JP2013194049A - Composition and method for amplifying human hematopoietic stem cell - Google Patents
Composition and method for amplifying human hematopoietic stem cell Download PDFInfo
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Abstract
Description
この発明は、ヒト造血幹細胞を増幅させるための組成物及び方法に関する。 The present invention relates to compositions and methods for amplifying human hematopoietic stem cells.
造血幹細胞を維持、未分化維持、自己複製又は増殖・増幅させる因子は長年にわたり探索されてきた。その結果、現在までに幹細胞因子(以下「ＳＣＦ（stem cell factor）」という。)、Flt3 ligand、TPO（トロンボポエチン）などの様々な因子やサイトカインを使って造血幹細胞を培養し増幅させる方法が考案されてきた（特許文献１〜５、非特許文献１〜４）。また、骨髄ストローマ細胞とヒト造血幹細胞を共培養することにより、ヒト造血幹細胞を増幅できることが知られており（特許文献１など）、ストローマ細胞に他のサイトカイン等を存在させた培地が提案されている（特許文献１、２、非特許文献２等）。 Factors that maintain hematopoietic stem cells, maintain undifferentiated, self-replicate or proliferate / amplify have been sought for many years. As a result, a method for culturing and amplifying hematopoietic stem cells using various factors and cytokines such as stem cell factor (hereinafter referred to as “SCF (stem cell factor)”), Flt3 ligand, TPO (thrombopoietin) has been devised. (Patent documents 1 to 5, non-patent documents 1 to 4). In addition, it is known that human hematopoietic stem cells can be amplified by co-culturing bone marrow stromal cells and human hematopoietic stem cells (Patent Document 1, etc.), and a medium in which other cytokines are present in stromal cells has been proposed. (Patent Documents 1 and 2, Non-Patent Document 2, etc.).
しかし、造血幹細胞を分化させずに幹細胞のまま増幅させることのできる、いわゆる「自己複製因子」は見つかっていない。更に、造血幹細胞を移植治療に十分な細胞数まで迅速に効率よく体外で増幅させる臨床応用可能な手法の確立には至っていない。
そのため、造血幹細胞を極力幹細胞のまま増幅させることができる因子の発見と、それを用いたヒト造血幹細胞を効率良く安全に増幅する移植治療に実用できる方法の開発が求められている。
However, a so-called “self-replicating factor” that can amplify hematopoietic stem cells without differentiation has been found. Furthermore, the establishment of a clinically applicable technique for rapidly and efficiently amplifying hematopoietic stem cells to a sufficient number of cells for transplantation treatment has not yet been established.
Therefore, discovery of a factor that can amplify hematopoietic stem cells as much as possible as stem cells and development of a method that can be practically used for transplantation treatment that efficiently and safely amplifies human hematopoietic stem cells using the same are required.
本発明者は、ヒト造血幹細胞をヒト骨髄ストローマ細胞（即ち、骨髄細胞を培養するとシャーレに付着して増殖する細胞集団）と共培養したが、ヒト造血幹細胞を維持・増幅することはできなかった。また、SCFはヒト造血幹細胞を維持・増幅できるものの、増幅率が十分ではなく更に増幅率を高める必要があった。しかし、造血幹細胞をSCF存在下で骨髄ストローマ細胞と共培養すると顕著な増幅が観察された。
そこで、本発明者は、ヒト骨髄ストローマ細胞を無血清培地で数日間培養した上清を、SCF存在下又は非存在下の無血清培地に添加して、ヒト造血幹細胞を培養した。その結果、骨髄ストローマ細胞培養上清だけでは造血幹細胞は増幅できなかったが、骨髄ストローマ細胞培養上清とSCFを同時に添加すると、造血幹細胞は十分に増幅できた。従って、ヒト骨髄ストローマ細胞から分泌されるタンパク質等とSCFだけでヒト造血幹細胞を増幅できることが分かった。
The present inventor co-cultured human hematopoietic stem cells with human bone marrow stromal cells (that is, a cell population that grows by attaching to a petri dish when bone marrow cells are cultured), but could not maintain or amplify human hematopoietic stem cells. . Moreover, although SCF can maintain and amplify human hematopoietic stem cells, the amplification rate is not sufficient, and it was necessary to further increase the amplification rate. However, significant amplification was observed when hematopoietic stem cells were co-cultured with bone marrow stromal cells in the presence of SCF.
Therefore, the present inventor added a supernatant obtained by culturing human bone marrow stromal cells for several days in a serum-free medium to a serum-free medium in the presence or absence of SCF, and cultured human hematopoietic stem cells. As a result, hematopoietic stem cells could not be amplified only by the bone marrow stromal cell culture supernatant, but when the bone marrow stromal cell culture supernatant and SCF were added simultaneously, the hematopoietic stem cells could be sufficiently amplified. Therefore, it was found that human hematopoietic stem cells can be amplified only with proteins and SCF secreted from human bone marrow stromal cells.
そこで、骨髄ストローマ細胞から分泌産生される造血幹細胞を増幅又は自己複製させる因子を同定し、より純化した系での効率良い体外での造血幹細胞の増幅法の開発を試みた。先ず、ヒト骨髄ストローマ細胞からpolyA+ mRNAを調製し、DNAマイクロアレイ（Affymetrix GeneChip Microarray）解析を行い、骨髄ストローマ細胞で高発現（又は他の細胞と比較して特異的に高発現）している分泌タンパク質のリストを作成した。リストには、既知の造血幹細胞の自己複製因子、増幅因子、サイトカインもあったが、機能が未だ解明されていない分泌タンパク質の中から、造血幹細胞を増幅又は自己複製させる可能性がある因子を絞り込み、候補因子リストを作成した。これら候補因子から、造血幹細胞を実際に維持・増幅する活性を指標に新規因子をスクリーニングした。ヒト臍帯血由来CD34陽性細胞（造血幹細胞）を、各候補因子を添加した無血清培地中で培養し、少しでも生存維持又は増殖させる活性のある因子をスクリーニングした。しかし、候補因子単独で造血幹細胞を維持又は増幅させるものはなかった。そこで、造血幹細胞の生存維持に最低限必要な因子であるSCFを無血清培地に低濃度で添加し、造血幹細胞がやっと生存維持できる環境を設定した。こうすることで、各候補因子の添加で造血幹細胞の維持・増幅効果を敏感に観測できる条件となり、造血幹細胞を維持又は増幅させる新規因子をスクリーニングした。その結果、IL31(interleukin-31)、PGF(placental growth factor、PLGFとも表記する)（胎盤成長因子）、VEGF-B(vascular endothelial growth factor B)（血管内皮増殖（成長）因子B）、IGFBP6(insulin-like growth factor binding protein 6)（インスリン様成長因子結合タンパク質６）、EFEMP1(EGF-containing fibulin-like extracellular matrix protein 1)、FGF8、FGF9、FGF10、FGF17、FGF19、FGF20(線維芽細胞増殖因子8, 9, 10, 17, 19, 20)の因子が新たに造血幹細胞をSCF存在下で維持・増幅させることができることを発見した。 Therefore, the present inventors have identified a factor that amplifies or self-replicates hematopoietic stem cells secreted and produced from bone marrow stromal cells, and attempted to develop an efficient method for amplifying hematopoietic stem cells in a more purified system. First, polyA + mRNA is prepared from human bone marrow stromal cells, analyzed by DNA microarray (Affymetrix GeneChip Microarray), and secreted protein that is highly expressed in bone marrow stromal cells (or specifically highly expressed compared to other cells) Created a list of The list includes known hematopoietic stem cell self-renewal factors, amplification factors, and cytokines, but narrows down the factors that may cause hematopoietic stem cells to amplify or self-replicate from secreted proteins whose functions have not yet been elucidated. A candidate factor list was created. From these candidate factors, novel factors were screened using as an index the activity of actually maintaining and amplifying hematopoietic stem cells. Human umbilical cord blood-derived CD34-positive cells (hematopoietic stem cells) were cultured in a serum-free medium supplemented with each candidate factor, and screened for factors having an activity to maintain or proliferate even a little. However, none of the candidate factors alone maintain or amplify hematopoietic stem cells. Therefore, SCF, which is the minimum factor necessary for maintaining the survival of hematopoietic stem cells, was added to a serum-free medium at a low concentration to set up an environment where hematopoietic stem cells can finally maintain their survival. In this way, the addition of each candidate factor provided a condition under which the effect of maintaining / amplifying hematopoietic stem cells could be observed sensitively, and a novel factor that maintains or amplifies hematopoietic stem cells was screened. As a result, IL31 (interleukin-31), PGF (also called placental growth factor, PLGF) (placental growth factor), VEGF-B (vascular endothelial growth factor B) (vascular endothelial growth (growth) factor B), IGFBP6 ( insulin-like growth factor binding protein 6), EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1), FGF8, FGF9, FGF10, FGF17, FGF19, FGF20 (fibroblast growth factor) 8, 9, 10, 17, 19, 20) were newly found to be able to maintain and amplify hematopoietic stem cells in the presence of SCF.
即ち、本発明は、下記（１）及び（２）の因子から成るヒト造血幹細胞を増幅させるための組成物である。
（１）幹細胞因子(SCF)
（２）IL31(interleukin-31)、PGF(placental growth factor、胎盤成長因子）、VEGF-B(vascular endothelial growth factor B、血管内皮増殖因子B）、IGFBP6(insulin-like growth factor binding protein 6、インスリン様成長因子結合タンパク質６）、EFEMP1(EGF-containing fibulin-like extracellular matrix protein 1)、FGF8、FGF9、FGF10、FGF17、FGF19及びFGF20(線維芽細胞増殖因子8, 9, 10, 17, 19及び20)から成る群から選択される少なくとも１種の因子
また本発明は、この組成物の存在下でヒト造血幹細胞を培養することから成る増幅したヒト造血幹細胞の製法である。
更に本発明は、この組成物を含む、無血清又はヒト血清から成るヒト造血幹細胞培養用の培地である。
That is, the present invention is a composition for amplifying human hematopoietic stem cells comprising the following factors (1) and (2).
(1) Stem cell factor (SCF)
(2) IL31 (interleukin-31), PGF (placental growth factor), VEGF-B (vascular endothelial growth factor B), IGFBP6 (insulin-like growth factor binding protein 6, insulin) Like growth factor binding protein 6), EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1), FGF8, FGF9, FGF10, FGF17, FGF19 and FGF20 (fibroblast growth factor 8, 9, 10, 17, 19 and 20 At least one factor selected from the group consisting of)) The present invention is also a method of producing amplified human hematopoietic stem cells comprising culturing human hematopoietic stem cells in the presence of this composition.
Further, the present invention is a medium for culturing human hematopoietic stem cells comprising this composition and comprising serum-free or human serum.
本発明の組成物により、造血幹細胞を体外で増幅することができるので、患者自身の骨髄（又は末梢血や出生時に保存した自家臍帯血）などから得た少量の造血幹細胞から移植治療に充分な量の造血幹細胞を調製することができ、HLA適合のドナーを待つことなく安全な移植治療が可能となる。即ち、自家移植が可能となり、移植に必要な幹細胞数も確保できる上に、拒絶反応の問題も解決できる。他家移植においても、細胞数が少なく移植治療できなかった臍帯血からでも、またドナーにリスクを与えない少量の骨髄採取からでも、成人の移植治療に必要な数の造血幹細胞の調達が可能となる。また、遺伝子治療に必要な造血幹細胞を容易に入手可能となり、リスクを伴うレトロウイルスを用いた遺伝子導入法を使わずとも、導入効率が悪くても安全な遺伝子導入法を用いることが可能となり、また、遺伝子導入した造血幹細胞を増幅することにも使用できることで、遅れている遺伝子治療の実用化と普及が期待できる。 The composition of the present invention allows hematopoietic stem cells to be amplified outside the body, and is sufficient for transplantation treatment from a small amount of hematopoietic stem cells obtained from the patient's own bone marrow (or peripheral blood or autologous umbilical cord blood preserved at birth). An amount of hematopoietic stem cells can be prepared, enabling safe transplantation treatment without waiting for an HLA-compatible donor. That is, autologous transplantation is possible, the number of stem cells necessary for transplantation can be secured, and the problem of rejection can be solved. It is possible to procure the number of hematopoietic stem cells necessary for transplantation treatment of adults, even from umbilical cord blood where the number of cells was small and could not be transplanted, or from a small amount of bone marrow that does not pose a risk to the donor. Become. In addition, hematopoietic stem cells necessary for gene therapy can be easily obtained, and it is possible to use a safe gene transfer method even if the transfer efficiency is low, without using a gene transfer method using a risky retrovirus, In addition, since it can be used to amplify gene-introduced hematopoietic stem cells, it can be expected that the delayed gene therapy will be put to practical use and spread.
本発明のヒト造血幹細胞を増殖させるための組成物は、（１）幹細胞因子(SCF)、及び
（２）IL31、PGF/PLGF、VEGF-B、EFEMP1、IGFBP6、FGF8、FGF9、FGF10、FGF17、FGF19及びFGF20から成る群から選択される少なくとも１種の因子から成る。
The composition for growing human hematopoietic stem cells of the present invention comprises (1) stem cell factor (SCF), and (2) IL31, PGF / PLGF, VEGF-B, EFEMP1, IGFBP6, FGF8, FGF9, FGF10, FGF17, It consists of at least one factor selected from the group consisting of FGF19 and FGF20.
本発明の組成物を構成する因子である幹細胞因子（SCF）は、造血幹細胞の細胞死を抑制し、単独でもある程度生存させ維持も可能であり、他の因子と共に自己複製あるいは増幅させる因子である（非特許文献１）。
ヒトSCFは、転写の違い（Alternative transcripts）により２つのアイソフォーム（isoform）があり、配列番号１及び２で表されるアミノ酸配列を有する。
Stem cell factor (SCF), which is a factor constituting the composition of the present invention, is a factor that suppresses cell death of hematopoietic stem cells and can survive and maintain to some extent alone, and can self-replicate or amplify together with other factors. (Non-Patent Document 1).
Human SCF has two isoforms due to differences in transcription (Alternative transcripts), and has amino acid sequences represented by SEQ ID NOs: 1 and 2.
本発明の組成物を構成する因子であるIL31はT細胞由来サイトカインであり、IL6ファミリー（IL6、IL11、IL27、IL31、Oncostatin M、Leukemia inhibitory factor(LIF)、Ciliary Neurotrophic Factor(CNTF), Cardiotrophin-1(CT-1)、neurotrophin-1/B cell stimulatory factor-3(NT-1/BCSF-3)からなる）の一員である。IL31はIL31RA/GPL(gp130-like)とOSMR(Oncostatin M receptor)のヘテロダイマーからなるレセプターに結合しシグナルを伝達する。過去にIL6、IL11、IL27、Oncostatin Mが造血幹細胞を増殖分化すると報告されている（Stem Cells 26, 2164-2172, 2008、Biochem. Biophys. Acta 1592, 313-321, 2002）。IL31の機能には免疫応答が知られているが、過去に造血幹細胞に対する作用は報告されておらず、本発明で初めて造血幹細胞に対する増幅活性が報告されることとなった。
ヒトIL31は、配列番号３で表されるアミノ酸配列を有する。
ヒトIL31として、各種データベースに、NM_001014336、AY499343、BC132998、BC133000等のcDNA塩基配列が登録され、Q6EBC2、NP_001014358、EAW98310、AAI32999、AAI33001、AAS86448、BAJ21082、AAI17083、AAI17085等のアミノ酸配列が登録されており、ゲノム遺伝子配列、人工合成遺伝子も登録されている。他に、マウス、ラット、チンパンジー、イヌ等の遺伝子も登録されており、本発明においては、下等生物遺伝子や人工合成遺伝子も含めてIL31としての特異的生理活性を持つタンパク質はいずれも利用することができる。
IL31, which is a factor constituting the composition of the present invention, is a T cell-derived cytokine, and the IL6 family (IL6, IL11, IL27, IL31, Oncostatin M, Leukemia inhibitory factor (LIF), Ciliary Neurotrophic Factor (CNTF), Cardiotrophin- 1 (CT-1), a member of neurotrophin-1 / B cell stimulatory factor-3 (NT-1 / BCSF-3). IL31 binds to a receptor composed of a heterodimer of IL31RA / GPL (gp130-like) and OSMR (Oncostatin M receptor) and transmits a signal. In the past, IL6, IL11, IL27, and Oncostatin M have been reported to proliferate and differentiate hematopoietic stem cells (Stem Cells 26, 2164-2172, 2008, Biochem. Biophys. Acta 1592, 313-321, 2002). Although an immune response is known for the function of IL31, no action on hematopoietic stem cells has been reported in the past, and amplification activity on hematopoietic stem cells has been reported for the first time in the present invention.
Human IL31 has an amino acid sequence represented by SEQ ID NO: 3.
As human IL31, cDNA base sequences such as NM_001014336, AY499343, BC132998, and BC133000 are registered in various databases, and amino acid sequences such as Q6EBC2, NP_001014358, EAW98310, AAI32999, AAI33001, AAS86448, BAJ21082, AAI17083, and AAI17085 are registered. Genomic gene sequences and artificial synthetic genes are also registered. In addition, genes such as mouse, rat, chimpanzee, and dog are also registered, and in the present invention, any protein having specific physiological activity as IL31 including lower organism genes and artificially synthesized genes is used. be able to.
本発明の組成物を構成する因子であるPGF/PLGFとVEGF-Bは、 VEGF-A、VEGF-B、VEGF-C、VEGF-D、PGF/PLGFからなるVEGFファミリーのメンバーであり、他のメンバーと同様に血管内皮細胞の増殖や透過性亢進活性を持つ。VEGFのレセプターにはVEGFR-1/Flt-1、VEGFR-2/Flk-1/KDR、VEGFR-3/Flt-4の３種があり、PGF/PLGFはVEGFR-1/Flt-1のみに結合する。VEGFR-1/Flt-1にはVEGF-A、VEGF-Bも結合するが他のVEGFは結合しない。VEGF-AはVEGFR-2/Flk-1/KDRにも結合する。胎児初期発生の段階で、VEGF-Aは造血を促進し、PGF/PLGFは造血幹細胞の維持や移動に作用しSCF産生を促すことで間接的に造血幹細胞を増やすことが報告されている（Nature Med. 8, 841-849, 2002）。しかし、PGF/PLGF及びVEGF-Bが造血幹細胞に直接作用し増幅させる活性は報告されていない。本発明でPGF/PLGFとVEGF-Bが新たに造血幹細胞の増幅活性を持つことを発見したことから、VEGF-A、VEGF-B、PGF/PLGFに共通のレセプターであるVEGFR-1/Flt-1を介したシグナルが造血幹細胞の増幅に関与していると考えられる。
ヒトPGF/PLGFには、３つのアイソフォーム（isoform）があり、配列番号４〜６で表されるアミノ酸配列を有する。
ヒトVEGF-Bには、２つのアイソフォーム（isoform）があり、配列番号７〜８で表されるアミノ酸配列を有する。
PGF / PLGF and VEGF-B, which are factors constituting the composition of the present invention, are members of the VEGF family consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF / PLGF. Similar to its members, it has vascular endothelial cell proliferation and permeabilization activities. There are three types of VEGF receptors: VEGFR-1 / Flt-1, VEGFR-2 / Flk-1 / KDR, and VEGFR-3 / Flt-4. PGF / PLGF binds only to VEGFR-1 / Flt-1 To do. VEGFR-1 / Flt-1 also binds VEGF-A and VEGF-B, but not other VEGF. VEGF-A also binds to VEGFR-2 / Flk-1 / KDR. It has been reported that VEGF-A promotes hematopoiesis and PGF / PLGF indirectly increases the number of hematopoietic stem cells by promoting SCF production by acting on the maintenance and migration of hematopoietic stem cells at the early fetal development stage (Nature Med. 8, 841-849, 2002). However, no activity has been reported that PGF / PLGF and VEGF-B directly act on hematopoietic stem cells and amplify them. In the present invention, it was discovered that PGF / PLGF and VEGF-B have a new hematopoietic stem cell amplification activity. Therefore, VEGFR-1 / Flt-, which is a receptor common to VEGF-A, VEGF-B, and PGF / PLGF. Signals mediated by 1 are thought to be involved in hematopoietic stem cell amplification.
Human PGF / PLGF has three isoforms and has an amino acid sequence represented by SEQ ID NOs: 4-6.
Human VEGF-B has two isoforms and has an amino acid sequence represented by SEQ ID NOs: 7-8.
ヒトPGF/PLGFの３種のスプライスバリアント（アイソフォーム）には、NM_001207012、NM_002632、BC007789、BC007255、BC001422、AK023843、AK057973、AK309886、AK310608、AK310886、EU332861等のcDNA塩基配列が登録されており、NP_002623、NP_001193941、AAH07789、AAH07255、AAH01422、ABY87550、EAW81209、EAW81208、EAW81207等のアミノ酸配列、ゲノム遺伝子、変異体やスプライスバリアント、人工合成遺伝子も登録されている。また、マウス、ラット、ウシ、ブタ、チンパンジー、アカゲザル等の遺伝子配列も同様に登録されている。本発明においては、PGF/PLGFとしての特異的生理活性がある限り、これらのいずれをも利用することができる。
ヒトVEGF-Bの２種のアイソフォームには、NM_003377、U52819、U48801、U43369、U43368、CR602359、BC008818等の塩基配列が登録されており、NP_003368、P49765、AAC50721、AAB06274、NP_001230662、AAL79001、AAL79000等のアミノ酸配列、ゲノム遺伝子、変異体やスプライスバリアントも登録されている。また、マウス、ラット、ウシ、カエル、人工合成遺伝子（タンパク質）等も同様に登録されている。本発明においては、VEGF-Bとしての特異的生理活性がある限り、これらのいずれをも利用することができる。
In the three splice variants (isoforms) of human PGF / PLGF, cDNA base sequences such as NM_001207012, NM_002632, BC007789, BC007255, BC001422, AK023843, AK057973, AK309886, AK310608, AK310886, EU332861 are registered, and NP_002623 NP_001193941, AAH07789, AAH07255, AAH01422, ABY87550, EAW81209, EAW81208, EAW81207, etc., amino acid sequences, genomic genes, mutants and splice variants, and artificially synthesized genes are also registered. In addition, gene sequences of mice, rats, cows, pigs, chimpanzees, rhesus monkeys, and the like are also registered. In the present invention, any of these can be used as long as it has a specific physiological activity as PGF / PLGF.
In the two isoforms of human VEGF-B, nucleotide sequences such as NM_003377, U52819, U48801, U43369, U43368, CR602359, BC008818 are registered, and NP_003368, P49765, AAC50721, AAB06274, NP_001230662, AAL79001, AAL79000, etc. Amino acid sequences, genomic genes, mutants and splice variants are also registered. In addition, mice, rats, cows, frogs, artificially synthesized genes (proteins) and the like are also registered. In the present invention, any of these can be used as long as it has specific physiological activity as VEGF-B.
本発明の組成物を構成する因子であるIGFBP6 (insulin-like growth factor（IGF） binding protein 6)の機能は不明である。幹細胞に関しては、IGFBP6が神経系幹細胞の細胞分化を抑制するとの報告があるが（Stem Cells Dev. 15, 407-421, 2006）、造血幹細胞への作用は報告がなく、本発明で初めて造血幹細胞への増幅作用が見出された。後述の実施例で示す通り、IGFBP6はIGFBP7/IGFBPrP1と同様に、IGF非存在下で活性があることから、IGFとの結合やそのレセプターを介さずに作用する。従って、IGFBP6のレセプターやその作用機序は不明であるが、IGFBP7/IGFBPrP1と共にIGFBP6の造血幹細胞に対する増幅活性が明らかとなった。
ヒトIGFBP6は、配列番号９で表されるアミノ酸配列を有する。
ヒトIGFBP6として、NM_002178、M62402、M69054、AY443494、CR541791、CR590705、AK313158、BC003507、BC005007、BC010162、BC011708等の塩基配列が登録されており、NP_002169、CAG46590、AAD04730、AAB06187、AAG37059、AAA88070、AAR05445、AAH11708、AAH10162、AAH05007、AAH03507、EAW96664等のアミノ酸配列、ゲノム遺伝子、変異体やスプライスバリアントも登録されている。また、マウス、ラット、ブタ、サケ、ヒツジ、ウシ、チンパンジー、イヌ、人工合成遺伝子（タンパク質）等も同様に登録されている。本発明においては、IGFBP6としての特異的生理活性がある限り、これらのいずれをも利用することができる。
The function of IGFBP6 (insulin-like growth factor (IGF) binding protein 6), which is a factor constituting the composition of the present invention, is unknown. Regarding stem cells, there is a report that IGFBP6 suppresses cell differentiation of neural stem cells (Stem Cells Dev. 15, 407-421, 2006), but no effect on hematopoietic stem cells has been reported, and for the first time in the present invention hematopoietic stem cells. Amplifying effect was found. As shown in the examples described later, IGFBP6 is active in the absence of IGF and acts without binding to IGF or its receptor, like IGFBP7 / IGFBPrP1. Therefore, although the receptor of IGFBP6 and the mechanism of its action are unknown, the amplification activity of IGFBP6 on hematopoietic stem cells together with IGFBP7 / IGFBPrP1 was revealed.
Human IGFBP6 has the amino acid sequence represented by SEQ ID NO: 9.
As human IGFBP6, base sequences such as NM_002178, M62402, M69054, AY443494, CR541791, CR590705, AK313158, BC003507, BC005007, BC010162, BC011708 are registered, and NP_002169, CAG46590, AAD04730, AAB06187, AAG37059, AAA88070, AA445 Amino acid sequences such as AAH10162, AAH05007, AAH03507, EAW96664, genomic genes, mutants and splice variants are also registered. In addition, mice, rats, pigs, salmon, sheep, cows, chimpanzees, dogs, artificially synthesized genes (proteins) and the like are also registered. In the present invention, any of these can be used as long as there is specific physiological activity as IGFBP6.
本発明の組成物を構成する因子であるEFEMP1の機能は不明であり、造血幹細胞との関係も過去に報告がない。唯一、Timp-3(Tissue inhibitor of metalloproteinase-3)という因子は、静止期にある造血幹細胞を細胞分裂期に入れる活性があるが（Blood 116, 4474-4482, 2010）、Timp-3はEFEMP1と結合するという報告がある（J. Biol. Chem. 279, 30469-30473, 2004）。しかしながら、本発明ではTimp-3存在下でも非存在下でもEFEMP1の活性に影響はなく、EFEMP1単独で造血幹細胞に対して増幅活性があることを見いだした。
ヒトEFEMP1には、４つのアイソフォーム（isoform）があり、配列番号１０〜１３で表されるアミノ酸配列を有する。
ヒトEFEMP1の４種のスプライスバリアントには、NM_001039348、NM_001039349、NM_004105、AH010389、NG_009098、BC098561、BC014410、AB209212、AK092854、AK290599、AK293058、AK301402、AK301531、AK312639、CR590528、CR595300、CR604757、CR607072、CR611721、CR620563、U03877等の塩基配列が登録されており、NP_001034438、NP_001034437、NP_004096、AAK11491、AAH98561、AAH14410等のアミノ酸配列、ゲノム遺伝子、変異体やスプライスバリアントも登録されている。また、マウス、ラット、ニワトリ、イヌ、ウシ、ブタ、セブラフィッシュ、アフリカゾウ、チンパンジー、パンダ、カニクイザル、人工合成遺伝子（タンパク質）等も同様に登録されている。本発明においては、EFEMP1としての特異的生理活性がある限り、これらのいずれをも利用することができる。
The function of EFEMP1, which is a factor constituting the composition of the present invention, is unknown, and there is no report on its relationship with hematopoietic stem cells. The only factor called Timp-3 (Tissue inhibitor of metalloproteinase-3) has the activity to bring hematopoietic stem cells in stationary phase into the cell division phase (Blood 116, 4474-4482, 2010). There are reports of binding (J. Biol. Chem. 279, 30469-30473, 2004). However, the present invention has found that EFEMP1 activity is not affected in the presence or absence of Timp-3, and that EFEMP1 alone has an amplifying activity on hematopoietic stem cells.
Human EFEMP1 has four isoforms and has an amino acid sequence represented by SEQ ID NOs: 10-13.
The four splice variants of human EFEMP1 include NM_001039348, NM_001039349, NM_004105, AH010389, NG_009098, BC098561, BC014410, AB209212, AK092854, AK290599, AK293058, AK301402, AK301531, AK312639, CR590528, CR595300, CR604757CR611707 , U03877, and the like, and amino acid sequences such as NP_001034438, NP_001034437, NP_004096, AAK11491, AAH98561, AAH14410, genomic genes, mutants, and splice variants are also registered. In addition, mice, rats, chickens, dogs, cows, pigs, sebrafish, African elephants, chimpanzees, pandas, cynomolgus monkeys, artificial synthetic genes (proteins), and the like are also registered. In the present invention, any of these can be used as long as it has specific physiological activity as EFEMP1.
本発明の組成物を構成する因子であるFGF（fibroblast growth factor）(繊維芽細胞増殖因子)ファミリーであるFGF8、FGF9、FGF10、FGF17、FGF19、FGF20は、過去に報告されているFGF1/aFGFとFGF2/bFGF(非特許文献２)に加えて、今回新たに造血幹細胞に対する増幅活性を持つことを発見した。一般にFGFは胚発生、創傷治癒、血管形成に関与し、骨、神経、血管組織への細胞分化を促進するが、今回発見した何れのFGFメンバーも、造血幹細胞に対する作用は報告が全くなく、今回初めて造血幹細胞を増幅させる活性の報告に至った。
FGFのレセプターには、FGFR1/CD331、FGFR2/CD332、FGFR3/CD333、FGFR4/CD334の４種が知られており、過去に報告されたFGF1/aFGF、FGF2/bFGF、FGF4に共通して強く結合するレセプターとして、FGFR3c及びFGFR4aがある。今回発見した6種のFGFメンバーもFGFR3c及びFGFR4aの両方又は何れかに強く結合することから、これらのレセプターを介したシグナルが造血幹細胞の増幅に関与していると考えられる。
ヒトFGF8には、４つのアイソフォーム（isoform）があり、配列番号１４〜１７で表されるアミノ酸配列を有する。
ヒトFGF9は、配列番号１８で表されるアミノ酸配列を有する。
ヒトFGF10は、配列番号１９で表されるアミノ酸配列を有する。
ヒトFGF17には、２つのアイソフォーム（isoform）があり、配列番号２０〜２１で表されるアミノ酸配列を有する。
ヒトFGF19は、配列番号２２で表されるアミノ酸配列を有する。
ヒトFGF20は、配列番号２３で表されるアミノ酸配列を有する。
The FGF (fibroblast growth factor) family, FGF8, FGF9, FGF10, FGF17, FGF19, and FGF20, which are factors constituting the composition of the present invention, have been reported in the past as FGF1 / aFGF. In addition to FGF2 / bFGF (Non-patent Document 2), the present inventors have newly discovered that it has amplification activity against hematopoietic stem cells. In general, FGF is involved in embryogenesis, wound healing, and blood vessel formation, and promotes cell differentiation into bone, nerve, and vascular tissue, but none of the FGF members discovered this time has been reported to have any effect on hematopoietic stem cells. For the first time, the activity of amplifying hematopoietic stem cells was reported.
There are four known FGF receptors, FGFR1 / CD331, FGFR2 / CD332, FGFR3 / CD333, and FGFR4 / CD334, and they bind strongly to FGF1 / aFGF, FGF2 / bFGF, and FGF4 reported in the past. Examples of receptors that can be used include FGFR3c and FGFR4a. The six FGF members discovered this time are strongly bound to either or both of FGFR3c and FGFR4a, and thus signals through these receptors are thought to be involved in the amplification of hematopoietic stem cells.
Human FGF8 has four isoforms and has an amino acid sequence represented by SEQ ID NOs: 14-17.
Human FGF9 has an amino acid sequence represented by SEQ ID NO: 18.
Human FGF10 has the amino acid sequence represented by SEQ ID NO: 19.
Human FGF17 has two isoforms and has amino acid sequences represented by SEQ ID NOs: 20-21.
Human FGF19 has an amino acid sequence represented by SEQ ID NO: 22.
Human FGF20 has an amino acid sequence represented by SEQ ID NO: 23.
各因子の塩基配列は、FGF8（４種のスプライスバリアント）としてNM_001206389、NM_006119、NM_033163、NM_0033164、NM_0033165、AB14615、BC069106、BC128235、BC128236、U36223、U46211、U46212、U46213、U46214等、FGF9としてNM_002010、AK095571、AK290792、BC069692、BC103978、BC103979、D4838等、FGF10としてNM_004465、AB002097、AF411527、AF508782、AK315314、BC069561、BC105021、BC105023、BC143772、CR541665、CR541688、GQ351295、U67918等、FGF17としてNM_003867、AB009249、AY358869、BC069475、BC105131、BC113489、BC143789等、FGF19としてNM_005117、AB18122、AF110400、AY358302、BC017664、BT006729、CR610453等、FGF20としてNM_019851、AB030648、AB044277、BC096720、BC098128、BC098153、BC098339、BC127125、BC137446、BC137447、BC141979、BC141980等が登録されている。アミノ酸配列としては、ヒトFGF8(４種のスプライスバリアント)としてNP_001193318、NP_006110、NP_149353、NP_149354、NP_149355等、FGF9としてNP_002001等、FGF10としてNP_004456等、FGF17としてNP_003858等、FGF19としてNP_005108等、FGF20としてNP_062825等が登録されている。他に各々、ゲノム遺伝子、変異体やスプライスバリアントも登録されている。更に、他の動物種、人工合成遺伝子も同様に登録されている。本発明においては、造血幹細胞に対する増幅活性がある限り、これらのいずれをも利用することができる。 The base sequence of each factor is NM_001206389, NM_006119, NM_033163, NM_0033164, NM_0033165, AB14615, BC069106, BC128235, BC128236, U36223, U46211, U46212, U46213, U46214, etc. as FGF8 (four splice variants), NM_002010, AK095571 , AK290792, BC069692, BC103978, BC103979, D4838, etc., NM10 as FGF10, AB002097, AF411527, AF508782, AK315314, BC069561, BC105021, BC105023, BC143772, etc. , BC105131, BC113489, BC143789, etc., NM19 as FGF19, AB18122, AF110400, AY358302, BC017664, BT006729, CR610453, etc. Etc. are registered. As amino acid sequences, human FGF8 (4 types of splice variants) NP_001193318, NP_006110, NP_149353, NP_149354, NP_149355, etc., FGF9, NP_002001, etc., FGF10, NP_004456, etc. Is registered. In addition, genomic genes, mutants and splice variants are also registered. Furthermore, other animal species and artificial synthetic genes are registered in the same manner. In the present invention, any of these can be used as long as they have amplification activity against hematopoietic stem cells.
本発明の造血幹細胞を増殖させるためには、本発明の組成物を含む適当な培地、好ましくは無血清培地で造血幹細胞を培養する。
培地は、造血幹細胞の生存や増殖が阻害されない限り特に限定されないが、例えば、StemSpan（Stem Cell technologies）、STEMα（STEM ALPHA）、StemPro-34無血清培地（Gibco Invitrogen）、StemPro MSC無血清培地（Invitorogen）、HSC-CFU培地(Miltenyl Biotech)、S-Clone無血清培地（SF-02、SF-03、CM-B、SF-B）（三光純薬）、HPGM培地（三光純薬）、AIM V培地（Invitorogen）、Marrow MAX骨髄培地（Invitrogen）、KnockOut DMEM/F-12培地（Invtrogen）、Stemline造血幹細胞増殖培地（Sigma）、SYN無血清培地（SYN H、SYN B）（AbCys SA）、SPE IV培地（AbCys SA）、MyeloCult培地（StemCell Technologies）、HPG無血清培地（Lonza）、UltraCULTURE培地（Lonza）、Opti-MEM培地（Gibco Invitrogen他）、MEM培地（Gibco Invitrogen他）、MEMα（Gibco Invitrogen他）、DMEM培地(Gibco Invitrogen他)、IMDM培地(Gibco Invitrogen他)、PRMI1640培地(Gibco Invitrogen他)、Ham F-12培地（Gibco他）、RD培地等を用いることができる。
培地には、造血幹細胞の増幅に効果があるLDLリポタンパク質、プロスタグランジンE1ないしE2、StemRegenin 1(SR１、アリル炭化水素受容体アンタゴニスト)、5-AzaD（5-aza-2'-deocycitidine D）、TSA（trichostatin）、TEPA（銅キレート剤）、等を添加してもよい。
In order to proliferate the hematopoietic stem cells of the present invention, the hematopoietic stem cells are cultured in a suitable medium containing the composition of the present invention, preferably a serum-free medium.
The medium is not particularly limited as long as the hematopoietic stem cell survival and proliferation are not inhibited. For example, StemSpan (Stem Cell technologies), STEMα (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium ( Invitorogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitorogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM / F-12 medium (Invtrogen), Stemline hematopoietic stem cell growth medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen et al.), MEM medium (Gibco Invitrogen et al.), MEMα (Gibco Invitrogen et al.), DMEM medium (Gibco Invitrogen et al.), IMDM medium (Gibco Invitrogen et al.), PRMI1640 medium (Gibco Invitrogen et al.), Ham F-12 medium (Gibco other), can be used RD medium and the like.
The medium contains LDL lipoprotein, prostaglandin E1 to E2, StemRegenin 1 (SR1, allyl hydrocarbon receptor antagonist), 5-AzaD (5-aza-2'-deocycitidine D), which is effective for the amplification of hematopoietic stem cells , TSA (trichostatin), TEPA (copper chelator), etc. may be added.
更に細胞の増幅維持等に効果があるインスリン、トランスフェリン、ラクトフェリン、２−メルカプトエタノール、エタノールアミン、亜セレン酸ナトリウム、HEPES、モノチオグリセロール、ピルビン酸ナトリウム、ポリエチレングリコール、各種ビタミン、各種アミノ酸、各種増殖因子、各種抗生物質、ヘパリン、ヘパラン硫酸、コンドロイチン硫酸、等を添加してもよい。
また、細胞外マトリックスであるコラーゲン（タイプI、III、IV、V、VI、VII、VIII等）、フィブロネクチン、バイグリカン、デコリン、ラミニン、等を添加してもよい。
なお、培地に動物由来の血清を添加してもよいが、血清を使う場合はヒト血清が好ましく、移植予定患者の血清を用いることがより好ましい。
Insulin, transferrin, lactoferrin, 2-mercaptoethanol, ethanolamine, sodium selenite, HEPES, monothioglycerol, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, various proliferation Factors, various antibiotics, heparin, heparan sulfate, chondroitin sulfate, etc. may be added.
Further, collagen (types I, III, IV, V, VI, VII, VIII, etc.), fibronectin, biglycan, decorin, laminin, etc., which are extracellular matrices, may be added.
In addition, animal-derived serum may be added to the medium, but when using serum, human serum is preferable, and it is more preferable to use the serum of the patient to be transplanted.
本発明の造血幹細胞を増殖させるためには、本発明の組成物を培地に添加し培養するが、骨髄ストローマ細胞など造血幹細胞を支持するフィーダー細胞等と共培養してもよいし、骨髄ストローマ細胞培養上清と共に培養してもよい。また、本発明の組成物を様々な担体を介して又は介さずにシャーレ等培養器（装置）に付着又は共有結合させて培養してもよいし、本発明の組成物の各因子を発現させたフィーダー細胞と共培養してもよい。
培地中のSCFの濃度は、0.1 ng/ml〜1 μg/ml、好ましくは5〜500 ng/ml、より好ましくは10〜200 ng/mlである。
培地中のIL31、PGF/PLGF、VEGF-B、IGFBP6、EFEMP1、FGF8、FGF9、FGF10、FGF17、FGF19、FGF20の濃度は、それぞれ、通常約0.1 ng/ml〜1 μg/ml、好ましくは5〜500 ng/ml、より好ましくは20〜200 ng/mlである。
造血幹細胞（未分画の造血幹細胞を含む細胞集団又は部分分画でもよい）は、培養用シャーレ、フラスコ、プレート、バッグ等、あるいは自動培養装置に、本発明の組成物やその他の因子や化合物を添加した上記記載の培地、好ましくは無血清培地に浮遊させ、5% CO2、37℃のインキュベーター内で、数日から１ヶ月ほど、好ましくは１週間から３週間程度、培地交換し容量を増やしながら培養することができる。また、酸素分圧を調整した環境で培養しても良い。
In order to proliferate the hematopoietic stem cells of the present invention, the composition of the present invention is added to the medium and cultured, but it may be co-cultured with feeder cells that support hematopoietic stem cells such as bone marrow stromal cells, or bone marrow stromal cells. You may culture with a culture supernatant. In addition, the composition of the present invention may be cultured while being attached or covalently bonded to a culture apparatus (apparatus) such as a petri dish with or without various carriers, or by expressing each factor of the composition of the present invention. Co-culture with feeder cells.
The concentration of SCF in the medium is 0.1 ng / ml to 1 μg / ml, preferably 5 to 500 ng / ml, more preferably 10 to 200 ng / ml.
The concentrations of IL31, PGF / PLGF, VEGF-B, IGFBP6, EFEMP1, FGF8, FGF9, FGF10, FGF17, FGF19, and FGF20 in the medium are usually about 0.1 ng / ml to 1 μg / ml, preferably 5 to 500 ng / ml, more preferably 20 to 200 ng / ml.
Hematopoietic stem cells (which may be a cell population or partial fraction containing unfractionated hematopoietic stem cells) may be added to a culture dish, flask, plate, bag, etc. In a 5% CO 2 , 37 ° C. incubator, the medium is changed for several days to about 1 month, preferably about 1 week to 3 weeks. It can be cultured while increasing. Moreover, you may culture | cultivate in the environment which adjusted oxygen partial pressure.
本発明の組成物は、造血幹細胞を増幅しうる他の因子（自己複製因子、サイトカイン、ケモカイン、増殖因子、増幅因子、分化因子、造血因子等）や他の化合物と共に培地に添加することにより、他の因子の増幅能を格段に増加させる効果がある。
このような他の因子は、培養容器に直接固定又は種々のタンパク質（ペプチド）等の担体を介して共有結合又は非共有結合で固定化して、無血清培地又はヒト血清を含む培地で造血幹細胞を体外で増幅することもできる。
このような他の因子は、造血細胞の生存維持、増殖、増幅、自己複製、未分化維持等を少なくとも促進させる活性のある因子である。このような因子として、例えば、Flt3 ligand、NOV、JAG1細胞外ドメイン、Pleiotrophin、Timp3、Oncostatin M、BMP4、IGF2等が挙げられる。これら以外にも、IL6（interleukin 6）とsIL6R（可溶性IL6レセプター）、IL1、IL2、IL3、IL5、IL7、IL8、IL10、IL11、IL16、IL27（C19orf10）、TPO（thrombopoietin）、Notch ligand（Jaggedファミリー／Deltaファミリー）キメラタンパク質、FGF１（fibroblast growth factor 1）、FGF2、FGF4、FGF8b、Ang1（Angiopoietin 1/Tie2 ligand）、IGF1、 IGFBP2（IGF binding protein 2）、IGFBP3、IGFBP7/IGFBPrP1、TGFβ (transforming growth factor)、Angpl（Angiopoietin-like protein）ファミリーのAngpl2、Angpl3、Angpl5、Angptl7やMfap4、PRG4（Hemangiopoietin, Lubricin）、Galectin-1（LGAS1）、VEGFA、VEGFC、VEGF-D、Wnt2、Wnt3a、Wnt5a、Wnt5b、Wnt7a、Wnt7b、Wnt10b、Wnt16、SDF-1、G-CSF、EPO、GM-CSF、CTC、CT-1、PDGF、PrP（prion protein）、Sonic hedgehog、PDGF、RANTES、種々のケモカイン、SDF-1、MIP-1α、EGF、LIF等が挙げられる。
また、他の化合物として、SB203580,SB239063、6-gingerol等のP38阻害剤、CHIR99021、Kenpaullone等のWntシグナル活性化剤（GSK3β阻害剤を含む）、SP600125、L-JNKi1 等のJNK阻害剤、2-Cl-C.OXT-A、dioleoyl phosphatidic acid(DOPA)、C6 ceramide、Trastuzumab等のERK活性化剤、ヒストンアセチルトランスフェラーゼ阻害剤、Valproic acid等のヒストンデアセチラーゼ阻害剤を添加してもよい。
The composition of the present invention is added to a medium together with other factors capable of amplifying hematopoietic stem cells (self-replicating factor, cytokine, chemokine, growth factor, amplification factor, differentiation factor, hematopoietic factor, etc.) It has the effect of significantly increasing the amplification ability of other factors.
Such other factors can be directly fixed to the culture vessel or covalently or non-covalently immobilized via a carrier such as various proteins (peptides), so that hematopoietic stem cells are cultured in a serum-free medium or a medium containing human serum. It can be amplified outside the body.
Such other factors are active factors that at least promote hematopoietic cell survival, proliferation, amplification, self-replication, undifferentiated maintenance, and the like. Examples of such factors include Flt3 ligand, NOV, JAG1 extracellular domain, Pleiotrophin, Timp3, Oncostatin M, BMP4, IGF2, and the like. Besides these, IL6 (interleukin 6) and sIL6R (soluble IL6 receptor), IL1, IL2, IL3, IL5, IL7, IL8, IL10, IL11, IL16, IL27 (C19orf10), TPO (thrombopoietin), Notch ligand (Jagged) Family / Delta family) Chimeric protein, FGF1 (fibroblast growth factor 1), FGF2, FGF4, FGF8b, Ang1 (Angiopoietin 1 / Tie2 ligand), IGF1, IGFBP2 (IGF binding protein 2), IGFBP3, IGFBP7 / IGFBPrP1, TGFβ (transforming growth factor), Angpl (Angiopoietin-like protein) family Angpl2, Angpl3, Angpl5, Angptl7, Mfap4, PRG4 (Hemangiopoietin, Lubricin), Galectin-1 (LGAS1), VEGFA, VEGFC, VEGF-D, Wnt2, Wnt3a, Wnt5a , Wnt5b, Wnt7a, Wnt7b, Wnt10b, Wnt16, SDF-1, G-CSF, EPO, GM-CSF, CTC, CT-1, PDGF, PrP (prion protein), Sonic hedgehog, PDGF, RANTES, various chemokines, SDF-1, MIP-1α, EGF, LIF and the like can be mentioned.
Other compounds include P38 inhibitors such as SB203580, SB239063 and 6-gingerol, Wnt signal activators (including GSK3β inhibitors) such as CHIR99021 and Kenpaullone, JNK inhibitors such as SP600125 and L-JNKi1, 2 An ERK activator such as -Cl-C.OXT-A, dioleoyl phosphatidic acid (DOPA), C6 ceramide and Trastuzumab, a histone acetyltransferase inhibitor, and a histone deacetylase inhibitor such as Valproic acid may be added.
ヒト造血幹細胞は、臍帯血、胎児肝臓、骨髄、胎児骨髄、末梢血、G-CSF等のサイトカインや抗癌剤の投与によって幹細胞を動員した末梢血、末梢血由来の細胞群等から純化することができる。ヒトES細胞、ヒトiPS細胞より造血幹細胞や造血系プロジェニター細胞に誘導された細胞、更にはヒト体細胞から遺伝子操作等で直接作成された造血幹細胞やプロジェニター細胞を用いることもできる。これらから、抗体を用いて免疫学的に染色し、セルソーター、磁気ビーズ等を用いて分離するか、ロゼット形成による細胞分離法や各種自動分離装置等を用いて分離することにより造血幹細胞を濃縮した分画を取得できる。
ヒト造血幹細胞のマーカーとしてはCD34陽性、CD38弱陽性（陰性）、CD133陽性、KDR陽性、CD90(Thy-1)陽性、CD117（c-Kit）陽性等が知られており、細胞分化抗原陰性等と組み合わせて用いることができる。さらに、ヒト骨髄や臍帯血や末梢血由来等の造血幹細胞としてSP(side population)細胞（Hoechst33342陰性細胞）を用いてもよい。
また、造血幹細胞を純化（単離）あるいは濃縮することなく、ヒト骨髄、臍帯血、抹消血等から赤血球等を除いた有核（又は単核）細胞又は幹細胞分画をそのまま培養に用いることもできる。ヒトES細胞やiPS細胞、またヒト体細胞から直接造血幹細胞や造血系プロジェニター細胞に誘導した細胞を純化することなく用いることもできるし、上記方法で純化した造血幹細胞やプロジェニター細胞を用いることもできる。
Human hematopoietic stem cells can be purified from umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, peripheral blood, peripheral blood mobilized by administration of cytokines and anticancer agents such as G-CSF, and peripheral blood-derived cell groups . Cells derived from human ES cells or human iPS cells into hematopoietic stem cells or hematopoietic progenitor cells, or hematopoietic stem cells or progenitor cells directly produced from human somatic cells by genetic manipulation or the like can also be used. From these, hematopoietic stem cells were concentrated by immunologically staining with antibodies and separating using a cell sorter, magnetic beads, etc., or by separating using a cell separation method by rosette formation, various automatic separation devices, etc. A fraction can be acquired.
As markers of human hematopoietic stem cells, CD34 positive, CD38 weak positive (negative), CD133 positive, KDR positive, CD90 (Thy-1) positive, CD117 (c-Kit) positive, etc. are known, such as cell differentiation antigen negative Can be used in combination. Furthermore, SP (side population) cells (Hoechst33342 negative cells) may be used as hematopoietic stem cells derived from human bone marrow, umbilical cord blood or peripheral blood.
In addition, nucleated (or mononuclear) cells or stem cell fractions obtained by removing red blood cells from human bone marrow, umbilical cord blood, peripheral blood, etc. may be used for culturing without purifying (isolating) or concentrating hematopoietic stem cells. it can. Human ES cells, iPS cells, and cells derived directly from human somatic cells into hematopoietic stem cells and hematopoietic progenitor cells can be used without purification, or hematopoietic stem cells and progenitor cells purified by the above method should be used. You can also.
以下、実施例にて本発明を例証するが本発明を限定することを意図するものではない。
以下の実施例において、全細胞数はヘマトサイトメーターで測定した。全細胞増幅倍率は、増幅前後の全細胞数の変化（倍率）として表す。増幅比は、基準（SCFのみで培養した場合）の全細胞数に対する因子を添加して増幅した全細胞数の比として表す。幹細胞の割合については、増幅した全細胞を、FITCラベルCD34抗体とPEラベルCD133抗体で反応させて、フローサイトメーター(ミリポア社製easyCyte5)によって全細胞中の造血幹細胞（CD34陽性細胞）の割合(%)を測定した。幹細胞増幅倍率は、培養前の造血幹細胞数に対して、体外で増幅した造血幹細胞数の増幅倍率を表す。増幅倍率や増幅比等は3回の実験における平均値を示し、±20％程度の誤差があった。
The following examples illustrate the invention but are not intended to limit the invention.
In the following examples, the total cell number was measured with a hematocytometer. The whole cell amplification factor is expressed as a change (magnification) of the total number of cells before and after amplification. The amplification ratio is expressed as the ratio of the total number of cells amplified by adding a factor to the total number of cells (when cultured with only SCF). Regarding the ratio of stem cells, the whole amplified cells were reacted with FITC-labeled CD34 antibody and PE-labeled CD133 antibody, and the ratio of hematopoietic stem cells (CD34 positive cells) in the total cells by flow cytometer (Millipore easyCyte5) ( %) Was measured. The stem cell amplification factor represents the amplification factor of the number of hematopoietic stem cells amplified outside the body with respect to the number of hematopoietic stem cells before culture. Amplification magnification, amplification ratio, etc. showed average values in three experiments, with an error of about ± 20%.
ヒト臍帯血CD34陽性細胞（Lonza社製、フローサイトメーター(FACS)解析で純度97％以上であることを確認）を造血幹細胞として用いた。無血清培地（StemCell Technologies社製StemSpan SFEM）に、ヒトSCF（Peprotech社）（20 ng/ml）を添加した。これに、ヒトIGFBP6（R&D Systems社）、ヒトPGF/PLGF（Peprotech社）、ヒトVEGF-B（Peprotech社）、ヒトEFEMP1（OriGene Technologies社）、ヒトIL31（Peprotech社）、ヒトFGF8（Peprotech社）、ヒトFGF9(Peprotech社)、ヒトFGF10(Peprotech社)、ヒトFGF17（Peprotech社）、ヒトFGF19（Peprotech社）、又はヒトFGF20（Peprotech社）を各100 ng/mlの濃度で添加した培地を準備した。FGFファミリーを添加した場合には、ヘパリン（1μg/ml）も添加した。
96穴プレート1穴に、上記各培地200μlにCD34陽性細胞を2.0〜6.0×104細胞（１.0〜3.0×105細胞/ml）を混ぜて、5%CO2、37℃で培養開始した。同じ成分を含む新鮮培地で適時部分交換し、48穴プレートに移し容量を増やしながら2週間培養した。
結果を表１に示す。
Human umbilical cord blood CD34 positive cells (manufactured by Lonza, confirmed to have a purity of 97% or more by flow cytometer (FACS) analysis) were used as hematopoietic stem cells. Human SCF (Peprotech) (20 ng / ml) was added to a serum-free medium (StemSpan SFEM manufactured by StemCell Technologies). Human IGFBP6 (R & D Systems), human PGF / PLGF (Peprotech), human VEGF-B (Peprotech), human EFEMP1 (OriGene Technologies), human IL31 (Peprotech), human FGF8 (Peprotech) Preparation of medium supplemented with human FGF9 (Peprotech), human FGF10 (Peprotech), human FGF17 (Peprotech), human FGF19 (Peprotech), or human FGF20 (Peprotech) at a concentration of 100 ng / ml did. When the FGF family was added, heparin (1 μg / ml) was also added.
In one well of a 96-well plate, mix 2.0-6.0 × 10 4 cells (1.0-3.0 × 10 5 cells / ml) with CD34-positive cells in 200 μl of the above medium and start culturing at 37 ° C. with 5% CO 2 . did. The fresh medium containing the same components was replaced in a timely manner, transferred to a 48-well plate, and cultured for 2 weeks while increasing the volume.
The results are shown in Table 1.
表１から、SCF存在下で、造血幹細胞は今回新たに発見したIGFBP6、IL31、PGF/PLGF、VEGF-B、EFEMP1、FGF8、FGF9、FGF10、FGF17、FGF19、FGF20の何れに因子の添加によっても、SCF単独と比較して全細胞の増幅倍率が増えることが分かる。造血幹細胞（CD34細胞）の割合はSCF単独とほぼ同じであり、結果として造血幹細胞の増幅倍率もSCF単独と比較して増加することが分かる。特に、SCF単独添加の場合に比べた増幅比が1.3以上、特に1.4以上の因子が好ましい。 From Table 1, in the presence of SCF, hematopoietic stem cells can be obtained by adding factors to any of the newly discovered IGFBP6, IL31, PGF / PLGF, VEGF-B, EFEMP1, FGF8, FGF9, FGF10, FGF17, FGF19, and FGF20. It can be seen that the amplification factor of all cells increases compared to SCF alone. It can be seen that the ratio of hematopoietic stem cells (CD34 cells) is almost the same as that of SCF alone, and as a result, the amplification factor of hematopoietic stem cells is also increased compared to that of SCF alone. In particular, a factor having an amplification ratio of 1.3 or more, particularly 1.4 or more, compared to the case of adding SCF alone is preferable.
Claims (8)
（１）幹細胞因子(SCF)
（２）IL31(interleukin-31)、PGF(placental growth factor）、VEGF-B(vascular endothelial growth factor B）、IGFBP6(insulin-like growth factor binding protein 6）、EFEMP1(EGF-containing fibulin-like extracellular matrix protein 1)、FGF8、FGF9、FGF10、FGF17、FGF19及びFGF20(線維芽細胞増殖因子8, 9, 10, 17, 19及び20)から成る群から選択される少なくとも１種の因子 A composition for amplifying human hematopoietic stem cells comprising the following factors (1) and (2).
(1) Stem cell factor (SCF)
(2) IL31 (interleukin-31), PGF (placental growth factor), VEGF-B (vascular endothelial growth factor B), IGFBP6 (insulin-like growth factor binding protein 6), EFEMP1 (EGF-containing fibulin-like extracellular matrix) at least one factor selected from the group consisting of protein 1), FGF8, FGF9, FGF10, FGF17, FGF19 and FGF20 (fibroblast growth factor 8, 9, 10, 17, 19 and 20)
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