JP5710634B2 - Method for differentiating human-derived pluripotent stem cells - Google Patents
Method for differentiating human-derived pluripotent stem cells Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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Description
本発明は、ヒト由来の多能性幹細胞を分化させる方法に関し、より詳しくは、前記ヒト由来の多能性幹細胞を、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞との共培養下で分化誘導する方法に関する。また、この方法に用いられるヒト由来の多能性幹細胞から樹立した間葉系幹細胞、並びにこの方法によって得られるヒト由来の多能性幹細胞から分化した細胞に関する。 The present invention relates to a method for differentiating human-derived pluripotent stem cells. More specifically, the human-derived pluripotent stem cells are co-cultured with mesenchymal stem cells established from human-derived pluripotent stem cells. Relates to a method for inducing differentiation. The present invention also relates to mesenchymal stem cells established from human-derived pluripotent stem cells used in this method, and cells differentiated from human-derived pluripotent stem cells obtained by this method.
ヒト胚性幹細胞（ＥＳ細胞）や人工多能性幹細胞（ｉＰＳ細胞）等の多能性幹細胞は種々の機能細胞に分化できる多能性を有しているため、再生医療への応用が期待されている。しかし、現在のところ、ヒト多能性幹細胞から機能細胞への分化誘導には、動物血清や動物由来のフィーダー細胞を必要としている。そのため、実際にヒト多能性幹細胞から分化誘導された機能細胞をヒトに投与する際には、外来性抗原となる動物細胞（異種細胞）の混入による未知の微生物やウィルス、プリオンによる感染等の危険性があり、これを回避する方法が必要とされている。 Since pluripotent stem cells such as human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have pluripotency that can be differentiated into various functional cells, application to regenerative medicine is expected. ing. However, at present, in order to induce differentiation of human pluripotent stem cells into functional cells, animal serum or animal-derived feeder cells are required. Therefore, when functional cells derived from human pluripotent stem cells are actually administered to humans, infection with unknown microorganisms, viruses, prions, etc. due to contamination with animal cells (heterologous cells) that are foreign antigens There is a danger and a way to avoid it is needed.
一方、ヒト多能性幹細胞から特定の細胞への分化、例えば、血液細胞への分化誘導に関しては、ヒト多能性幹細胞から胚樣体を介する分化誘導法があり、胚様体内に赤血球等の血液細胞が見出されたとする報告もある。しかしながら、これらの赤血球の大部分はβグロビンを発現しない一次造血を起源とする胚型赤血球で、βグロビンを発現する成人型の二次造血に由来する赤血球はほとんど認められず、成人型の二次造血への分化誘導の効率が極めて悪い（非特許文献１〜２）。 On the other hand, regarding differentiation from human pluripotent stem cells to specific cells, for example, differentiation induction into blood cells, there is a differentiation induction method from human pluripotent stem cells via embryonic rods, such as erythrocytes in the embryoid body There are reports that blood cells were found. However, most of these erythrocytes are embryonic erythrocytes originating from primary hematopoiesis that do not express β-globin. Almost no erythrocytes derived from adult-type secondary hematopoiesis that express β-globin are observed. The efficiency of differentiation induction into secondary hematopoiesis is extremely poor (Non-Patent Documents 1 and 2).
また、ヒト多能性幹細胞から成人型の血液細胞を分化誘導するためには、ヒト多能性幹細胞をフィーダー細胞と共培養する方法が報告されている。しかしながら、従来のヒト由来フィーダー細胞との共培養系ではヒト多能性幹細胞から血液細胞への分化誘導は困難であったことから、マウス由来のフィーダー細胞と共培養する方法が利用されており（非特許文献３〜５）、そうした方法で分化誘導された細胞は、前述のような危険性があるため、ヒトに投与することはできなかった。 In order to induce differentiation of adult blood cells from human pluripotent stem cells, a method of co-culturing human pluripotent stem cells with feeder cells has been reported. However, since it was difficult to induce differentiation from human pluripotent stem cells to blood cells in a conventional co-culture system with human-derived feeder cells, a method of co-culturing with mouse-derived feeder cells has been used ( Non-patent documents 3 to 5), cells induced to differentiate by such a method could not be administered to humans because of the risks described above.
本発明は、上記従来技術の有する課題に鑑みてなされたものであり、異種細胞を用いずにヒト由来の多能性幹細胞を分化させる方法を提供することを目的とする。 This invention is made | formed in view of the subject which the said prior art has, and it aims at providing the method of differentiating a human-derived pluripotent stem cell, without using a heterogeneous cell.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、ヒト由来の多能性幹細胞を共培養するためのフィーダー細胞として、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞（ｍｅｓｅｎｃｈｙｍａｌ ｓｔｒｏｍａｌ ｃｅｌｌ、ＭＳＣ）を用いた場合には、ヒト由来の多能性幹細胞を分化させることができることを見出した。このことは、従来、ヒト由来の多能性幹細胞とヒト由来フィーダー細胞との共培養系ではヒト多能性幹細胞を分化させることが困難であったことに鑑みれば、驚くべき知見である。 As a result of intensive studies to achieve the above object, the present inventors have established mesenchymal stem cells established from human-derived pluripotent stem cells as feeder cells for co-culturing human-derived pluripotent stem cells. It was found that human-derived pluripotent stem cells can be differentiated when (mesenchymal stromal cell, MSC) is used. This is a surprising finding in view of the fact that it has conventionally been difficult to differentiate human pluripotent stem cells in a co-culture system of human-derived pluripotent stem cells and human-derived feeder cells.
また、本発明者らは、ヒト由来の多能性幹細胞からの間葉系幹細胞の樹立においても、異種動物由来の血清やフィーダー細胞を用いることなく、ヒト由来の血小板溶解液を用いることによって行うことができることを見出した。さらに、前記間葉系幹細胞と多能性幹細胞とが同一人に由来するものであっても、該多能性幹細胞を分化誘導させることができることを見出した。 In addition, the present inventors also perform the establishment of mesenchymal stem cells from human-derived pluripotent stem cells by using human-derived platelet lysate without using heterologous animal-derived serum or feeder cells. I found that I can do it. Furthermore, it has been found that even if the mesenchymal stem cell and the pluripotent stem cell are derived from the same person, the pluripotent stem cell can be induced to differentiate.
このように本発明者らは、異種細胞の使用を一切排除して、ヒト由来の多能性幹細胞を分化誘導する系を確立することに世界で初めて成功し、本発明を完成するに至った。 Thus, the present inventors have succeeded for the first time in the world in establishing a system for inducing differentiation of human-derived pluripotent stem cells by eliminating the use of heterologous cells, and have completed the present invention. .
本発明は、より詳しくは、以下の発明を提供するものである。
（１）ヒト由来の多能性幹細胞を分化させる方法であって、前記ヒト由来の多能性幹細胞を、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞との共培養下で分化誘導する方法。
（２）前記多能性幹細胞が、ＥＳ細胞及びｉＰＳ細胞からなる群から選択される少なくとも一の細胞である（１）に記載の方法。
（３）前記間葉系幹細胞が、ヒト由来の血小板溶解液の存在下でヒト由来の多能性幹細胞から樹立した細胞である（１）又は（２）に記載の方法。
（４）前記間葉系幹細胞と前記ヒト由来の多能性幹細胞とが同一人に由来する（１）〜（３）のうちのいずれかに記載の方法。
（５）前記分化誘導が、血液細胞への分化誘導である（１）〜（４）のうちのいずれかに記載の方法。
（６）（１）〜（５）のうちのいずれかに記載の方法に用いられる、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞。
（７）（１）〜（６）のうちのいずれかに記載の方法によって得られる、ヒト由来の多能性幹細胞から分化した細胞。More specifically, the present invention provides the following inventions.
(1) A method for differentiating human-derived pluripotent stem cells, wherein the human-derived pluripotent stem cells are induced to differentiate under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. how to.
(2) The method according to (1), wherein the pluripotent stem cell is at least one cell selected from the group consisting of ES cells and iPS cells.
(3) The method according to (1) or (2), wherein the mesenchymal stem cell is a cell established from a human-derived pluripotent stem cell in the presence of a human-derived platelet lysate.
(4) The method according to any one of (1) to (3), wherein the mesenchymal stem cell and the human-derived pluripotent stem cell are derived from the same person.
(5) The method according to any one of (1) to (4), wherein the differentiation induction is differentiation induction into blood cells.
(6) A mesenchymal stem cell established from a human-derived pluripotent stem cell used in the method according to any one of (1) to (5).
(7) A cell differentiated from a human-derived pluripotent stem cell obtained by the method according to any one of (1) to (6).
本発明によれば、異種細胞を用いることなく、ヒト多能性幹細胞から分化誘導された種々の細胞を産生することが可能となる。従って、本発明により、ヒトに投与する場合でも高い安全性を有する形態で、ヒト多能性幹細胞から分化誘導された種々の細胞を提供することが可能となる。 According to the present invention, various cells induced to differentiate from human pluripotent stem cells can be produced without using heterologous cells. Therefore, according to the present invention, it is possible to provide various cells that have been induced to differentiate from human pluripotent stem cells in a form that is highly safe even when administered to humans.
本発明は、ヒト由来の多能性幹細胞を分化させる方法であって、前記ヒト由来の多能性幹細胞を、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞との共培養下で分化誘導する方法である。 The present invention is a method for differentiating human-derived pluripotent stem cells, wherein the human-derived pluripotent stem cells are differentiated under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. It is a way to guide.
本発明において分化させる対象とするヒト由来の多能性幹細胞は、ヒトを構成する種々の細胞に分化できる多能性と自己複製能とを有する細胞であり、例えば、ヒト由来の胚性幹細胞（ＥＳ細胞）、ヒト由来の人工多能性幹細胞（ｉＰＳ細胞）、ヒト由来の胚性腫瘍細胞（ＥＣ細胞）、ヒト由来の胚性生殖細胞（ＥＧ細胞）が挙げられる。これらの中では、生物学的特性の解析が格段に進んでいるという観点から、ＥＳ細胞及びｉＰＳ細胞からなる群から選択される少なくとも一の細胞であることが好ましい。また、胚を壊すことなく作製することができるという倫理的な観点から、さらに再生医療等に用いる際に、多能性幹細胞から分化した細胞を移植する患者と血液型の点において適合させ易いという観点から、本発明にかかるヒト由来の多能性幹細胞としてヒト由来のｉＰＳ細胞を用いることがより好ましい。さらにまた、多能性幹細胞から分化した細胞を移植する患者と血液型が完全に一致するという観点から、本発明にかかるヒト由来の多能性幹細胞として患者由来の体細胞から樹立したｉＰＳ細胞を用いることが特に好ましい。なお、本発明における血液型とは、赤血球の型（ＡＢＯ、ＲＨ）のみならず、白血球の型（ＨＬＡ）をも意味する。 The human-derived pluripotent stem cells to be differentiated in the present invention are cells having pluripotency capable of differentiating into various cells constituting human and self-replicating ability. For example, human-derived embryonic stem cells ( ES cells), human-derived artificial pluripotent stem cells (iPS cells), human-derived embryonic tumor cells (EC cells), and human-derived embryonic germ cells (EG cells). Among these, at least one cell selected from the group consisting of ES cells and iPS cells is preferable from the viewpoint that the analysis of biological characteristics is progressing remarkably. In addition, from the ethical point of view that it can be produced without breaking the embryo, it can be easily adapted in terms of blood type to patients transplanted with cells differentiated from pluripotent stem cells when used in regenerative medicine etc. From the viewpoint, it is more preferable to use a human-derived iPS cell as the human-derived pluripotent stem cell according to the present invention. Furthermore, iPS cells established from patient-derived somatic cells are used as human-derived pluripotent stem cells according to the present invention from the viewpoint that the blood type completely matches that of a patient transplanting cells differentiated from pluripotent stem cells. It is particularly preferable to use it. The blood type in the present invention means not only the type of red blood cells (ABO, RH) but also the type of white blood cells (HLA).
本発明においては、ヒト由来の多能性幹細胞と共培養を行うための「間葉系幹細胞」として、それ自体が、ヒト由来の多能性幹細胞（例えば、前述のｉＰＳ細胞、ＥＳ細胞等）から樹立した間葉系幹細胞を用いることを特徴とする。当該間葉系幹細胞は、ヒト由来の多能性幹細胞を分化誘導させる際のフィーダー細胞として機能する。間葉系幹細胞は、脂肪細胞、軟骨細胞、骨芽細胞、筋細胞等の種々の間葉系の細胞へ分化する能力と自己複製能とを有する細胞である。また、本発明に用いる間葉系幹細胞としては、再生医療等に用いる際に、多能性幹細胞から分化した細胞を移植する患者への拒絶反応が起こらないようにするという観点から、血液型の点において、分化させるヒト由来の多能性幹細胞と適合している間葉系幹細胞であることが好ましく、分化させるヒト由来の多能性幹細胞と同一人に由来する間葉系幹細胞であることがより好ましい。 In the present invention, “mesenchymal stem cells” for co-culture with human-derived pluripotent stem cells are themselves human-derived pluripotent stem cells (for example, the aforementioned iPS cells, ES cells, etc.) It is characterized by using mesenchymal stem cells established from The mesenchymal stem cells function as feeder cells when differentiation-inducing human-derived pluripotent stem cells. Mesenchymal stem cells are cells having the ability to differentiate into various mesenchymal cells such as adipocytes, chondrocytes, osteoblasts, and myocytes, and self-replicating ability. Moreover, as a mesenchymal stem cell used in the present invention, when used for regenerative medicine and the like, from the viewpoint of preventing rejection of a patient transplanted with a cell differentiated from a pluripotent stem cell, In that respect, it is preferably a mesenchymal stem cell that is compatible with the human-derived pluripotent stem cell to be differentiated, and may be a mesenchymal stem cell derived from the same person as the human-derived pluripotent stem cell to be differentiated. More preferred.
本発明にかかる間葉系幹細胞をヒト由来の多能性幹細胞から樹立する方法としては特に制限されることなく、例えば、「Ｗａｎｇら、Ｓｔｅｍ Ｃｅｌｌｓ、２００５年、２３巻、１２２１〜１２２７ページ」や「Ｈｗａｎｇら、ＰＮＡＳ、２００８年、１０５巻、２０６４１〜２０６４６ページ」に記載されているような、胚樣体を形成した後、異種動物由来の血清を含む培養液を用いて間葉系幹細胞に誘導する方法、「Ｌｉａｎら、Ｃｉｒｃｕｌａｔｉｏｎ、２０１０年、１２１巻、１１１３〜１１２３ページ」に記載されているような、最初に無血清の状態で培養した細胞から抗ヒトＣＤ２４抗体及び抗ヒトＣＤ１０５抗体を用いてＣＤ３４−ＣＤ１０５＋細胞を分画した後、異種動物由来の血清を用いて間葉系幹細胞に誘導する方法が挙げられる。また「Ｄｏｕｃｅｔら、Ｊ Ｃｅｌｌ Ｐｈｙｓｉｏｌ、２００５年、２０５巻、２２８〜２３６ページ」や「Ｍｉｒａｂｅｔら、Ｃｅｌｌ Ｔｉｓｓｕｅ Ｂａｎｋ、２００８年、９巻、１〜１０ページ」に記載されているような、ヒト由来の血小板溶解液（ＰＬ）を用いて間葉系幹細胞に誘導する方法や、後述の実施例において示すような、ヒト由来の血小板溶解液（ＰＬ）を用いて、ヒト由来の多能性幹細胞から間葉系幹細胞を樹立する方法（「Ｅｂｉｈａｒａら、「Ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｓｔｅｍ（ＥＳ） ｃｅｌｌ−ｄｅｒｉｖｅｄ ｍｅｓｅｎｃｈｙｍａｌ ｓｔｅｍ ｃｅｌｌｓ ｃａｐａｂｌｅ ｏｆ ｅｆｆｉｃｉｅｎｔｌｙ ｍａｉｎｔａｉｎｉｎｇ ｈｕｍａｎ ＥＳ ａｎｄ ｉｎｄｕｃｅｄ ｐｌｕｒｉｐｏｔｅｎｔ ｓｔｅｍ ｃｅｌｌｓ ｕｎｄｅｒ ａｎｉｍａｌ ｓｅｒｕｍ−ｆｒｅｅ ｃｏｎｄｉｔｉｏｎｓ．」、７ｔｈ Ｍｅｅｔｉｎｇ ｏｆ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｓｏｃｉｅｔｙ ｆｏｒ Ｓｔｅｍ Ｃｅｌｌ Ｒｅｓｅａｒｃｈ、２００９年」、「海老原ら、「ヒトＥＳ細胞維持能を有するヒトＥＳ細胞由来ストローマ細胞」、第８回日本再生医療学会、２００９年」等 参照）が挙げられる。The method for establishing mesenchymal stem cells according to the present invention from human-derived pluripotent stem cells is not particularly limited. For example, “Wang et al., Stem Cells, 2005, Vol. 23, pages 1221-1227” After the formation of embryonic rods as described in “Hwang et al., PNAS, 2008, 105, 20641-20646”, a mesenchymal stem cell was prepared using a culture solution containing serum derived from a heterologous animal. A method for inducing anti-human CD24 antibody and anti-human CD105 antibody from cells initially cultured in a serum-free state as described in “Lian et al., Circulation, 2010, 121, 1113 to 1123”. A method for fractionating CD34 − CD105 + cells using the method and then inducing mesenchymal stem cells using serum derived from a heterologous animal Is mentioned. It is also derived from humans, as described in “Douchet et al., J Cell Physiol, 2005, 205, pp. 228-236” and “Mirabet et al., Cell Tissue Bank, 2008, vol. 9, 1-10”. From a pluripotent stem cell derived from a human using a platelet lysate (PL) derived from human, as shown in the examples described later, Methods for establishing mesenchymal stem cells (“Ebihara et al.,“ Human embryonic stem (ES) cell-derived mesenchymal stem cells capable of efficient maintenance in human estenting human espe ells under animal serum-free conditions. ", 7th Meeting of International Society for Cell Cell Research, 2009", "Ebihara et al.," Human ES cell-derived stromal cells with human ES cell maintenance ability, 8th ""Academic Society, 2009").
これらの方法の中では、異種動物由来の血清を用いることなく間葉系幹細胞を樹立することができるという観点から、フィーダー細胞を用いることなく、ヒト由来の血清、ヒト由来の血漿、ヒト由来の血小板溶解液（ｐｌａｔｅｌｅｔ ｌｙｓａｔｅ、ＰＬ）からなる群から選択される少なくとも一の血液成分を用いて、ヒト由来の多能性幹細胞から間葉系幹細胞を樹立する方法であることが好ましい。さらに、得られる間葉系幹細胞において染色体異常の発生する可能性が非常に低いこという観点から、血小板溶解液を用いて樹立する方法であることがより好ましく、また、血液型（特にＨＬＡの型）を完全に一致させるという観点から、多能性幹細胞から分化した細胞を移植する患者由来の血小板溶解液や血清等を用いて樹立する方法であることが特に好ましい。さらに、かかる間葉系幹細胞の増殖を抑えるという観点から、ヒト由来の多能性幹細胞との共培養に際しては、放射線（例えば、１５〜１８Ｇｙ）を照射しておくことが好ましい。 Among these methods, from the viewpoint that mesenchymal stem cells can be established without using sera from different animals, human-derived sera, human-derived plasma, human-derived sera are used without using feeder cells. A method of establishing mesenchymal stem cells from human-derived pluripotent stem cells using at least one blood component selected from the group consisting of platelet lysate (PL) is preferable. Furthermore, from the viewpoint that the possibility of occurrence of chromosomal abnormality in the obtained mesenchymal stem cells is very low, it is more preferable that the method is established using a platelet lysate, and the blood type (particularly the type of HLA) From the viewpoint of completely matching), it is particularly preferable to establish a method using a platelet lysate or serum derived from a patient transplanted with cells differentiated from pluripotent stem cells. Furthermore, from the viewpoint of suppressing the proliferation of such mesenchymal stem cells, it is preferable to irradiate with radiation (for example, 15 to 18 Gy) in co-culture with human-derived pluripotent stem cells.
本発明は、本発明の方法に用いられる、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞をも提供するものである。 The present invention also provides mesenchymal stem cells established from human-derived pluripotent stem cells used in the method of the present invention.
本発明においては、こうしてヒト由来の多能性幹細胞から樹立した間葉系幹細胞との共培養下でヒト由来の多能性幹細胞を分化誘導する。多能性幹細胞を血液細胞に分化誘導する場合、例えば、所望の血液細胞に分化させ得る因子を培地中に添加して培養すればよい。このような因子としては、例えば、幹細胞因子（ＳＣＦ）、血管内皮増殖因子（ＶＥＧＦ）、トロンボポエチン（ＴＰＯ）、顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）、マクロファージコロニー刺激因子（Ｍ−ＣＳＦ）、顆粒球・マクロファージコロニー刺激因子（ＧＭ−ＣＳＦ）、エリスロポエチン（ＥＰＯ）、塩基性線維芽細胞増殖因子（ｂＦＧＦ）、血小板由来成長因子（ＰＤＧＦ）、上皮成長因子（ＥＧＦ)、白血病抑制因子（ＬＩＦ)、骨形成タンパク質４（ＢＭＰ−４）、ＴＮＦ−α、Ｆｌｔ３リガンド、ヘパリン、インターロイキン（ＩＬ−１α、ＩＬ−２、ＩＬ−３、ＩＬ−４、ＩＬ−５、ＩＬ−７、ＩＬ−９、ＩＬ−１１、ＩＬ−１５、ＩＬ−６、融合蛋白質−６（ＩＬ−６と可溶性ＩＬ−６受容体との複合体）等）からなる群より選択される少なくとも一のサイトカインが挙げられる。 In the present invention, human-derived pluripotent stem cells are induced to differentiate under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. When inducing differentiation of pluripotent stem cells into blood cells, for example, a factor that can be differentiated into desired blood cells may be added to the medium and cultured. Examples of such factors include stem cell factor (SCF), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granule Sphere / macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), Bone morphogenetic protein 4 (BMP-4), TNF-α, Flt3 ligand, heparin, interleukin (IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, A group consisting of IL-11, IL-15, IL-6, fusion protein-6 (complex of IL-6 and soluble IL-6 receptor), etc. Ri least one cytokine and the like are selected.
本発明においては、１０〜５００ｎｇ／ｍＬ ヒト幹細胞因子（ｈＳＣＦ）、１０〜５００ｎｇ／ｍＬ ヒト血管内皮増殖因子（ｈＶＥＧＦ）、１０〜１０００ｎｇ／ｍＬ ヒト融合蛋白質−６（ヒトインターロイキン−（ＩＬ−）６とヒト可溶性ＩＬ−６受容体との複合体、ｈＦＰ−６）、５〜１００ｎｇ／ｍＬ ｈＩＬ−３、５〜１００ｎｇ／ｍＬ ヒトトロンボポエチン（ｈＴＰＯ）、５〜１００ｎｇ／ｍＬ 顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）、１〜２０Ｕ／ｍＬ ヒトエリスロポエチン（ｈＥＰＯ）、１〜１００ｎｇ／ｍＬ ｈｂＦＧＦ、及び、１〜１００ｎｇ／ｍＬ ヒト骨形成タンパク質（ｈＢＭＰ）−４からなるサイトカインカクテルを添加して培養する方法が特に好ましい。 In the present invention, 10-500 ng / mL human stem cell factor (hSCF), 10-500 ng / mL human vascular endothelial growth factor (hVEGF), 10-1000 ng / mL human fusion protein-6 (human interleukin- (IL-)) 6 and human soluble IL-6 receptor complex, hFP-6), 5-100 ng / mL hIL-3, 5-100 ng / mL human thrombopoietin (hTPO), 5-100 ng / mL granulocyte colony stimulating factor ( G-CSF), 1-20 U / mL human erythropoietin (hEPO), 1-100 ng / mL hbFGF, and 1-100 ng / mL human bone morphogenetic protein (hBMP) -4. Is particularly preferred.
そして、これらの因子を添加した培地中において本発明にかかる間葉系幹細胞と共培養することにより、ヒト由来の多能性幹細胞を、赤血球、白血球、血小板といった血液細胞の基となる造血幹細胞や種々の造血前駆細胞に分化誘導することができる。さらに、得られた造血幹細胞や造血前駆細胞を、例えば、後述の実施例に示すような血清やサイトカイン等が添加されているメチルセルロース培地（例えば、ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ．社製のＭｅｔｈｏｃｕｌｔ Ｈ４４３５）を用いた血液コロニ―培養や、「Ｓｕｉら、Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ、１９９５年、９２巻、２８５９〜２８６３ページ」、「Ｓｕｉら、Ｊ Ｅｘｐ Ｍｅｄ、１９９６年、１８３巻、８３７〜８４５ページ」に記載の無血清の条件下での血液コロニ―培養法や浮遊血液培養法によって、成熟した血液細胞とすることができる。また、後述の実施例に示す通り、多能性幹細胞から分化した細胞を移植する患者由来の血清（自家血清）等を用いた血液コロニー培養法によっても成熟した血液細胞とすることができる。これらの方法の中では、異種動物血清（ＦＢＳ等）のみならず、ａｌｌｏｇｅｎｅｉｃ（同種異系）抗原をも混入していない成熟した血液細胞が得られるという観点から、自家血清等を用いた血液コロニ―培養法が好ましい。 Then, by co-culturing with the mesenchymal stem cells according to the present invention in a medium supplemented with these factors, human-derived pluripotent stem cells are converted into hematopoietic stem cells that are the basis of blood cells such as red blood cells, white blood cells, and platelets, Differentiation can be induced into various hematopoietic progenitor cells. Furthermore, the obtained hematopoietic stem cells and hematopoietic progenitor cells were used, for example, in a methylcellulose medium (for example, Methocult H4435 manufactured by StemCell Technologies Inc.) to which serum, cytokines and the like as shown in Examples described later were added. Blood colony culture or “Sui et al., Proc Natl Acad Sci USA, 1995, 92, 2859-2863”, “Sui et al., J Exp Med, 1996, 183, 837-845” Mature blood cells can be obtained by blood colony culture or suspension blood culture under serum-free conditions. Moreover, as shown in the below-mentioned Example, it can be set as a mature blood cell also by the blood colony culture method using the serum (autologous serum) etc. from the patient who transplants the cell which differentiated from the pluripotent stem cell. Among these methods, blood colonies using autologous serum and the like are obtained from the viewpoint of obtaining mature blood cells not contaminated with not only allogeneic animal serum (FBS and the like) but also allogeneic (allogeneic) antigens. -Culture methods are preferred.
多能性幹細胞を血液細胞以外の細胞に分化誘導する方法としては、ドーパミン産生神経細胞に分化誘導する場合には、例えば、「Ｚｅｎｇら、Ｓｔｅｍ Ｃｅｌｌｓ、２００４年、２２巻、９２５〜９４０ページ」に記載の、ヒトＥＳ細胞と、マウス骨髄間質細胞由来の前脂肪細胞であるＰＡ６細胞とを共培養する方法が挙げられる。また、血管内皮細胞に分化誘導する場合には、例えば、「Ｓｏｎｅら、Ａｒｔｅｒｉｏｓｃｌｅｒ Ｔｈｒｏｍｂ Ｖａｓｃ Ｂｉｏｌ．、２００７年、２７巻、２１２７〜２１３４ページ」に記載のヒトＥＳ細胞と、マウス新生児の頭蓋冠から分離したＯＰ９細胞とを共培養し、その後出現してくるＶＥＧＦ−Ｒ２（＋）ＴＲＡ１−６０（−）ＶＥ−ｃａｄｈｅｒｉｎ（＋）細胞を分離し、コラーゲンIVコーティング上で培養する方法が挙げられる。さらに、心筋細胞に分化誘導する場合には、例えば、「Ｙａｍａｓｈｉｔａら、ＦＡＳＥＢ Ｊ．、２００５年、１９巻、１５３４〜１５３６ページ」に記載のマウスＥＳ細胞をコラーゲンIVコーティング上において、ＬＩＦを含有していない培養液中で培養し、その後出現してくるＦｌｋ−１（＋）細胞とＯＰ−９細胞とを共培養する方法が挙げられる。そして、これらの方法において用いられる異種動物由来のフィーダー細胞（ＰＡ６細胞やＯＰ９細胞）の代わりに、本発明にかかる間葉系幹細胞を用いることにより、異種細胞を用いずにヒト由来の多能性幹細胞をドーパミン産生神経細胞、血管内皮細胞、心筋細胞等の機能細胞に分化することが可能となる。 As a method for inducing differentiation of pluripotent stem cells into cells other than blood cells, in the case of inducing differentiation into dopaminergic neurons, for example, “Zeng et al., Stem Cells, 2004, Vol. 22, pages 925-940” And a method of co-culturing human ES cells and PA6 cells, which are preadipocytes derived from mouse bone marrow stromal cells. In the case of inducing differentiation into vascular endothelial cells, for example, human ES cells described in “Sone et al., Arterioscler Thromb Vas Biol., 2007, 27, 2127-2134” and the calvaria of a newborn mouse are used. Examples include a method in which the separated OP9 cells are co-cultured and then VEGF-R2 (+) TRA1-60 (−) VE-cadherin (+) cells appearing thereafter are separated and cultured on a collagen IV coating. Further, in the case of inducing differentiation into cardiomyocytes, for example, mouse ES cells described in “Yamashita et al., FASEB J., 2005, Vol. 19, pages 1534 to 1536” contain LIF on a collagen IV coating. A method of co-culturing Flk-1 (+) cells and OP-9 cells appearing after culturing in a non-cultured medium. Then, by using the mesenchymal stem cells according to the present invention instead of the feeder cells (PA6 cells and OP9 cells) derived from different animals used in these methods, human-derived pluripotency without using the different cells Stem cells can be differentiated into functional cells such as dopaminergic neurons, vascular endothelial cells, cardiomyocytes.
間葉系幹細胞との共培養下でヒト由来の多能性幹細胞の分化誘導を行う段階においては、ヒト由来の多能性幹細胞は、高い増殖能を有しているという観点から、未分化コロニ―を形成している状態となっていることが好ましい。ヒト由来の多能性幹細胞の未分化コロニ―の形成は、例えば、ヒト由来のＥＳ細胞を用いる場合は、後述のＨＥＳ１培養液中で、ヒト由来のｉＰＳ細胞を用いる場合は、後述のＨＥＳ２培養液中で、６〜１０日程度培養することにより行うことができる。 In the stage of inducing differentiation of human-derived pluripotent stem cells in the co-culture with mesenchymal stem cells, human-derived pluripotent stem cells are highly differentiated from the viewpoint of having high proliferative ability. It is preferable that “−” is formed. The formation of undifferentiated colonies of human-derived pluripotent stem cells is, for example, in the case of using human-derived ES cells, in the HES1 culture medium described below, and in the case of using human-derived iPS cells, the below-described HES2 culture. It can be performed by culturing in a liquid for about 6 to 10 days.
本発明は、上記の本発明の方法により得られる、ヒト由来の多能性幹細胞から分化した細胞をも提供するものである。 The present invention also provides a cell differentiated from a human-derived pluripotent stem cell obtained by the above-described method of the present invention.
以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.
＜ヒトＥＳ細胞及びｉＰＳ細胞の維持培養＞
ヒトＥＳ細胞としてはＨ１ヒトＥＳ細胞（ＷｉＣｅｌｌ Ｒｅｓｅａｒｃｈ Ｉｎｓｔｉｔｕｔｅ製）又は、京都大学中辻憲夫教授から供与されたＫｈＥＳ−１細胞を用いた。ヒトｉＰＳ細胞としては、京都大学山中伸弥教授から供与されたヒトｉＰＳ細胞（２５３Ｇ１ヒトｉＰＳ細胞）を用いた。そして、これらヒトＥＳ細胞及びｉＰＳ細胞は、マウス胎仔繊維芽細胞（ＭＥＦ）との共培養により維持し、継代は６〜８日毎に実施した。<Maintenance culture of human ES cells and iPS cells>
As human ES cells, H1 human ES cells (manufactured by WiCell Research Institute) or KhES-1 cells provided by Professor Norio Nakajo of Kyoto University were used. As human iPS cells, human iPS cells (253G1 human iPS cells) provided by Professor Shinya Yamanaka of Kyoto University were used. These human ES cells and iPS cells were maintained by co-culture with mouse fetal fibroblasts (MEF), and passage was performed every 6 to 8 days.
Ｈ１ヒトＥＳ細胞の共培養のための培養液としては、ダルベッコ変法イーグル培地（ＤＭＥＭ）とＨａｍ’ｓ ｎｕｔｒｉｅｎｔ ｍｉｘｔｕｒｅ Ｆ−１２（Ｓｉｇｍａ社製）とを１：１で混合し、これに０．１ｍＭ ２−メルカプトエタノール（Ｗａｋｏ社製）、２００ｍＭ Ｌ−グルタミン（Ｉｎｖｉｔｒｏｇｅｎ社製）、１Ｍ ＨＥＰＥＳ（Ｉｎｖｉｔｒｏｇｅｎ社製）、最小必須培地（ＭＥＭ）−非必須アミノ酸溶液（Ｉｎｖｉｔｒｏｇｅｎ社製）、５ｎｇ／ｍＬ ヒト組み換え塩基性線維芽細胞増殖因子（ｂＦＧＦ）（Ｒ＆Ｄ社製）と２０％ ノックアウト血清代替物（ＫＳＲ）（Ｉｎｖｉｔｒｏｇｅｎ社製）を加えたものを用いた（以下、ＨＥＳ１培養液とも称する）。 As a culture solution for co-culture of H1 human ES cells, Dulbecco's modified Eagle's medium (DMEM) and Ham's neutral mixture F-12 (manufactured by Sigma) were mixed in a 1: 1 ratio. 1 mM 2-mercaptoethanol (Wako), 200 mM L-glutamine (Invitrogen), 1M HEPES (Invitrogen), minimal essential medium (MEM) -non-essential amino acid solution (Invitrogen), 5 ng / mL human A recombinant basic fibroblast growth factor (bFGF) (manufactured by R & D) and 20% knockout serum substitute (KSR) (manufactured by Invitrogen) were used (hereinafter also referred to as HES1 culture medium).
ＫｈＥＳ−１細胞又はヒトｉＰＳ細胞（２５３Ｇ１ヒトｉＰＳ細胞）の維持培養のための培養液としては、ＤＭＥＭとＨａｍ’ｓ ｎｕｔｒｉｅｎｔ ｍｉｘｔｕｒｅ Ｆ−１２とを１：１で混入し、これに０．１ｍＭ ２−メルカプトエタノール、２００ｍＭ Ｌ−グルタミン、１Ｍ ＨＥＰＥＳ、ＭＥＭ−非必須アミノ酸溶液、４ｎｇ／ｍＬ ヒト組み換えｂＦＧＦと２０％ ＫＳＲとを加えものを用いた（以下、ＨＥＳ２培養液とも称する）。 As a culture solution for maintenance culture of KhES-1 cells or human iPS cells (253G1 human iPS cells), DMEM and Ham's neutral mixture F-12 were mixed at 1: 1, and 0.1 mM 2 -Mercaptoethanol, 200 mM L-glutamine, 1 M HEPES, MEM-non-essential amino acid solution, 4 ng / mL human recombinant bFGF and 20% KSR were used (hereinafter also referred to as HES2 culture medium).
また、未分化なヒトＥＳ細胞コロニーであることの確認は、組織免疫染色によるＯｃｔ−４、Ｓｏｘ−２、ＴＲＡ−１−６０、Ｎａｎｏｇの発現、及び免疫不全ＮＯＤ−ｓｃｉｄマウスの腎被膜下への移植によるテラトーマ形成能によった。 In addition, confirmation of undifferentiated human ES cell colonies was confirmed by the expression of Oct-4, Sox-2, TRA-1-60, Nanog by tissue immunostaining, and under the kidney capsule of immunodeficient NOD-scid mice. It depends on teratoma formation ability by transplantation.
＜ヒトＥＳ細胞及びｉＰＳ細胞から間葉系幹細胞への分化誘導＞
血小板採取装置により得られたヒト血小板濃厚血漿を−８０℃で冷凍した後解凍することにより血小板を破壊した後、９００ｇで遠心分離して上清を回収し、血小板溶解液（ｐｌａｔｅｌｅｔ ｌｙｓａｔｅ、ＰＬ）を作製した。ヒト多能性幹細胞から間葉系幹細胞を分化誘導するために、ＭＥＦ上で維持培養されていたヒトＥＳ細胞やｉＰＳ細胞を回収して、又は凍結されていたヒトＥＳ細胞やｉＰＳ細胞を解凍して、フィーダー細胞を敷かないゼラチンコートされた１０ｃｍ培養皿に播種し、ＨＥＳ１培養液又はＨＥＳ２培養液に、５％ ＰＬと１０，０００Ｕ ノボ−ヘパリン（持田製薬株式会社製）とを添加し（以下、ＰＬ培養液とも称する）、３７℃、５％ＣＯ２の条件下で培養した。培養液交換は週２回行い、２〜３週毎に継代培養し、培養６〜８週目に、ヒトＥＳ細胞やｉＰＳ細胞を均一な紡錘形の細胞へと分化させた。得られた結果を図１〜３に示す。<Induction of differentiation from human ES cells and iPS cells into mesenchymal stem cells>
Platelet lysate (PL) is obtained by destroying platelets by freezing and thawing human platelet-rich plasma obtained by a platelet collection device at −80 ° C., and then centrifuging at 900 g to collect the supernatant. Was made. In order to induce differentiation of mesenchymal stem cells from human pluripotent stem cells, human ES cells and iPS cells maintained on MEF are collected, or frozen human ES cells and iPS cells are thawed. 5% PL and 10,000 U Novo-heparin (manufactured by Mochida Pharmaceutical Co., Ltd.) are added to the HES1 culture solution or the HES2 culture solution (hereinafter referred to as “Mochida Pharmaceutical Co., Ltd.”). , Also referred to as PL culture solution), and cultured under conditions of 37 ° C. and 5% CO 2 . Culture medium exchange was performed twice a week, subcultured every 2-3 weeks, and human ES cells and iPS cells were differentiated into uniform spindle-shaped cells at 6-8 weeks of culture. The obtained results are shown in FIGS.
＜間葉系幹細胞の性状の解析＞
前記の通りにして得られた均一な紡錘形の細胞の細胞表面マーカーをＦＡＣＳ（製品名：ＦＡＣＳＣａｌｉｂｕｒ ｉｎｓｔｒｕｍｅｎｔ、ＢＤ Ｍｅｄｉｃａｌ Ｓｙｓｔｅｍｓ社製）を用いて検出し、ＦｌｏｗＪｏソフトウェア（Ｔｏｍｙ Ｄｉｇｉｔａｌ Ｂｉｏｌｏｇｙ社製）を用いて解析した。また、得られた均一な紡錘形の細胞の遺伝子発現を、表１に示すプライマーを用いてＲＴ−ＰＣＲで検討した。さらに、ＮＨ ＯｓｔｅｏＤｉｆｆ Ｍｅｄｉｕｍ、ＮＨ ＡｄｉｐｏＤｉｆｆ Ｍｅｄｉｕｍ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社製）を用いて、得られた均一な紡錘形の細胞から脂肪細胞や骨芽細胞への分化誘導を行い、各々オイルレッドＯ（Ｏｉｌ ｒｅｄ Ｏ）染色、アルカリフォスファターゼ（ＡＬＰ）染色により各細胞に分化誘導されていることを確認した。得られた結果を図４〜７に示す。さらに、Ｈ１ヒトＥＳ細胞又は２５３Ｇ１ヒトｉＰＳ細胞から分化誘導された均一な紡錘形の細胞（５０細胞）については、染色体検査を実施した。<Analysis of characteristics of mesenchymal stem cells>
Cell surface markers of uniform spindle-shaped cells obtained as described above were detected using FACS (product name: FACSCalibur instrument, manufactured by BD Medical Systems), and using FlowJo software (manufactured by Tomy Digital Biology). Analyzed. In addition, gene expression of the obtained uniform spindle-shaped cells was examined by RT-PCR using the primers shown in Table 1. Further, using NH OsteoDiff Medium and NH AdipoDiff Medium (Miltenyi Biotec), differentiation of the obtained spindle-shaped cells into adipocytes and osteoblasts was performed, and oil red O (Oil red O) was used. It was confirmed that differentiation was induced in each cell by staining and alkaline phosphatase (ALP) staining. The obtained results are shown in FIGS. Further, chromosome examination was performed on uniform spindle-shaped cells (50 cells) induced to differentiate from H1 human ES cells or 253G1 human iPS cells.
図１に示した結果から明らかなように、ＭＥＦ上で維持されていたヒトＥＳ細胞（Ｈ１ヒトＥＳ細胞）を回収して、フィーダー細胞を用いず、ゼラチンをコートした１０ｃｍ培養皿に播種し、ＰＬ培養液で３７℃、５％ＣＯ２の条件下で培養すると、６〜８週後に均一な紡錘形の細胞、すなわち間葉系幹細胞が分化誘導された。また、同様の方法を用いて、ｋｈＥＳ−１細胞や２５３Ｇ１ヒトｉＰＳ細胞からも、図２〜３に示した結果から明らかなように、間葉系幹細胞を分化誘導することができた。As is clear from the results shown in FIG. 1, human ES cells (H1 human ES cells) maintained on MEF were collected and seeded in a 10 cm culture dish coated with gelatin without using feeder cells. When cultured in a PL culture solution at 37 ° C. and 5% CO 2 , after 6 to 8 weeks, uniform spindle-shaped cells, ie, mesenchymal stem cells, were induced to differentiate. In addition, using the same method, it was possible to induce differentiation of mesenchymal stem cells from khES-1 cells and 253G1 human iPS cells, as is apparent from the results shown in FIGS.
さらに、これらの細胞の表面抗原をＦＡＣＳ法にて解析すると、図４に示した結果から明らかなように、前記均一な紡錘形の細胞は、血液細胞、血管内皮細胞、未分化なＥＳ細胞のマーカーであるＣＤ４５、ＣＤ３４、ＣＤ１４、ＣＤ３１、ＳＳＥＡ−４は発現しておらず、間葉系幹細胞のマーカーであるＣＤ１０５、ＣＤ１６６を発現していた。さらに、骨芽細胞や脂肪細胞へ分化誘導すると、図５〜６に示した結果から明らかなように、ＡＬＰ染色陽性の骨芽細胞やオイルレッド染色陽性の脂肪細胞に分化したことより、前記の方法によって樹立された細胞は間葉系幹細胞であることが確認された。また、樹立された間葉系幹細胞のＲＴ−ＰＣＲ法による解析では、図７に示した結果から明らかなように、未分化なヒトＥＳ細胞のマーカーであり、ｈＥＳＣ（Ｈ１ヒトＥＳ細胞）においては遺伝子発現が確認されたＯｃｔ−４の発現は認められなかった。また、マウス細胞由来のｍＨＰＲＴの発現もＨ１ヒトＥＳ細胞から樹立された間葉系幹細胞（ｈＥＳＣ由来ＭＳＣ）においては認められなかったため、かかる間葉系幹細胞において、未分化なヒトＥＳ細胞やＭＥＦの残存又は混入はないことが確認された。 Further, when the surface antigens of these cells are analyzed by the FACS method, as is clear from the results shown in FIG. 4, the uniform spindle-shaped cells are markers for blood cells, vascular endothelial cells, and undifferentiated ES cells. CD45, CD34, CD14, CD31 and SSEA-4 were not expressed, and CD105 and CD166, which are markers for mesenchymal stem cells, were expressed. Furthermore, when differentiation was induced into osteoblasts and adipocytes, as clearly shown from the results shown in FIGS. 5 to 6, the differentiation into ALP staining positive osteoblasts and oil red staining positive adipocytes, The cells established by the method were confirmed to be mesenchymal stem cells. Further, in the analysis of the established mesenchymal stem cells by RT-PCR, as is apparent from the results shown in FIG. 7, it is a marker for undifferentiated human ES cells, and in hESC (H1 human ES cells) The expression of Oct-4 in which gene expression was confirmed was not observed. In addition, since mHPRT expression derived from mouse cells was not observed in mesenchymal stem cells established from H1 human ES cells (hESC-derived MSCs), in these mesenchymal stem cells, undifferentiated human ES cells and MEFs It was confirmed that there was no residue or contamination.
さらに、図には示さないが、Ｈ１ヒトＥＳ細胞、２５３Ｇ１ヒトｉＰＳ細胞から分化誘導された間葉系幹細胞については染色体検査を実施し、いずれも、解析された５０細胞全てが正常核型であることを確認した。 Further, although not shown in the figure, the mesenchymal stem cells differentiated from H1 human ES cells and 253G1 human iPS cells were subjected to chromosomal examination, and all of the analyzed 50 cells were normal karyotypes. It was confirmed.
従って、ヒトＰＬ（血小板溶解液）を用いて６〜８週間培養することにより、異種動物由来の血清を用いることなく、ヒトＥＳ細胞から間葉系幹細胞へ分化誘導でき、これらには未分化なヒトＥＳ細胞やＭＥＦも混入していないことが確認された。また、かかるヒトＰＬを用いたヒト多能性幹細胞から間葉系幹細胞への分化誘導法は、ヒト由来のＥＳ細胞、ヒト由来のｉＰＳ細胞いずれにも適応可能で有り、染色体異常の発生する可能性は非常に低いことを示している。 Therefore, by culturing for 6 to 8 weeks using human PL (platelet lysate), differentiation from human ES cells to mesenchymal stem cells can be induced without using sera from different animals, and these are undifferentiated. It was confirmed that human ES cells and MEF were not mixed. In addition, the differentiation induction method from human pluripotent stem cells to mesenchymal stem cells using human PL can be applied to both human-derived ES cells and human-derived iPS cells, and may cause chromosomal abnormalities. The sex is very low.
＜ヒトＥＳ細胞及びｉＰＳ細胞の未分化コロニーの形成＞
前記の通りにして、６ｗｅｌｌプレート上でヒトＥＳ細胞やｉＰＳ細胞から分化誘導された間葉系幹細胞を、１５〜１８Ｇｙの放射線照射した後にヒトＥＳ細胞やｉＰＳ細胞と共培養した。すなわち、この際の培養液として、間葉系幹細胞上に播種する細胞がＨ１ヒトＥＳ細胞であればＨＥＳ１培養液を、２５３Ｇ１ヒトｉＰＳ細胞であればＨＥＳ２培養液を用いて、間葉系幹細胞との共培養を行い、ヒトＥＳ細胞及びｉＰＳ細胞の未分化コロニーの形成を行った。得られた結果を図８〜１０に示す。また、一部の実験では、ヒト多能性幹細胞由来間葉系幹細胞と共培養されたヒト多能性幹細胞の未分化性を確認するために、未分化なヒトＥＳ細胞のマーカーであるＯｃｔ−４、Ｓｏｘ−２、ＴＲＡ−１−６０、Ｎａｎｏｇの発現を検討した。すなわち、蛍光標識した各々のマーカータンパク質を認識する抗体を用いて、形成されたコロニ―を染色し、蛍光顕微鏡を用いて観察した。得られた結果を図１１〜１４に示す。さらに、形成されたコロニ―を構成する細胞が未分化性を維持していることを確認するため、Ｈ１ヒトＥＳ細胞由来間葉系幹細胞上で培養されたＨ１ヒトＥＳ細胞又は２５３Ｇ１ヒトｉＰＳ細胞をＮＯＤ−ｓｃｉｄマウスに移植し、テラトーマが形成されるかどうかを検証した。<Formation of undifferentiated colonies of human ES cells and iPS cells>
As described above, mesenchymal stem cells differentiated from human ES cells and iPS cells on 6-well plates were irradiated with 15-18 Gy of radiation and then co-cultured with human ES cells and iPS cells. That is, as the culture medium at this time, if the cells to be seeded on the mesenchymal stem cells are H1 human ES cells, the HES1 culture medium is used. If the cells are 253G1 human iPS cells, the HES2 culture medium is used. Were co-cultured to form undifferentiated colonies of human ES cells and iPS cells. The obtained results are shown in FIGS. In some experiments, in order to confirm the undifferentiation of human pluripotent stem cells co-cultured with human pluripotent stem cell-derived mesenchymal stem cells, Oct-- which is a marker for undifferentiated human ES cells. 4, expression of Sox-2, TRA-1-60, Nanog was examined. That is, the formed colonies were stained with an antibody that recognizes each marker protein that was fluorescently labeled, and observed using a fluorescence microscope. The obtained results are shown in FIGS. Furthermore, in order to confirm that the cells constituting the formed colony maintain undifferentiation, H1 human ES cells or 253G1 human iPS cells cultured on H1 human ES cell-derived mesenchymal stem cells are used. It transplanted to the NOD-scid mouse | mouth and it was verified whether teratoma was formed.
図８〜１０に示した結果から明らかなように、前記の方法を用いてヒトＥＳ細胞やｉＰＳ細胞から間葉系幹細胞を分化誘導し、１５〜１８Ｇｙの放射線照射した後、その上に自己、非自己のヒトＥＳ細胞やｉＰＳ細胞を播種して６〜１０日間共培養すると、ＭＥＦによる継代培養中のヒトＥＳ細胞やｉＰＳ細胞、あるいは解凍直後のヒトＥＳ細胞やｉＰＳ細胞であっても、未分化コロニーの形成を認めた。また、図１１〜１４に示した結果から明らかなように、Ｈ１ヒトＥＳ細胞由来間葉系幹細胞上で培養されたＨ１ヒトＥＳ細胞は、いずれも未分化なヒトＥＳ細胞マーカーを発現していた。さらに、図には示さないが、Ｈ１ヒトＥＳ細胞由来間葉系幹細胞上で培養されたＨ１ヒトＥＳ細胞、２５３Ｇ１ヒトｉＰＳ細胞をＮＯＤ−ｓｃｉｄマウスに移植すると、内胚樣、中胚葉、外胚葉由来の細胞からなるテラトーマが形成され、これらのヒト多能性幹細胞では未分化性が維持されていることが確認できた。 As is clear from the results shown in FIGS. 8 to 10, the mesenchymal stem cells were induced to differentiate from human ES cells and iPS cells using the above-described method, and after irradiation with 15 to 18 Gy, self, When non-self human ES cells or iPS cells are seeded and co-cultured for 6 to 10 days, human ES cells or iPS cells that have been subcultured with MEF, or human ES cells or iPS cells that have just been thawed, Formation of undifferentiated colonies was observed. Moreover, as is clear from the results shown in FIGS. 11 to 14, all of the H1 human ES cells cultured on the H1 human ES cell-derived mesenchymal stem cells expressed an undifferentiated human ES cell marker. . Furthermore, although not shown in the figure, when H1 human ES cells and 253G1 human iPS cells cultured on mesenchymal stem cells derived from H1 human ES cells are transplanted into NOD-scid mice, endoderm fistula, mesoderm, ectoderm Teratoma consisting of cells derived from the cells was formed, and it was confirmed that these human pluripotent stem cells were maintained undifferentiated.
（実施例１）
＜ヒトＥＳ細胞及びｉＰＳ細胞から血液細胞への分化誘導＞
前記の通りにして、ヒトＥＳ細胞やｉＰＳ細胞の未分化コロニーの形成が認められる培養６〜１０日目に、２ｍＭ グルタミンと、４×１０−４Ｍ モノチオグリセロール（ＭＴＧ、Ｓｉｇｍａ社製）と５０ｍｇ／ｍＬ アスコルビン酸（Ｓｉｇｍａ社製）とを含む無血清培地であるＳｔｅｍＰｒｏ−３４（Ｉｎｖｉｔｒｏｇｅｎ社製）培養液に、１００ｎｇ／ｍＬ ヒト幹細胞因子（ｈＳＣＦ）、１００ｎｇ／ｍＬ ヒト血管内皮増殖因子（ｈＶＥＧＦ）、１００ｎｇ／ｍＬ ヒト融合蛋白質−６（ヒトインターロイキン−（ＩＬ−）６とヒト可溶性ＩＬ−６受容体との複合体、ｈＦＰ−６）、２０ｎｇ／ｍＬ ｈＩＬ−３、１０ｎｇ／ｍＬ ヒトトロンボポエチン（ｈＴＰＯ）、１０ｎｇ／ｍＬ 顆粒球コロニー刺激因子（Ｇ−ＣＳＦ）、５Ｕ／ｍＬ ヒトエリスロポエチン（ｈＥＰＯ）、１０ｎｇ／ｍＬ ｈｂＦＧＦ、１０ｎｇ／ｍＬ ヒト骨形成タンパク質（ｈＢＭＰ）−４からなるサイトカインカクテルを添加して、培養液を週２回交換しながら、培養を継続した。得られた結果を図１５〜１７、２６に示す。(Example 1)
<Induction of differentiation from human ES cells and iPS cells into blood cells>
As described above, 2 mM glutamine and 4 × 10 −4 M monothioglycerol (MTG, manufactured by Sigma) were cultured on days 6 to 10 where formation of undifferentiated colonies of human ES cells and iPS cells was observed. 100 ng / mL human stem cell factor (hSCF), 100 ng / mL human vascular endothelial growth factor (hVEGF) was added to StemPro-34 (Invitrogen) culture medium, which is a serum-free medium containing 50 mg / mL ascorbic acid (manufactured by Sigma). ), 100 ng / mL human fusion protein-6 (complex of human interleukin- (IL-) 6 and human soluble IL-6 receptor, hFP-6), 20 ng / mL hIL-3, 10 ng / mL human thrombopoietin (HTPO), 10 ng / mL granulocyte colony stimulating factor (G-CSF), 5 U / mL The culture was continued while a cytokine cocktail consisting of human erythropoietin (hEPO), 10 ng / mL hbFGF, 10 ng / mL human bone morphogenetic protein (hBMP) -4 was added, and the culture medium was changed twice a week. The obtained results are shown in FIGS.
＜血液細胞の同定＞
前記の通りにして、１０〜１４日間共培養して得られた細胞を、ウシ胎児血清（ＦＢＳ）等を含むＭｅｔｈｏｃｕｌｔ Ｈ４４３５（ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ．社製）を用いて、血液コロニー培養し、血液コロニーの形成を行った。また、形成された血液コロニーについては、各コロニーのサイトスピン標本を作製し、Ｍａｙ−Ｇｒｕｎｗａｌｄ−Ｇｉｅｍｓａ染色又は組織免疫染色して、コロニーを構成している細胞の同定を行った。なお、組織免疫染色は、前記の通りにして得られた細胞を、４％ パラホルムアルデヒド（ＰＦＡ）にて固定した後、Ｏｃｔ−４、Ｓｏｘ−２、ＴＲＡ−１−６０、Ｎａｎｏｇ、ｇlｙｃｏｐｈｏｒｉｎ Ａ（ＧＰＡ）、ヘモグロビン（Ｈｂ）、βグロビンの発現を、免疫染色により検討した。また、核染色を、Ｈｏｅｃｈｓｔ３３３４２（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社製）を用いて行った。得られた結果を図１８〜２５、２７〜３１に示す。<Identification of blood cells>
As described above, the cells obtained by co-culture for 10 to 14 days were subjected to blood colony culture using Methocult H4435 (manufactured by StemCell Technologies Inc.) containing fetal bovine serum (FBS) and the like. Was formed. Moreover, about the formed blood colony, the cytospin sample of each colony was produced, May-Grunwald-Giemsa dyeing | staining or tissue immunostaining was performed, and the cell which comprises the colony was identified. For tissue immunostaining, cells obtained as described above were fixed with 4% paraformaldehyde (PFA), and then Oct-4, Sox-2, TRA-1-60, Nanog, glycophorin A ( The expression of GPA), hemoglobin (Hb), and β globin was examined by immunostaining. In addition, nuclear staining was performed using Hoechst 33342 (Molecular Probes). The obtained results are shown in FIGS.
図１５〜１７に示した結果から明らかなように、前述のように１５〜１８Ｇｙの放射線照射したＨ１ヒトＥＳ細胞由来の間葉系幹細胞との共培養によりＨ１ヒトＥＳ細胞の未分化コロニー（図１５参照）が形成される培養６〜１０日目に、前記サイトカインカクテル等を含むＳｔｅｍＰｒｏ−３４培養液に変更して、培養を継続し、培養液を変更してから１０〜１４日目にヒトＥＳ細胞から形成されたコロニーの中に、未分化な血液細胞が間葉系幹細胞下で増殖していることを示す敷石状細胞群（造血幹細胞や種々の造血前駆細胞等）が確認された（図１６参照）。また、その後造血細胞、または血液細胞と推測される小型円形細胞の増殖が確認された（図１７参照）。 As is clear from the results shown in FIGS. 15 to 17, undifferentiated colonies of H1 human ES cells (FIG. 15) by co-culture with 15 to 18 Gy-irradiated H1 human ES cell-derived mesenchymal stem cells as described above. 15) is formed on day 6 to day 10 of the culture, changing to the StemPro-34 culture medium containing the cytokine cocktail and the like, continuing the culture, and changing the culture medium on day 10 to 14 Among colonies formed from ES cells, a set of cobblestone cells (hematopoietic stem cells, various hematopoietic progenitor cells, etc.) indicating that undifferentiated blood cells proliferate under mesenchymal stem cells were confirmed ( (See FIG. 16). In addition, the growth of hematopoietic cells or small round cells presumed to be blood cells was confirmed (see FIG. 17).
さらに、図１８〜２３に示した結果から明らかなように、それらの細胞を回収して、血液コロニー培養すると、赤血球系細胞から構成される赤血球系コロニー、好中球、マクロファージや単球等の骨髄球系細胞から構成される骨髄球系コロニー、赤血球系細胞、骨髄球系細胞及び巨核球系細胞から構成される混合コロニーが形成された。 Furthermore, as apparent from the results shown in FIGS. 18 to 23, when those cells are collected and cultured with blood colonies, erythroid colonies composed of erythroid cells, neutrophils, macrophages, monocytes, etc. A mixed colony composed of myeloid colonies composed of myeloid cells, erythroid cells, myeloid cells and megakaryocytes was formed.
また、図２４及び図２５に示した結果から明らかなように、これらの赤血球系コロニーに含まれる細胞は、赤血球系細胞のマーカーであるＧＰＡやＨｂを発現する赤血球系細胞であることを確認した。さらに、これらの赤血球系細胞が、未熟な一次造血を起源とする胚性赤血球であるか、二次造血を起源とする成人型赤血球であるかを検討したところ、これらの赤血球の９５％以上が、二次造血に特異的なβグロビンの発現する成人型赤血球であることを確認した（図２４参照）。なお、図には示さないが、Ｏｃｔ−４、Ｓｏｘ−２、ＴＲＡ−１−６０、Ｎａｎｏｇといった未分化マーカーはこれら細胞において発現していないことも確認された。 Further, as is clear from the results shown in FIGS. 24 and 25, it was confirmed that the cells contained in these erythroid colonies were erythroid cells expressing GPA and Hb, which are markers for erythroid cells. . Furthermore, when examining whether these erythroid cells are embryonic erythrocytes originating from immature primary hematopoiesis or adult erythrocytes originating from secondary hematopoiesis, more than 95% of these erythrocytes It was confirmed that it was an adult type erythrocyte expressing β-globin specific for secondary hematopoiesis (see FIG. 24). Although not shown in the figure, it was also confirmed that undifferentiated markers such as Oct-4, Sox-2, TRA-1-60, and Nanog were not expressed in these cells.
また、図２６〜２８に示した結果から明らかなように、２５３Ｇ１ヒトｉＰＳ細胞由来間葉系幹細胞と２５３Ｇ１ヒトｉＰＳ細胞との共培養においても、２５３Ｇ１ヒトｉＰＳ細胞から形成されたコロニー中に敷石状細胞群が確認され（図２６参照）、１０〜１４日後に、それらの細胞を回収して血液コロニー培養すると、血液コロニーが形成され（図２７参照）、マクロファージ、骨髄球系細胞等の血液細胞が含まれていた（図２８参照）。 As is clear from the results shown in FIGS. 26 to 28, in the co-culture of 253G1 human iPS cell-derived mesenchymal stem cells and 253G1 human iPS cells, a cobblestone is formed in the colony formed from 253G1 human iPS cells. A cell group is confirmed (see FIG. 26), and 10 to 14 days later, when those cells are collected and blood colony cultured, a blood colony is formed (see FIG. 27), and blood cells such as macrophages and myeloid cells (See FIG. 28).
さらに、図２９〜３１に示した結果から明らかなように、２５３Ｇ１ヒトｉＰＳ細胞由来間葉系幹細胞とＨ１ヒトＥＳ細胞との共培養においても、Ｈ１ヒトＥＳ細胞から形成されたコロニー中に敷石状細胞群（造血幹細胞や種々の造血前駆細胞等）が確認され（図示せず）、１０〜１４日後に、それらの細胞を回収して血液コロニー培養すると、赤血球コロニー、混合コロニー、骨髄球系コロニー等様々な血液コロニーが形成された。 Furthermore, as is clear from the results shown in FIGS. 29 to 31, even in the co-culture of 253G1 human iPS cell-derived mesenchymal stem cells and H1 human ES cells, a cobblestone is formed in the colony formed from H1 human ES cells. Cell groups (hematopoietic stem cells, various hematopoietic progenitor cells, etc.) are confirmed (not shown), and after 10-14 days, when these cells are collected and blood colony cultured, erythrocyte colonies, mixed colonies, myeloid colonies Various blood colonies were formed.
従って、ヒト由来の多能性幹細胞を、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞との共培養下で分化誘導することによって、血液細胞といった分化した細胞を得ることができることが明らかになった。さらには、従前のヒト多能性幹細胞から胚樣体を介して血液細胞に分化誘導する方法（非特許文献１〜２ 参照）では得ることが困難だった二次造血に特異的な成人型赤血球も、前述の通り、本発明の方法では９５％以上と極めて効率良く得ることができることも明らかになった。 Therefore, it is clear that differentiated cells such as blood cells can be obtained by inducing differentiation of human-derived pluripotent stem cells under co-culture with mesenchymal stem cells established from human-derived pluripotent stem cells. Became. Furthermore, adult-type erythrocytes specific for secondary hematopoiesis that were difficult to obtain by conventional methods for inducing differentiation from human pluripotent stem cells into blood cells via embryonic rods (see Non-Patent Documents 1 and 2). However, as described above, it has also been clarified that the method of the present invention can be obtained very efficiently at 95% or more.
また、分化誘導させられる多能性幹細胞と、かかる間葉系幹細胞とが同一人由来のものであっても（前記、Ｈ１ヒトＥＳ細胞由来の間葉系幹細胞とＨ１ヒトＥＳ細胞との共培養の例、及び、２５３Ｇ１ヒトｉＰＳ細胞由来間葉系幹細胞と２５３Ｇ１ヒトｉＰＳ細胞との共培養の例 参照）、異なる人由来のものであっても（前記、２５３Ｇ１ヒトｉＰＳ細胞由来間葉系幹細胞とＨ１ヒトＥＳ細胞との共培養の例 参照）、同様にヒト由来の多能性幹細胞から分化した細胞を得ることができることも明らかになった。 Further, even if the pluripotent stem cell that is induced to differentiate and the mesenchymal stem cell are derived from the same person (the above-mentioned co-culture of the H1 human ES cell-derived mesenchymal stem cell and the H1 human ES cell) And the example of co-culture of 253G1 human iPS cell-derived mesenchymal stem cells and 253G1 human iPS cells), and those derived from different persons (the 253G1 human iPS cell-derived mesenchymal stem cells and Similarly, it was revealed that differentiated cells can be obtained from human-derived pluripotent stem cells (see Example of co-culture with H1 human ES cells).
次に、ヒトＰＬ（血小板溶解液）の代わりに自家血清を含む培地にて培養することにより、ヒトｉＰＳ細胞から自家由来の間葉系幹細胞を樹立し、さらに当該ヒトｉＰＳ細胞を当該間葉系幹細胞との共培養下で、自家血清を用いて血球系細胞に分化させることができるかどうかを以下に示す通り検証した。 Next, by culturing in a medium containing autologous serum instead of human PL (platelet lysate), autologous mesenchymal stem cells are established from human iPS cells, and the human iPS cells are further converted into the mesenchymal system. Whether it can be differentiated into blood cells using autologous serum under co-culture with stem cells was verified as shown below.
＜ヒトｉＰＳ細胞の維持培養＞
先ず、Ｎ Ｔａｋａｙａｍａら、Ｊｏｕｒｎａｌ ｏｆ Ｅｘｐｅｒｉｍｅｎｔａｌ Ｍｅｄｉｃｉｎｅ、２０１０年１２月、２０７巻、１３号、２８１７〜２８３０ページの記載に従って、健常成人由来の繊維芽細胞からヒトｉＰＳ細胞（ＳＰＨ−０１０３）を樹立し、＜ヒトＥＳ細胞及びｉＰＳ細胞の維持培養＞に記載の方法と同様の方法にて、ＭＥＦ上で維持した（図３２ 参照）。<Maintenance culture of human iPS cells>
First, human iPS cells (SPH-0103) were established from fibroblasts derived from healthy adults according to the description of N Takayama et al., Journal of Experimental Medicine, December 2010, Vol. 207, No. 13, pages 2817-2830, It was maintained on MEF by the same method as described in <Maintenance culture of human ES cells and iPS cells> (see FIG. 32).
＜ヒトｉＰＳ細胞（ＳＰＨ−０１０３）から間葉系幹細胞への分化誘導＞
次に、ＭＥＦ上にて維持されていたヒトｉＰＳ細胞（ＳＰＨ−０１０３）を回収して、フィーダー細胞を敷かないゼラチンコートした１０ｃｍ培養皿に移し、５％前記健常成人由来の血清（自家血清）を加えたＨＥＳ１培養液（自家培地）にて、３７℃、５％ＣＯ２の条件下で培養した。その結果、培養６〜８週ぐらいで図３３に示すような均一な紡錘形の細胞（間葉系幹細胞）が分化誘導された。<Induction of differentiation from human iPS cells (SPH-0103) to mesenchymal stem cells>
Next, human iPS cells (SPH-0103) maintained on MEF were collected and transferred to a gelatin-coated 10 cm culture dish without feeder cells, and 5% serum from the healthy adult (autologous serum) In a HES1 culture solution (autologous medium) to which was added, the cells were cultured at 37 ° C. and 5% CO 2 . As a result, uniform spindle-shaped cells (mesenchymal stem cells) as shown in FIG. 33 were induced to differentiate in about 6 to 8 weeks of culture.
＜ヒトｉＰＳ細胞（ＳＰＨ−０１０３）の未分化コロニーの形成＞
そして、ヒトｉＰＳ細胞（ＳＰＨ−０１０３）から分化誘導された間葉系幹細胞に１５〜１８Ｇｙの放射線を照射した後に、その上で自家ｉＰＳ細胞（ＳＰＨ−０１０３）を共培養することにより、図３４に示すように、自家ｉＰＳ細胞はコロニーを形成し、未分化な状態で維持された。<Formation of undifferentiated colonies of human iPS cells (SPH-0103)>
Then, after irradiating 15-18 Gy of radiation to mesenchymal stem cells induced to differentiate from human iPS cells (SPH-0103), autologous iPS cells (SPH-0103) were co-cultured thereon to obtain FIG. As shown, autologous iPS cells formed colonies and were maintained in an undifferentiated state.
また、前記未分化コロニーついて、未分化なヒトＥＳ細胞のマーカーであるＯｃｔ−４、Ｓｏｘ−２、ＳＳＥＡ−４、ＴＲＡ−１−６０、Ｎａｎｏｇの発現を＜ヒトＥＳ細胞及びｉＰＳ細胞の未分化コロニーの形成＞に記載の方法と同様に検討した。その結果、図３５に示す通り（ＴＲＡ−１−６０については図には示さないが）、ヒトｉＰＳ細胞（ＳＰＨ−０１０３）由来の間葉系幹細胞上で培養されたヒトｉＰＳ細胞（ＳＰＨ−０１０３）は、いずれも未分化なヒトＥＳ細胞マーカーを発現しており、未分化性が維持されていることが確認できた。 Further, for the undifferentiated colonies, expression of Oct-4, Sox-2, SSEA-4, TRA-1-60, Nanog, which is a marker of undifferentiated human ES cells, is expressed as follows: <Undifferentiation of human ES cells and iPS cells Colony formation> was examined in the same manner as described above. As a result, human iPS cells (SPH-0103) cultured on mesenchymal stem cells derived from human iPS cells (SPH-0103) as shown in FIG. 35 (TRA-1-60 is not shown in the figure). ) All expressed an undifferentiated human ES cell marker, and it was confirmed that the undifferentiated state was maintained.
（実施例２）
＜ヒトｉＰＳ細胞（ＳＰＨ−０１０３）から血液細胞への分化誘導＞
前記の通りにして、ヒトｉＰＳ細胞（ＳＰＨ−０１０３）の未分化コロニーの形成が認められる培養６〜１０日目に、１００ｎｇ／ｍＬ ｈＳＣＦ、１００ｎｇ／ｍＬ ｈＶＥＧＦ、１００ｎｇ／ｍＬ ｈＦＰ−６、２０ｎｇ／ｍＬ ｈＩＬ−３、１０ｎｇ／ｍＬ ｈＴＰＯ、１０ｎｇ／ｍＬ Ｇ−ＣＳＦ、５Ｕ／ｍＬ ｈＥＰＯ、１０ｎｇ／ｍＬ ｈｂＦＧＦ、１０ｎｇ／ｍＬ ｈＢＭＰ−４からなるサイトカインカクテルを添加した、２ｍＭ グルタミン及び４×１０−４Ｍ ＭＴＧと５０ｍｇ／ｍＬ アスコルビン酸を含む無血清培地（ＳｔｅｍＰｒｏ−３４培養液）に変更して培養を継続した。培養液を変更してから１０〜１４日目の結果を図３６に示す。(Example 2)
<Induction of differentiation from human iPS cells (SPH-0103) into blood cells>
As described above, 100 ng / mL hSCF, 100 ng / mL hVEGF, 100 ng / mL hFP-6, 20 ng / day were observed on days 6 to 10 of culture in which formation of undifferentiated colonies of human iPS cells (SPH-0103) was observed. 2 mM glutamine and 4 × 10 −4 M supplemented with a cytokine cocktail consisting of mL hIL-3, 10 ng / mL hTPO, 10 ng / mL G-CSF, 5 U / mL hEPO, 10 ng / mL hbFGF, 10 ng / mL hBMP-4 The culture was continued after changing to a serum-free medium (StemPro-34 culture solution) containing MTG and 50 mg / mL ascorbic acid. FIG. 36 shows the results on the 10th to 14th days after changing the culture solution.
図３６に示した結果から明らかなように、培養液を変更してから１０〜１４日目の、ヒトｉＰＳ細胞（ＳＰＨ−０１０３）から形成されたコロニーの中に、小型円形細胞の増殖が確認された（図３６のａ）。また、一部には未分化な血液細胞が間葉系幹細胞下にて増殖していることを示す敷石状細胞群が確認された（図３６のｂの矢印が示す箇所）。 As is clear from the results shown in FIG. 36, the growth of small round cells was confirmed in colonies formed from human iPS cells (SPH-0103) on the 10th to 14th day after the change of the culture medium. (A in FIG. 36). In addition, a set of cobblestone cells indicating that undifferentiated blood cells are proliferating under mesenchymal stem cells was confirmed (part indicated by an arrow b in FIG. 36).
さらに、その後、それらの細胞を回収して、ＦＢＳの代わりに自家血清を用いて血液コロニー培養を行った。すなわち、当該細胞を、３０％ 自家血清、０．９％ メチルセルロース、１％ ＢＳＡ（純度１００％）、５０μＭ ２−メルカプトエタノール、１００ｎｇ／ｍｌ ｈＳＣＦ、２０ｎｇ／ｍｌ ｈＩＬ−３、１００ｎｇ／ｍｌ ｈＩＬ−６、１０ｎｇ／ｍｌ ヒトＧ−ＣＳＦ、５Ｕ／ｍｌ ｈＥＰＯ、１０ｎｇ／ｍｌ ｈＴＰＯ及びαメディウムからなる培養液にて、３７℃、５％ＣＯ２の条件下で血液コロニー培養を行った。得られた結果を図３７及び３８に示す。Furthermore, those cells were then collected, and blood colony culture was performed using autologous serum instead of FBS. That is, the cells were treated with 30% autologous serum, 0.9% methylcellulose, 1% BSA (purity 100%), 50 μM 2-mercaptoethanol, 100 ng / ml hSCF, 20 ng / ml hIL-3, 100 ng / ml hIL-6. Blood colonies were cultured in a culture solution consisting of 10 ng / ml human G-CSF, 5 U / ml hEPO, 10 ng / ml hTPO and α-medium under conditions of 37 ° C. and 5% CO 2 . The obtained results are shown in FIGS.
図３７に示した結果から明らかなように、前記自家血清を用いた血液コロニー培養によって、赤血球系細胞から構成される赤血球系コロニー（図３７ a、ｄ）、好中球、マクロファージ・単球等の骨髄球系細胞から構成される骨髄球系コロニー（図３７ ｂ、ｅ）、赤血球系細胞、骨髄球系細胞及び巨核球系細胞から構成される混合コロニー（図３７ ｃ、ｆ）が形成された。また、表２に、独立して４回施行した自家間葉系幹細胞との共培養によって形成された血液コロニーについての結果を示す。なお表中、「ｉＰＳコロニーの数」は、１回の前記自家血清を用いた血液コロニー培養に供したｉＰＳ細胞のコロニーの数を示す。 As is clear from the results shown in FIG. 37, erythroid colonies composed of erythroid cells (FIGS. 37a and 37d), neutrophils, macrophages / monocytes, etc. by blood colony culture using the autologous serum. A myeloid colony (Fig. 37b, e) composed of the myeloid cells of the cell, and a mixed colony (Fig. 37c, f) composed of the erythroid cells, myeloid cells and megakaryocytes. It was. Table 2 shows the results for blood colonies formed by co-culture with autologous mesenchymal stem cells, which were performed four times independently. In the table, “number of iPS colonies” indicates the number of colonies of iPS cells subjected to blood colony culture using one autologous serum.
また、これらの赤血球コロニーに含まれる赤血球系細胞におけるβグロビン等の発現を＜血液細胞の同定＞に記載の方法と同様の方法にて検討したところ、ほぼ１００％の赤血球系細胞にαグロビン、γグロビンが、また約７０％の赤血球系細胞にβグロビンが発現していた（図３８ 参照）。従って、これらの血液コロニーは二次造血を起源としており、約７０％の赤血球系細胞は成人型ヘモグロビンを合成していることが明らかになった。 In addition, when the expression of β globin and the like in erythroid cells contained in these erythroid colonies was examined by the same method as described in <Identification of blood cells>, α globin, γ globin and β globin were expressed in about 70% of erythroid cells (see FIG. 38). Therefore, it was revealed that these blood colonies originated from secondary hematopoiesis, and about 70% of erythroid cells synthesized adult hemoglobin.
従って、上述のようにヒトＰＬを用いずとも、自家血清を用いて、間葉系幹細胞を分化誘導し、分化誘導された間葉系幹細胞と自家ｉＰＳ細胞とを共培養することで、ヒトｉＰＳ細胞から異種動物血清（ＦＢＳ等）やａｌｌｏｇｅｎｅｉｃ（同種異系）血清を用いずに、血液細胞を誘導できることが明らかになった。 Therefore, without using human PL as described above, autologous serum is used to induce differentiation of mesenchymal stem cells, and by coculturing the differentiation-induced mesenchymal stem cells and autologous iPS cells, human iPS It was revealed that blood cells can be induced from cells without using heterologous animal serum (such as FBS) or allogeneic (allogeneic) serum.
また、実際に、一人のドナーから樹立されたｉＰＳ細胞（ＳＰＨ−０１０３）から、そのドナーの血清を用いて、動物細胞・動物血清を用いることなく、赤血球、白血球、巨核球等の血液細胞が分化誘導できることも明らかになった。 In addition, blood cells such as red blood cells, white blood cells, megakaryocytes, etc. can be obtained from iPS cells (SPH-0103) established from a single donor without using animal cells or animal serum. It was also revealed that differentiation can be induced.
以上説明したように、本発明によれば、ヒト由来のＥＳ細胞、ｉＰＳ細胞にかかわらず、ヒト由来の多能性幹細胞から樹立した間葉系幹細胞と共培養することにより、自己又は自己以外のヒト由来の多能性幹細胞から、血液細胞等の機能細胞に分化誘導ができる。 As described above, according to the present invention, by co-culturing with mesenchymal stem cells established from human-derived pluripotent stem cells regardless of human-derived ES cells and iPS cells, self or other than self Differentiation can be induced from human-derived pluripotent stem cells to functional cells such as blood cells.
本発明の方法により分化誘導することによって得られる血液細胞等の機能細胞は、異種細胞の混入がなく、さらにヒト由来の血清溶解液を用いて樹立した間葉系幹細胞の共培養下で分化誘導すればり、異種血清を混入させることもない。このため本発明の方法により得られた各種細胞を用いれば、動物細胞の混入、未知の微生物やウィルスやプリオンによる感染等の危険性のない、極めて安全性の高い再生医療等を実現することができる。 Functional cells such as blood cells obtained by inducing differentiation by the method of the present invention are free of foreign cell contamination, and further induced to differentiate under co-culture of mesenchymal stem cells established using human-derived serum lysate. In other words, no heterogeneous serum is mixed. For this reason, if various cells obtained by the method of the present invention are used, it is possible to realize extremely safe regenerative medicine without risk of contamination with animal cells, infection with unknown microorganisms, viruses, and prions. it can.
配列番号１〜６
＜２２３＞ 人工的に合成されたプライマーの配列SEQ ID NOs: 1-6
<223> Artificially synthesized primer sequences
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| WO2015112581A1 (en) | 2014-01-21 | 2015-07-30 | The Medical College Of Wisconsin, Inc. | Methods for selective inhibition of pluripotent stem cells |
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| US10246679B2 (en) | 2015-03-02 | 2019-04-02 | The University Of Tokyo | Method for producing pluripotent stem cell having improved chimera forming ability from primed pluripotent stem cell, and composition and combination therefor |
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