mice Origin of cells 細胞 Number of transferred mouse embryos 25 ───────────── 8.0 Day 8 of fertilizationBefore culture 0/2 After co-culture of 0/2 PAS (4 days) 2/2 3/3 8.0 Day of fertilization 8.0 days before culture 0/2 0/2 egg yolk Sac After co-culture (4 days) 2/2 4/4 ────────────────────────────────────

When cells immediately after isolation from PAS or yolk sac were transplanted into irradiated mice, no engraftment was observed in all individuals. On the other hand, in the cell group co-cultured with AGM-S3 cells, engraftment was confirmed in all individuals in both cell groups derived from yolk sac and PAS. That is, engraftment ability could be imparted to cells that did not have the ability to engraft adults.

On the 8th day of the embryo, the vascular system circulating between the yolk sac and the embryo has not been established. Therefore, in this example, changes in cells that independently occurred in the yolk sac and PAS sites are analyzed.

No findings have been reported to confirm the ability of hematopoietic cells derived from the yolk sac to adhere to adults. In particular, it has not been clarified whether hematopoietic cells that develop in the yolk sac can differentiate into hematopoietic stem cells.However, according to the present example, fetal hematopoietic cells that develop in the yolk sac also have AGM.
It was shown that coculture with the derived stromal cells can convert them into adult hematopoietic stem cells. This indicates the high ability of AGM-derived stromal cells to convert hematopoietic cells derived from yolk sac to hematopoietic stem cells.

[0055]

According to the present invention, mammalian cells such as fetal hematopoietic cells can be converted into hematopoietic stem cells. The hematopoietic stem cells produced by the method of the present invention have an ability to transplant into adult mammals and can be used for transplantation of hematopoietic stem cells in the medical field.

Claims (7)

[Claims] 1. Mammalian cells that can be converted to hematopoietic stem cells by co-culture with a cell line that is isolated and established from the mammalian AGM region and that supports the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells. A method for producing hematopoietic stem cells, comprising converting mammalian cells into hematopoietic stem cells by co-culturing with a cell line. 2. The method according to claim 1, wherein the mammalian cell is selected from cord blood cells, embryonic stem cells, cells derived from para-aortic mesoderm or yolk sac, neural stem cells, vascular endothelial cells, or embryonic stem cells. . 3. The method according to claim 1, wherein the mammalian cell is a cell derived from para-aortic mesoderm or yolk sac. 4. The method according to claim 1, wherein said cell line is derived from a mouse or a human. 5. The method according to claim 1, wherein the method involves proliferation of hematopoietic stem cells converted from the mammalian cells. 6. The method according to claim 1, wherein the cell line is adheredly grown on an inner wall of an incubator to form a layer, and the cell line is cultured with a mammalian cell layered thereon. Method. 7. The method according to claim 1, wherein the hematopoietic stem cells have an ability to transplant into an adult mammal.