【０００１】
【発明の属する技術分野】
本発明は、ヒト血液を含有するキメラ動物及びその製造方法に関する。
【０００２】
【従来の技術】
マウスにおいて造血幹細胞活性はin vivoの実験系、Long term marrow repopulation法によって測定される。この方法を用いることで、マウス造血幹細胞はほぼ純化されたと考えられ、これを材料として更なる研究が行えるようになっている1)。ヒト造血幹細胞のアッセイとしては最近まで適当な動物モデルが存在せず、in vitroの骨髄培養によりコロニー形成能を測定する方法2-4)が一般的であった。しかしcolony-forming cells(CFCs)やlong-term culture-initiating cells (LTC-IC)で観察されるコロニー形成能を持つ細胞のin vivo repopurating capasityにについては不明確である。1983年のscidマウスの発見5)により、ヒトの造血系をin vivoで再構築できることが示され、以後その研究法は急速に普及した。造血幹細胞の検出にはlong term multilineage engraftmentを測定する必要がある。最近ではより効率よくヒト造血系を再構築させうるNOD/SCIDマウスが開発6)されアッセイに頻用されるようになった7-10)。このマウスを用いたシステムはヒト血液細胞が容易に生着する点で優れた系といえるが、放射線照射により移植を成立させることによるstromaなど周辺組織への影響、小動物の限界としてヒト血液をアッセイするには寿命が短いこと、ヒト造血幹細胞(hematopoietic stem cell, 以下HSC)の増殖能をみるのにもscaleが小さいこと、一般にT細胞系への分化は観察できない11-13)ことが問題点としてあげられる。
【０００３】
ヒト造血幹細胞をアッセイするために大動物を用いることができれば、さらにヒト造血系を正確にアッセイすることが可能となると考えられるが、これまでヒト血液系を導入できるような免疫不全を持つ大動物は知られていない。Zanjaniらはこの問題をin utero transplantationにより解決した14)。免疫系が成熟する以前の時期の胎仔期であればzenograftを拒絶できない可能性があり、大動物に異種移植を行える可能性がある。彼らはヒト造血細胞をヒツジ胎仔に移植することによりzeno-transplantationにもかかわらずキメラを作出し、ヒト造血幹細胞に関する優れた研究をおこなっている15-18)。最近ではnod/scidマウス13)やイヌ胎仔19)に同様の手法でin uteroヒト造血幹細胞移植を行いキメラを作出しえたとする報告もなされている。
【０００４】
この手法は造血幹細胞のアッセイにとどまらず、さまざまな応用が可能である。近年の産科学領域における急速な出生前診断技術の向上により、現在では1st trimesterに胎児異常を同定することも可能となりつつある20-23)。遺伝的背景を持つある種の疾患については出生直後の骨髄移植が治療に有効であるが、これにはmyeloabrationの必要や、intensiveな免疫抑制、適合ドナーの問題など、解決されていないさまざまな問題がある。一方子宮内移植により骨髄移植を行う方法が完成すれば、このような問題が解決される可能性がある。alloの子宮内造血幹細胞移植の実験はげっ歯動物24),ヤギ25-28), 非ヒト霊長類29-32)で検討され、ヒトでも臨床で数例行われるようになった33-34)。さらに移植造血幹細胞に治療遺伝子を導入しておくことにより子宮内胎児遺伝子治療の可能性も期待できる35)。
【０００５】
また動物実験や様々な臨床報告によると、血液キメリズムの達成は移植免疫の寛容をもたらすことが知られている36-39)。この手法により大動物にマイクロキメリズムが達成されると、その動物はヒト臓器に関して免疫寛容がもたらされている可能性があり、ヒト臓器のincubatorのように用いることもできるかもしれない40)。
【０００６】
【発明が解決しようとする課題】
本発明は、ヒトの血液を含有するキメラ動物を提供することを目的とする。
【０００７】
【課題を解決するための手段】
本発明者は、上記課題を解決するため鋭意研究を行った結果、動物の胎児にヒトの血液幹細胞を移植することにより、ヒトの血液が循環する動物を作製することに成功し、本発明を完成するに至った。
【０００８】
すなわち、本発明は、ヒト血液を含有するキメラ動物である。該動物としては、例えばブタなどの家畜動物が挙げられる。
さらに、本発明は、造血幹細胞を動物の胎児に移植することを特徴とするキメラ動物の製造方法である。ここで、胎児としては妊娠３０日から６０日のものが挙げられる。
以下、本発明を詳細に説明する。
【０００９】
【発明の実施の形態】
本発明者は、ヒト造血幹細胞をブタ胎仔に移植する異種移植の実験系でヒト血液キメラを作出できるかを調べる実験を行い、造血幹細胞がmultilineageに分化し、microchimeraを長期にわたって維持できることを明らかにした。
【００１０】
本発明は、ヒト型の血液を体内で循環し得るキメラ動物に関するものであり、特に家畜又は非ヒト霊長類を対象とするものである。
本発明において移植する造血幹細胞としては、ヒト臍帯血および骨髄由来のものが挙げられる。臍帯血及び骨髄からの造血幹細胞は、比重遠心法（例えばFicoll 、Lymphoprepなどを用いた方法）、パニング法などの一般に行われている手法により採取することができる。
【００１１】
上記の通り採取されたヒト造血幹細胞のうち、本発明ではCD34陽性かつCD3陰性の有核細胞を使用することが好ましい。
CD34陽性有核細胞は、採取された細胞を抗ヒトCD34モノクローナル抗体で標識し、MACSまたはFACS装置を用いて選別することができる。一方、CD3陰性有核細胞は、採取された細胞に、ビオチンを結合した抗-CD3 モノクローナル抗体を反応させて結合し、得られる複合体にストレプトアビジンを結合させて標識する。そして磁気ビーズで上記複合体を除去することにより、CD3陰性有核細胞を得ることができる。
【００１２】
移植するための造血幹細胞は、適当な緩衝液（例えばPBS）に希釈して使用する。細胞濃度は3.9x105〜1.5x106個、好ましくは一匹あたり1x106個以上である。
上記の通り得られた造血幹細胞を動物（ヒトを除く実験動物）の胎児に移植する。動物としては、ウマ、ブタ、イノシシ、ロバ、ヤギなどの比較的大型の家畜動物と、サルのような非ヒト霊長類を使用することができるが、ブタが好ましい。ブタの品種は特に限定されるものではなく、メイシャン、モンカイ、ユカタンマイクロブタなどの黒色品種、六白と呼ばれるバークシャー種、デュロックなどの茶色品種、 大ヨークシャー、ランドレースなどの白色品種が挙げられる。
【００１３】
移植は以下の通り行う。
まず、家畜動物を人工授精により妊娠させ、妊娠の診断は人工授精後超音波により行った。移植日は、妊娠後30日〜81日、好ましくは30〜60日、さらに好ましくは35日〜52日である。
【００１４】
移植当日、妊娠動物に麻酔導入後、腹部を剃毛し充分に石鹸水等で腹部を洗浄及び消毒する。
細胞は、超音波断層装置を用いて、モニター上に写し出される映像を観察しながら胎仔に移植する。細胞の移植は注射針又は穿刺針により行い、その注入は、胎仔一匹あたり0.2mlの生理食塩水に懸濁した造血幹細胞を穿刺針を通じて注入する。移植に使用される穿刺針の太さは、22〜23ゲージ、好ましくは22ゲージである。細胞は胎仔腹腔内に注入されるが、これと同時に被移植胎仔を生下時に同定する目的で、レントゲンで同定されるマーカーを挿入することが好ましい。その後、必要により抗生物質を注入し、細胞の移植が完了する。
【００１５】
術後は、感染症を予防する目的で抗生物質の投与することが好ましい。
移植後の胎児を母体内で生育させ、通常分娩又は帝王切開によりキメラ動物を得ることができる。得られた動物にはヒトの血液が循環しているため、ヒト疾患の治療用医薬を開発するための実験動物等として有用である。
【００１６】
また、本発明は、ヒトの血液細胞（あるいは造血幹細胞）の増殖系として利用できるため、輸血用血液、骨髄移植用骨髄を生産するために有用である。例えば、白血病患者に対し、ブタの骨髄から当該患者のの造血幹細胞を回収して自家移植を行うことが可能である。
さらに、本発明のキメラ動物には、ヒトに対して免疫寛容が誘導されるため、ヒトの臓器移植を行うにあたり、臓器の維持・再生の場所としてキメラブタを使えることができる。
【００１７】
【実施例】
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれら実施例にその技術的範囲が限定されるものではない。
〔実施例1〕 ヒト血液キメラブタの作製
(1) 造血幹細胞の調製
造血幹細胞の供給源としてはヒト臍帯血および骨髄を用いた。これら血液細胞を比重遠心法により分離し、有核細胞を得た。この有核細胞はphosphate-buffered saline(PBS)にて洗浄した。造血幹細胞移植のための分画としては、有核細胞全て、CD34陽性細胞、CD3陰性有核細胞をそれぞれ用いた。
【００１８】
▲１▼ CD34陽性細胞の純化
有核細胞を抗ヒトCD34モノクローナル抗体で標識した。標識には蛍光、もしくは磁気ビーズをもちいた。有核細胞からヒトCD34陽性細胞を純化するために、MACS（Direct CD34 isolation kit Miltenyi Biotec Bergisch Gladbach, Germany）またはFACS Vantage(Becton Dickinson, San Jose, CA)を用いた。
【００１９】
▲２▼ CD3陰性有核細胞の純化
有核細胞をbiotin - conjugated anti-CD3 mAbs (PharMingen, San Diego, CA)により標識し、洗浄後BigMag streptavidin ultra-load particles (PerSeptive Biosystems, Inc. Framingham, MA) でさらにこれを標識、 Magnetic Particle Concentrator (Dynal MPC-1 Oslo, Norway)によりCD3陽性細胞を除去した。
【００２０】
(2) ブタ胎仔へのヒト造血幹細胞移植
▲１▼ 麻酔
移植当日、妊娠ブタはケタラールの筋注により前麻酔をおこない、その後マスクによる吸入麻酔をおこなった。吸入麻酔には笑気、酸素、イソフルレンを用いた。
【００２１】
▲２▼ 消毒
妊娠ブタに麻酔導入後、腹部を剃毛し充分に石鹸水で腹部を洗浄した後、イソジンにて充分に消毒した。
【００２２】
▲３▼ 超音波によるブタ胎仔の描出
ブタ胎仔への細胞移植には超音波断層装置Aloka ultrasound diagnostic equipment SSD-1000(ALOKA CO.,LTD Tokyo, Japan)をもちいた。超音波プローブには滅菌カバーを装着した7.5MHz electronic convex sector probe(Aloka; UST-987-7-5)を使用した。プローブに穿刺用ガイド(puncture adapter; Aloka MP-2458)を装着し、胎仔をモニター上に描出した。
【００２３】
▲４▼ 穿刺針
胎仔へのヒト造血幹細胞導入のためにもちいた穿刺針は22-gauge, 150mm P.T.C.D. needle(Top Tokyo, Japan)である。細胞は胎仔腹腔内に注入されるが（後述）、このさい同時に被移植胎仔を生下時に同定する目的に、レントゲンで同定されるマーカーを挿入した。マーカーはP.T.C.D. needleのスタイレットを3mmの長さに細切し、オートクレーブにて滅菌することにより作成した。胎仔穿刺時にはP.T.C.D. needleのスタイレットを抜去し、この無菌マーカーを外筒内に挿入後胎仔腹腔を穿刺した。その後スタイレットを挿入圧出することでマーカーを胎仔腹腔内に挿入した。
【００２４】
▲５▼ 細胞の注入
再度スタイレットを抜去し、胎仔一匹あたり0.2mlの生理食塩水に懸濁した造血幹細胞をP.T.C.D. needleを通じて注入した。その後抗生物質を胎仔腹腔内に0.2ml、羊水腔に0.3ml注入後、穿刺針を抜去した。この手技により最大４胎仔に細胞の移植が可能であった。
なお、移植は多くは超音波ガイド下におこなったが、一部では全身麻酔下に開腹し、子宮を直視下に胎仔を同定し、胎仔腹部に細胞移植をおこなった。
【００２５】
▲６▼ 術後の抗生物質の投与
妊娠ブタにはその後２日間にわたり抗生物質を筋注により投与した。
▲７▼ 分娩
分娩は自然経腟分娩、もしくは帝王切開分娩によりおこなった。ブタ妊娠期間は通常114日である。生下時、出生児および死産児を軟X線撮影し、マーカーを同定することにより被移植仔を同定した。
【００２６】
〔実施例2〕 出生仔の解析
(1) ヒト造血幹細胞移植を受けた新生仔の末梢血、骨髄、臓器の採取
ケタラールによる麻酔科に経静脈より末梢血を、骨盤蝶形骨より骨髄細胞採取した。また麻酔科に安楽死させた後ブタ各種臓器を採取した。
(2) 有核血球細胞の分離
得られた血液はin 0.83% ammonium chloride with 0.1% sodium bicarbonate(pH7.0)を用いて赤血球を溶血、PBSにて洗浄し有核細胞とした。
【００２７】
(3) 有核細胞中のヒト血液細胞の同定
ヒト血液細胞の同定にはフローサイトメトリーおよびPCR法を用いた。
▲１▼ フローサイトメトリー
得られた有核細胞を、抗ヒトCD45抗体、抗ブタpan-tissue抗体で標識した。抗ヒトCD45抗体陽性かつ抗ブタpan-tissue抗体陰性細胞がヒト血液細胞と判定される。ここにさらにヒト血球分化抗原抗体、CD3,CD19, CD13,CD14, CD33,CD34,CD56,CD41 and CD61を加え、血球細胞の分化についても観察した。
【００２８】
▲２▼ PCR
得られた有核細胞よりゲノムDNAをSepaGene (Sanko-jyunyaku Tokyo, Japan)により回収した。ヒトゲノムの同定にはAlu配列に特異的なプライマーを設計し、PCRにより増幅することでおこなった。
プライマーは以下の２種である。
5'-CTGGGCGCAAGAACGAGATTCTAT-3' （配列番号１）
5'-CTCACTACTTTGTGACAGGTTCA-3' （配列番号２）
PCRは94℃60秒の反応後、94℃20秒＋58℃20秒＋72℃30秒の反応を43サイクルおこない、最後に72℃７分の反応後終了した。得られたPCR産物はアガロースゲル上で臭化ブロマイドにたいする紫外線照射で観察した。
【００２９】
(4) ブタ胎仔中に存在するヒト造血幹細胞のコロニー形成能の測定
ブタ骨髄有核細胞を分離後、抗ヒトCD34抗体、抗ヒトCD45抗体で標識し、そのダブルポジティブ細胞をFACSにより分離回収した。同細胞をヒト血液増殖因子を含む軟寒天培地cells (Methocult GF H4434; StemCell Technologies Inc. Vancouver, B.C.)上で培養し、２週間後のコロニー形成能を観察した。
【００３０】
(5) 結果
最初に本発明者は、ヒツジやイヌで報告されている方法を踏襲し、開腹後子宮外より胎児腹部を穿刺することにより子宮内造血幹細胞移植を行った。このメリットは全妊娠胎仔に細胞移植を行いうることである。しかし、その結果移植した６例中４匹が術後5日以内に感染によると思われる流産に至った。
【００３１】
そこで本発明者は、開腹術を施行せず、超音波ガイド下に母体の体外から胎仔に移植を行う方法を開発した。この方法では母体の腸管ガスのために全ての胎仔を超音波下に同定することは困難であり、実際には妊娠胎仔の平均4匹に移植が可能であった。しかしブタ腹壁は非常に細菌が多いと考えられ、この方法を用いても、徹底的な感染コントロールが流産防止に必要であった。具体的には腹壁の消毒を十分に行った後に移植を行い、更に胎児、羊水腔、母体に抗生物質を投与することで流産は顕著に減少した。
【００３２】
ブタ胎仔は妊娠30日でCRLが約20mmとなる。本発明の方法で技術的に移植が可能となるのはこの時期からで、妊娠40日以降は腹部前後径、左右径が約20mmとなるため容易に超音波ガイド下の穿刺腹腔穿刺を施行することが可能である(図１)。但し、この時期でも心腔内や臍帯血管等、血中に移植を行うことは困難であった。
【００３３】
ブタは多胎妊娠動物であり、平均12匹が出産する。本発明者が移植を行いうるブタはそのうちの一部であり、死産が生じた場合に被移植仔の同定が困難となる。そこで被移植仔に細胞とともにマーカーを移植することで、被移植仔を軟X線撮影によって他から区別することを試みた。その結果、新生児にマーカーが同定され（図２）、このマーカーがあっても胎児発育に影響のないことも確認された。
【００３４】
少ない頻度のヒト細胞を効率よくflow cytmetryで同定するために、anti-pig pan tissue 抗体を使用した。この抗体はブタ血球のほぼ全てのlineageと反応するため、これに標識されるブタ細胞をgate outすることで、ヒト血液との分離が良好となり、0.01%の頻度であっても正確にヒト細胞を同定できた。さらに低いキメリズムの場合のヒト細胞の同定法としてPCRも併用した。この検出感度は0.0001％以上で可能であった。
In utero 移植の結果のまとめを示す（表１）。
【００３５】
【表１】
妊娠ブタ３５匹の９１胎仔にヒト臍帯血由来造血幹細胞移植を行った。その結果１０例３５移植胎仔が流産、分娩した移植胎仔５２例中１５例(%)にヒト細胞が同定可能であった。移植時の妊娠時期とキメラ成立の関係を表2に示した。
【００３６】
【表２】
移植が成立した胎児は60日で移植後5日で解析しキメラであった1例と、妊娠５２日で移植しPCRレベルでキメラであった１例を除き、すべて妊娠５０日以前に移植を行ったものであった。これよりzanotransplantationが可能となるのはブタ胎児の場合妊娠50日以前が好ましいと考えられる。
生着例を表3にまとめた。
【００３７】
【表３】
移植細胞はMNCs, T deplete, CD34すべてにおいて生着が認められ、最長では移植後110日でもヒト細胞が同定された。しかしほぼすべてのブタでそのキメリズムは低く、最高でも0.6%の頻度であった。D3585、D3564はCD3 depleteの細胞を移植したが、血中にはＴ細胞のdominat expansionが認められ（図３）、GvHの存在を疑わせた。また1例は大量の無菌性腹水の貯留が認められ、分娩中死亡した胎児があり、また移植後20日ごろに子宮内胎児死亡となった児も認めたことから、T細胞の混入はGvHDを引き起こしている可能性があった。
【００３８】
生着したヒト細胞は末梢血、骨髄、胸腺、肝臓、脾臓の全ての解析臓器に認められた（図４〜７）。骨髄と肝臓ではB cell, Myeloid, NK cell, MegakaryocyteとT細胞を除く全てのlineageが同定された。一方、胸腺ではCD3陽性のヒトT細胞のみが同定され、他のlineageを発現している細胞は認めなかった。この胸腺中に存在するT細胞はCD3陽性、CD4陽性で、αβTCRをもつT細胞であった。またこの骨髄中にはヒトCD34陽性細胞が存在し、これをsortingにより回収しコロニーアッセイを施行したところその0.8％がコロニーを形成能を持っていた。形成されたコロニーは赤血球系とマクロファージ系コロニーであった。
【００３９】
(6) 考察
ブタ胎児へ開腹することなく非侵襲的に超音波ガイド下に造血幹細胞移植を行い、異種移植であってもキメラが成立する方法を開発した。ヒトCD34陽性細胞はmultilineageにengraftし、またコロニー形成能を持つ細胞もブタ骨髄中に維持されていた。ブタ胸腺ではCD34陽性細胞から分化したヒトT細胞が集積していた。
【００４０】
ブタ胎仔へのin uteroヒト造血幹細胞移植により、免疫抑制やmyeloabrationなしにヒト血液キメラを作成できることが明らかとなった。キメラ状態は最長で移植後110日まで持続しており、long term engraftment が確認された。一方で、そのヒト血液キメラのキメリズムは低く、多くはPCR levelのマイクロキメリズムであった。
【００４１】
ヒト細胞が長期に生着した例の移植時期は妊娠50日以前であった。このことは免疫系が発育する前に移植をすることが重要であることを示している。ブタ免疫系について検討した報告では41)、lymphocytesはd28に出現するが、T cellとして認められるのは脾臓や末梢血ではd50までに出現するとされている。sIgM 陽性細胞は44日に最初に肝臓に同定される42)。この報告は本実験の移植成立時期に矛盾しない。
【００４２】
移植したヒトCD34陽性細胞はブタ胎仔の中でT細胞を含むmultilineageに分化することが示された。scidマウスを用いた系では一般的にはT細胞への分化は認められない。このことは、ブタという大動物の系は、ヒトT細胞をin vivoでアッセイする上で有用である可能性があることを示している。胸腺に移動したヒト血液細胞はブタ胸腺で教育されている可能性がある43-45)。
【００４３】
一方、ヒト血液細胞を行うことで、GvHDが疑われる胎仔も存在した。出産時すでに死後長期経過している胎仔にマーカーが確認される例があり、また一例では出産時に大量の腹水が存在し死亡した例があった。妊娠中の解析ではMNCsを移植したブタではT細胞がdominantに増殖しており、これらのことから移植細胞からT細胞を除くことが望ましいかもしれない。またはこれを逆に利用することにより、GvHDモデルとして利用できる可能性もある。
【００４４】
ヒツジの系ではT細胞の存在はヒト細胞の生着を促進する作用と、GVHDをもたらす作用の両者があるとされている46)。本発明の方法においては、ヒツジやイヌを用いた系と異なり、開腹しないことにより非侵襲的である点が特徴的である。また、in utero 移植は、myeloabrationなしに安全に新生仔キメラを作出する事ができる。この点も臨床応用を考える上で重要と考えられる。キメラの作成は生後のintensiveな管理を必要とする新生児期骨髄移植と比較し簡便である。移植実験系に於いてはPCRレベルでのマイクロキメリズムでも免疫寛容に有効とされている。そのためキメラになったrecipientはヒト移植臓器にも寛容となっている可能性があり、将来的な応用も可能である。ブタは多胎動物なので、様々な細胞をマーカーを変えて移植することで同時に様々なアッセイを行い得るメリットがある。
【００４５】
参考文献
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7. Ziegler BL, Valtieri M, Porada GA, De Maria R, Muller R, Masella B, Gabbianelli M, Casella I, Pelosi E, Bock T, Zanjani ED, Peschle C: KDR receptor: a key marker defining hematopoietic stem cells. Science
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【００４６】
【発明の効果】
本発明により、ヒト血液を含有するキメラ家畜動物及びその製造方法が提供される。本発明の動物は、ヒトの疾患に対する医薬等を開発するための実験動物等として有用である。
【００４７】
【配列表】
SEQUENCE LISTING
<110> Japan Science and Technology Corporation
<120> Chimeric animal having human blood
<130> P00-0112
<140>
<141>
<160> 2
<170> PatentIn Ver. 2.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic DNA
<400> 1
ctgggcgcaa gaacgagatt ctat 24<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic DNA
<400> 2
ctcactactt tgtgacaggt tca 23
【００４８】
【配列表フリーテキスト】
配列番号１：合成DNA
配列番号２：合成DNA
【図面の簡単な説明】
【図１】実際の造血幹細胞移植操作を示す図である。
【図２】被移植仔中のマーカーを示す図である。
【図３】 CD3ヒトT細胞を部分的に除いたCD3 deplete細胞を移植したブタ胎仔の肝臓の解析結果を示す図である。妊娠45日にヒトCD3 depleted CB 3x107個を移植、移植後38日に早産したため解析。骨髄、肝臓、脾臓にヒト細胞は同定され、その大半はCD3陽性T細胞であった。このことは、CD3を部分的に除去しても、残存T細胞によりGVHDが生じ得ることを示している。
【図４】キメラブタの胸腺中ヒトT細胞の表面抗原の解析結果を示す図である。妊娠37日にヒトCD34陽性細胞を1.1x106個移植し、移植後47日に胎仔胸腺をFACSで解析した。胸腺中にはヒトCD3陽性T細胞が同定された。その表面抗原はCD4又はCD8のシングルポジティブであり、αβTCRをもっていた。このことは、ヒトCD34陽性細胞がブタ胸腺に移動し、ヒトT細胞に分化し得ることを示している。
【図５】ヒトCD34陽性細胞がブタの体内で多様な系列の血球細胞に分化することを示す図である。 CD34陽性細胞を1.1x106個移植し、46日後にFACSで分析した。ヒトCD34陽性細胞は骨髄球系（CD13、CD14、CD33）、Bリンパ球系（CD19）、NK細胞系（CD56）、血小板系（CD41、CD61）とmultilineageに分化し生着した。また骨髄前駆細胞（CD34）も認め、このことはCD34陽性細胞がブタ血中で自己複製又は維持されていることを示す。胸腺にはCD3陽性細胞を認め、ブタ体内でCD34陽性細胞がヒトT細胞に分化し得ることを示している。
【図６】ヒト血球の組織での分布を示す図である。 CD34陽性細胞を1.1x106個移植し、46日後に解析した。ヒト血球はブタ骨髄、肝臓、胸腺に主に分布していた。末梢血、脾臓にも存在していたが、その頻度は他に比べて低かった。
【図７】ヒトCD34陽性細胞移植後出生した仔の骨髄の解析結果を示す図である。妊娠35日にヒトCD34陽性細胞を移植したところ、キメラブタが出生した。キメリズムは骨髄で0.6％（移植後88日目）であった。このブタはその後正常に発育した。ヒト細胞は骨髄系、B細胞系の両者とともに、CD34陽性の骨髄前駆細胞にも分化していた。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chimeric animal containing human blood and a method for producing the same.
[0002]
[Prior art]
In mice, hematopoietic stem cell activity is measured by an in vivo experimental system, Long term marrow repopulation method. By using this method, it is considered that mouse hematopoietic stem cells have been almost purified, and this can be used as a material for further research. 1) . There is no suitable animal model for human hematopoietic stem cell assay until recently, and a method for measuring colony forming ability by in vitro bone marrow culture 2-4) Was common. However, the in vivo repopurating capasity of cells with colony-forming ability observed in colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-IC) is unclear. Discovery of scid mouse in 1983 Five) Showed that the human hematopoietic system can be reconstructed in vivo, and since then the research method has rapidly spread. To detect hematopoietic stem cells, it is necessary to measure long term multilineage engraftment. Recently, NOD / SCID mice that can reconstruct human hematopoiesis more efficiently have been developed. 6) And is now frequently used in assays 7-10) . This mouse system is an excellent system in that human blood cells can easily engraft, but human blood is assayed as an effect on surrounding tissues such as stroma by establishing a transplant by irradiation, and as a limitation of small animals. To have a short life span, a small scale to see the ability of human hematopoietic stem cells (HSC) to proliferate, and generally differentiation into T cell lines cannot be observed 11-13) This is a problem.
[0003]
If large animals can be used to assay human hematopoietic stem cells, it is thought that human hematopoietic system can be assayed more accurately, but large animals with immunodeficiency that can introduce human blood system so far Is not known. Zanjani et al. Solved this problem by in utero transplantation 14) . Zenograft may not be rejected during the fetal period before the immune system matures, and large animals may be able to perform xenotransplantation. They have created a chimera in spite of zeno-transplantation by transplanting human hematopoietic cells into sheep fetuses and are doing excellent research on human hematopoietic stem cells 15-18) . Recently nod / scid mice 13) Or dog fetus 19) In addition, it has been reported that chimeras can be produced by transplanting in utero human hematopoietic stem cells using the same technique.
[0004]
This technique is not limited to the assay of hematopoietic stem cells, and can be applied in various ways. Due to the rapid improvement in prenatal diagnosis technology in the field of obstetrics in recent years, it is now possible to identify fetal abnormalities in the 1st trimester 20-23) . Immediately after birth, bone marrow transplantation is effective for treatment of certain diseases with a genetic background, but there are various unsolved problems such as the need for myeloabration, intensive immunosuppression, and the problem of compatible donors. There is. On the other hand, if a method for bone marrow transplantation by intrauterine transplantation is completed, such a problem may be solved. Allo intrauterine hematopoietic stem cell transplantation experiment is rodent twenty four) ,Goat 25-28) , Non-human primate 29-32) And several clinical cases have been conducted in humans. 33-34) . Furthermore, by introducing a therapeutic gene into transplanted hematopoietic stem cells, fetal gene therapy potential can be expected. 35) .
[0005]
In addition, according to animal experiments and various clinical reports, achievement of blood chimerism is known to result in tolerance of transplant immunity 36-39) . If microchimerism is achieved in a large animal by this technique, the animal may have been tolerated with respect to a human organ and may be used like an incubator in a human organ 40) .
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a chimeric animal containing human blood.
[0007]
[Means for Solving the Problems]
As a result of earnest research to solve the above problems, the present inventor succeeded in producing an animal in which human blood circulates by transplanting human blood stem cells into the fetus of the animal. It came to be completed.
[0008]
That is, the present invention is a chimeric animal containing human blood. Examples of the animal include livestock animals such as pigs.
Furthermore, the present invention is a method for producing a chimeric animal, wherein hematopoietic stem cells are transplanted into an animal fetus. Here, examples of the fetus include those from the 30th to the 60th day of pregnancy.
Hereinafter, the present invention will be described in detail.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present inventor conducted an experiment to examine whether a human blood chimera can be created in a xenotransplantation experimental system in which human hematopoietic stem cells are transplanted into a pig fetus, and revealed that hematopoietic stem cells can differentiate into multilineage and maintain microchimera for a long period of time. did.
[0010]
The present invention relates to a chimeric animal that can circulate human blood in the body, and is particularly intended for livestock or non-human primates.
Hematopoietic stem cells to be transplanted in the present invention include those derived from human umbilical cord blood and bone marrow. Hematopoietic stem cells from umbilical cord blood and bone marrow can be collected by a commonly used technique such as specific gravity centrifugation (for example, a method using Ficoll, Lymphoprep, etc.), panning method or the like.
[0011]
Of the human hematopoietic stem cells collected as described above, CD34 positive and CD3 negative nucleated cells are preferably used in the present invention.
CD34-positive nucleated cells can be selected by labeling the collected cells with an anti-human CD34 monoclonal antibody and using a MACS or FACS apparatus. On the other hand, CD3-negative nucleated cells are labeled by reacting the collected cells with a biotin-conjugated anti-CD3 monoclonal antibody and binding the resulting complex with streptavidin. Then, by removing the complex with magnetic beads, CD3-negative nucleated cells can be obtained.
[0012]
Hematopoietic stem cells for transplantation are used after diluting in an appropriate buffer (for example, PBS). Cell concentration is 3.9x10 Five ~ 1.5x10 6 1x10 per animal, preferably 1 6 It is more than one.
The hematopoietic stem cells obtained as described above are transplanted into fetuses of animals (experimental animals excluding humans). As animals, relatively large livestock animals such as horses, pigs, wild boars, donkeys, goats, and non-human primates such as monkeys can be used, but pigs are preferred. Pig varieties are not particularly limited, and examples include black varieties such as Meishan, Monkai, and Yucatan micropig, Berkshire varieties called six white, brown varieties such as Duroc, and white varieties such as large Yorkshire and Landrace.
[0013]
Transplantation is performed as follows.
First, livestock animals were made pregnant by artificial insemination, and the diagnosis of pregnancy was performed by ultrasound after artificial insemination. The transplantation day is 30 to 81 days after pregnancy, preferably 30 to 60 days, and more preferably 35 to 52 days.
[0014]
On the day of transplantation, after anesthesia is introduced into the pregnant animal, the abdomen is shaved and the abdomen is thoroughly washed and disinfected with soapy water.
The cells are transplanted into the fetus using an ultrasonic tomography apparatus while observing an image projected on the monitor. Cell transplantation is performed with an injection needle or a puncture needle, and the injection is performed by injecting hematopoietic stem cells suspended in 0.2 ml of physiological saline per fetus through the puncture needle. The thickness of the puncture needle used for transplantation is 22 to 23 gauge, preferably 22 gauge. The cells are injected into the fetal abdominal cavity, and at the same time, it is preferable to insert a marker identified by X-rays for the purpose of identifying the transplanted fetus at birth. Thereafter, antibiotics are injected as necessary to complete cell transplantation.
[0015]
After surgery, it is preferable to administer antibiotics for the purpose of preventing infection.
The transplanted fetus can be grown in the mother's body, and a chimeric animal can be obtained by normal delivery or caesarean section. Since human blood circulates in the obtained animal, it is useful as an experimental animal or the like for developing a medicine for treating human diseases.
[0016]
Further, the present invention can be used as a proliferation system of human blood cells (or hematopoietic stem cells), and thus is useful for producing blood for blood transfusion and bone marrow for bone marrow transplantation. For example, for a leukemia patient, hematopoietic stem cells of the patient can be collected from porcine bone marrow and autotransplanted.
Furthermore, since the chimera animal of the present invention induces immune tolerance to humans, chimera pigs can be used as a place for organ maintenance / regeneration when transplanting human organs.
[0017]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1] Production of human blood chimeric pig
(1) Preparation of hematopoietic stem cells
Human umbilical cord blood and bone marrow were used as a source of hematopoietic stem cells. These blood cells were separated by specific gravity centrifugation to obtain nucleated cells. The nucleated cells were washed with phosphate-buffered saline (PBS). As fractions for hematopoietic stem cell transplantation, all nucleated cells, CD34 positive cells, and CD3 negative nucleated cells were used.
[0018]
(1) Purification of CD34 positive cells
Nucleated cells were labeled with anti-human CD34 monoclonal antibody. The label was fluorescent or magnetic beads. To purify human CD34 positive cells from nucleated cells, MACS (Direct CD34 isolation kit Miltenyi Biotec Bergisch Gladbach, Germany) or FACS Vantage (Becton Dickinson, San Jose, Calif.) Was used.
[0019]
(2) Purification of CD3-negative nucleated cells
Nucleated cells are labeled with biotin-conjugated anti-CD3 mAbs (PharMingen, San Diego, CA), washed, and then labeled with BigMag streptavidin ultra-load particles (PerSeptive Biosystems, Inc. Framingham, MA), Magnetic Particle Concentrator CD3 positive cells were removed by (Dynal MPC-1 Oslo, Norway).
[0020]
(2) Human hematopoietic stem cell transplantation into pig fetuses
▲ 1 ▼ Anesthesia
On the day of transplantation, the pregnant pigs were pre-anaesthetized with ketal injection, and then inhaled with a mask. For inhalation anesthesia, laughing gas, oxygen, and isoflurane were used.
[0021]
▲ 2 ▼ Disinfection
After anesthesia was introduced into the pregnant pig, the abdomen was shaved and the abdomen was thoroughly washed with soapy water and then thoroughly disinfected with isodine.
[0022]
▲ 3 ▼ Drawing of pig fetus by ultrasound
For cell transplantation into pig fetuses, an ultrasonic tomograph Aloka ultrasound diagnostic equipment SSD-1000 (ALOKA CO., LTD Tokyo, Japan) was used. A 7.5 MHz electronic convex sector probe (Aloka; UST-987-7-5) equipped with a sterilization cover was used as the ultrasonic probe. A puncture adapter (Aloka MP-2458) was attached to the probe, and the fetus was depicted on a monitor.
[0023]
▲ 4 ▼ Puncture needle
The puncture needle used to introduce human hematopoietic stem cells into the fetus is a 22-gauge, 150 mm PTCD needle (Top Tokyo, Japan). Cells were injected into the fetal abdominal cavity (described later). At the same time, a marker identified by X-rays was inserted for the purpose of identifying the transplanted fetus at birth. The marker was prepared by chopping a PTCD needle stylet to a length of 3 mm and sterilizing in an autoclave. At the time of fetal puncture, the stylet of the PTCD needle was removed, this sterile marker was inserted into the outer tube, and the fetal abdominal cavity was punctured. Thereafter, the marker was inserted into the fetal abdominal cavity by extruding the stylet.
[0024]
▲ 5 ▼ Cell injection
The stylet was removed again, and hematopoietic stem cells suspended in 0.2 ml of physiological saline per fetus were injected through a PTCD needle. Thereafter, 0.2 ml of antibiotics was injected into the fetal abdominal cavity and 0.3 ml into the amniotic fluid cavity, and then the puncture needle was removed. This procedure allowed transplantation of cells into up to 4 fetuses.
In many cases, transplantation was performed under ultrasound guidance, but in some cases, the abdomen was opened under general anesthesia, the fetus was identified under direct observation of the uterus, and cell transplantation was performed in the fetal abdomen.
[0025]
(6) Postoperative antibiotic administration
Pregnant pigs received antibiotics intramuscularly over the next 2 days.
▲ 7 ▼ Delivery
Delivery was either spontaneous vaginal delivery or cesarean delivery. The gestation period for pigs is usually 114 days. At birth, the pups were born and born dead by soft X-ray and the markers were identified.
[0026]
[Example 2] Analysis of offspring
(1) Collection of peripheral blood, bone marrow, and organs of newborns that have undergone human hematopoietic stem cell transplantation
Peripheral blood was collected from a vein and bone marrow cells were collected from a pelvic sphenoid bone in anesthesia with Ketalar. In addition, various organs were collected after euthanasia in anesthesiology.
(2) Separation of nucleated blood cells
The obtained blood was hemolyzed with red blood cells using 0.83% ammonium chloride with 0.1% sodium bicarbonate (pH 7.0) and washed with PBS to give nucleated cells.
[0027]
(3) Identification of human blood cells in nucleated cells
Flow cytometry and PCR were used to identify human blood cells.
(1) Flow cytometry
The obtained nucleated cells were labeled with anti-human CD45 antibody and anti-porcine pan-tissue antibody. Anti-human CD45 antibody-positive and anti-porcine pan-tissue antibody-negative cells are determined as human blood cells. Furthermore, human blood cell differentiation antigen antibodies, CD3, CD19, CD13, CD14, CD33, CD34, CD56, CD41 and CD61 were added to observe the differentiation of blood cells.
[0028]
(2) PCR
Genomic DNA was recovered from the obtained nucleated cells by SepaGene (Sanko-jyunyaku Tokyo, Japan). The human genome was identified by designing primers specific to the Alu sequence and amplifying by PCR.
The following two types of primers are used.
5'-CTGGGCGCAAGAACGAGATTCTAT-3 '(SEQ ID NO: 1)
5'-CTCACTACTTTGTGACAGGTTCA-3 '(SEQ ID NO: 2)
PCR was performed at 94 ° C. for 60 seconds, followed by 43 cycles of 94 ° C. for 20 seconds + 58 ° C. for 20 seconds + 72 ° C. for 30 seconds. Finally, the reaction was completed after 72 ° C. for 7 minutes. The resulting PCR product was observed on an agarose gel by ultraviolet irradiation against bromide bromide.
[0029]
(4) Measurement of colony-forming ability of human hematopoietic stem cells present in pig fetuses
Porcine bone marrow nucleated cells were separated, labeled with anti-human CD34 antibody and anti-human CD45 antibody, and the double positive cells were separated and collected by FACS. The cells were cultured on soft agar medium cells containing human blood growth factor (Methocult GF H4434; StemCell Technologies Inc. Vancouver, BC), and colony-forming ability after 2 weeks was observed.
[0030]
(5) Result
First, the present inventor followed the method reported in sheep and dogs, and performed intrauterine hematopoietic stem cell transplantation by puncturing the fetal abdomen from outside the uterus after laparotomy. The merit is that all pregnant fetuses can be transplanted with cells. However, as a result, 4 out of 6 transplanted cases resulted in miscarriage that was thought to be caused by infection within 5 days after the operation.
[0031]
Therefore, the present inventor has developed a method for transplanting a fetus from outside the mother's body under ultrasound guidance without performing laparotomy. With this method, it was difficult to identify all fetuses under ultrasound due to maternal intestinal gas, and in fact, an average of 4 pregnant fetuses could be transplanted. However, the porcine abdominal wall is thought to be very bacterial, and even with this method, thorough infection control was necessary to prevent miscarriage. Specifically, after the abdominal wall was sufficiently disinfected, transplantation was performed, and antibiotics were administered to the fetus, amniotic fluid cavity, and mother's body.
[0032]
The pig fetus has a CRL of about 20 mm after 30 days of gestation. It is from this time that transplantation is technically possible with the method of the present invention, and after the 40th day of pregnancy, the abdominal anteroposterior diameter and the left and right diameter are about 20 mm. Is possible (FIG. 1). However, even at this time, it was difficult to perform transplantation into the blood, such as in the heart chamber and umbilical cord blood vessels.
[0033]
Pigs are multiple pregnant animals, with an average of 12 giving birth. The pigs that can be transplanted by the inventor are a part of them, and when a stillbirth occurs, it becomes difficult to identify the transplanted offspring. Therefore, we tried to distinguish the transplanted child from others by soft X-ray photography by transplanting the marker with the cell into the transplanted child. As a result, a marker was identified in the newborn (FIG. 2), and it was confirmed that the presence of this marker had no effect on fetal growth.
[0034]
An anti-pig pan tissue antibody was used to efficiently identify low-frequency human cells by flow cytmetry. Since this antibody reacts with almost all lineage of porcine blood cells, by separating out the porcine cells labeled to this, separation from human blood is improved and human cells are accurately detected even at a frequency of 0.01%. Could be identified. PCR was also used as a method for identifying human cells in the case of lower chimerism. This detection sensitivity was possible at 0.0001% or more.
A summary of the results of In utero transplantation is shown (Table 1).
[0035]
[Table 1]
Human umbilical cord blood-derived hematopoietic stem cells were transplanted into 91 fetuses of 35 pregnant pigs. As a result, human cells could be identified in 15 (%) of 52 transplanted fetuses in which 10 cases and 35 transplanted fetuses were miscarried and delivered. Table 2 shows the relationship between the pregnancy time at the time of transplantation and the formation of the chimera.
[0036]
[Table 2]
All transplanted fetuses were analyzed at 60 days and 5 days after transplantation, except for one case that was chimera and one case that was transplanted at 52 days of pregnancy and chimera at the PCR level. It was what I did. From this, it is considered that zanotransplantation is possible before the 50th day of pregnancy in the case of a pig fetus.
Examples of engraftment are summarized in Table 3.
[0037]
[Table 3]
The transplanted cells were engrafted in all MNCs, T deplete, and CD34, and human cells were identified up to 110 days after transplantation. However, almost all pigs had low chimerism, with a frequency of at most 0.6%. D3585 and D3564 transplanted CD3 deplete cells, but T cell dominat expansion was observed in the blood (FIG. 3), and the presence of GvH was suspected. In one case, a large amount of sterile ascites was observed, a fetus died during delivery, and a child who died in utero around 20 days after transplantation. There was a possibility that it was causing.
[0038]
Engrafted human cells were found in all analyzed organs of peripheral blood, bone marrow, thymus, liver, and spleen (FIGS. 4-7). In the bone marrow and liver, all lineage except B cell, Myeloid, NK cell, Megakaryocyte and T cell were identified. On the other hand, only CD3-positive human T cells were identified in the thymus, and no cells expressing other lineage were found. The T cells present in the thymus were CD3 positive, CD4 positive, and T cells with αβTCR. In addition, human CD34-positive cells were present in the bone marrow, and when these were collected by sorting and subjected to a colony assay, 0.8% of them had the ability to form colonies. The colonies formed were erythroid and macrophage colonies.
[0039]
(6) Discussion
A method has been developed in which hematopoietic stem cell transplantation is performed non-invasively under ultrasonic guidance without laparotomy into a pig fetus, and a chimera can be established even in xenotransplantation. Human CD34 positive cells were engrafted in multilineage, and cells with colony forming ability were also maintained in porcine bone marrow. In the porcine thymus, human T cells differentiated from CD34 positive cells were accumulated.
[0040]
It has been clarified that human blood chimeras can be created without immunosuppression or myeloabration by transplantation of in utero human hematopoietic stem cells into pig fetuses. The chimera state lasted up to 110 days after transplantation, and long term engraftment was confirmed. On the other hand, the chimerism of the human blood chimera was low, and most were microchimerism at the PCR level.
[0041]
The transplantation time for human cells engrafted for a long time was 50 days before pregnancy. This indicates that it is important to transplant before the immune system develops. In a report examining the porcine immune system 41) Lymphocytes appear in d28, but T cells are recognized as appearing by d50 in spleen and peripheral blood. sIgM positive cells are first identified in the liver on day 44 42) . This report is consistent with the time of transplantation in this experiment.
[0042]
The transplanted human CD34 positive cells were shown to differentiate into multilineage containing T cells in pig fetuses. In the system using scid mice, differentiation into T cells is generally not observed. This indicates that the large animal system of pigs may be useful for assaying human T cells in vivo. Human blood cells that have migrated to the thymus may be educated in the porcine thymus 43-45) .
[0043]
On the other hand, there were fetuses with suspected GvHD using human blood cells. There were cases where the marker was confirmed in fetuses that had already passed since death at the time of birth, and in one case, there was a case where a large amount of ascites was present at the time of birth and died. Analysis during pregnancy shows that T cells proliferate dominantly in pigs transplanted with MNCs, and it may be desirable to remove T cells from the transplanted cells. Or by using this in reverse, there is a possibility that it can be used as a GvHD model.
[0044]
In sheep systems, the presence of T cells is believed to have both an effect of promoting human cell engraftment and an effect of causing GVHD. 46) . Unlike the system using sheep and dogs, the method of the present invention is characterized in that it is non-invasive by not laparotomy. Also, in utero transplantation can safely produce newborn chimeras without myeloabration. This point is also considered important when considering clinical application. Chimera creation is simpler than neonatal bone marrow transplantation, which requires intensive management after birth. In transplantation experiment systems, microchimerism at the PCR level is also effective for immune tolerance. Therefore, the chimeric recipient may be tolerated by human transplanted organs and can be applied in the future. Since pigs are multiple animals, there are advantages in that various assays can be performed simultaneously by transplanting various cells with different markers.
[0045]
References
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[0046]
【The invention's effect】
According to the present invention, a chimeric livestock animal containing human blood and a method for producing the same are provided. The animal of the present invention is useful as an experimental animal for developing medicines for human diseases.
[0047]
[Sequence Listing]
SEQUENCE LISTING
<110> Japan Science and Technology Corporation
<120> Chimeric animal having human blood
<130> P00-0112
<140>
<141>
<160> 2
<170> PatentIn Ver. 2.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic DNA
<400> 1
ctgggcgcaa gaacgagatt ctat 24 <210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic DNA
<400> 2
ctcactactt tgtgacaggt tca 23
[0048]
[Sequence Listing Free Text]
SEQ ID NO: 1 synthetic DNA
Sequence number 2: Synthetic DNA
[Brief description of the drawings]
FIG. 1 is a diagram showing an actual hematopoietic stem cell transplantation operation.
FIG. 2 is a view showing markers in a transplanted pup.
FIG. 3 shows the results of analysis of the liver of a pig fetus transplanted with CD3 deplete cells from which CD3 human T cells have been partially removed. Human CD3 depleted CB 3x10 on the 45th day of pregnancy 7 Individuals were transplanted and analyzed because they were delivered prematurely 38 days after transplantation. Human cells were identified in the bone marrow, liver, and spleen, most of which were CD3-positive T cells. This indicates that GVHD can be generated by residual T cells even if CD3 is partially removed.
FIG. 4 shows the results of analysis of surface antigens of human T cells in the thymus of chimeric pigs. 1.1x10 human CD34 positive cells on gestation day 37 6 One piece was transplanted, and the fetal thymus was analyzed by FACS 47 days after the transplantation. Human CD3-positive T cells were identified in the thymus. The surface antigen was CD4 or CD8 single positive and had αβTCR. This indicates that human CD34 positive cells migrate to the porcine thymus and can differentiate into human T cells.
FIG. 5 shows that human CD34-positive cells differentiate into various types of blood cells in the pig body. 1.1x10 CD34 positive cells 6 Individuals were transplanted and analyzed by FACS after 46 days. Human CD34 positive cells differentiated into multilineage and engrafted into myeloid lineage (CD13, CD14, CD33), B lymphocyte lineage (CD19), NK cell lineage (CD56), and platelet lineage (CD41, CD61). Bone marrow progenitor cells (CD34) are also observed, indicating that CD34 positive cells are self-replicating or maintained in porcine blood. CD3-positive cells are observed in the thymus, indicating that CD34-positive cells can differentiate into human T cells in the pig body.
FIG. 6 is a diagram showing the distribution of human blood cells in tissues. 1.1x10 CD34 positive cells 6 Individual transplants were analyzed after 46 days. Human blood cells were mainly distributed in porcine bone marrow, liver and thymus. It was also present in peripheral blood and spleen, but the frequency was lower than others.
FIG. 7 is a view showing the analysis results of bone marrow of a pup born after transplantation of human CD34 positive cells. When human CD34 positive cells were transplanted on the 35th day of pregnancy, chimeric pigs were born. Chimerism was 0.6% in the bone marrow (88 days after transplantation). The pig subsequently developed normally. Human cells were differentiated into CD34 positive bone marrow progenitors as well as myeloid and B cell lines.
