JP2004350601A - Method for differentiating embryonic stem cell of primatial animal to hemopoietic cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for supplying a hemopoietic cell, a differentiated cell or the like of a primatial animal. <P>SOLUTION: The method for differentiating the embryonic stem cell of the primatial animal to the hemopoietic cell comprises keeping the embryonic stem cell of the primatial animal in a condition suitable for differentiation induction of the hemopoietic cell, transplanting the obtained cell in a fetus in the uterus of a pregnant sheep, and obtaining the hemopoietic cell of the primatial animal from the born fetus of the sheep. The method for producing the hemopoietic cell of the primatial animal comprises using a stroma cell strain from which a macrophage colony-stimulating factor is deleted as a feeder cell, keeping the embryonic cell of the primatial animal on the feeder cell, transplanting the obtained cell derived from the primatial animal in the fetus in the uterus of the pregnant sheep, growing the fetus so as to be born, and separating the hemopoietic cell of the primatial animal differentiated from the embryonic stem cell of the primatial animal from the born fetus. The hemopoietic cell obtained by the production method, and the method for creating a chimera sheep producing the hemopoietic cell of the primatial animal by keeping the embryonic stem cell of the primatial animal in a condition suitable for the differentiation induction, and transplanting the obtained cell in the fetus in the uterus of the pregnant sheep are also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０１３６】
また、ヒト臍帯血ＣＤ３４＋細胞を移植したヒツジ胎仔を対照として用いた。このとき、β２−ミクログロブリンに対するプライマー対として、外部プライマー対：
ｈＢ２ＭＧ−２Ｆ ５’−ＧＴＣＴＧＧＡＴＴＴＣＡＴＣＣＡＴＣＴＧ−３’ （配列番号：７）と前記ｈＢ２ＭＧ ５Ｒとからなるプライマー対、及び
内部プライマー対：
ｈＢ２ＭＧ−２Ｆ ５’−ＧＴＣＴＧＧＧＴＴＴＣＡＴＣＣＡＴＣＣＧ−３’ （配列番号：３と同じ）と前記ｈＢ２ＭＧ−３Ｒとからなるプライマー対を用い、前記ＤＮＡを鋳型として、半定量的ＰＣＲを行なった。なお、ＰＣＲ条件は、 反応液〔組成：Ｈ_２０ ３０．７５μｌ、１０×ＰＣＲ緩衝液 ５μｌ、ｄＮＴＰ（各２．５ｍＭ） ４μｌ、Ｅｘ Ｔａｑ（５Ｕ／μｌ） ０．２５μｌ、プライマー（各１０ｐＭ） 各２．５μｌ、試料由来ＤＮＡ（２５０ｎｇ相当量） ５μｌ〕を用い、９５℃で５分間のインキュベーションの後、９５℃で１分間と５８℃で１分間と７２℃で１分間とを１サイクルとして、外部増幅時には、２５サイクル、内部増幅時には、３０サイクルを行ない、７２℃で５分間インキュベーションして、４℃で維持する条件とした。
【０１３７】
なお、定量のために、下記表２に示す希釈系列の試料を作製し、用いた。
【０１３８】
【表２】

【０１３９】
ＰＣＲにより生成した産物を、２％ アガロースゲル（０．０１％ ＥｔＢｒ含有）による電気泳動に供した。
【０１４０】
その結果、ヒト臍帯血ＣＤ３４＋細胞（対照群）をヒツジ胎仔の肝実質内に移植後、約４ヶ月の個体の末梢血からのみ０．０１％未満のキメラ率でサル由来のβ２−ミクログロブリンの塩基配列が検出された。
【０１４１】
実施例６ 造血系組織及び細胞のコロニーアッセイ及びコロニー解析
初代培養用に分離した細胞で造血コロニーアッセイを行なった。
【０１４２】
凍結保存されていた細胞のクライオチューブを３７℃の恒温槽で溶解し、９ｍｌの２％ ＦＢＳ−ＩＭＤＭを入れた１５ｍｌ コニカルチューブ中に懸濁し、得られた懸濁液の一部を用いて細胞数を計数した。ついで、懸濁液を、１３００ｒｐｍで４分間遠心分離し、得られたペレットを、２％ ＦＢＳ−ＩＭＤＭで１×１０^６細胞／ｍｌとなるように懸濁し、細胞浮遊液を得た。さらに、２％ ＦＢＳ−ＩＭＤＭで１０倍希釈して、１×１０^５細胞／ｍｌの細胞浮遊液を調製した。
【０１４３】
１４ｍｌのポリスチレン丸底チューブに、商品名：Ｍｅｔｈｏｃｕｌｔ ＧＦ＋〔ステムセルテクノロジーズ（ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ）社製、カタログ番号：ＳＴ−０４４３５〕を４ｍｌ入れ、細胞浮遊液を４００μｌ添加し、キャップを閉めて激しく振って攪拌した。その後、泡が消えるまで約５分間静置した。
【０１４４】
ついで、得られた細胞を含む培地 １．１ｍｌを、１８Ｇ針を付けた２．５ｍｌ 注射器で、３．５ｍｍ ディッシュの中央に注入し、ディッシュの底に均一に広げた。
【０１４５】
１５ｃｍ ペトリ皿に、３．５ｃｍ ディッシュを６枚と、リン酸緩衝化生理食塩水 ３ｍｌを含む ３．５ｃｍ ディッシュを保湿用として入れ、３７℃、５％ ＣＯ_２の条件下でインキュベーションした。
【０１４６】
コロニーの計数は、インキュベーション開始から、１４日目に行なった。
【０１４７】
しかしながら、かかる方法では、サル由来の造血前駆細胞だけでなくヒツジ由来の造血前駆細胞もコロニーを形成していることがわかった。
【０１４８】
そこで、コロニーを形成している培地からコロニーを取り、各コロニーからＤＮＡを抽出し、得られたＤＮＡを鋳型とし、サルβ２−ミクログロブリンに対するプライマー対を用いて、ＰＣＲを行ない、サル由来の造血前駆細胞の存在を確認した。
【０１４９】
サルβ２−ミクログロブリンに対するプライマー対として、外部プライマー対：前記ＣＢ２ＭＧ−２ＦとｈＢ２ＭＧ ５Ｒとからなるプライマー対、及び
内部プライマー対：前記ＣＢ２ＭＧ−２ＦとｈＢ２ＭＧ−３Ｒとからなるプライマー対を用い、前記ＤＮＡを鋳型として、ＰＣＲを行なった。なお、ＰＣＲ条件は、 反応液〔組成：Ｈ_２０ ３０．７５μｌ、１０×ＰＣＲ緩衝液 ５μｌ、ｄＮＴＰ（各２．５ｍＭ） ４μｌ、Ｅｘ Ｔａｑ（５Ｕ／μｌ） ０．２５μｌ、プライマー（各１０ｐＭ） 各２．５μｌ、試料由来ＤＮＡ（２５０ｎｇ相当量） ５μｌ〕を用い、９５℃で５分間のインキュベーションの後、９４℃で３０秒間と５７℃で１分間と７２℃で１分間とを１サイクルとして、外部増幅時及び内部増幅時ともに、２５サイクルを行ない、７２℃で５分間インキュベーションして、４℃で維持する条件とした。得られた産物について、２％ アガロースゲルによる電気泳動により解析した。
【０１５０】
その結果、造血系分化誘導サルＥＳ細胞を用いた場合、図２に示されるように、造血系分化誘導処理をしたＥＳ細胞を腹腔内に移植後１ヶ月の個体の胎仔肝臓で約３０％（ｎ＝１）であり、造血系分化誘導処理をしたＥＳ細胞を肝実質内に移植した出生後（移植後３〜６ヶ月）の個体の骨髄で約１％（ｎ＝３）の頻度でサル由来の造血コロニーがあることがわかった。すなわち、ＥＳ細胞を、造血系分化誘導処理し、ヒツジ胎仔に導入することにより、該ヒツジ胎仔は、サル由来の造血系細胞を産生することがわかった。
【０１５１】
配列表フリーテキスト
配列番号：１は、プライマーＣＢ２ＭＧ−２Ｆの配列である。
【０１５２】
配列番号：２は、プライマーｈＢ２ＭＧ ５Ｒの配列である。
【０１５３】
配列番号：３は、プライマーＣＢ２ＭＧ−２ＦＣＢ又はｈＢ２ＭＧ−２Ｆの配列である。
【０１５４】
配列番号：４は、プライマーｈＢ２ＭＧ−３Ｒの配列である。
【０１５５】
配列番号：５は、プライマーＣβ１の配列である。
【０１５６】
配列番号：６は、プライマーＣβ２の配列である。
【０１５７】
配列番号：７は、プライマーｈＢ２ＭＧ−２Ｆの配列である。
【０１５８】
【発明の効果】
本発明の霊長類動物の胚性幹細胞からの造血系細胞への分化方法によれば、霊長類の造血系細胞を安定に供給することができ、霊長類動物の胚性幹細胞への外来遺伝子の導入等の遺伝子工学的操作を行なうことなく、該霊長類動物の胚性幹細胞を造血系細胞に分化させることができ、霊長類動物の個体にとって異種動物において、該個体にとって同種である造血系細胞を得ることができるという優れた効果を奏する。また、本発明の霊長類動物の造血系細胞の製造方法によれば、霊長類の造血系細胞を安定に供給することができ、霊長類の造血系細胞を大量に供給することができ、霊長類動物の個体にとって異種動物において、該個体にとって同種である造血系細胞を得ることができ、霊長類動物の輸血用血小板を安定に供給することができ、霊長類動物の衣装供養のＣＤ３４＋細胞を安定に供給することができるという優れた効果を奏する。また、本発明の霊長類の造血系細胞を産生するキメラヒツジの作製方法によれば、霊長類の造血系細胞を安定に供給することができるキメラヒツジを得ることができるという優れた効果を奏する。
【０１５９】
【配列表】

【図面の簡単な説明】
【図１】図１は、霊長類動物の胚性幹細胞を、造血系細胞の分化誘導に適した条件で維持し、得られた細胞を示す。パネル（Ａ）は、未分化胚性幹細胞を示し、スケールバーは、１００μｍを示す。パネル（Ｂ）は、ＯＰ９細胞上ＢＭＰ−４の存在下での培養時の細胞を示す。パネル（Ｃ）は、胚性幹細胞を、造血系分化誘導条件に維持して得られた細胞を示し、スケールバーは、１００μｍを示す。
【図２】図２は、移植後に得られたヒツジ胎仔において、サルβ２−ミクログロブリンを検出した結果を示す図である。図中、パネル（Ａ）は、胎仔肝臓、パネル（Ｂ）は、骨髄の結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for supplying hematopoietic cells, differentiated cells and the like of primates. More specifically, the present invention relates to a method for differentiating primate embryonic stem cells into hematopoietic cells, a hematopoietic cell obtained by the differentiation method, a method for producing a chimeric sheep that produces hematopoietic cells of a primate, The present invention relates to a method for producing a chimeric sheep that produces differentiated cells of primates, and a method for producing differentiated cells of primates.
[0002]
[Prior art]
For patients with diseases such as aplastic leukemia, hematopoietic stem cell transplantation represented by bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood stem cell transplantation, etc. is performed to produce blood cells in the patient's body. ing.
[0003]
However, in the above-mentioned hematopoietic stem cell transplantation, it is difficult to stably supply hematopoietic stem cells suitable for the patient to be transplanted, and there are cases where the donor is physically or physically damaged when hematopoietic stem cells are collected. There are disadvantages.
[0004]
Therefore, in order to stably supply hematopoietic stem cells, attempts have been made to transplant hematopoietic stem cells transuterinely into pig fetuses to amplify hematopoietic progenitor cells in the pig body (Non-patent Document 1).
[0005]
On the other hand, in vitro research on the differentiation of embryonic stem cells (hereinafter also referred to as ES cells) for application to regenerative medicine for the purpose of functional recovery, repair, and regeneration of dysfunctional tissues and organs Has been done.
[0006]
As an example of an in vitro differentiation from embryonic stem cells to blood cells, the phenotype of the final hematopoietic stem cells was determined from mouse ES cells into which HoxB4, a homeotic selector gene related to self-renewal of hematopoietic stem cells, was introduced. Differentiation into cells shown (Non-Patent Document 2), differentiation from mouse ES cells to cells presumed to be hematopoietic progenitor cells using OP9 stromal cells (Non-Patent Documents 3 and 4), Rhesus monkey ES cells are cultured on mouse S17 stromal cells and differentiated into hematopoietic stem cells (Non-patent Document 5), and from mouse ES cells to hematopoietic progenitor cells (Non-patent Document 6).
[0007]
Further, Non-Patent Document 5 discloses that by culturing rhesus monkey ES cells in the presence of bone morphogenetic protein-4 (BMP-4), the differentiation of hematopoietic stem cells is 15 as compared to the case in the absence of BMP-4. Doubled increase is disclosed. Furthermore, Non-Patent Document 6 discloses that culturing mouse ES cells in the presence of BMP-4 increases the differentiation of hematopoietic progenitor cells as compared to the case in the absence of BMP-4. Yes.
[0008]
However, in general, cells (neural cells, cardiomyocytes, fetal hematopoietic cells, etc.) appearing early in the development of hematopoietic stem cells, hepatocytes, pancreatic β cells, etc., compared to in vitro differentiation. Cells appearing in a “field” inductive in the later stage have a drawback that differentiation from the ES cells is difficult.
[0009]
Further, in the differentiation method described in Non-Patent Document 2, since ES cells into which HoxB4 related to self-renewal of hematopoietic stem cells is used are used as ES cells, the applicable range of the obtained hematopoietic stem cells is limited. There is a drawback that there is.
[0010]
Furthermore, since the differentiation methods described in Non-Patent Documents 3 and 4 use OP9 stromal cells, it is considered that the yolk sac hematopoiesis (primary hematopoiesis) is actually reproduced and the hematopoietic system is reconstructed. However, it is difficult to induce differentiation of hematopoietic stem cells.
[0011]
[Non-Patent Document 1]
Hiromitsu Nakauchi, 1 other, “3. Establishment of human blood chimera pig and its application”, “Record of 117th Medical Society Symposium Stem Cell and Cell Therapy-[III] Tissue / Organ Stem Cell: Development of Foundation for Clinical Application (2) August 4-6, 2000, p99-106 [online], Internet <URL: http://www.med.or.jp/jams/symposium/kiroku/117/pdf/1170099.pdf >
[Non-Patent Document 2]
Michael Kyba et al., Cell, Vol. 109, April 5, 2002, p29-37.
[Non-Patent Document 3]
Toru Nakano, “5. Differentiation of embryonic stem cells into blood cells”, edited by Takashi Yokota et al., Experimental Medicine Special Issue “Forefront of Stem Cell Research and Application to Regenerative Medicine, Elucidation of Developmental and Differentiation Mechanisms, and Clinical” Published September 25, 2001, Vol. 19, No. 15, p 1966-971
[Non-Patent Document 4]
Toru Nakano et al., Science, Vol. 265, August 19, 1994, p1098-1101
[Non-Patent Document 5]
Fei Li, et al., Blood, Vol. 98, issued July 15, 2001, p335-342
[Non-Patent Document 6]
Brit M. Johansson et al., Molecular and Cellular Biology, Vol. 15, No. 1, published in January 1995, p141-151
[0012]
[Problems to be solved by the invention]
The present invention provides a stable supply of primate hematopoietic cells and the introduction of embryonic stem cells of the primate animal without performing genetic engineering operations such as introduction of a foreign gene into the embryonic stem cell of the primate animal. It is an object of the present invention to provide a method for differentiating hematopoietic cells from primate embryonic stem cells, which enables at least one of differentiation into hematopoietic cells. The present invention also provides a stable supply of primate hematopoietic cells, a large supply of primate hematopoietic cells, a stable supply of primate blood transfusion platelets, It is an object of the present invention to provide a method for producing hematopoietic cells of a primate animal that enables at least one of stably supplying CD34 + cells for transplantation. Furthermore, this invention aims at providing the hematopoietic cell obtained by the said manufacturing method. It is another object of the present invention to provide a method for producing a chimeric sheep that can stably supply primate hematopoietic cells.
[0013]
[Means for Solving the Problems]
That is, the gist of the present invention is as follows.
[1] Embryonic stem cells of primates are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into fetuses in the uterus of pregnant sheep, and then primates from the born sheep fetuses. A method of differentiating primate embryonic stem cells into hematopoietic cells, characterized by obtaining hematopoietic cells of the primate,
[2] (I) using a stromal cell line deficient in macrophage colony-stimulating factor as a feeder cell, maintaining a primate embryonic stem cell on the feeder cell; and
(II) transplanting the cells derived from the primate obtained in the step (I) to a fetus in the uterus of a pregnant sheep, and growing the fetus until birth.
The differentiation method according to [1] above,
[3] The differentiation method according to [2] above, wherein in step (I), embryonic stem cells of primate animals are maintained on feeder cells in the presence of bone morphogenetic protein-4.
[4] (I) using a stromal cell line deficient in macrophage colony-stimulating factor as a feeder cell, and maintaining primate embryonic stem cells on the feeder cell;
(II) transplanting the cells derived from the primate obtained in the step (I) into a fetus in a uterus of a pregnant sheep, and allowing the fetus to grow to birth; and
(III) A step of separating hematopoietic cells of the primate animal differentiated from embryonic stem cells of the primate from the fetus born in the step (II)
A method for producing hematopoietic cells of primates,
[5] Hematopoietic cells obtained by the production method of [4] above, and
[6] A primate animal characterized in that embryonic stem cells of a primate animal are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into a fetus in the uterus of a pregnant sheep. A method for producing a chimeric sheep producing hematopoietic cells of
About.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, embryonic stem cells are treated under conditions for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into a sheep fetus in the uterus of a pregnant sheep. Based on the inventors' surprising discovery that monkey-derived hematopoietic cells are produced.
[0015]
The method for differentiating primate embryonic stem cells from hematopoietic cells of the present invention comprises maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells, and subjecting the resulting cells to pregnancy sheep. A primate hematopoietic cell is obtained from the born sheep fetus.
[0016]
The differentiation method of the present invention has one major feature in maintaining primate embryonic stem cells under conditions suitable for induction of differentiation of hematopoietic cells (hereinafter also referred to as hematopoietic differentiation induction conditions).
[0017]
In the differentiation method of the present invention, in order to maintain embryonic stem cells of primate animals under the above-described conditions for inducing hematopoietic differentiation, even if the undifferentiated embryonic stem cells are transplanted as they are, hematopoietic cells from the undifferentiated embryonic stem cells Surprisingly, the primate embryonic stem cells can be differentiated into hematopoietic cells in spite of the fact that differentiation into the cells does not occur substantially.
[0018]
Further, in the differentiation method of the present invention, since the primate embryonic stem cells are maintained in the hematopoietic differentiation induction conditions, when the primate embryonic stem cells are transplanted into a sheep fetus, It exhibits an excellent effect of being able to differentiate by utilizing the internal environment of the sheep fetus more effectively.
[0019]
Furthermore, in the differentiation method of the present invention, in order to differentiate embryonic stem cells of a primate animal into hematopoietic cells, a foreign gene (for example, a homeotic selector gene) that induces differentiation in the embryonic stem cell of the primate animal. It is advantageous in that it is not substantially required to perform genetic engineering operations such as introduction of. Therefore, the differentiation method of the present invention does not require advanced techniques, expensive machines, reagents and the like, and is useful for supplying cells and tissues for transplantation.
[0020]
In addition, the differentiation method of the present invention has one major feature in that a sheep fetus in the uterus of a pregnant sheep is used.
[0021]
Since the sheep fetus is used in the differentiation method of the present invention, the cells obtained by maintaining the embryonic stem cells of the primate animal under the hematopoietic differentiation induction conditions are surprisingly and essentially primate animals. It exhibits an excellent effect of being able to differentiate into hematopoietic cells derived from.
[0022]
Further, according to the differentiation method of the present invention, since the sheep fetus is used, an excellent effect that the chimera ratio with the primate is increased can be obtained. Therefore, according to the differentiation method of the present invention, the excellent effect that hematopoietic cells derived from primate animals can be obtained in the sheep more efficiently.
[0023]
Furthermore, according to the differentiation method of the present invention, since the fetus in the uterus of the pregnant sheep is used, it has an excellent feature that embryonic stem cells of primate animals can be further transplanted after birth. . Therefore, according to the differentiation method of the present invention, hematopoietic cells derived essentially from primates can be supplied more efficiently and stably.
[0024]
The differentiation method of the present invention is one of the major features of transplanting cells obtained by maintaining embryonic stem cells of primate animals under the above-mentioned conditions for inducing differentiation of hematopoietic systems to sheep fetuses in the uterus of pregnant sheep. There is.
[0025]
The differentiation method of the present invention is derived from a primate animal in order to transplant a cell obtained by maintaining embryonic stem cells of a primate animal under the above-mentioned hematopoietic differentiation induction conditions to a sheep fetus in the uterus of a pregnant sheep. Hematopoietic cells can be supplied efficiently and stably. Furthermore, the differentiation method of the present invention is a method for transplanting cells obtained by maintaining embryonic stem cells of primate animals under the conditions for inducing differentiation of hematopoietic system to a sheep fetus in the uterus of a pregnant sheep. Thus, the non-acting primate cytokine exerts an excellent effect that it can selectively stimulate embryonic stem cells of transplanted primates or hematopoietic cells derived essentially from primates. . Therefore, hematopoietic cells derived from primates can be supplied more efficiently and stably.
[0026]
In this specification, primate animals refer to humans, monkeys, and the like. Examples of the monkey include cynomolgus monkey, rhesus monkey, Japanese monkey, marmoset, and the like.
[0027]
In the present specification, embryonic stem cells (hereinafter also referred to as ES cells) refer to undifferentiated cells having multipotency and self-renewal ability.
[0028]
Specific examples of primate embryonic stem cells used in the differentiation method of the present invention include CMK6 cells.
[0029]
Such embryonic stem cells can be maintained and supplied by using cells such as mouse embryo fibroblasts, which have stopped dividing proliferation, as feeder cells and culturing them in a medium for ES cells.
[0030]
The medium for ES cells may be any medium suitable for embryonic stem cells depending on the type of embryonic stem cells. Specifically, for example, in the case of cynomolgus monkey-derived embryonic stem cell line CMK6, the composition per 200 ml is DMEM / F12 163 ml, fetal calf serum 30 ml (final concentration 15%), L-glutamine 2 ml (final concentration 2 mM), penicillin (100 U / ml) -Streptomycin (100 μg / ml) 2 ml, non-essential amino acid solution 2 ml, 2-mercaptoethanol 1 ml (final concentration 0.1 mM).
[0031]
The maintenance and proliferation of the undifferentiated state of the embryonic stem cells can be performed, for example, by culturing under conditions such as the presence of leukemia inhibitory factor (LIF), depending on the type of embryonic stem cells.
[0032]
In addition, the culture conditions for maintaining and proliferating the undifferentiated state of the embryonic stem cells vary depending on the type of embryonic stem cells. For example, 5% CO₂In the gas phase, it may be maintained at 36 to 38 ° C, preferably 37 ° C.
[0033]
The feeder cells used for maintaining and proliferating the undifferentiated state of the embryonic stem cells vary depending on the cell type, and can be maintained, proliferated, etc. in a D10 medium, for example.
[0034]
The culture conditions of the feeder cells vary depending on the cell type, but for example, 5% CO2.₂In the gas phase, it may be maintained at 36 to 38 ° C, preferably 37 ° C.
[0035]
In the present specification, examples of hematopoietic cells include hematopoietic stem cells and cell groups obtained by differentiation from the hematopoietic stem cells, that is, cells having properties such as erythroblasts, myeloid cells, megakaryocytes, and lymphocytes. Specific examples include erythroblasts and myelocytes.
[0036]
In the present invention, conditions suitable for induction of differentiation of hematopoietic cells, that is, conditions for inducing differentiation of hematopoietic cells include, for example, conditions for culturing in the presence of type IV collagen, α-minimum essential medium (α-MEM), etc. Various conditions such as conditions for culturing in the presence, conditions for culturing on feeder cells suitable for hematopoietic differentiation, conditions for adhering to the dish after preparation of the embryoid body, conditions for any combination of these, and the like can be mentioned.
[0037]
In the present invention, "the cells obtained by maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells" are evaluated when hematopoietic colony forming ability and expression of surface markers are evaluated. It is a cell in the differentiation stage that becomes negative. Such cells may be so-called mesoderm cells or cells close to the state, properties, etc. of the mesoderm cells. Specifically, cells obtained by maintaining the cynomolgus monkey-derived embryonic stem cell line CMK6 for about 6 days under conditions suitable for inducing differentiation of hematopoietic cells can be mentioned.
[0038]
The hematopoietic colony forming ability can be evaluated by, for example, a colony assay described later.
[0039]
As the surface marker, CD45, TER119, α₄-Integrin, VE-cadherin, c-Kit, Sca-1, CD34, CD13, CD14, GPIIb / IIIa, CD3, CD4, CD8, sIgM, CD19 and the like.
[0040]
Examples of feeder cells used for maintaining hematopoietic differentiation induction conditions include stromal cell lines lacking macrophage stimulating factor, mouse bone marrow cell line lacking macrophage stimulating factor, and mouse yolk sac lacking macrophage stimulating factor. Examples include cells obtained by treating cell lines and the like with mitomycin C treatment or X-ray treatment. The stromal cell line deficient in the macrophage stimulating factor is OP9 cell line, and the mouse bone marrow cell line is S17 cell line [Collins et al. Immunol. 1987, Vol. 138, p1082-1087], the mouse yolk sac cell line is C166 cell line [Wang et al., In Vitro Cell. Dev. Biol. Anim. 1996, Vol. 32, p292-299].
[0041]
The feeder cells used for maintaining the hematopoietic differentiation induction conditions can be prepared by, for example, seeding in a gelatin-coated culture vessel using a MEM medium (Minimum Essential medium Eagle). The feeder cells may be seeded to such an extent that the culture vessel is covered without any gap.
[0042]
The culture conditions of the feeder cells vary depending on the type of cells used, but in the case of the stromal cell line OP9 cells, for example, 5% CO 2₂In the gas phase, it is preferably maintained at 36 to 38 ° C, particularly preferably 37 ° C.
[0043]
Specifically, the feeder cells used when maintaining the hematopoietic differentiation induction conditions, when using OP9 cells as feeder cells,
-Medium for OP9 cells {eg, composition per 1250 ml: α-MEM (Gibco, catalog number: 12000-022) 980 ml, fetal calf serum 250 ml (final concentration 20%), penicillin (100 U / ml) -Streptomycin (100 μg / ml) 10 ml, 200 mM L-glutamine solution 10 ml} at 37 ° C., 5% CO₂Cultured under conditions,
-The cells that have adhered to the culture and grown to confluence or immediately before are washed twice with phosphate buffered saline;
-Treating the cells on the dish after washing with trypsin;
-Adding the OP9 cell culture medium to recover the medium containing the cells;
-The obtained cells are subcultured 1: 4-5 in the medium for OP cells,
-37 ° C, 5% CO₂Incubate until confluent under conditions,
The cells obtained are inactivated with mitomycin C,
-Obtained cells (eg 1 × 10⁵Cells / ml) on a gelatin-coated culture dish and seeded in the OP9 cell culture medium.
Can be obtained.
[0044]
The pregnancy sheep used in the present invention is 40 days or more after pregnancy, preferably 50 days or more, more preferably 60 days or more from the viewpoint of safety, for example, prevention of miscarriage, to reduce immune rejection, From the viewpoint of obtaining a sufficient survival rate of transplanted cells, it is desirable that it is 85 days or less after pregnancy, preferably 75 days or less, more preferably 60 days or less. Therefore, it is desirable that the fetus in the uterus be 50 to 80 days pregnant.
[0045]
As a differentiation method of the present invention, more specifically,
(I) using a stromal cell line deficient in macrophage colony-stimulating factor as a feeder cell, and maintaining primate embryonic stem cells on the feeder cell; and
(II) transplanting the cells derived from the primate obtained in the step (I) to a fetus in the uterus of a pregnant sheep, and growing the fetus until birth.
The method containing is mentioned.
[0046]
In the step (I), it is desirable to seed the embryonic stem cells of the primate animal so as to increase the density from the viewpoint of further differentiation.
[0047]
In the step (I), it is desirable to culture on feeder cells in a medium corresponding to embryonic stem cells of primate animals.
[0048]
In addition, what is necessary is just to change a culture medium suitably during culture | cultivation.
[0049]
In the step (I), the embryonic stem cell of the primate is 5% CO 2 on the feeder cell.₂In the gas phase, it is preferably maintained at 36 to 38 ° C, particularly preferably 37 ° C.
[0050]
Specifically, for example, in the case of a cynomolgus monkey embryonic stem cell line CMK6, a differentiation medium (composition per 100 ml: IMDM (Iscove's Modified Dulbecco's Medium) 84 ml, 8% horse serum 8 ml, 8% fetal calf serum 8 ml 5 × 10^-6M Hydrocortisone 0.18 μg, BMP-4 2 μg (final concentration 20 ng / ml), SCF 2 μg (final concentration 20 ng / ml), IL-3 2 μg (final concentration 20 ng / ml), IL-6 1 μg (final concentration 10 ng / ml) ), VEGF 2 μg (final concentration 20 ng / ml), G-CSF 2 μg (final concentration 20 ng / ml), Flt. 3 ligand 2 μg (final concentration 10 ng / ml), EPO 200 U (2 U / ml)] 5 ml, and the resulting cell suspension was placed on feeder cells at 5 × 10 5⁵Cells are seeded with an appropriate amount of cells, then 37 ° C., 5% CO 2₂The conditions which culture | cultivate on conditions for 6 days are mentioned.
[0051]
The cells obtained by carrying out the step (I) correspond to the “cells obtained by maintaining primate embryonic stem cells under conditions suitable for induction of hematopoietic cell differentiation”.
[0052]
In step (I), from the viewpoint of obtaining hematopoietic cells more efficiently, it is preferable that the primate be added in the presence of bone morphogenetic protein-4, on feeder cells, and in the absence of leukemia inhibitory factor, in addition to the above-mentioned hematopoietic differentiation induction conditions It is desirable to maintain embryonic stem cells of primates.
[0053]
The cells obtained by carrying out the step (I) are then transplanted into a fetus in the uterus of a pregnant sheep, and the fetus is grown until it reaches birth [step (II)].
[0054]
The cells used for transplantation are treated with trypsin and, for example, 0.1% BSA (bovine serum albumin) / HBSS (Hanks balanced salt aqueous solution) is used to improve the engraftment rate, chimera rate, etc., for example, 1 × 10⁶Cells or more, preferably 1 × 10⁶Cells to 1 × 10⁹Cells, preferably 1 × 10⁷Cells to 1 × 10⁹It can be prepared by suspending into cells.
[0055]
In step (II), transplantation of cells into the fetus can be performed, for example, by puncture injection of transplantation cells into the liver, liver parenchyma, intraperitoneal cavity, heart, umbilical cord, or the like.
[0056]
Transplant surgery is, for example,
-Pregnant sheep on the 60th day of gestation are accustomed to the breeding environment at the time of transplantation 1 week to 10 days before transplantation,
-Anesthetize the sheep mother,
-Fix the sheep in the proper position for the treatment,
-In order to facilitate transplantation of cells to the transplant site with a clean operation, for example, when transplanting cells into the abdominal cavity or liver parenchyma, the abdominal midline incision is followed by uterine maternal extraperitoneal Reversing
Or the like.
[0057]
When transplanting cells into the abdominal cavity, for example, the ovine myometrium is incised, the sheep fetus is driven into the exposed egg membrane, and the cells for transplantation into the fetal abdominal cavity are directly viewed through the transparent egg membrane. And then the myometrium, peritoneal muscle layer and skin are closed and closed. In addition, when transplanting cells into the liver parenchyma, if the cells for transplantation are punctured and injected into the liver parenchyma of the sheep fetus under ultrasound guide from the uterine wall, the peritoneal muscle layer and the skin are sutured and closed in layers. Good.
[0058]
In step (II), after the transplantation operation, the pregnant sheep is raised until birth, for example, under the same conditions as normal breeding.
[0059]
The fact that the sheep fetus obtained in step (II) is essentially producing hematopoietic cells derived from primates is that appropriate tissues such as liver, bone marrow, etc. are collected from the sheep fetus. Obtaining a cell for primary culture, and (1) conducting a hematopoietic colony assay on the obtained cell, (2) a marker gene specific to a primate animal for the hematopoietic colony formed in (1) above. It can be assessed by examining expression.
[0060]
The (1) hematopoietic colony assay is, for example,
(1) Obtain a tissue biopsy of the liver, bone marrow, etc. from a sheep fetus,
(2) The obtained tissue is treated appropriately according to the tissue such as trypsin treatment, DNase I treatment, lysis treatment, etc., to obtain cells,
(3) The obtained cells are suspended in 2% FBS (fetal bovine serum) -IMDM to obtain a cell suspension.
(4) The obtained cell suspension and MeC medium {for example, trade name: MethoCultGF + [manufactured by StemCell Technologies, catalog number: ST-04435] etc.} are mixed and stirred.
(5) Next, the medium containing the obtained cells was injected into the center of the dish, and the culture was performed at 37 ° C., 5% CO 2.₂Incubate under conditions of 14 days
Is done.
[0061]
In addition, the expression of the marker gene specific to the primate animal of the above (2) is performed by extracting the DNA from the colony formed by the hematopoietic colony assay by a conventional method. It can be evaluated by detecting the gene by hybridization using a probe specific to a specific marker gene, PCR using a specific primer pair, or the like.
[0062]
In the differentiation method of the present invention, the cells obtained in step (I) may be further transplanted into the sheep fetus obtained in step (II). By further transplanting the cells obtained in step (I), primate hematopoietic cells can be supplied efficiently and stably.
[0063]
In the differentiation method of the present invention, a cytokine specific to a primate animal may be administered to the sheep fetus obtained in the step (II). Administration of cytokines to sheep fetuses enables selective stimulation of differentiation into hematopoietic cells derived essentially from primates within the sheep fetus.
[0064]
Examples of the cytokine include stem cell factor (SCF), basic fibroblast growth factor (bFGF), oncostane M (OSM), flk2 / flk3 ligand, interleukin-1, interleukin-3, granulocyte colony stimulating factor ( G-CSF), chemokine, erythropoietin, thrombopoietin, etc., and any combination thereof.
[0065]
Evaluation of hematopoietic cells of primate animals obtained by the differentiation method of the present invention is, for example, CD45, TER119, α₄-Integrin, VE-cadherin, c-Kit, Sca-1, CD34 (stem cell marker), CD13 (myelocyte marker), CD14 (myelocyte marker), GpIIb / IIIa (megakaryocyte marker), CD3 (T cell marker) ), CD4 (T cell marker), CD8 (T cell marker), sIgM (B cell marker), CD19 (B cell marker) and the like.
[0066]
The differentiation method of the present invention is used for production of primate hematopoietic cells. Accordingly, the present invention provides a method for producing primate hematopoietic cells.
[0067]
The method for producing hematopoietic cells of the primate animal of the present invention comprises:
(I) using a stromal cell line deficient in a macrophage colony-stimulating factor as a feeder cell, and maintaining embryonic stem cells of a primate animal on the feeder cell;
(II) transplanting the cells derived from the primate obtained in the step (I) into a fetus in a uterus of a pregnant sheep, and allowing the fetus to grow to birth; and
(III) A step of separating hematopoietic cells of the primate animal differentiated from embryonic stem cells of the primate from the fetus born in the step (II)
It is a method including.
[0068]
The production method of the present invention has one major characteristic in maintaining embryonic stem cells of primate animals under conditions for inducing hematopoietic differentiation. Therefore, according to the production method of the present invention, in order to maintain the embryonic stem cells of primate animals under the hematopoietic differentiation induction conditions, even if the undifferentiated embryonic stem cells are transplanted as they are, the undifferentiated embryonic stem cells In spite of the fact that differentiation into hematopoietic cells does not occur substantially, it is surprisingly effective that hematopoietic cells can be produced from embryonic stem cells of the primate animal.
[0069]
Further, in the differentiation method of the present invention, when embryonic stem cells of a primate animal are transplanted into a sheep fetus, the embryonic stem cell can more effectively utilize the internal environment of the sheep fetus, It exhibits the excellent effect of being able to stably supply hematopoietic cells of primates.
[0070]
In addition, the production method of the present invention has one major feature in that a sheep fetus in the womb of a pregnant sheep is used.
[0071]
Since the sheep fetus is used in the production method of the present invention, the chimera rate with the primate is increased, whereby the primate hematopoietic cells are converted from the primate embryonic stem cells. It exhibits an excellent effect that a large amount of hematopoietic cells can be supplied.
[0072]
Furthermore, according to the production method of the present invention, primate embryonic stem cells can be further transplanted after birth, and the primate embryo transplanted by a primate cytokine that does not act on sheep. Because hematopoietic stem cells or hematopoietic cells derived essentially from primates can be selectively stimulated, hematopoietic cells derived essentially from primates can be supplied more efficiently and stably. Can do.
[0073]
Further, according to the production method of the present invention, blood transfusion platelets and transplanted CD34 + cells can be stably supplied.
[0074]
The steps (I) and (II) in the production method of the present invention are the same as the steps (I) and (II) in the differentiation method of the present invention.
[0075]
In the step (III), hematopoietic cells of the primate differentiated from the embryonic stem cells of the primate are separated from the fetus born in the step (II).
[0076]
In step (III), the sheep fetus is desirably an individual as early as possible after birth from the viewpoint of chimera rate.
[0077]
Further, as in the case of the differentiation method, from the viewpoint of efficiently and stably supplying primate hematopoietic cells, the cells obtained in step (I) were introduced into the sheep fetus born in step (II). May be further transplanted, and the above cytokines specific to primates may be administered.
[0078]
In step (III), the separation of primate hematopoietic cells is, for example,
1) Collecting liver, bone marrow, blood (peripheral blood, umbilical cord blood), thymus, spleen, etc. from sheep fetuses,
2) From the obtained organ, obtain a cell group by an appropriate method,
3) Isolating hematopoietic cells derived from primates from the obtained cell population.
Or the like.
[0079]
In the step 3), for example, a flow cytometry using a marker specific to a primate animal or an antibody against the marker, a method using an immunobead, and the like are performed.
[0080]
The “marker specific to a primate animal” may be any marker that crosses primates and does not cross sheep, and includes HLA-ABC, β2-microglobulin, CD45 and the like.
[0081]
The evaluation of primate hematopoietic cells obtained by the production method of the present invention can be performed by, for example, CD45, TER119, α, as in the differentiation method.₄-It can be performed by using the presence or absence of expression of integrin, VE-cadherin, c-Kit, Sca-1, CD34, CD13, CD14, GPIIb / IIIa, CD3, CD4, CD8, sIgM, CD19, etc. as an index.
[0082]
In addition, hematopoietic cells of primate animals obtained by the production method of the present invention are also included in the present invention. Such hematopoietic cells are not particularly limited, but are expected to be used as, for example, transplantation materials for angiogenesis therapy, and can be applied to ischemic diseases such as myocardial infarction.
[0083]
In addition, chimeric sheep that produce hematopoietic cells of primates can be produced by the same operation as the differentiation method of the present invention. Accordingly, a method for producing a chimeric sheep that produces hematopoietic cells of a primate animal is also included in the present invention.
[0084]
The method for producing a chimeric sheep producing hematopoietic cells of a primate animal according to the present invention comprises maintaining embryonic stem cells of a primate animal under conditions suitable for inducing differentiation of hematopoietic cells, and obtaining the obtained cells from a pregnant sheep. One major feature is the transplantation into fetuses in the womb.
[0085]
According to the method for producing a chimeric sheep of the present invention, a chimeric sheep capable of supplying primate hematopoietic cells more stably can be obtained.
[0086]
Since the chimeric sheep obtained by the method for producing a chimeric sheep of the present invention has cells derived from primates in an immunologically tolerant state, the chimeric sheep has differentiation induction of hematopoietic cells. The cells obtained can be further transplanted by maintaining the conditions suitable for the above. In addition, the chimeric sheep obtained by the method for producing a chimeric sheep of the present invention selectively differentiates into hematopoietic cells derived from primates in the body by administering a specific cytokine to a primate animal. Can irritate.
[0087]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by this Example. In the following examples, “%” represents “% by weight” unless otherwise specified. CO₂, O₂And N₂The “%” notation in FIG.
[0088]
Example 1 Culture of monkey embryonic stem cells
(1) Preparation of feeder cells
Mouse embryo fibroblasts BALB / cAJc1 (H2 antigen haplotype H-2d) (supplied by Claire Japan) up to 5 passages were added to D10 medium [composition: 10% FCS, containing mitomycin C at a final concentration of 10 μg / ml] By culturing in DMEM] for 2 to 4 hours, cell division and growth were stopped and inactivated. Subsequently, the medium containing mitomycin C was removed. The treated cells were washed with phosphate buffered saline. The washed cells were treated with trypsin / EDTA (0.05% trypsin, 1 mM EDTA) to obtain a cell suspension, and then the cell suspension was centrifuged to obtain cells.
[0089]
Subsequently, the obtained cells were suspended in D10 medium and gelatin-coated 6 cm culture dish (manufactured by FALCON, trade name: Tissue culture dish) 1 × 10⁶Seeds to be individual. Cells obtained by culturing until adhering to the dish were used as feeder cells.
[0090]
(2) Culture of cynomolgus monkey-derived ES cell line CMK6
The D10 medium was removed from the feeder cell culture of Example 1 (1), and the feeder cells were washed with phosphate buffered saline.
[0091]
Next, cynomolgus monkey-derived ES cells CMK6 that had been cryopreserved were transferred to ES cell medium [composition per 200 ml: DMEM-F12 (manufactured by Gibco)] 163 ml, fetal calf serum 30 ml (final concentration 15%), L- Glutamine 2 ml (final concentration 2 mM), penicillin (100 U / ml) -streptomycin (100 μg / ml) 2 ml, non-essential amino acid solution [manufactured by Gibco] 2 ml, 2-mercaptoethanol 1 ml (final concentration 0.1 mM)] Suspended in 5 ml. The obtained cell suspension was seeded on the feeder cells. The culture is performed at 37 ° C. and 5% CO.₂Performed under conditions. The ES cell medium was changed once every two days.
[0092]
After culturing for 7 days, the old medium was aspirated and removed, and the cells were washed with phosphate buffered saline. Then, 1 ml of 0.25% trypsin / HBSS (Hanks balanced salt solution) was added to the cells on the dish and incubated at 37 ° C. for 4 to 5 minutes. Thereafter, the bottom of the dish was tapped at room temperature to detach the cell colonies.
[0093]
Next, undifferentiated cell colonies were recovered from the cell colonies by pipetting with a 5 ml pipette using the ES cell medium. The collected cells were suspended in the ES cell medium, and 5 ml of the obtained cell suspension was seeded on feeder cells on a 6 cm dish. Thereafter, the cells were passaged every 2 to 4 days according to the size of the colonies.
[0094]
The obtained undifferentiated ES cells were used for transplantation into sheep fetuses. Such undifferentiated ES cells were used for the following induction of hematopoietic differentiation.
[0095]
The undifferentiated ES cells were cultured in a medium [composition: 15% FCS / DMEM-F12] to obtain embryoid bodies. Such embryoid bodies were used for transplantation into sheep.
[0096]
Example 2 Induction of hematopoietic differentiation of monkey embryonic stem cells
(1) Preparation of feeder cells
Macrophage colony-stimulating factor dysfunctional mouse (C57BL / 6 × C3H) F2-op / op mouse stromal cell line prepared from neonatal calvaria, preadipocyte line OP9 cells for OP9 cells Medium {Composition per 1250 ml: α-MEM [manufactured by Gibco, catalog number: 12000-022] 980 ml, fetal calf serum 250 ml (final concentration 20%), penicillin (100 U / ml) -streptomycin (100 μg / ml) ) 10 ml, 200 mM L-glutamine solution 10 ml} at 37 ° C., 5% CO₂Cultured under conditions. Thereafter, the cells that adhered in the culture and grew to confluence or immediately before that were washed twice with phosphate buffered saline. Subsequently, 0.1% trypsin-EDTA (2 ml / 10 cm dish) was added to the cells on the dish after washing, and the mixture was incubated for 5 minutes in an incubator.
[0097]
The OP9 cell culture medium was added to the dish, and the medium containing the cells was collected in a 50 ml conical tube. The medium containing the collected cells was centrifuged at 1500 rpm for 5 minutes. The obtained cells were added to the OP cell culture medium at 1: 4 to 1: 5 (about 2.5 × 10 5⁵Cells ~ 4 x 10⁵Seeded with cells, 37 ° C, 5% CO₂Under conditions, the cells were cultured until they became confluent.
[0098]
By culturing the obtained cells on a 10 cm dish (manufactured by FALCON) in 10 ml of the OP9 cell medium containing mitomycin C at a final concentration of 10 μg / ml for 2 to 4 hours, cell division and growth were stopped. And inactivated. Subsequently, the medium containing mitomycin C was removed. The treated cells were washed with phosphate buffered saline. The washed cells were treated with trypsin / EDTA (0.05% trypsin, 1 mM EDTA) to obtain a cell suspension, and then the cell suspension was centrifuged to obtain cells.
[0099]
The obtained cells were placed in a gelatin-coated 6 cm culture dish (manufactured by FALCON, trade name: Tissue Culture Dish) in the above OP9 cell culture medium 1: 2 (about 1 × 10 per medium).⁵Cells / ml). The cells obtained by culturing were used as feeder cells in the following hematopoietic differentiation induction culture.
[0100]
(2) Hematopoietic differentiation induction culture
In Example 1, the cynomolgus monkey-derived ES cell lines CMK6 and CMK6 / SeV recovered in 1 ml of 0.25% trypsin / HBSS were differentiated from the medium for differentiation (composition per 100 ml: 84 ml of IMDM, 8 ml of 8% horse serum, 8% fetal calf serum 8ml, 5x10^-6M Hydrocortisone 0.18 μg, BMP-4 2 μg (final concentration 20 ng / ml), SCF 2 μg (final concentration 20 ng / ml), IL-3 2 μg (final concentration 20 ng / ml), IL-6 1 μg (final concentration 10 ng / ml) ), VEGF 2 μg (final concentration 20 ng / ml), G-CSF 2 μg (final concentration 20 ng / ml), Flt. 3 ligand 2 μg (final concentration 10 ng / ml), EPO 200 U (2 U / ml)] suspended in 5 ml, and the resulting cell suspension was used in the hematopoietic differentiation induction culture obtained in Example 2 (1). Seed 1: 3 on feeder cells.
[0101]
Then, 37 ° C, 5% CO₂Cultured under conditions for 6 days. During culture, the medium was changed as appropriate. The obtained cells were used for the following transplantation and the like as hematopoietic differentiation induction-treated cells (FIG. 1).
[0102]
(3) Cell processing for transplantation
The medium is aspirated and removed from the dish of the hematopoietic differentiation-inducing cells obtained in Example 2 (2), and 1 ml of 0.25% trypsin / HBSS is added to the dish at 37 ° C., 5% CO₂Incubated for 4 minutes under the following conditions.
[0103]
Subsequently, the cells on the feeder cells were collected in a 50 ml conical tube containing 20 ml of the differentiation medium together with the feeder cells with a scraper [trade name: cell scraper-M, Sumilon (manufactured by SUMILON)], 800 rpm, Centrifuged for 4 minutes.
[0104]
The supernatant was aspirated and removed, and the cells were suspended in 20 ml of the differentiation medium in a 50 ml conical tube to obtain a cell suspension.
[0105]
Immediately before transplantation, the cell suspension was centrifuged at 800 rpm for 4 minutes. The supernatant was aspirated and removed, and then the cells obtained were washed by adding 20 ml of 0.1% BSA / HBSS, suspending and centrifuging at 800 rpm for 4 minutes.
[0106]
Thereafter, the supernatant was aspirated and removed, and the cells were suspended in 0.4 ml of 0.1% BSA / HBSS. The resulting suspension was placed in a 1.5 ml assist tube and stored on ice.
[0107]
Example 3 Cell transplantation
Pregnant sheep purchased from Japan Lamb, an experimental sheep handling company, was used. Transplantation was scheduled on the 60th day of pregnancy, and it was made accustomed to the rearing environment at the time of transplantation 1 week to 10 days before transplantation. In addition, fetuses were confirmed by abdominal ultrasonography.
[0108]
Sheep mothers were anesthetized with ketamine intramuscular injection (15 mg / kg body weight). Lay the sheep on its back on the operating table, position and fix it, and then intubate the oral trachea.₂Surgery was performed under general anesthesia with / air / halothane.
[0109]
The operation was performed with a clean operation. After laparotomy with a midline incision in the lower abdomen, the uterus was turned into the abdominal cavity.
[0110]
The cells for transplantation were injected in two ways: (1) in the fetus and (2) in the liver parenchyma.
[0111]
In the case of transplantation into the abdominal cavity, the myometrium of the sheep is incised, the sheep fetus is driven into the exposed egg membrane while being preserved, and the 23G needle is inserted into the abdominal cavity of the fetus under direct vision through the transparent egg membrane. 1 undifferentiated ES cells, embryoid bodies, transplant cells obtained in Example 2 (1 × 10⁷~ 1x10⁸Cells) were injected through puncture, and the myometrium, peritoneal muscle layer, and skin were closed in layers. In the case of transplantation into the liver parenchyma, cells for transplantation (1 × 10 6) are carried out with ultrasound from the uterine wall and into the sheep fetal liver parenchyma using a 25 G catalan needle.⁷~ 1x10⁸Cells) was punctured and injected, and the peritoneal muscle layer and the skin were closed and closed.
[0112]
After the operation, antibiotics (mycillin sol) were injected intramuscularly. Subsequently, the tube was extubated, and anesthesia awakening of the sheep was confirmed.
[0113]
Example 4 Sample collection from fetus
The fetuses 1 month after transplantation were removed by cesarean section under maternal general anesthesia and dissected, and whole body tissues including hematopoietic tissues were collected as samples.
[0114]
Specifically, each of the two uterus (transplanted fetus (tail-cut), control fetus), and a total of 4 fetuses, from the mother who is pregnant, tissue collection and processing in the following procedure At the same time.
[0115]
The collected tissues are shown below.
Endoderm tissue
1. Liver (including bile duct, liver parenchyma, hepatic artery, portal vein area)
2. Pancreatic head (pancreatic duct, pancreatic parenchyma)
3. Thymus
4). Thyroid
5). Lung (bronchi, alveoli)
6). Small intestine
7). Omentum (visceral peritoneum)
Ectodermal tissue
8). Cerebrum
9. Spinal cord
10. Skin (appendix; hair, sweat glands, sebaceous glands)
11. Tail amputation (skin) [collected from a cell transplanted fetus]
Mesodermal tissue
12 Fat (connective tissue)
13. Cartilage
14 Bone marrow
15. Lymph nodes
16. Skeletal muscle
17. Myocardium
18. Aorta
19. spleen
20. kidney
21. Gonadal
The following samples were collected for primary culture.
22. Peripheral blood (umbilical artery blood)
23. Umbilical cord blood (umbilical cord venous blood)
24. Bone marrow (after reflux)
25. Liver (after reflux)
26. Ascites (after reflux)
[0116]
Sheep maternal, spontaneously breathing, O by oral intubation₂Induction of anesthesia was started with / air / halocene. After 15 minutes, the maternal body was cesarean sectioned, and two unilateral uterus fetuses were removed, and the tissue was collected and processed. In addition, the body weight of the fetus transplanted with cells (transplanted fetus) was 950 g, and the body weight of the control fetus was 1040 g.
[0117]
Subsequently, umbilical cord venous blood (umbilical cord blood sample) and umbilical arterial blood (peripheral blood sample) were collected with the umbilical cord connected to the mother. Here, the amount of blood collected was 10 ml for the cord blood sample of the transplanted fetus, 20 ml for the peripheral blood sample of the transplanted fetus, 20 ml for the cord blood sample of the control fetus, and 20 ml for the peripheral blood sample of the control fetus.
[0118]
The umbilical cord was dissected and then the umbilical vein was cannulated with a 6G atom tube. As the umbilical artery could not be cannulated, the cut end was opened.
[0119]
A reflux solution (cold lactec 500 ml) was connected to the cannula on the umbilical vein side, and reflux was started by infusion. Reflux was performed using the umbilical artery side as a blood removal path until blood flowed out (about 1000 ml in total).
[0120]
Each of the transplanted fetus and the control fetus was laparotomized, and the partial liver and femur were collected as a primary culture sample by a clean operation. The organ was removed. The extracted organ was divided into petri dishes on ice.
[0121]
Three tissue specimens of 5 mm × 5 mm × 3 mm were cut out. Two of them were placed in a 15 ml sample bottle containing 4% buffered paraformaldehyde (PFA) / 8% sucrose and fixed overnight. This sample was a 4% PFA fixed sample.
[0122]
The bone marrow sample was decalcified.
[0123]
In addition, the 4% PFA-fixed sample is sequentially maintained in 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol for 1 to 2 hours, and 100% ethanol for 1 to 2 hours, Furthermore, it maintains for 1 to 2 hours with 100% ethanol, and then dehydrates by maintaining overnight with 100% ethanol, and further with 100% ethanol overnight, soaking in benzene (twice for 30 minutes), Subsequently, it was immersed in paraffin (45 minutes × twice) to obtain a paraffin-embedded section sample after fixing with 4% PFA.
[0124]
The remaining one specimen was placed in Cryomold No. 1 and embedded and frozen. The embedding and freezing were carried out by embedding with OCT compound and freezing with liquid nitrogen. This sample was used as a specimen for a fresh frozen section.
[0125]
Several small pieces were cut out from the remaining organs, placed one by one into three cryotubes, and the tubes were frozen with liquid nitrogen. Small pieces of these organs were used as samples for DNA extraction or RNA extraction.
[0126]
In case of miscarriage, stillbirth or death after birth, the fetus is taken out, peripheral blood (heparin blood sampling) about 20 ml, bone marrow (collected in heparin saline (10 units / ml)) appropriate amount, umbilical cord blood (heparin blood sampling) about 20 ml, liver (Collected during heparinized diet) Approximately 10 g was collected, and the body surface, intraperitoneal cavity, intrathoracic cavity, and brain were necropsied to confirm that there was no teratoma or other tumor formation. When tumor formation was confirmed, the tumor was removed and fixed with 4% paraformaldehyde.
[0127]
In addition, in cases analyzed after birth, peripheral blood and bone marrow were collected at a rate of about once a month after birth. Specifically, about 20 ml of umbilical cord blood (heparin blood collection) was collected at birth, and about 20 ml of peripheral blood (heparin blood collection) was collected at birth or within 7 days after birth. After birth, about 20 ml of peripheral blood (heparin blood collection) was collected every 2 weeks during the first few months, and an appropriate amount of bone marrow (collected during heparin diet) was collected every month. The collected sample was put in a sterile conical tube and stored by shaking at room temperature, and nucleated cells were separated and stored frozen.
[0128]
Hematopoietic tissues and cells such as liver, umbilical cord blood, bone marrow, and peripheral blood were separated into leukocyte differentiation fractions by hemolysis, and cryopreserved as DNA extraction samples or RNA extraction samples, primary culture samples, and FACS analysis samples. .
[0129]
As the primary culture sample, the liver was subjected to cell treatment as follows. A sample is collected under aseptic procedure, the sample is appropriately minced and collected in a 50 ml conical tube, 20 ml of 1% trypsin / EDTA / phosphate buffered saline is added, and the suspension is well suspended at 37 ° C. Incubated for 10 minutes with shaking. To the obtained product, 200 μl of DNase I solution [solution of 10 mg DNase I dissolved in 1 ml 0.15M NaCl] was added, and further 1M MgCl₂100 μl was added and incubated at 37 ° C. for 10 minutes. Thereafter, the product was filtered through a 70 mm diameter nylon mesh (manufactured by FALCON, trade name: cell strainer), and the same amount of D10 medium as the product was added. Thereafter, the mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed by suction. If the pellet is red due to red blood cell contamination, add approximately 10 times more ACK lysing buffer to the pellet, maintain on ice for 15 to 0 minutes, and then centrifuge at 1500 rpm for 5 minutes at 4 ° C. did. The pellet was washed twice with about 40 ml of phosphate buffered saline. The supernatant is aspirated and removed, and the resulting pellet is 10⁶-10⁷The cells / vials were suspended in a cell banker and stored frozen at −80 ° C.
[0130]
Further, as a sample for primary culture, umbilical cord blood, peripheral blood and bone marrow were subjected to cell treatment as follows. A sample was collected by aseptic operation following heparin blood collection, and 35 ml of blood was placed in a 50 ml conical tube and centrifuged at 1500 rpm for 10 minutes. Plasma was aspirated and removed, and the pellet was dispensed into five 50 ml conical tubes. Then, 40 ml (about 10 times the amount of pellet) ACK lysing buffer was added to each conical tube, mixed, and incubated on ice for 15 minutes. After centrifugation at 1500 rpm for 5 minutes at 4 ° C., the supernatant was aspirated and removed. When the pellet was red, the centrifugation was repeated at 1500 rpm for 10 minutes. Next, 40 ml of phosphate buffered saline was added to the pellet, suspended, and each tube was combined into one tube and washed by centrifugation at 1500 rpm for 5 minutes at 4 ° C. Such washing was performed twice. Aspirate the supernatant and remove the pellet 5 × 10⁶The cells / vials / ml were suspended in a cell banker and stored frozen.
[0131]
Example 5 Analysis of genomic DNA from each tissue
DNA was extracted from the sample obtained in Example 4 using the QIAamp DNA Mini Kit and the QIAamp DNA Blood Mini Kit according to the manufacturer's protocol attached to the kit.
[0132]
For analysis of sheep embryos transplanted with monkey-derived cells, as a primer pair for monkey β2-microglobulin, an external primer pair:
A primer pair consisting of CB2MG-2F: 5'-GTCTGGATTTCCATCCATCT-3 '(SEQ ID NO: 1) and hB2MG 5R: 5'-GGCTGTGACAAAGTCACATGG-3' (SEQ ID NO: 2); and
Internal primer pair:
Primer pair consisting of CB2MG-2F: 5'-GTCTGGGTTTCATCCATCCCG-3 '(SEQ ID NO: 3) and hB2MG-3R: 5'-GGTGAATTCAGGTGTAGTACAA G-3' (SEQ ID NO: 4)
Using the DNA as a template, semiquantitative PCR was performed. PCR conditions are as follows: reaction solution [composition: H₂0 30.75 μl, 10 × PCR buffer 5 μl, dNTP (2.5 mM each) 4 μl, Ex Taq (5 U / μl) 0.25 μl, primer (10 pM each) 2.5 μl each, sample-derived DNA (equivalent to 250 ng) 5 μl], after incubation at 95 ° C. for 5 minutes, 95 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 1 minute as one cycle, 25 cycles for external amplification, 30 cycles for internal amplification The cycle was performed, and the condition was maintained at 4 ° C. by incubating at 72 ° C. for 5 minutes.
[0133]
Also, a primer pair for β-actin:
Cβ1: 5'-CATTGTCCATGGACTCTGGCGACGG-3 '(SEQ ID NO: 5)
Cβ2: 5'-CATCTCCCTGCTCGAAGTCTAGGGC-3 '(SEQ ID NO: 6)
PCR was performed in the same manner using a primer pair consisting of
[0134]
For quantification, samples of the dilution series shown in Table 1 below were prepared and used.
[0135]
[Table 1]

[0136]
Sheep fetuses transplanted with human umbilical cord blood CD34 + cells were used as a control. At this time, as a primer pair for β2-microglobulin, an external primer pair:
a primer pair consisting of hB2MG-2F 5'-GTCTGGATTTCCATCCATTG-3 '(SEQ ID NO: 7) and hB2MG 5R, and
Internal primer pair:
Semi-quantitative PCR was performed using a primer pair consisting of hB2MG-2F 5'-GTCTGGGTTTCATCATCATCG-3 '(same as SEQ ID NO: 3) and hB2MG-3R, and using the DNA as a template. PCR conditions are as follows: reaction solution [composition: H₂0 30.75 μl, 10 × PCR buffer 5 μl, dNTP (2.5 mM each) 4 μl, Ex Taq (5 U / μl) 0.25 μl, primer (10 pM each) 2.5 μl each, sample-derived DNA (equivalent to 250 ng) 5 μl], after incubation at 95 ° C. for 5 minutes, 95 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 1 minute as one cycle, 25 cycles for external amplification, 30 cycles for internal amplification The cycle was performed, and the condition was maintained at 4 ° C. by incubating at 72 ° C. for 5 minutes.
[0137]
For quantification, samples of the dilution series shown in Table 2 below were prepared and used.
[0138]
[Table 2]

[0139]
The product generated by PCR was subjected to electrophoresis on a 2% agarose gel (containing 0.01% EtBr).
[0140]
As a result, after transplantation of human umbilical cord blood CD34 + cells (control group) into the liver parenchyma of sheep fetuses, the β2-microglobulin derived from monkeys has a chimera rate of less than 0.01% only from the peripheral blood of individuals about 4 months old. The base sequence was detected.
[0141]
Example 6 Colony assay and colony analysis of hematopoietic tissue and cells
Hematopoietic colony assays were performed on cells isolated for primary culture.
[0142]
The cryo-tube of the cryopreserved cell is lysed in a 37 ° C. constant temperature bath, suspended in a 15 ml conical tube containing 9 ml of 2% FBS-IMDM, and a portion of the resulting suspension is used to produce cells. Numbers were counted. The suspension was then centrifuged at 1300 rpm for 4 minutes and the resulting pellet was washed with 1% 10% with 2% FBS-IMDM.⁶It suspended so that it might become a cell / ml, and the cell suspension was obtained. Furthermore, it is diluted 10 times with 2% FBS-IMDM, and 1 × 10⁵A cell / ml cell suspension was prepared.
[0143]
Place 4 ml of trade name: Methocult GF + [manufactured by StemCell Technologies, catalog number: ST-04435] into a 14 ml polystyrene round bottom tube, add 400 μl of the cell suspension, close the cap and shake vigorously. did. Then, it was left still for about 5 minutes until the foam disappeared.
[0144]
Next, 1.1 ml of the medium containing the obtained cells was injected into the center of a 3.5 mm dish with a 2.5 ml syringe equipped with an 18G needle and spread evenly on the bottom of the dish.
[0145]
In a 15 cm Petri dish, put 3.5 cm dish containing 6 pieces of 3.5 cm dish and 3 ml of phosphate buffered saline for moisturizing, 37 ° C, 5% CO₂Incubation was performed under the following conditions.
[0146]
Colony counting was performed on the 14th day from the start of incubation.
[0147]
However, in this method, it was found that not only monkey-derived hematopoietic progenitor cells but also sheep-derived hematopoietic progenitor cells formed colonies.
[0148]
Therefore, colonies are taken from the medium forming the colonies, DNA is extracted from each colony, PCR is performed using the obtained DNA as a template and a primer pair for monkey β2-microglobulin, and hematopoiesis derived from monkeys The presence of progenitor cells was confirmed.
[0149]
As a primer pair for monkey β2-microglobulin, an external primer pair: a primer pair consisting of CB2MG-2F and hB2MG 5R, and
Internal primer pair: PCR was performed using the primer pair consisting of the CB2MG-2F and hB2MG-3R and the DNA as a template. PCR conditions are as follows: reaction solution [composition: H₂0 30.75 μl, 10 × PCR buffer 5 μl, dNTP (2.5 mM each) 4 μl, Ex Taq (5 U / μl) 0.25 μl, primer (10 pM each) 2.5 μl each, sample-derived DNA (equivalent to 250 ng) 5 μl] and after incubation at 95 ° C. for 5 minutes, 94 cycles of 30 seconds, 57 ° C. for 1 minute and 72 ° C. for 1 minute are used as 25 cycles for both external amplification and internal amplification. The test was carried out at 72 ° C. for 5 minutes and maintained at 4 ° C. The obtained product was analyzed by electrophoresis on a 2% agarose gel.
[0150]
As a result, when hematopoietic differentiation-inducing monkey ES cells were used, as shown in FIG. 2, about 30% of the embryonic liver of the individual one month after transplantation of hematopoietic differentiation induction treatment into the peritoneal cavity ( n = 1), monkeys with a frequency of about 1% (n = 3) in the bone marrow of individuals after birth (3-6 months after transplantation) transplanted into the liver parenchyma with ES cells that have undergone hematopoietic differentiation induction treatment It was found that there is a hematopoietic colony derived from. That is, it was found that when the ES cells were induced to induce hematopoietic differentiation and introduced into sheep fetuses, the sheep fetuses produced monkey-derived hematopoietic cells.
[0151]
Sequence listing free text
SEQ ID NO: 1 is the sequence of primer CB2MG-2F.
[0152]
SEQ ID NO: 2 is the sequence of primer hB2MG 5R.
[0153]
SEQ ID NO: 3 is the sequence of primer CB2MG-2FCB or hB2MG-2F.
[0154]
SEQ ID NO: 4 is the sequence of primer hB2MG-3R.
[0155]
SEQ ID NO: 5 is the sequence of primer Cβ1.
[0156]
SEQ ID NO: 6 is the sequence of primer Cβ2.
[0157]
SEQ ID NO: 7 is the sequence of primer hB2MG-2F.
[0158]
【The invention's effect】
According to the method for differentiating hematopoietic cells from embryonic stem cells of primate animals of the present invention, hematopoietic cells of primates can be stably supplied, and foreign genes can be transferred to primate embryonic stem cells. Hematopoietic cells that can differentiate embryonic stem cells of primate animals into hematopoietic cells without performing genetic engineering operations such as introduction, and are heterogeneous for primate animals and allogeneic to the individuals There is an excellent effect that can be obtained. Further, according to the method for producing hematopoietic cells of primate animals of the present invention, primate hematopoietic cells can be stably supplied, and a large amount of primate hematopoietic cells can be supplied. Hematopoietic cells that are allogeneic to the individual can be obtained in a heterogeneous animal for the ape-like animal, platelets for blood transfusion of the primate animal can be stably supplied, and CD34 + cells for primate animal costume feeding can be obtained. There is an excellent effect that it can be supplied stably. Further, according to the method for producing a chimeric sheep producing primate hematopoietic cells of the present invention, there is an excellent effect that a chimeric sheep capable of stably supplying primate hematopoietic cells can be obtained.
[0159]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows cells obtained by maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells. Panel (A) shows undifferentiated embryonic stem cells and the scale bar shows 100 μm. Panel (B) shows cells during culture in the presence of BMP-4 on OP9 cells. Panel (C) shows cells obtained by maintaining embryonic stem cells under hematopoietic differentiation induction conditions, and the scale bar indicates 100 μm.
FIG. 2 is a diagram showing the results of detection of monkey β2-microglobulin in an ovine fetus obtained after transplantation. In the figure, panel (A) shows the result of fetal liver and panel (B) shows the result of bone marrow.

Claims (6)

The primate embryonic stem cells are maintained under conditions suitable for induction of hematopoietic cell differentiation. A method for differentiating primate embryonic stem cells into hematopoietic cells, characterized by obtaining hematopoietic cells. (I) using a stromal cell line deficient in macrophage colony-stimulating factor as a feeder cell, and maintaining embryonic stem cells of a primate animal on the feeder cell; and (II) a primate obtained in step (I) Transplanting animal-derived cells into a fetus in the uterus of a pregnant sheep and allowing the fetus to grow to birth;
The differentiation method according to claim 1, comprising: The differentiation method according to claim 2, wherein in step (I), embryonic stem cells of primate animals are maintained on feeder cells in the presence of bone morphogenetic protein-4. (I) a step of maintaining a stromal cell line deficient in a macrophage colony-stimulating factor as a feeder cell, and maintaining embryonic stem cells of a primate animal on the feeder cell;
(II) transplanting the cells derived from the primate animal obtained in the step (I) to a fetus in the uterus of a pregnant sheep and growing the fetus until birth, and (III) the step (II) A method of producing hematopoietic cells of a primate animal, comprising isolating hematopoietic cells of a primate animal differentiated from embryonic stem cells of the primate animal from a fetus born in step 1). A hematopoietic cell obtained by the production method according to claim 4. A primate hematopoietic system characterized by maintaining embryonic stem cells of a primate animal under conditions suitable for inducing differentiation of hematopoietic cells and transplanting the obtained cells into a fetus in the uterus of a pregnant sheep A method for producing a chimeric sheep producing cells.

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