WO2011071085A1

WO2011071085A1 - Method for producing cells induced to differentiate from pluripotent stem cells

Info

Publication number: WO2011071085A1
Application number: PCT/JP2010/072044
Authority: WO
Inventors: 啓光中内; 聡英紙谷; 奈穂鈴木; 慶一伊藤; 聡山▲崎▼
Original assignee: 国立大学法人東京大学
Priority date: 2009-12-08
Filing date: 2010-12-08
Publication date: 2011-06-16
Also published as: JP5896457B2; JPWO2011071085A1

Abstract

Disclosed is a method for producing target cells that have been induced to differentiate from pluripotent stem cells, which involves a process for implanting pluripotent stem cells derived from a mammalian individual into a non-human mammal, a process for dosing the aforementioned non-human mammal with an agent for inducing differentiation into target cells, a process for raising the aforementioned non-human mammal long enough for the implanted pluripotent stem cells to form teratomas in vivo in said mammal and for inducing the aforementioned pluripotent stem cells to differentiate into the target cells, and a process for recovering the target cells derived from the aforementioned mammalian individual from the aforementioned non-human mammal.

Description

Method for producing cells induced to differentiate from pluripotent stem cells

The present invention relates to a method for producing target cells differentiated from pluripotent stem cells, and more specifically, a step of administering a differentiation inducer to target cells to the non-human mammal, and transplanted pluripotent stem cells Growing the animal for a period of time sufficient to form teratomas in the living body of the non-human mammal and inducing differentiation of the pluripotent stem cells into target cells, and from the non-human mammal to the mammal And a method for producing a target cell induced to differentiate from a pluripotent stem cell, and a target cell obtained by the production method.

Stem cells are undifferentiated cells that have both self-renewal ability and pluripotency. In particular, ES cells and iPS cells form teratomas (benign tumors) when administered to a living body, including three germ layers (digestive tract, liver, pancreas, bladder, lung, tonsils, pharynx, parathyroid gland, etc.) Including mesoderm system, nerve cells and skin cells, inner ear, eyes, mammary gland, nails, teeth, spinal cord and brain, such as germ layer, blood cells and muscle cells, bone cells, heart, gonadal, urinary system, fat, spleen Because it contains fetal tissues and mature tissue structures derived from the ectoderm system, which becomes the nervous system, etc., it must be versatile enough to differentiate into all cell lines including germ cells in vivo. Are known (Non-Patent Documents 1 to 4).

Therefore, in the field of regenerative medicine, etc., there is an expectation for the development of a technique for producing in vitro or injured cells or tissues from pluripotent stem cells such as ES cells and iPS cells. Even if stem cells are used, it is difficult to induce differentiation into functional cells efficiently under in vitro conditions.

For example, in the induction of differentiation into hematopoietic stem / progenitor cells (HSPC), there is a report that transplantable hematopoietic stem cells (HSC) can be produced from mouse ES cells by overexpressing HoxB4 (Non-patent Document 5). To 6). However, it has been reported that HSCs derived from ES cells by HoxB4 have a phenotype different from adult HSCs (HSCs that have developed to an intermediate position between fetal and adult HSCs) (non- As is clear from Patent Documents 7 to 8), it is difficult to induce differentiation of pluripotent stem cells into functional cells under in vitro conditions.

On the other hand, in teratomas formed from ES cells or the like in vivo, as described above, various cells are formed, and the formed cells are expected to be functional. However, since cells are produced randomly and in a wide variety, it has been difficult to efficiently induce differentiation of pluripotent stem cells into desired functional cells even under in vivo conditions.

Thus, although development of a method for producing a target cell necessary for treating a disease or injury is required, a method for efficiently inducing differentiation from a pluripotent stem cell to a desired functional cell is: At present, it has not been put into practical use.

The present invention has been made in view of the above-described problems of the prior art, and is capable of efficiently inducing differentiation of pluripotent stem cells into desired functional cells, and an object of differentiation induction from pluripotent stem cells. It aims at providing the production method of a cell.

As a result of intensive studies to achieve the above-mentioned object, the present inventors utilize the teratoma-forming ability of pluripotent stem cells and control it in vivo, so that the desired functional cells and tissues, A method for creating an organ has been found and the present invention has been completed.

That is, by transplanting mouse or human-derived iPS cells into mice and administering cytokines or the like for inducing differentiation into the target cells, the target differentiated cells derived from the iPS cells are formed in the formed teratoma or in the mouse body. Has been found to be formed with high efficiency. In addition, transplanting the differentiated cells induced in this way to a dysfunctional animal restores its function, and it is clear that the induced differentiated cells have almost the same function as the original cells. did. Furthermore, pluripotent stem cells are administered to various treated individuals, such as organ-deficient animals or immunodeficient animals, so that the target cells (including target tissues and target organs) can be more efficiently and transspecies. It also showed that it becomes possible to obtain.

It was also shown that only target cells can be efficiently induced by pre-treatment of pluripotent stem cell genes and controlling the expression of genes such as suicide genes in a time-specific manner.

Furthermore, by using pluripotent stem cells that have been induced to differentiate to some extent from universal cells such as ES cells and iPS cells, a teratoma containing more target cells and the like can be formed. The inventors have also found that the influence can be suppressed, and have completed the present invention.

More specifically, the present invention provides the following inventions.
(1) transplanting pluripotent stem cells derived from a mammal individual to a non-human mammal;
Administering to the non-human mammal a differentiation-inducing agent for target cells;
Growing the animal for a time sufficient for the transplanted pluripotent stem cells to form teratomas in the living body of the non-human mammal, and inducing differentiation of the pluripotent stem cells into target cells;
Recovering a target cell derived from the mammal individual from the non-human mammal;
A method for producing a target cell induced to differentiate from a pluripotent stem cell.
(2) The method according to (1), wherein the pluripotent stem cell is an induced pluripotent stem (iPS) cell prepared using a somatic cell of the mammal individual.
(3) The method according to (1), wherein the pluripotent stem cell is an embryonic stem (ES) cell prepared from a fertilized egg derived from the mammal.
(4) The pluripotent stem cell is transplanted into at least one tissue selected from the group consisting of subcutaneous, testis, and renal capsule of the non-human mammal (1) to (3) The method described in 1.
(5) The method according to any one of (1) to (4), wherein the target cell is a hepatocyte or pancreatic cell, and the target cell is recovered from teratoma formed in the non-human mammal.
(6) The method according to (5), wherein the pancreatic cells are pancreatic Langerhans islet cells.
(7) The method according to any one of (1) to (4), wherein the target cell is a hematopoietic cell, and the hematopoietic cell is recovered from the bone marrow of the non-human mammal.
(8) The method according to (7), wherein the pluripotent stem cell is a Lnk-deficient cell.
(9) The method according to (1) to (8), wherein the step of inducing differentiation of the pluripotent stem cell into a target cell is performed in the presence of co-cultured cells.
(10) The method according to (9), wherein the co-cultured cells are OP-9 cells.
(11) The method according to any one of (1) to (10), wherein the differentiation-inducing agent is continuously administered subcutaneously to the non-human mammal for a predetermined period.
(12) The method according to any one of (1) to (11), wherein the non-human mammal lacks the ability to form a target cell.
(13) The method according to any one of (1) to (12), wherein the non-human mammal is an immunodeficient animal.
(14) The method according to any one of (1) to (13), wherein in the step of inducing differentiation of the pluripotent stem cell derived from the animal individual into a target cell, an operation for removing cells other than the target cell to be mixed is performed. the method of.
(15) The method according to (14), wherein the cells other than the target cell are pluripotent stem cells that remain in an undifferentiated state.
(16) The method according to (14) or (15), wherein the removing operation is achieved by using a suicide gene at a desired time.
(17) A target cell obtained by the method according to any one of (1) to (16).

The present invention has made it possible to efficiently induce differentiation of target cells from pluripotent stem cells. Moreover, unlike in vitro differentiation-inducing systems, differentiation induction into cells having almost the same functions as the original cells (for example, cells capable of restoring the functions when transplanted into dysfunctional animals). Became possible.

Murine iPS cells are transplanted into nude mice, differentiation inducers such as cytokines are administered to the nude mice, and teratomas are formed in the living body of the nude mice, differentiation induced hepatocytes, pancreatic cells, or hematopoietic system It is the schematic which shows the process of producing a cell. It is a microscope picture of the immunostaining which uses the anti albumin antibody or the anti- CK19 antibody which shows that hepatocytes are produced in the teratoma produced by administering a differentiation inducer. It is a microscope picture of the immuno-staining using an anti- CYP7A1 antibody which shows that hepatocytes are produced in the teratoma produced by administering a differentiation inducer. It is the schematic which shows the process of indocyanine green adsorption reaction, and the photograph which shows that hepatocytes are produced in the teratoma produced by administering the differentiation-inducing agent dye | stained by this reaction. It is a microscope picture of the immuno-staining using an anti-insulin antibody which shows that the pancreatic cell (pancreatic Langerhans islet cell) is produced in the teratoma produced by administering a differentiation-inducing agent. It is a plot figure which shows the result of having analyzed the expression of CD45 and GFP in the peripheral blood of the mouse | mouth in which the teratoma was formed with the flow cytometer. Schematic diagram showing that iPS cells have differentiated into hematopoietic stem cells in the mouse body and home to bone marrow, and flow cytometer analysis shows that hematopoietic progenitor cells derived from iPS cells in the bone marrow of mice with teratomas formed by lineage-c FIG. 5 is a plot diagram showing that −Kit + Sca-1 + (KSL) exists. Schematic diagram showing transplantation of mouse-derived bone marrow cells in which teratoma is formed into wild type C57 / BL6 mice, and by peripheral cytoplasm in wild type C57 / BL6 mice 4 weeks after transplantation by flow cytometer analysis. It is a plot figure which shows that the various hematopoietic system cell derived from an iPS cell exists. A vector encoding a suicide gene (TK gene) used to remove cells other than target cells (hepatocytes) mixed in the teratoma, and Cre under the control of the Alb promoter which is a marker gene for hepatocytes. It is the schematic which shows the vector which expresses recombinase, and the schematic which shows the screening method of the Alb expression cell (hepatocyte) by ganciclovir administration using these 2 types of vectors. It is the microscope picture of a cell line which shows that the screening system by a suicide gene is effective in removal of cells other than the target cell mixed in the teratoma. FIG. 2 is a schematic diagram showing the process of establishing iPS cells from Lnk ^{− / −} GFP transgenic (Tg) mice or GFP Tg mice. It is a microscope picture which shows the result of the immunostaining of Nanog and SSEA-1 in a Lnk ^{− / −} GFP iPS cell. In the figure, the scale bar indicates 100 μm. It is a photograph showing a chimeric mouse prepared from Lnk ^{− / −} GFP iPS cells. FIG. 5 is a photograph showing nude mice in which teratomas derived from Lnk ^{− / −} GFP iPS cells are formed. 2 is a photograph showing teratomas derived from Lnk ^{− / −} GFP iPS cells collected from nude mice. In the figure, the scale bar indicates 1.25 cm. It is a microscope picture which shows the various structure | tissues containing the three germ layers derived from iPS cell in a teratoma. It is the schematic which shows the process of transplanting the hematopoietic stem cell (HSC) induction | guidance | derivation from iPS cell through teratoma formation, and the bone marrow derived from the mouse | mouth which formed this teratoma. That is, iPS cells were injected subcutaneously into nude mice with or without OP9 cells. Cytokines were placed in an osmotic pump and administered for 2 weeks. And it is the schematic which shows having transplanted the bone marrow cell from which the iPS cell origin HSC was detected to the irradiation mouse | mouth. It is a plot figure which shows the result of having analyzed the peripheral blood of the teratoma formation nude mouse 12 weeks after inject | pouring an iPS cell by the flow cytometry. Note in the figure, the numbers indicate the percentage of GFP ⁺ cells in CD45 ⁺ cells ^{^{(GFP + / CD45 + cells (}} %)). Change in the percentage of GFP ⁺ cells in CD45 ⁺ cells ^{^{(GFP + / CD45 + cells (}} %)) is a graph showing that iPS cell injection period after, and is dependent on teratoma size. It is a graph which shows the ratio of the GFP ⁺ cell derived from iPS cell in the peripheral blood and the bone marrow of the mouse | mouth which formed the teratoma under each condition. In addition, 12 weeks after iPS cell injection, 5 teratoma-forming mice under each condition were analyzed. In the figure, the vertical axis on the left indicates the ratio of GFP ⁺ cells in CD45 ⁺ cells (GFP ⁺ / CD45 ⁺ cells (%)) in the peripheral blood of teratoma-forming nude mice. The right vertical axis shows the ratio of GFP ⁺ cells in KSL cells (GFP ⁺ / KSLcells (%)) in the bone marrow of teratoma-forming nude mice. Further, in the bar graph showing the results under each condition, the left side shows the result of analyzing peripheral blood, and the right side shows the result of analyzing bone marrow. Error bars represent standard deviation. Those marked with three asterisks (***) indicate a significantly different value (p <0.001) compared to untreated (None, iPS cells only) injected samples. It is a plot figure which shows the result of having analyzed the bone marrow of the mouse | mouth with which the teratoma was formed by administering a cytokine and OP9 cell 12 weeks after iPS cell injection | pouring by flow cytometry. ² is a photomicrograph showing the results of analyzing colonies derived from iPS cells formed by dividing GFP ⁺ CD34 ⁻ KSL cells derived from teratoma-forming mouse bone marrow cells one by one and adding a cytokine that induces hematopoietic differentiation. In the figure, the left side (Phase) shows the result of phase difference observation, and the right side (GFP) shows the result of fluorescence observation. A graph showing the results of evaluation of the quantity and type of CFC-nmEM by dividing 100 GFP ⁺ CD34 ⁻ KSL cells derived from teratoma-forming mouse bone marrow one by one, adding a cytokine that induces hematopoietic differentiation, and culturing for 10 days. is there. CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte). In the figure, “nm” on the horizontal axis represents a colony composed of two strains of neutrophil / macrophage, “nmM” represents a colony composed of three strains of neutrophil / macrophage / megakaryocyte, and “nmE” represents neutrophil. "NmEM" indicates a colony consisting of 4 strains of neutrophil / macrophage / erythroblast / megakaryocyte. FIG. 6 is a micrograph of a cytospin sample of a CD34 ⁻ KSL cell-derived colony observed by leuco staining (Leukostein). In the figure, GM represents granulocytes / macrophages, Meg represents megakaryocytes, and E represents erythroblasts. It is a graph which shows the result of a bone marrow transplantation assay. That is, GFP derived from Lnk ^{− / −} GFP iPS cells in the peripheral blood of a primary mouse transplanted with bone marrow cells derived from teratoma-forming mice into irradiated mice and a secondary transplant recipient mouse 12 weeks after the primary transplantation. It is a graph which shows the chimerism of ⁺ / CD45 ⁺ cell. Four animals were analyzed in the primary transplantation and 10 animals were analyzed in the secondary transplantation. In the figure, error bars indicate standard deviation. In each bar graph, “myeloid”, “B cell”, and “T cell” are shown from the top. 12 is a graph showing the chimerism of spleen and bone marrow Lnk ^{− / −} GFP iPS cell-derived GFP ⁺ hematopoietic cells / CD45 ⁺ cells of recipient mice (analysis number: 4 animals) 12 weeks after the primary transplantation. In the figure, error bars indicate standard deviation (SD). In each bar graph, “myeloid”, “B cell”, and “T cell” are shown from the top. GFP ⁺ cells in hematopoietic progenitor cells (HPC, Lin ⁻ ) or hematopoietic stem cells (HSC, CD34 ⁻ KSL) in bone marrow cells of recipient mice (analysis number: 4) 12 weeks after primary transplantation It is a graph which shows a ratio. In the figure, error bars indicate standard deviation (SD). It is a plot figure which shows the result of having analyzed the peripheral blood of the teratoma formation nude mouse 12 weeks after inject | pouring an iPS cell by the flow cytometry. Note in the figure, the numbers indicate the percentage of GFP ⁺ cells in CD45 ⁺ cells ^{^{(GFP + / CD45 + cells (}} %)). It is a plot figure which shows the correspondence between the number of GFP ⁺ CD45 ⁺ cells derived from GFP iPS cells and the period after iPS cell injection in peripheral blood of mice in which teratomas are formed by administration of cytokines and OP9 cells. It is a graph which shows the ratio (GFP ^<+ > / CD45 ^<+> cells (%)) of GFP ^< ^+> cell in CD45 ^<+> cell in the peripheral blood of a teratoma formation nude mouse. It should be noted that four mice under each condition were analyzed 12 weeks after iPS cell injection. In the figure, error bars represent standard deviation. Those marked with two asterisks (**) indicate a significantly different value (p <0.01) compared to the untreated sample. It is a plot figure which shows the result of having analyzed the bone marrow cell derived from the mouse | mouth with which the teratoma was formed by administering a cytokine and OP9 cell 12 weeks after inject | pouring an iPS cell, by the flow cytometry. It is a graph which shows the result of a bone marrow transplantation assay. That is, GFP iPS cell-derived in the peripheral blood of primary recipient mice transplanted with all bone marrow cells of teratoma-forming mice and secondary recipient mice transplanted with 40 GFP ⁺ CD34 ⁻ KSL cells selected from the bone marrow and transplanted It is a graph which shows the chimerism of GFP ^<+ > / CD45 ^<+> cell. In addition, 6 animals were analyzed in the primary transplantation and 5 animals were analyzed in the secondary transplantation. Error bars represent standard deviation. In each bar graph, “myeloid”, “B cell”, and “T cell” are shown from the top. It is a plot figure which shows the chimerism of the GFP ⁺ hematopoietic cell / CD45 ⁺ cell derived from a GFP iPS cell in the spleen and the bone marrow of a recipient mouse (analysis number: 6) 12 weeks after primary transplantation. GFP ⁺ cells in hematopoietic progenitor cells (HPC, Lin ⁻ ) or hematopoietic stem cells (HSC, CD34 ⁻ KSL) in the spleen and bone marrow of recipient mice (analysis number: 6) 12 weeks after primary transplantation It is a graph which shows a ratio. In the figure, error bars indicate standard deviation (SD). Results were obtained by dividing 100 GFP ⁺ CD34 ⁻ KSL cells derived from teratoma-forming mouse bone marrow one by one, adding cytokines that induce hematopoietic differentiation, culturing, and culturing for 10 days to evaluate the quantity and type of CFC-nmEM. It is a graph to show. CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte). In the figure, “nm” on the horizontal axis represents a colony composed of two strains of neutrophil / macrophage, “nmM” represents a colony composed of three strains of neutrophil / macrophage / megakaryocyte, and “nmE” represents neutrophil. "NmEM" indicates a colony consisting of 4 strains of neutrophil / macrophage / erythroblast / megakaryocyte. FIG. 3 is a schematic diagram showing a method for producing mγc-iPS cells into T cells in X-SCID mice. iPS cells were established from X-SCID mice, and mγc gene was introduced into the iPS cells to prepare mγc-iPS cells. Then, mγc-iPS cells and OP9 cells were injected into X-SCID mice for teratoma formation. It is an electrophoresis photograph showing the PCR analysis result of mγc-iPS cells. In the figure, mγc-iPS cells (mγc-iPSC) # 1 to 5 are obtained by introducing 3 factors (Oct3 / 4, Klf4, and Sox2) into cells derived from X-SCID mice having IL-2Rg mutation. Cell. 4F B6 iPS cells (4F B6 iPS iPSC) are cells having a c-Myc transgene and having no common gamma chain (γc) mutation. Pure water (Water) represents a negative control. 2 is an electrophoresis photograph showing the results of analyzing the expression of an ES cell marker gene in mγc-iPS cells by RT-PCR. Pure water (Water) and MEF represent negative controls. ES cells (ESC) represent a positive control. FIG. 4 is a plot and histogram showing expression of GFP and mouse gamma chain (γc) in mγc-iPS cell # 4 (mγc-iPSC # 4). In the figure, B6 iPS cells (B6 iPSC) represent a negative control. The filled histogram on the right side shows the results for mγc-iPSC # 4, and the white histogram on the left side shows the results for the negative control (B6 iPS cells). It is a plot figure which shows the result of having analyzed the peripheral blood of the teratoma formation mouse | mouth 12 weeks after inject | pouring an iPS cell by the flow cytometry. In the figure, the numerical value indicates the ratio (%) of GFP ⁺ cells in each cell. It is the schematic which shows the induction | guidance | derivation method from a human iPS cell to a hematopoietic stem cell (HSC) through teratoma formation. That is, human iPS cells were injected into the testes of NOD / SCID mice together with OP9 cells. Cytokines were placed in an osmotic pump and administered for 2 weeks. And it is the schematic which shows having transplanted the bone marrow cell of the teratoma formation mouse | mouth to the irradiation NOD / SCID mouse or the irradiation NOD / SCID / JAK3-deficient mouse. It is a plot figure which shows the result of having analyzed the bone marrow cell of the teratoma formation nude mouse 12 weeks after inject | pouring a human iPS cell by flow cytometry. In the figure, “m” indicates that it is derived from a mouse, and “h” indicates that it is derived from a human. It is a plot figure which shows the chimerism in the peripheral blood of a recipient mouse 8 weeks after bone marrow transplantation. In the figure, “Sorted” indicates the result of transplantation of bone marrow cells from which mCD45 ⁺ cells of teratoma-forming mice were removed. “Total” indicates the result of transplanting all bone marrow cells of teratoma-forming mice. “M” indicates that it is derived from a mouse, and “h” indicates that it is derived from a human. It is a microscope picture which shows the result of having investigated the expression of CD45, CD34, and osteocalcin in the teratoma section derived from a human iPS cell by immunostaining. In the figure, “Merge” indicates a superposition of these three expressions. 2 is a photomicrograph showing the results of examining the expression of CD45, CD34, and VE-cadherin by immunostaining in teratoma sections derived from human iPS cells. In the figure, “Merge” indicates a superposition of these three expressions. It is a microscope picture which shows the result of having investigated the expression of CD45, CD34, and GFAP in the teratoma section derived from a human iPS cell by immunostaining. In the figure, “Merge” indicates a superposition of these three expressions. It is a plot figure which shows the result of having analyzed the cell derived from the human iPS cell in teratoma by flow cytometry. “M” indicates that it is derived from a mouse, and “h” indicates that it is derived from a human. It is a microscope picture which shows the result of the immuno-staining of CD45 ⁺ cell in the teratoma derived from a GFP iPS cell. In the figure, “DAPI + merge” shows the expression of CD45 and GFP and the result of DAPI staining superimposed, and the arrow shows GFP ⁺ CD45 ⁺ cells derived from GFP iPS cells. The scale bar indicates 150 μm. It is a plot figure which shows the result of having analyzed iPS cell origin GFP ⁺ CD45 ⁺ cell and KSL cell in teratoma by flow cytometry. Note in the figure, numerical values on the left indicate the percentage of ^GFP + CD45 ⁺ cells teratoma cells, the second number is the cell ^c-Kit of (teratoma ^GFP + CD45 ⁺ cells in a cell) in + SCA- It shows 1 ⁺ fraction of cells (%). It is a microscope picture which shows the result of the immunostaining of osteocalcin (Osteocalcin), VE-cadherin (VE-cadherin), and GFAP in teratoma. In the figure, the scale bar indicates 75 μm. It is a microscope picture which shows the result of the immunostaining of CD45, c-Kit, and HSC niche marker: osteocalcin in teratoma. In the figure, “DAPI + merge” indicates a superposition of these three expressions and the results of DAPI staining, and the arrow indicates CD45 ⁺ c-Kit ⁺ cells. The scale bar indicates 75 μm. FIG. 2 is a photomicrograph showing the results of immunostaining of CD45, c-Kit, and HSC niche marker: VE-cadherin in teratoma. In the figure, “DAPI + merge” indicates a superposition of these three expressions and the results of DAPI staining, and the arrow indicates CD45 ⁺ c-Kit ⁺ cells. The scale bar indicates 75 μm. It is the photograph which shows the teratoma formed in the mouse | mouth (the left side in a figure) which transplanted the undifferentiated ES cell, and the mouse | mouth (right side in the figure) transplanted after carrying out differentiation induction to the endoderm progenitor cell. It is a microscope picture which shows the result of the immunohistochemical dyeing | staining in the formed teratoma. In the figure, “Merge” indicates the result of overlapping the expression of CK19 and Foxa2 with the result of DAPI staining. It is a graph which shows the number of intestinal tract-like structures of CK19 positive in the produced teratoma tissue section.

The present invention
(A) transplanting pluripotent stem cells derived from a mammal individual to a non-human mammal;
(B) administering to the non-human mammal an agent for inducing differentiation into a target cell;
(C) growing the animal for a time sufficient for the transplanted pluripotent stem cells to form a teratoma in the living body of the non-human mammal, and inducing differentiation of the pluripotent stem cells into target cells;
(D) recovering a target cell derived from the mammal individual from the non-human mammal;
A method for producing target cells induced to differentiate from pluripotent stem cells.

In the present invention, the “mammal” is not particularly limited, and examples thereof include humans, mice, rats, cows, horses, pigs, sheep, monkeys, dogs, and cats. In the present invention, the “non-human mammal” is not particularly limited, and examples thereof include mice, rats, cows, horses, pigs, sheep, monkeys, dogs, and cats.

In addition, the “target cell” according to the present invention is one or a plurality of cells selected from various cells constituting the mammal individual, for example, hematopoietic cells, hepatocytes, pancreas Examples include cells, intestinal cells, thymocytes, and bone / chondrocytes. Furthermore, the “target cell” according to the present invention includes various cell groups (for example, tissues and organs) constituting the mammal individual.

Here, “hematopoietic cell” means a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), and a cell obtained by differentiation from HSC or HPC, that is, erythroblast, myeloid cell, megakaryocyte system, lymphocyte. And the like, and examples thereof include HSC, HPC, neutrophils, macrophages, erythroblasts, and megakaryocytes.

The term “pancreatic cell” means a cell constituting the pancreas, that is, an exocrine cell constituting the exocrine part, a Langerhans islet cell constituting the Langerhans islet (endocrine part), and the like, for example, an acinar cell, A Examples include cells (α cells), B cells (β cells), D cells (δ cells), and PP cells.

First, (a) the step of transplanting pluripotent stem cells derived from the mammal individual to the non-human mammal will be described.

The “pluripotent stem cell” to be differentiated in the present invention is a cell having pluripotency capable of differentiating into various cells constituting the mammal individual and self-replicating ability, for example, an embryonic stem cell ( Embryonic stem cells (ES cells), induced pluripotent stem cells (Induced pluripotent stem cells, iPS cells), embryonic tumor cells (Embryonic carcinoma cells, EC cells), embryonic germ cells (EmbrycellG cells) Universal cells such as multipotent germline stem cell (mGS cell), fertilized egg, inner cell mass (inner cell mass, ICM), ectoderm progenitor cells, endoderm progenitor cells, mesoderm Such systems progenitor cells, differentiable cell series include stem cells / progenitor cells are limited.

Among these, from the ethical viewpoint that embryos can be produced without breaking them, and when used for regenerative medicine, etc., patients transplanted with cells differentiated from pluripotent stem cells and blood types (erythrocyte type, From the viewpoint of easy adaptation in terms of leukocyte type), it is preferable to use iPS cells prepared using somatic cells of the individual mammal as the pluripotent stem cells according to the present invention.

The “iPS cell” according to the present invention is a cell that has acquired ES cell (Embryonic stem cells) -like differentiation pluripotency by introducing a cell reprogramming factor into the somatic cell of the individual mammal. In addition, the “cell reprogramming factor” used in the present invention, when introduced into a somatic cell, imparts pluripotency to the somatic cell alone or in cooperation with other pluripotent factors. It can be any factor that can be used, and is not particularly limited. However, Oct3 / 4, c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, Eras, ECAT7, ECAT8, Gdf3, Sox15, It is preferably at least one protein selected from the group consisting of ECAT15-1, ECAT15-2, Fthl17, Sal14, Rex1, Utf1, Tcl1, Stella, β-catenin, Stat3 and Grb2. Further, among these proteins, it is more preferable to introduce Oct3 / 4, c-Myc, Sox2 and Klf4 (4 factors) into the somatic cells from the viewpoint that iPS cells can be established efficiently with few factors. Further, from the viewpoint of reducing the risk of canceration of the resulting pluripotent stem cells, it is more preferable to introduce Oct3 / 4, Sox2 and Klf4 (3 factors) excluding c-Myc into the somatic cells.

In addition, as shown in the examples described later, from the viewpoint that a teratoma containing more target cells can be formed and the influence of the increase in teratomas on the health condition of the host can be suppressed, some differentiation is induced from universal cells. It is preferable to use the pluripotent stem cell (for example, the stem cell / progenitor cell in which the differentiable cell line is limited) as the pluripotent stem cell according to the present invention. As a method for establishing such pluripotent stem cells, for example, for endoderm progenitor cells, pluripotent cells are seeded in a culture dish coated with Collagen-Type4 as shown in the Examples below, and activin A, BMP4, etc. And then culturing in a serum-free medium supplemented with activin A, EGF, FGF4, and the like.

Note that, as described in Non-Patent Documents 5 to 8, for example, it is generally difficult to induce differentiation of pluripotent stem cells into functional cells without genetic modification. However, in the present invention, as shown in Examples described later, functional cells induced to differentiate can be efficiently obtained even using pluripotent stem cells that are not genetically modified.

However, pluripotent stem cells that have been genetically modified can also be used as pluripotent stem cells according to the present invention from the viewpoint of increasing the number of target cells produced and enhancing the function of the target cells. In such pluripotent stem cells, genetic modification is not particularly limited, and known techniques can be appropriately employed. For example, in the case of enhancing the function or expression of a protein that induces an increase in the number of target cells produced or enhancement of function, a method of introducing a gene encoding the protein into a pluripotent stem cell as a transgene, And a method for introducing into the pluripotent stem cell a mutation that enhances the expression of the gene in the DNA sequence of the expression control region of the gene. Furthermore, in the case of suppressing the expression or function of a protein that inhibits the increase in the number of target cells produced or the enhancement of function, for example, a method of knocking out or knocking in a gene encoding the protein in pluripotent stem cells, A method of introducing a mutation that suppresses the expression of the gene into the DNA sequence of the gene expression regulatory region, or a transgene encoding siRNA or shRNA for the gene is introduced into the pluripotent stem cell. A method is mentioned.

Furthermore, when the target cell is a hematopoietic cell, a mouse lacking the Lnk protein overproduces a hematopoietic stem cell (HSC) having a high self-renewal capacity, and the Lnk protein is deficient as shown in Examples described later. Since the pluripotent stem cell-derived HSC has a very high replication ability, it is preferable to use Lnk-deficient cells as the pluripotent stem cells according to the present invention.

The Lnk protein is an intercellular adapter protein that is known as a factor that negatively regulates the TPO / c-mpl pathway in HSC and the like. Typically, as an Lnk derived from human, Accession No. The protein (gene) specified by NP_005466 (No. NM_005474) is mentioned, and Access No. Examples include proteins (genes) specified by NP_032533 (No. NM_008507).

The “Lnk deficient cell” according to the present invention is a cell deficient in the expression and / or function of the Lnk protein, and the “Lnk deficient cell” is, for example, as described above, knocking out the Lnk gene, It can be prepared by introducing siRNA against the Lnk gene.

In the present invention, a method for transplanting the pluripotent stem cells into a non-human mammal is not particularly limited, and a known technique can be appropriately used.

In the present invention, the tissue for transplanting the pluripotent stem cells into a non-human mammal is not particularly limited, and examples thereof include subcutaneous, testis, renal capsule, and bone marrow. Among these, it is preferable to transplant subcutaneously from the viewpoint that it is frequently used as a place where teratomas derived from mouse iPS cells and the like are formed. In addition, when transplanting human iPS cells into a non-human mammal, the frequency of teratoma formation is low even when injected into mice or the like subcutaneously, and from the viewpoint that it is a tissue with more blood flow, It is preferable to transplant to the testis. For the purpose of induction into hematopoietic stem cells, transplantation into bone marrow is preferable. Furthermore, from the viewpoint that the donor-derived tissue and the recipient tissue are not easily mixed, it is preferable to transplant to a tissue (ectopic) other than the tissue in which the target cell exists.

In the present invention, when the pluripotent stem cells are transplanted into a non-human mammal, the pluripotent stem cells may be used by mixing with other components. Such other components are not particularly limited, and include physiological saline such as phosphate buffered saline (PBS), medium, buffer, and preservative.

Furthermore, as shown in the examples described later, from the viewpoint of increasing the efficiency of differentiation induction into hematopoietic cells, and based on the results of in vitro research, the efficiency of differentiation induction into nerve cells, vascular endothelial cells, and cardiomyocytes is increased. In view of the in vitro research results, “Zeng et al., Stem Cells, 2004, Vol. 22, pp. 925 to 940 are preferable. ”,“ Sone et al., Arterioscler Thromb Vas Biol., 2007, 27, 2127-2134 ”,“ Yamashita et al., FASEB J., 2005, 19, 1534-1536 ”).

The term “co-cultured cell” according to the present invention means a cell that can be used in a culture system for pluripotent stem cells to prepare culture conditions for inducing proliferation or differentiation of pluripotent stem cells. Cells, stromal cells, and more specifically OP-9 cells, PA6 cells, MEF (mouse fetal fibroblasts), NIH3T3 cells, M15 cells, and 10T / 2 cells. Among these, the “co-culture cells” according to the present invention, as shown in the examples described later, can increase the induction efficiency of the pluripotent stem cells to the target cells by mixing with the pluripotent stem cells. From the viewpoint, OP-9 cells are preferably used.

Further, as shown in the examples described later, when differentiation is induced into intestinal cells, hepatocytes, nerve cells, vascular endothelial cells, etc., laminin, collagen IV, heparan sulfate proteoglycan, entactin /

nidogen

1, 2, TGF- at least selected from the group consisting of β, epidermal growth factor, insulin-like growth factor, fibroblast growth factor,

tissue plasminogen activator

3, 4 and other growth factors naturally produced in EHS tumors One component is preferably mixed in the pluripotent stem cell.

Furthermore, in the present invention, the non-human mammal to which the pluripotent stem cells are transplanted is not particularly limited, and includes the aforementioned mice, etc., but a space for inducing target cells derived from pluripotent stem cells is secured. Thus, from the viewpoint of increasing the efficiency of differentiation induction, an animal lacking the ability to form target cells is preferable. In addition, even in a heterogeneous relationship between the mammal from which the pluripotent stem cell is derived and the non-human mammal into which the pluripotent stem cell is transplanted, the target cell differentiated from the pluripotent stem cell is produced. From the viewpoint of being able to do so, it is preferably an immunodeficient animal.

Next, (b) the step of administering an agent for inducing differentiation into a target cell to the non-human mammal will be described.

In the present invention, the "differentiation inducer to target cells" administered to the non-human mammal is not particularly limited as long as it is a physiologically active substance that works when differentiation induction from pluripotent stem cells to target cells is performed, Examples thereof include polypeptides and proteins (cytokines, hormones, enzymes, etc.), vitamins, saccharides, fatty acids, amino acids, nucleic acids, minerals and the like.

More specifically, the “differentiation inducer to target cells” according to the present invention is more specifically, when the target cells are hepatocytes, retinoic acid, fibroblast growth factor 1 (FGF1), FGF4, hepatocyte proliferation Factor (HGF), oncostatin M (OsM), and when the target cell is a pancreatic cell, activin A, epidermal growth factor (EGF), Noggin, insulin-like growth factor-2 (IGF-2), When nicotinamide is used and the target cell is a hematopoietic cell, stem cell factor (SCF) and thrombopoietin (TPO) are used. When the target cell is an enteric cell, activin A and bone morphogenetic factor 4 ( BMP4), EGF, FGF4, NOGGIN.

As shown in the examples described later, in the differentiation induction of pancreatic Langerhans islet cells, first, activin A or the like is added to iPS cells to induce differentiation into endoderm progenitor cells, and EGF, After inducing endoderm cells committed to the pancreas by adding bFGF and Noggin, nicotinamide or IGF-2 is added to the endoderm cells to induce differentiation of the pancreas into Langerhans islet cells. Thus, in the differentiation induction of the target cell, the differentiation stage at which the “differentiation inducer” acts varies depending on the type of differentiation inducer. Therefore, in the present invention, the predetermined period for administering the differentiation-inducing agent to the non-human mammal is a period sufficient for each differentiation-inducing agent to act and induce the desired differentiation. means. Each differentiation-inducing agent may be administered to cells transiently or continuously as long as each target differentiation can be achieved. From the viewpoint of mimicking the development of an original organ, it is preferable to administer the differentiation inducer continuously.

In the present invention, the site for administering the differentiation-inducing agent in the non-human mammal is not particularly limited, but is preferably subcutaneous from the viewpoint that the procedure required for administration is simple.

Furthermore, in the present invention, the method for administering the differentiation inducer to the non-human mammal is not particularly limited, and a known method (for example, a method using an osmotic pump) can be appropriately selected and used.

Next, (c) the transplanted pluripotent stem cells are allowed to grow for a sufficient period of time to form teratomas in the living body of the non-human mammal, and the pluripotent stem cells are induced to differentiate into target cells. The process will be described.

In the present invention, there is no particular limitation as “a sufficient time for the transplanted pluripotent stem cells to form teratomas in the living body of the non-human mammal”, but it is preferably 4 to 12 weeks after the transplantation, More preferably, it is 8-12 weeks. If the time is less than the lower limit, the transplanted pluripotent stem cells tend not to be sufficiently induced to differentiate into target cells. On the other hand, if the time exceeds the upper limit, the health of the non-human mammal due to teratoma increase It tends to adversely affect the condition.

Further, in the step of inducing differentiation of the pluripotent stem cell derived from the animal individual according to the present invention into the target cell, cells other than the target cell to be mixed are removed from the viewpoint of efficiently inducing differentiation of only the target cell. It is preferable to perform the operation to do.

In the present invention, the “cell other than the target cell” is not particularly limited. For example, a pluripotent stem cell (undifferentiated cell) that does not induce differentiation and remains in an undifferentiated state or other than the target cell. Cells derived from pluripotent stem cells that have differentiated into other cells (cells that have differentiated into other cell lineages).

In addition, the operation for removing cells other than the target cells to be mixed is not particularly limited. For example, it is preferable to cause the suicide gene to function in cells other than the target cells, as shown in Examples described later. Thereby, only the target cell can be highly purified and used for transplantation.

In addition, a “suicide gene” used to cause a suicide gene to function in cells other than the target cell is cell death (apoptosis) in cells expressing the protein under conditions that allow the protein encoded by the gene to function. , Necrosis, etc.) can be used. For example, thymidine kinase (TK) gene, herpesvirus thymidine kinase (HSVtk) gene (Proc. Natl. Acad. Sci, USA 78 (1981), pages 1441-1445. Reference), cytosine deaminase gene (EG11326 codA 355395.356678 E. coli), uracil phosphoribosyltransferase gene (EG11332 up 26618894.26618268 E.c) li), guanine phosphoribosyl transferase (gpt) gene (EG10414 gpt 255977..256435 E.coli), a nitro reductase gene.

TK is a microorganism-derived metabolic enzyme gene, which metabolizes ganciclovir (GCV) to produce ganciclovir 5'-triphosphate. This ganciclovir 5'-triphosphate inhibits DNA synthesis, thereby inducing cell death of cells expressing TK. That is, by adding GCV, the condition is such that TK can function, and cell death is induced in cells in which TK is expressed.

Further, in the operation achieved by working the suicide gene at such a desired time, there is no particular limitation as to the method of `` working the suicide gene '', and any method can be used as long as the protein encoded by the suicide gene can function. Examples include a method in which a TK gene is introduced into a pluripotent stem cell and the TK gene is removed only by a cell differentiated into a target cell by a Cre-LoxP system, as shown in the Examples described later. More specifically, when the target cell is a hepatocyte, as shown in FIG. 9, a viral vector in which an HSV-derived TK gene is sandwiched between LoxP sequences, and control of an Alb promoter that is a marker gene for hepatocytes. A viral vector expressing Cre recombinase is prepared below. After the cells are infected with the two viruses, differentiation induction into hepatocytes is performed in the present invention. Thereafter, by administering GCV, only Alb-producing cells from which the TK gene has been removed by the expression of Cre can survive, and cell death of undifferentiated cells or cells differentiated into other cell lineages can be induced. In the same manner as described above, even when the target cell is a cell other than a hepatocyte, by selecting a promoter specific to the target cell (for example, when a pancreatic lineage cell is used as the target cell). By selecting the Pdxl promoter, cells other than the target cell can be removed.

Furthermore, when the cells other than the target cells are pluripotent stem cells that remain in an undifferentiated state, for example, the pluripotent stem cells can be used as promoters of undifferentiated marker genes (Nanog, Oct3 / 4 gene, etc.). Using a gene to which TK, which is a suicide gene, has been introduced, and inducing differentiation of the pluripotent stem cells into target cells according to the present invention, then selectively removing only undifferentiated cells by administering GCV. Can do.

Next, (d) a step of collecting target cells derived from the individual mammal from the non-human mammal will be described.

In the present invention, the method for recovering the target cells derived from the individual mammal is not particularly limited, and examples thereof include a method for recovering from the teratoma formed in the living body of the non-human mammal. In addition, when the target cell is a hematopoietic cell, as shown in the examples below, the hematopoietic cell induced to differentiate by the method of the present invention surprisingly moves from the teratoma and engrafts in the bone marrow. It can also be recovered from the bone marrow of the non-human mammal.

The present invention also provides target cells obtained by the above-described method. That is, the present invention also provides one or a plurality of cells selected from various cells constituting the mammalian individual, which are induced to differentiate from pluripotent stem cells by the method described above. Furthermore, a tissue or organ composed of one or a plurality of cells selected from various cells constituting the mammal individual is also provided. Such “target cells” are not particularly limited, and examples thereof include hematopoietic cells, hepatocytes, pancreatic cells, intestinal cells, thymocytes, and bone / chondrocytes.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. In Examples 1 to 3, the following materials were used and the following methods were used.

<Cell>
As mouse iPS cells, three genes of Oct3 / 4, Sox2 and Klf4 were introduced into the tail tip fibroblasts (TTF, tail tip fibroblast) of Kusabila Orange transgenic mice (129 / Sv mice derived) using a retroviral vector. Established and Lnk knockout mice (derived from C57BL / 6 mice) were used by introducing the above 3 genes with a lentiviral vector. In addition, it was confirmed before the experiment that both the cell lines can produce chimeric mice as blastocyst hosts for wild-type mice.

<Cell culture>
Mouse iPS cells were cultured in E14.1 KSR medium by co-culture with mouse fetal fibroblasts (MEF) treated with mitomycin C. The composition of the E14.1 KSR medium is as follows.
Dulbecco's modified Eagle medium (DMEM, manufactured by Invitrogen), 15% knockout serum substitute (KSR, manufactured by Invitrogen) as an additive, 2 mM L-glutamine-penicillin-streptomycin (manufactured by Invitrogen), 1 × non-essential amino acid ( 1 mM HEPES (manufactured by Invitrogen), 0.1 mM 2-mercaptoethanol (manufactured by Gibco), 1000 IU / ml leukemia inhibitory factor (Leukemia Inhibitory Factor, LIF).

<Animals>
KSN / Slc-nu / nu mice and C57BL / 6 mice were purchased from SLC Japan.

<Cell administration>
5 × 10 ⁶ iPS cells were administered subcutaneously to the nude mice. In the differentiation experiment into blood cells and hematopoietic stem cells, 1 × 10 ⁶ OP9 cells were simultaneously administered.

<Teratoma formation and differentiation into target cells>
Mouse iPS cells were peeled off from the dish by trypsin treatment and suspended in PBS at about 5 × 10 ⁶ cells / 50 μL and injected subcutaneously into KSN / Slc-nu / nu mice. The day of iPS cell injection was set to “day 1”, and cytokine administration was started on the same day. A total amount of 100 μL of cytokine was placed in alzet micro-osmotic pump model 1002 (manufactured by DURECT Corporation), and a pump was implanted subcutaneously into the mouse. For hepatocytes and pancreatic cells (Islet cells, pancreatic Langerhans islet cells), empty pumps were removed from mice subcutaneously on day 14 and day 28, and pumps containing new cytokines were implanted. Examples of cytokine type, dose, and administration timing are shown in FIG. In FIG. 1, “KSN / Slc-nu / nu” indicates a nude mouse in the KSN background, and the names of cytokines and the like injected into the mouse are as follows.
RA: Retinoic Acid (retinoic acid)
FGF1: Fibroblast Growth Factor 1 (fibroblast growth factor 1)
FGF4: Fibroblast Growth Factor 4 (fibroblast growth factor 4)
HGF: Hepatocyte Growth Factor (Hepatocyte Growth Factor) OsM: Oncostatin M (Oncostatin M, a cytokine belonging to leukemia inhibitory factor, has multiple functions)
Activin A (Activin A)
EGF: Epidermal Growth Factor (epidermal growth factor)
bFGF: basic FGF (basic fibroblast growth factor)
Nicotinamide (Nicotinamide)
IGF-2: Inslin Like Growth Factor-2 (insulin-like growth factor-2)
OP9: Mouse bone marrow stromal cell line SCF: Stem Cell Factor (stem cell factor)
TPO: Thrombopoietin (thrombopoietin, a hematopoietic factor involved in the proliferation and differentiation of progenitor cells of platelets).

<Immunostaining>
The excised teratoma and the liver and pancreas of a wild type mouse were frozen with liquid nitrogen after excision. C. T.A. A frozen section was prepared by embedding with a compound (manufactured by Tissue-TeK). Sections were fixed with 4% paraformaldehyde (PFA), treated with acetone, blocked with MAXBlock Blocking Medium (registered trademark, manufactured by Active Motif) for 1 hour at room temperature, washed twice with PBS and applied with primary antibody. The reaction was allowed to proceed overnight at 4 ° C. Primary antibodies include goat anti-mouse ALB Ab, Rabbit anti-mouse CK19 Ab (manufactured by Invitrogen), Rat anti-mouse CYP7A1 Ab (manufactured by Santa Cruz), mouse anti-mouseIns It was. Next, after washing 3 times with PBS, the reaction was allowed to proceed with the secondary antibody for 1 hour at room temperature. As secondary antibodies, donkey anti-goat IgG Alexa 647, goat anti-rabbit IgG Alexa 488, goat anti-rat IgG Alexa 488, and goat anti-mouse IgG Alexa 488 (manufactured by Invitrogen) were used.

<Indocyanine green adsorption reaction>
Indocyanine green (Indocyanine green, manufactured by Sigma) was dissolved in 5 mg / ml DMSO (manufactured by Sigma) and prepared with PBS to 1 mg / ml. 500 μL of this solution was intravenously injected into mice, and after 30 minutes, the teratomas and liver were excised and the color was observed. Alternatively, the teratoma and liver excised from the mouse were immersed in an indocyanine green solution, and the color was observed after incubation at 37 ° C. for 30 minutes.

<Flow cytometry analysis>
For determination of blood cell differentiation ability, peripheral blood and bone marrow cells were analyzed by flow cytometry (FACS Aria). The antibodies used were anti-mouse CD45-APC, anti-mouse CD4, CD8, Gr-1, Mac-1, B220, IL-7R-Biotin, anti-Streptavidin-APC / Cy7, anti-mouse Sca-1- Pacific Blue, anti-mouse c-Kit-APC, anti-mouse CD3-PE / Cy5, anti-mouse B220-Pacific Blue, anti-mouse Gr-1-APC (manufactured by Anti-mouse Mac-1-APC) ) Was used. Peripheral blood was collected from the mouse's inferior vein and analyzed by reacting with antibodies after hemolysis. Bone marrow cells were collected from the femur and tibia of mice and reacted with antibodies for analysis.

Example 1
<Differentiation into the liver>
As described above, frozen sections were prepared from teratomas differentiated into hepatocytes and immunostained with albumin, CK19, and CYP7A1, which are hepatocyte markers. The obtained results are shown in FIGS. As is clear from the results shown in FIG. 2 and FIG. 3, only the teratoma that received differentiation induction by cytokines was confirmed to express the same marker as that of hepatocytes.

Also, as described above, in the adsorption reaction of indocyanine green in teratoma differentiated into hepatocytes, only the teratoma induced to differentiate showed the same adsorption as in the liver. That is, it was suggested that the teratoma induced to differentiate has a function similar to that of the liver (see FIG. 4).

(Example 2)
<Differentiation into pancreas>
As described above, frozen sections were prepared from teratomas differentiated into pancreatic cells, and immunostaining for insulin, which is a marker for pancreatic islets of Langerhans, was performed. The obtained results are shown in FIG. As is clear from the results shown in FIG. 5, only the teratoma that received differentiation induction by cytokines was confirmed to express insulin similar to pancreatic cells.

(Example 3)
<Differentiation into blood cells and hematopoietic stem cells>
As described above, as a result of analyzing the peripheral blood of the teratoma-producing mouse using a flow cytometer, blood cells derived from Lnk − / − iPS cells were confirmed in the blood of the nude mouse (see FIG. 6). Moreover, the ratio of blood cells derived from iPS cells was higher under the conditions of SCF + TPO + OP9 than SCF + TPO. Furthermore, as a result of analyzing bone marrow cells of nude mice that produced teratomas, hematopoietic progenitor cells Lineage-c-Kit + Sca-1 + (KSL) derived from iPS cells were confirmed in the bone marrow (see FIG. 7). Further, when this bone marrow cell was transplanted into a wild type C57 / BL6 mouse and peripheral blood after 4 weeks was analyzed, it was confirmed that almost 100% of the blood was derived from iPS cells and differentiated into various cells. (See Figure 8). That is, it was suggested that iPS cells differentiated into hematopoietic stem cells in nude mice and home to bone marrow. Furthermore, by using ES cells and iPS cells deficient in the protein Lnk that negatively regulates the functions of hematopoietic stem cells and progenitor cells, a larger amount of hematopoietic stem cells and progenitor cells was amplified compared to normal ES cells iPS cells. .

Example 4
<Removal of undifferentiated cells to be mixed and method of removing other than target cells>
When efficient differentiation induction of pluripotent stem cells as described above is performed in an in vivo environment, a problem of tumor formation due to pluripotency may occur. In addition, when transplanting differentiation-induced cells in vitro, there is a risk that the mixed undifferentiated cells form a tumor. Therefore, in the present invention, it is preferable to use a screening system that allows only the target differentiated cells to survive.

One method that can solve these problems is a screening system using suicide genes. For example, Thymidine Kinase (TK), which is a microorganism-derived metabolic enzyme gene that does not originally exist in mammals, produces its metabolite ganciclovir 5'-triphosphate by the addition of ganciclovir (GCV). This ganciclovir 5'-triphosphate inhibits DNA synthesis, thereby inducing cell death of cells expressing TK. As one aspect of the present invention, a virus-derived TK gene is introduced into an ES cell or iPS cell, and a system in which the TK gene is removed by the Cre-LoxP system only in cells that have differentiated into target cells is constructed. Thus, it is considered that efficient differentiation of ES and iPS cells into target cells using an in vivo organ formation environment can be performed.

Specifically, a retroviral vector in which an HSV-derived TK gene is sandwiched between LoxP sequences and a lentiviral vector that expresses Cre recombinase under the control of the Alb promoter, which is a marker gene for hepatocytes, are prepared. In addition, by using a VSV-G envelope for virus production, it can be used for both mouse ES / iPS cells and human ES / iPS cells (see FIG. 9). After infecting cells with two viruses, hepatocyte differentiation is induced as described above. Thereafter, by administration of GCV, it is considered that only Alb-producing cells from which the TK gene has been removed by the expression of Cre can survive, and cell death of undifferentiated cells or cells differentiated into other cell lineages can be induced.

Therefore, the above-mentioned possibility was verified. That is, genes such as Cre recombinase and TK under the control of the Alb promoter were introduced into HuH7 cells (cell line derived from human liver cancer) and NIH3T3 cells (mouse fetal epithelial cell line) using the viral vector shown in FIG. When GCV is administered after this, only the AlH-expressing HuH7 cells survive, and efficient cell death is induced in the NIH3T3 cells (see FIG. 10), so that the present method can be suitably used in the present invention. Was confirmed. Therefore, the establishment of this system makes it possible to survive only differentiated cells of interest such as Alb-expressing cells by administering GCV after transplanting undifferentiated cells into the living body.

Next, the method for producing hematopoietic stem cells and hematopoietic progenitor cells of the present invention shown in Example 3 and the functions of these cells obtained by the method were analyzed in more detail. In Examples 5 to 8, the following materials were used and the following methods were used.

<Mouse>
C57BL / 6 (B6) mice, KSN / Slc nude mice, and green fluorescent protein (GFP) transgenic mice were purchased from Japan SLC Corporation. Lnk ^{− / −} GFP transgenic mice were bred and maintained at the laboratory animal research facility of the Institute of Medical Science, University of Tokyo. In addition, the production and evaluation of X-SCID mice having a genetic background of B6 were carried out according to the description in “Ohbo, K et al., Blood, 1996, Vol. 87, pages 956-967”. Furthermore, NOD / SCID mice were purchased from Nippon Claire Co., Ltd. NOD / SCID / JAK3-deficient mice were purchased from Sankyo Lab Service Co., Ltd. In addition, the care of these mice was performed according to the guidance regarding the recombinant DNA experiments and experimental animals of the University of Tokyo.

<Cell lines and culture conditions>
Mouse iPS cells include Lnk ^{− / −} GFP transgenic C57BL / 6 (B6) mice, or tail tip fibroblasts (TTF, tail tip fibroblasts) derived from GFP transgenic B6 mice, Oct3 / 4, Sox2, and Klf4. These three genes were introduced by lentivirus all-in-one vector and re-programmed and used. The characteristics of the obtained iPS cells were confirmed as shown in FIGS. 11 to 16 described later.

In addition, mouse iPS cells maintained an undifferentiated state by co-culture with mouse fetal fibroblasts (MEF). The composition of the medium used for the co-culture is as follows.
Dulbecco's modified Eagle medium (DMEM, manufactured by GIBCO), 15% knockout serum substitute (Knockout SR, manufactured by GIBCO), 20 mM HEPES buffer solution (manufactured by Invitrogen), 0.1 mM MEM non-essential amino acid solution (Manufactured by Invitrogen), 0.1 mM L-glutamine (manufactured by Invitrogen), 100 U / ml penicillin, 100 μg / ml streptomycin (manufactured by Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (manufactured by GIBCO), and 1000 U / Ml ESGRO (manufactured by GIBCO). The medium was changed daily and the cells were passaged every 2-3 days to avoid abnormal growth and differentiation.

As human iPS cells, those established by reprogramming by introducing 3 genes of Oct3 / 4, Sox2 and Klf4 into normal human epidermal keratinocytes (Lonza) by using a lentiviral vector were used. In addition, human iPS cells maintained an undifferentiated state by co-culture with MEF. The composition of the human iPS cell culture medium used for co-culture is as follows.
Dulbecco's Modified Eagle Medium-F12 (Sigma-Aldrich), 20% Knockout SR (GIBCO), 0.1 mM MEM non-essential amino acid solution (Invitrogen), 0.2 mM L-glutamine (additives) Invitrogen), 0.1 mM 2-mercaptoethanol (GIBCO), and 5 ng / ml bFGF (Peprotech). The medium was changed every day and the cells were passaged every 7 days.

OP9 cells were maintained in a growth medium consisting of minimal essential medium α (α-MEM, manufactured by Invitrogen) supplemented with 20% fetal calf serum (FBS, manufactured by HyClone).

<Histopathology and immunostaining>
Tissue sections were evaluated by paraffin-embedding teratoma tissue and staining with hematoxylin and eosin (H & E).

Fluorescent immunostaining of Nanog and SSEA-1 was performed using anti-mouse Nanog antibody (diluted to 1/100, Cosmo Bio) and anti-mouse SSEA-1 antibody (diluted to 1/100, Abcam) And then Alexa Fluor 546-labeled anti-rabbit IgG antibody (diluted to 1/300, manufactured by Invitrogen), and allophycocyanin (APC) -labeled anti-mouse IgM antibody (1/100) Used by diluting and incubating with eBioscience). The counterstaining of the nuclei was performed using DAPI (manufactured by Sigma-Aldrich) according to the manufacturer's instructions. Subsequently, the sections immunofluorescently stained were visualized with a microscope (BX-51) and a digital camera system (DP-71) (both from Olympus) and photographed.

The teratoma tissue is rapidly frozen using dry ice, embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek), and using CM3050 cryostat (Leica Microsystems), Prepared in 7-8 μm sections. These tissue sections were fixed with ethanol and immunostained. That is, each section was incubated with the primary antibody for 24 hours at 4 ° C. and then with the secondary antibody for 30 minutes at room temperature.

The primary antibodies are anti-mouse CD45 antibody (diluted to 1/50, used by BD Bioscience), Alexa Fluor488 labeled anti-mouse CD117 (c-Kit) antibody (diluted to 1/10, used by BioLegend) ), Anti-mouse osteocalcin antibody (Ostecalcin, BGLAP) (diluted to 1/200, LifeSpan Biosciences), and anti-mouse VE-cadherin antibody (diluted to 1/200, used, Abcam) ) Was used. As secondary antibodies, Alexa Fluor 546-labeled goat-derived anti-rat IgG antibody and Alexa Fluor 647-labeled goat-derived anti-rabbit IgG antibody (both manufactured by Invitrogen) were used.

After the treatment with such an antibody, the section was sealed with a fluorescent staining mounting medium (manufactured by Dako) containing 4,6-diamidino-2-phenylindole (DAPI) for counterstaining the nucleus, and both TCS SP2 and AOBS were encapsulated. Observation was carried out with a focal laser scanning microscope (manufactured by Leica Microsystems).

<Blastocyst injection>
iPS cells were trypsinized with a 0.25% trypsin-EDTA solution (GIBCO). Trypsinized iPS cells and MEFs were replated on non-coating dishes and incubated for 30 minutes to remove MEFs. To create a chimeric mouse, 10 iPS cells were injected into blastocysts derived from ICR mice, and the blastocysts injected with iPS cells were transplanted into the uterus of pseudopregnant mice.

<Teratoma formation and differentiation from iPS cells to hematopoietic stem cells (HSC)>
Differentiation induction from mouse iPS cells to HSC is as follows. That is, 5 × 10 ⁶ mouse iPS cells were injected subcutaneously into KSN / Slc mice (4-5 weeks old). And differentiation induction to HSC was performed under the following conditions.
1) As a control, only iPS cells were injected.
2) Hematopoietic cytokines containing 200 ng stem cell factor (SCF, manufactured by Peprotech) and 200 ng thrombopoietin (TPO, manufactured by Peprotech) were placed in a micro osmotic pump (manufactured by ALZET), and the pump was implanted subcutaneously for 2 weeks.
3) iPS cells were transplanted with 1 × 10 ⁶ OP9 stromal cells.
4) The hematopoietic cytokine and the OP9 stromal cells were administered.

Further, differentiation induction from human iPS cells to HSC is as follows. That is, 1 × 10 ⁶ human iPS cells and 5 × 10 ⁵ OP9 stromal cells were injected into the testis of NOD / SCID mice (5-7 weeks old). Furthermore, hematopoietic cytokines containing 200 ng human SCF (Peprotech) and 200 ng TPO (Peprotech) were placed in a micro-osmotic pump (ALZET), and the pump was implanted subcutaneously for 2 weeks.

<Flow cytometry analysis and sorting>
Regarding blood cells derived from mouse iPS cells, hematopoietic stem cells (HSC) were analyzed using flow cytometry as described below.

Peripheral blood cells and spleen cells of mice were APC-labeled anti-CD45 antibody (BD Biosciences), APC-Cy7-labeled anti-CD34 antibody (manufactured by eBioscience), Pacific Blue-labeled anti-CD45R / B220 antibody (manufactured by eBioscience), phycoerythrin ( Phycoerythrin (PE) -Cy7-labeled anti-Gr-1 antibody (BioLegend) and PE-Cy7-labeled anti-Mac-1 antibody (BioLegend) were used for staining.

The mouse bone marrow cells in which the teratoma was formed were analyzed according to the description of Osawa, M, et al., Science, 1996, 273, 242-245. That is, the bone marrow cells are biotinylated anti-Gr-1 antibody, anti-Mac-1 antibody, anti-CD45R / B220 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-IL-7R antibody, and anti-TER119 antibody (manufactured by eBioscience). ).

These cells were then labeled with MACS anti-biotin microbeads (Miltenyi Biotec), and lineage ⁺ cells (Lineage ⁺ cell) were removed by LS-MACS system (Miltenyi Biotec).

Further, the cells were stained with Alexa Fluor 700-labeled anti-CD34 antibody, Pacific Blue-labeled anti-Sca-1, and APC-labeled anti-c-Kit antibody (all manufactured by eBioscience). The biotinylated antibody was detected using streptavidin-APC-Cy7 (manufactured by eBioscience). Four-color analysis and sorting were performed by FACSAria (Becton Dickinson).

Regarding blood cells derived from human iPS cells, hematopoietic stem cells (HSC) were analyzed using flow cytometry as described below.

Bone marrow cells of teratoma-formed mice were treated with APC-labeled anti-CD45 antibody (BD Biosciences), Pacific Blue-labeled anti-human CD45 antibody (BioLegend), and FITC-labeled anti-human CD34 antibody (BD Biosciences). Stained.

Peripheral blood of recipient mice was APC-labeled anti-mouse CD45 antibody (BD Biosciences), Pacific Blue-labeled anti-human CD45 antibody (BioLegend), Alexa Fluor 488-labeled anti-human CD3 antibody (BD Biosciences), APC- Staining was performed using an H7-labeled anti-human CD19 antibody (BD, Biosciences) and a PE-Cy7-labeled anti-human CD33 antibody (BD, Biosciences).

And these analyzes and sorting were performed using FACSAria (made by BD).

<Single cell culture>
Purified CD34 ⁻ KSL cells derived from iPS cells were seeded in 96-well plates containing 200 μL of medium in each well for single clones. In addition, the composition of the used culture medium is as follows.
S-clone SF-O3 medium (manufactured by Sanko Junyaku Co., Ltd.), as additives, 1% bovine serum albumin (BSA), mouse SCF (50 ng / mL), mouse TPO (50 ng / mL), mouse IL-3 ( 10 ng / mL), and mouse EPO (1 U / mL) (all manufactured by PeproTech).
The cells were cultured at 37 ° C. in a humidified atmosphere and 5% CO ₂ environment. Then, 10 days after the start of the culture, the cells were attached on a slide glass using cytospin, and subjected to blood smear staining using a hema color (registered trademark, manufactured by MERCK).

<Bone marrow transplantation assay>
For Lnk ^{− / −} GFP iPS cell-derived HSCs, 1 × 10 ⁷ BM cells of mice with teratoma formation were transplanted into lethal radiation treated (9.5 Gy) wild type B6 recipient mice. Then, 4 weeks and 12 weeks after transplantation, PB cells of recipient mice were analyzed by flow cytometry.

In addition, 12 weeks after the primary BM transplantation, 1 × 10 ⁷ bone marrow cells derived from the primary transplant recipient mouse (primary recipient mouse) are secondary transplanted to another recipient mouse (secondary recipient mouse). Transplanted as. And the ratio of chimerism was derived by the calculation of (GFP ⁺ cell number / CD45 ⁺ cell number) × 100%.

In addition, for GFP iPS cell-derived HSCs, 40 GFP ⁺ CD34 ⁻ KSL cells were selected from the primary recipient mouse and secondary to another recipient mouse (secondary recipient mouse) along with 2 × 10 ⁵ B6 bone marrow cells. Transplanted as a transplant.

In addition, for human iPS cell-derived HSCs, 1 × 10 ⁷ BM cells of mice with teratoma formed were transplanted into radiation-treated (2 Gy) NOD / SCID recipient mice or NOD / SCID / JAK3-deficient recipient mice. did. And 8 weeks after transplantation, PB cells of recipient mice were analyzed by flow cytometry.

(Example 5)
<Method of HSC induction using Lnk ^{− / −} GFP iPS cells>
As described above, iPS cells were first established from Lnk ^{− / −} GFP transgenic mice. That is, as shown in FIG. 11, the TTF of Lnk ^{− / −} GFP transgenic mice was reprogrammed by introducing 3 factors (Oct3 / 4, Klf4, and Sox2) using a lentiviral all-in-one vector. did. The obtained iPS cells (Lnk ^{− / −} GFP iPS cells) were confirmed to express GFP, Nanog, and SSEA-1 by immunofluorescence analysis (see FIG. 12). Furthermore, Lnk ^{− / −} GFP iPS cells have the ability to form teratomas in nude mice and can contribute to chimeric mice by injecting into blastocysts, confirming that they have pluripotency. (See FIGS. 13-16).

Then, by preparing teratomas using Lnk protein-deficient iPS cells, whether or not induction into HSC is possible, and further examining conditions suitable for the induction, Lnk ^{− / −} GFP iPS cells were analyzed using KSN / Slc nude mice were injected subcutaneously and induced to differentiate into HSC under the following conditions (see FIG. 17).
Condition 1: As a control, iPS cells were injected into nude mice.
Condition 2: Hematopoietic cytokines (SCF and TPO) were placed in a micro osmotic pump and administered continuously for 2 weeks.
Condition 3: OP9 stromal cell line was transplanted with iPS cells.
Condition 4: The hematopoietic cytokine and the OP9 stromal cell line were administered.

Subsequently, the ratio of Lnk ^{− / −} iPS cell-derived GFP ⁺ CD45 ⁺ cells in peripheral blood and bone marrow cells of teratoma-forming mice was analyzed by flow cytometry at a predetermined time. The obtained results are shown in FIGS. 18 to 21 As is clear from the results shown in FIG. 18, CD45 ⁺ cells derived from Lnk ^{− / −} GFP iPS cells were detected in most peripheral blood of teratoma-forming mice. It was also revealed that the proportion of CD45 ⁺ cells derived from Lnk ^{− / −} GFP iPS cells gradually increased along with the growth (size) of teratomas (see FIG. 19). As is clear from the results shown in FIGS. 18 to 20, the ratio was highest when cytokine and OP9 cells were administered (the average value after 12 weeks after iPS cell introduction was the condition 1: 0. 002 ± 0.01%, Condition 2: 1.02 ± 1.15%, Condition 3: 0.87 ± 0.79%, Condition 4: 4.26 ± 3.79%).

As a result of analyzing bone marrow cells of teratoma-forming mice 12 weeks after introduction of iPS cells, as shown in FIG. 21, Lineage (Lin) ⁻ cells as pluripotent progenitors (MPPs) ⁻ Lin ⁻ as HSPC GFP ⁺ cells were detected in c-Kit ⁺ Sca-1 ⁺ (KSL) cells and CD34 ⁻ KSL cells as long-term HSCs (LT-HSCs). In addition, when cytokines and OP9 cells were administered, the ratio of LNK ^{− / −} GFP iPS cell-derived KSL cells was the highest (see FIG. 20).
That is, based on these results, the induction of hematopoietic cells defined as an immunophenotype including a BM primitive cell population is similar to the results described in Example 3 through Lnk ^{− / −.} It has also been found that this can be done from GFP iPS cells and that hematopoietic cytokines and co-cultured cells (eg, OP9 cells) promote this induction.

Next, it was examined by colony assay whether functional HSC was contained in the HSC derived from Lnk ^{− / −} GFP iPS cells thus obtained. Specifically, GFP ⁺ CD34 ⁻ KSL cells in the bone marrow were divided into individual cells, each seeded in a 96-well plate, and cultured for 10 days with cytokines for colony formation (cytokines that induce hematopoietic differentiation). Then, the quantity and type of CFC-nmEM after 10 days of culture were evaluated. CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte). The obtained results are shown in FIGS.

As is apparent from the results shown in FIGS. 22 to 24, 22.9% of the seeded CD34 ⁻ KSL cells formed large colonies containing nmEM colonies (see FIG. 22), and CD34 ⁻ KSL cells were neutrophils. The formation of all blood cell lineages with spheres, macrophages, erythroblasts and megakaryocytes demonstrated that HSCs derived from Lnk ^{− / −} GFP iPS cells are multipotent, ie functional HSCs (See FIG. 23 and FIG. 24). .

In order to obtain direct evidence that hematopoietic stem / progenitor cells (HSPC) derived from Lnk ^{− / −} GFP iPS cells have bone marrow reconstruction ability (chimerism), as shown in FIG. Murine bone marrow cells were transplanted into lethal irradiated B6 mice. Next, the ratio of HSPC derived from Lnk ^{− / −} GFP iPS cells in the peripheral blood, spleen, and bone marrow of such B6 mice (recipient mice) was examined. The obtained results are shown in FIGS.

Table 1 shows the chimerism of iPS cell-derived hematopoietic cells in recipient mice 12 weeks after bone marrow transplantation, and the T cell chimerism is the ratio of GFP ⁺ CD3 ⁺ / CD45 ⁺ cells (% B cell chimerism is the ratio (%) of GFP ⁺ B220 ⁺ / CD45 ⁺ cells, and myeloid chimerism is the ratio (%) of GFP ⁺ Gr-1 ⁺ Mac-1 ⁺ / CD45 ⁺ cells (Mean ± se, Lnk ^{− / −} GFP iPS cell group: n = 4, GFPiPS cell group: n = 6).

As is apparent from the results shown in FIG. 25 and Table 1, as a result of analysis of the peripheral blood cells of the recipient mouse, hematopoietic cells derived from Lnk ^{− / −} GFP iPS cells having multi-lineage reconstruction ability were frequently found. was detected.

Further, as apparent from the results shown in FIG. 26 and Table 1, as a result of analysis of the spleen and bone marrow of the recipient mouse, hematopoietic cells derived from Lnk ^{− / −} GFP iPS cells having multi-lineage reconstruction ability were found. Detected frequently. Further, as is apparent from the results shown in FIG. 27, cells derived from Lnk ^{− / −} GFP iPS cells are bone marrow cells, and the average proportion of GFP ⁺ cells in the CD34 ⁻ KSL cell fraction (HSC cell fraction) is It was as high as 45%.

In order to obtain direct evidence that HSCs derived from Lnk ^{− / −} GFP iPS cells have long-term bone marrow reconstruction ability, lethal bone marrow cells of primary recipient mice as shown in FIG. Transplanted into B6 mice (secondary recipient mice) irradiated with radiation (secondary transplantation). The obtained results are shown in FIG.

From the results shown in FIG. 25, in secondary recipient mice at least 12 weeks after transplantation, cells derived from Lnk ^{− / −} GFP iPS cells are resident in multilineage cells (after 4 weeks: 76.6). ± 9.5%, after 12 weeks: 60.6 ± 13.2%, where these numerical values (%) represent mean values ± standard deviations, and the numbers examined are each n = 10), It became clear that LSC ^{− / −} GFP iPS cells can be used to produce HSCs capable of long-term self-renewal and multi-lineage hematopoietic reconstruction through teratoma formation. During the observation period of a series of transplantation experiments, leukemia and other abnormalities (blood abnormalities) are not observed in recipient mice, and HSCs derived from Lnk ^{− / −} GFP iPS cells have normal hematopoietic ability. Became clear.

(Example 6)
<Production of functional HSC from GFP iPS cells not having Lnk mutation>
Next, in order to evaluate whether functional HSC can be induced even in iPS cells not accompanied by Lnk deficiency, iPS cells (GFP iPS cells) were established from GFP transgenic mice as shown in FIG. Then, HSC induction via teratoma formation was examined under the same conditions as when Lnk ^{− / −} GFP iPS cells were used. The obtained results are shown in FIGS.

As is clear from the results shown in FIGS. 28 and 29, GFP ⁺ CD45 ⁺ cells increased with time in the peripheral blood of teratoma-forming mice. Further, the ratio of GFP ⁺ CD45 ⁺ cells was highest in the presence of cytokines and OP9 cells 12 weeks after iPS cell transplantation (condition 1: 0.003 ± 0.006%, condition 2: 0.013 ±). 0.01%, condition 3: 0.025 ± 0.01%, condition 4: 0.16 ± 0.09%, these numerical values (%) indicate the average value ± standard deviation, and the number examined is n = 4) (see FIG. 30). Furthermore, as is apparent from the results shown in FIG. 31, GFP ⁺ cells were detected in Lin ⁻ cells, KSL cells, and CD34 ⁻ KSL cells in the bone marrow of teratoma-forming mice 12 weeks after iPS cell transplantation.

Further, as is apparent from the results shown in FIGS. 32 and 33, as a result of transplantation of bone marrow cells obtained from teratoma-forming mice, GFP iPS cell-derived cells were obtained in the peripheral blood, spleen, and bone marrow cells of primary recipient mice. It was detected that blood cells were engrafted. Furthermore, GFP ⁺ cells were also detected in the hematopoietic primitive cell fraction in the recipient bone marrow, including Lin ⁻ cells, KSL cells, and CD34 ⁻ KSL cells (see FIG. 34).

Furthermore, as is clear from the results shown in FIG. 35, in the colony assay, CD34 ⁻ KSL cells formed all the blood cell lineages with neutrophils, macrophages, erythroblasts, megakaryocytes, and thus derived from GFP iPS cells. It has been demonstrated that HSCs are pluripotent, ie functional HSCs.

Further, as is apparent from the results shown in FIG. 32, in the secondary recipient mice obtained by transplantation of these series of bone marrow cells, high chimerism was confirmed from 4 to 12 weeks after transplantation, and GFP was observed. ^{+ The} strong engrafting ability of the cells was maintained.

Therefore, it has been clarified that the method of the present invention is a method capable of producing a functional HSC having long-term bone marrow reconstruction ability even from iPS cells without Lnk mutation.

(Example 7)
<Gene therapy via iPS cells in X-SCID mice>
X-linked severe combined immunodeficiency (X-SCID) is one of severe combined immunodeficiencies (SCID) characterized by severe impairment in T cell and B cell immunity. Luo patients are known to have mutations in a gene encoding a common gamma chain (γc) shared by many cytokine receptors that play important functions in the immune system. (See Buckley, RH, et al., Journal of Pediatrics, 1997, 130, 378-387).

Therefore, although development of gene therapy using disease-specific iPS cells is expected (see Hanna, J. et al., Science, 2007, 318, 1920-1923), functional hematopoietic stem cells have been developed so far. Since the production method of (HSC) has not been put to practical use, this point has been a major obstacle in the development of such gene therapy.

As described above, by the method of the present invention, hematopoietic progenitor cells that can be transplanted and can reconstruct the lymph-myeloid lineage can be prepared from iPS cells. Then, next, an attempt was made to apply the present invention to an X-SCID treatment model using gene therapy and disease-specific iPS cells.

36. First, as shown in FIG. 36, iPS cells were prepared from X-SCID mice, mouse γC gene and the like were introduced via retrovirus, and then clone selection was performed, so that mouse γC was highly expressed. Cell line (mγc-iPSC # 4) was established. (See Figures 37-39).

In order to prepare teratomas, mγc-iPSC # 4 was injected together with OP9 cells subcutaneously in X-SCID mice. Twelve weeks after the injection of iPS cells, GFP ⁺ CD45 ⁺ cells derived from mγc-iPS cells (mγc-iPSC) were detected in the peripheral blood of one teratoma-formed X-SCID mouse ( (See FIG. 40).

However, as expected from clinical experience, by competing with the overwhelming majority of allogeneic cells (B cells and myeloids) remaining in the bone marrow derived from the host, GFP-expressing B cells and myeloid lineages The expansion of cell progeny was most likely limited, and the cells only appeared to a trace (see FIG. 40).

In contrast, in the expression of mature T cells derived from functionally corrected mγc-iPSC # 4, normal distribution of CD4 / CD8 cells and generation of naive T cells (CD4 ⁺ CD62L ⁺ ) Was clearly recognized (see FIG. 40).

From these results, combining therapeutic genetic recombination using disease-specific iPS cells and the method of producing hematopoietic stem / progenitor cells (HSPC) corresponding to in vivo lymphocyte production via teratoma formation of the present invention. It was shown that it can be a model for X-SCID gene therapy.

In addition, in conventional gene therapy, a heterogeneous cell population (such as HSPC derived from a patient) into which a viral vector is randomly integrated must be transplanted, and there is a risk that the transplanted patient suffers from leukemia. It was. On the other hand, in the above method, since the gene is introduced into the iPS cell, the iPS cell into which the gene is introduced at a risk-free position on the genomic DNA can be selected, cloned and proliferated. Therefore, by using hematopoietic stem cells derived from iPS cells whose safety has been confirmed for transplantation, the present invention can also provide a gene therapy method capable of avoiding the risk of leukemia.

(Example 8)
<Induction of engraftable HSCs from human iPS cells via teratoma formation>
Next, in order to examine whether functional HSCs can be induced from human iPS cells through teratoma formation, human iPS cells are injected subcutaneously into NOD / SCID mice as shown in FIG. 41 to produce teratomas. Then, HSC was induced by the same method as mouse HSC induction. Then, 12 weeks after the injection of human iPS cells, peripheral blood and bone marrow of teratoma-forming mice were analyzed. The obtained results are shown in FIG.

As is apparent from the results shown in FIG. 42 and Table 2, a human-derived cell population (mCD45 ⁻ hCD45 ⁺ ) was clearly detected in the peripheral blood of 1 of 15 mice examined, and 10 teratomas were found. In the marrow of forming mice, fractions of human-derived cells (hCD45 ⁻ hCD34 ⁺ , hCD45 ^dull hCD34 ⁺ , and hCD45 ⁺ hCD34 ⁻ ) could be detected in the mCD45 negative cell population.

Further, in order to confirm that human iPS cell-derived HSCs having replication ability exist, as shown in FIG. 41, whole bone marrow cells obtained from teratoma-forming mice or bone marrow cells from which mCD45 has been removed were obtained. , Irradiated NOD / SCID mice, or NOD / SCID / JAK3-deficient mice. The obtained results are shown in FIG.

As is apparent from the results shown in FIG. 43 and Table 2, the survival of blood cells derived from human iPS cells having multilineage replication ability in the peripheral blood of recipient mice 8 weeks after transplantation. The arrival was confirmed. Moreover, the chimerism derived from human iPS cells was generally higher in mice transplanted with mCD45-removed cells (Sorted) than in mice transplanted with whole bone marrow cells (Total).

Therefore, it has been clarified that the present invention can produce engraftable HSCs from human iPS cells without any genetic modification.

Further, in order to examine how blood cells or HSCs derived from iPS cells are produced and exist in the teratomas, the cells constituting iPS cell-derived teratomas formed in mice are analyzed, and the HSC niche-like Whether cells (HSCs niche-like cells) were present was examined.

Note that the HSC niche is a microenvironment necessary for maintaining the HSC. Although the actual state of HSC has not yet been clarified (Calvi, LM, et al., Nature, 2003, 425, 841-846, Kiel, MJ, et al., Cell, 2005, 121, Based on recent research results, other cell types included in osteoblasts, endothelial cells, and bone marrow resident glial cells (BM-residential glial cells) contribute to HSC niche formation. (For osteoblasts and endothelial cells, see the above two documents (“Calvi, LM et al., 2003”, “Kiel, MJ et al., 2005”).) Bone marrow resident glial cells Is based on unpublished observation results of the present inventors.)

Therefore, in a teratoma tissue section 12 weeks after human iPS cell injection, osteocalcin expression as an osteoblast marker, VE-cadherin expression as an endothelial marker, and glial fibrillary acidic protein (GFAP), as a glial marker ) Was examined. The obtained results are shown in FIGS.

As is clear from the results shown in FIGS. 44 to 46, many cells expressing osteocalcin, VE-cadherin, or GFAP were detected in the teratoma derived from human iPS cells. It was also revealed that CD45 ⁺ CD34 ⁺ HSC cells are frequently present in the vicinity of these HSC niche-like cells.

Furthermore, it was confirmed by FACS analysis that teratoma mCD45 ⁻ cells contained human iPS cell-derived cell fractions (hCD45 ⁺ hCD34 ⁻ , hCD45 ⁺ hCD34 ⁺ , hCD45 ⁻ hCD34 ⁺ ) (see FIG. 47). ).

Further, in the teratoma derived from mouse iPS cells, it was confirmed by immunostaining that GFP ⁺ CD45 ⁺ cells and GFP ⁻ CD45 ⁺ cells were present 12 weeks after the GFP iPS cells were injected (FIG. 48). reference).

Furthermore, FACS analysis was able to identify GFP ⁺ KSL cells in CD45 ⁺ cells (see FIG. 49), and these results revealed that adult hematopoietic stem cells were produced from iPS cells in teratomas. ..

In addition, even in teratomas derived from mouse iPS cells, the presence of HSC niche-like cells such as osteocalcin + cells, VE-cadherin + cells, and GFAP + cells can be confirmed (see FIG. 50). Furthermore, it was confirmed that CD45 ⁺ c-Kit ⁺ cells containing mouse iPS cell-derived HSC were present in the vicinity of VE-cadherin + cells and osteocalcin + cells (see FIGS. 51 and 52).

Therefore, in the teratoma formation process according to the present invention, an environment corresponding to bone marrow is formed in the teratoma by producing various cells that can constitute the HSC niche in an environment suitable for hematopoietic differentiation, and as a result. It was suggested that HSC is produced.

Example 9
<Organogenesis in mouse individuals using germinal progenitor cells>
In a method for organ transplantation by directly transplanting ES cells or iPS cells as described above into a mouse individual,
1. Since pluripotent stem cells retain the differentiation ability of various cell lineages, cells other than the target cells may be formed.
2. When the teratoma-forming ability of the transplanted pluripotent stem cells is too high, there is a possibility that the health condition of the host is harmed by the increase in teratomas before the target cells are formed.
This can be a problem.

Therefore, in order to solve such problems, as one aspect thereof, ES cells and iPS cells are first induced to differentiate in vitro, and endoderm progenitors having high differentiation potential to endoderm system while maintaining pluripotency. Cells were prepared and transplanted into mice. That is, it was performed according to the materials and methods described below. The obtained results are shown in FIGS.

<Culture of mouse ES cells>
E14tg2a, a MEF-independent ES cell line, was cultured in a medium having the following composition in a 0.1% gelatin-coated culture dish.
Glasgow Modified Eagle Medium (Glasgow Modified Eagle Medium, GMEM), 10% FCS, 1% L-glutamine-penicillin-streptomycin solution (manufactured by SIGMA), 0.1 mM non-essential amino acid (manufactured by GIBCO), 1 mM Sodium pyruvate, 0.1 mM 2-mercaptoethanol, leukemia inhibitory factor (LIF) (1000 U / ml).

<Induction of differentiation from E14Tg2a to foregut endoderm (endodermal progenitor cells)>
Collagen-Type4 coated culture dishes were seeded with E14tg2a at a density of 1 × 10 ⁴ cells / ml. 20 ng / ml human activin A (manufactured by Peprotech) and 10 ng / ml human BMP4 (manufactured by Peprotech) were added to an SFO3 serum-free medium (manufactured by Sanko Junyaku Co., Ltd.) and cultured for 2 days. After 2 days, the medium was changed to one in which 20 ng / ml human activin A (manufactured by Peprotech), 20 ng / ml mouse EGF (manufactured by Peprotech) and 10 ng / ml FGF4 (manufactured by SIGMA) were added to the SFO3 medium. The culture was further continued for 5 days.

<Transplantation into mouse>
Undifferentiated ES cells and endoderm progenitor cells obtained by inducing differentiation from ES cells were transplanted into the renal capsule of 8-week-old KSN nude mice, 2 × 10 ⁶ cells each. At the time of transplantation, each cell was suspended in 100 μl of ice-cooled DMEM serum-free medium and Matrigel (manufactured by BD Biosciences) in an equal volume and transplanted under the mouse kidney capsule.

Matrigel is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma rich in extracellular matrix protein. The main components are laminin, collagen IV, heparan sulfate proteoglycan, And entactin /

nidogen

1 and 2, which are naturally produced by TGF-β, epidermal growth factor, insulin-like growth factor, fibroblast growth factor,

tissue plasminogen activator

3, 4 and EHS tumors Other growth factors.

<Immunohistochemical staining>
Teratomas formed under the renal capsule of KSN nude mice were cut into appropriate sizes, shaken overnight in 4% PFA, and then shaken overnight in a 10% sucrose solution and in a 30% sucrose solution for 2 days. . Using Cliomold 2 (manufactured by Sakura Finetek Japan), O.I. C. T.A. It was embedded in a block form in a compound (manufactured by Sakura Finetek Japan) and frozen on dry ice. Then, the block was cut into a thickness of 7 μm using Cryostat CM 3050SIV (manufactured by Leica) and pasted on a slide glass coated with MAS to prepare a tissue specimen. The tissue specimen was washed twice with PBS and immersed in methanol (MtOH) cooled to −20 ° C. for 10 minutes. After washing MtOH twice with PBS, blocking with 5% donkey serum (manufactured by SIGMA) / PBS was performed at room temperature for 30 minutes. Primary antibodies (anti-FoxA2 and CK19 antibodies) diluted in blocking buffer were added and allowed to react overnight at 4 ° C. After the primary antibody was reacted, it was washed three times with PBS, and as a secondary antibody, donkey-derived anti-goat Alexa 546 antibody (manufactured by Invitrogen) and donkey-derived anti-rabbit Alexa 488 antibody (manufactured by Invitrogen) were blocked in buffer. Each was diluted 500 times and allowed to react at room temperature for 45 minutes. Finally, the plate was washed twice with PBS, and 4 ', 6-diamidino-2-phenyllinole (DAPI, manufactured by Roche) was reacted for 10 minutes. After DAPI was reacted, washing with PBS was performed once. Then, PBS was removed, and it embedded and observed with Dako Fluorescent Mounting Medium (made by Dako).

In addition, three sections are randomly selected from undifferentiated ES cells and teratomas obtained by injecting endoderm progenitor cells obtained by inducing differentiation from ES cells, and CK19-positive intestinal tract present in tissue sections. The number of like structures was counted.

As is apparent from the results shown in FIGS. 53 to 55, when the endoderm progenitor cells were transplanted, the size of the teratoma formed was small compared to the case of transplanting ES cells (see FIG. 53). ). Furthermore, in teratomas derived from endoderm progenitor cells, the number of FoxA2-positive endoderm cells, particularly CK19-positive intestinal-like cells, increased compared to that derived from ES cells (FIGS. 54 and 55). reference). Therefore, in the present invention, by using pluripotent stem cells that have been induced to differentiate to some extent from universal cells such as ES cells, a teratoma containing more cells constituting the target organ can be formed, and a host due to an increase in teratoma It has become clear that the effects on the health status of can be suppressed.

As described above, according to the present invention, pluripotent stem cells can be efficiently induced to differentiate into desired functional cells. Therefore, the method for producing target cells induced to differentiate from pluripotent stem cells of the present invention is useful in regenerative medicine, transplantation medicine, cell medicine and the like.

Claims

Transplanting pluripotent stem cells derived from a mammal individual to a non-human mammal;
Administering to the non-human mammal a differentiation-inducing agent for target cells;
Growing the animal for a time sufficient for the transplanted pluripotent stem cells to form teratomas in the living body of the non-human mammal, and inducing differentiation of the pluripotent stem cells into target cells;
Recovering a target cell derived from the mammal individual from the non-human mammal;
A method for producing a target cell induced to differentiate from a pluripotent stem cell.
The method according to claim 1, wherein the pluripotent stem cells are artificial pluripotent stem (iPS) cells prepared using somatic cells of the individual mammal.
The method according to claim 1, wherein the pluripotent stem cell is an embryonic stem (ES) cell prepared from a fertilized egg derived from the mammal.
The pluripotent stem cell is transplanted into at least one tissue selected from the group consisting of subcutaneous, testis, and renal capsule of the non-human mammal. Method.
The method according to any one of claims 1 to 4, wherein the target cell is a hepatocyte or a pancreatic cell, and the target cell is recovered from teratoma formed in the non-human mammal.
The method according to claim 5, wherein the pancreatic cells are pancreatic islets of Langerhans.
The method according to any one of claims 1 to 4, wherein the target cell is a hematopoietic cell, and the hematopoietic cell is recovered from the bone marrow of the non-human mammal.
The method according to claim 7, wherein the pluripotent stem cell is a Lnk-deficient cell.
The method according to any one of claims 1 to 8, wherein the step of inducing differentiation of the pluripotent stem cells into the target cells is performed in the presence of co-cultured cells.
The method according to claim 9, wherein the co-cultured cells are OP-9 cells.
The method according to any one of claims 1 to 10, wherein the differentiation-inducing agent is continuously administered subcutaneously to the non-human mammal for a predetermined period.
The method according to any one of claims 1 to 11, wherein the non-human mammal is deficient in the ability to form target cells.
The method according to any one of claims 1 to 12, wherein the non-human mammal is an immunodeficient animal.
The method according to any one of claims 1 to 13, wherein in the step of inducing differentiation of pluripotent stem cells derived from the animal individual into target cells, an operation is performed to remove cells other than the target cells to be mixed. .
The method according to claim 14, wherein the cells other than the target cells are pluripotent stem cells that remain in an undifferentiated state.
The method according to claim 14 or 15, wherein the removing operation is achieved by causing a suicide gene to function at a desired time.
A target cell obtained by the method according to any one of claims 1 to 16.