JP2004502425A - Culture of pancreatic stem cells with designated intermediate developmental stages - Google Patents

Abstract

The present invention relates to the finding that there is an intermediate differentiation stage pancreatic stem cell that can be grown in a stable manner in successive serial passages while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture, and are advantageous in that they can be induced into a late developmental stage, ie, prototypical islet cells. The present invention further provides culture techniques for selecting cells at these intermediate differentiation stages and for selectively removing early or late stage pancreatic cells.

Description

（インスリンｍＲＮＡ発現）
膵臓細胞の正体、分化または成熟度を特徴付けるために用いられ得る１つのマーカーは、インスリンｍＲＮＡのレベルである。例えば、本発明の中間の細胞集団は、規定された範囲内のインスリンｍＲＮＡの発現を示す。インスリンｍＲＮＡを定量するための方法としては、ノーザンブロット、ヌクレアーゼ保護およびプライマー伸長が挙げられる。１つの実施形態では、ＲＮＡは、培養細胞の集団から抽出され、そしてプロインスリンメッセージの量が定量的逆転写ＰＣＲによって測定される。逆転写の後、インスリンｃＤＮＡは、インスリンｃＤＮＡ配列にハイブリダイズするプライマー、および増幅産物の量がサンプル中に存在するｍＲＮＡの量に関連する増幅条件を用いてサンプルから特異的かつ定量的に増幅される（例えば、Ｚｈｏｕら，Ｊ　Ｂｉｏｌ　Ｃｈｅｍ　２７２：２５６４８−５１（１９９７）を参照のこと）。反応速度定量手順は、出発ｍＲＮＡレベルが決定され得る精度に起因して好ましい。
【００４３】
頻繁に、インスリンｍＲＮＡの量は、構成的に発現されるｍＲＮＡ（例えば、アクチン）に対して正規化され、アクチンは、アクチン特異的プライマーを用いて同じＲＮＡサンプルから特異的に増幅される。従って、インスリンｍＲＮＡの発現レベルは、インスリンｍＲＮＡ増幅産物の、アクチンｍＲＮＡ増幅産物に対する比として、または単に、インスリン：アクチンｍＲＮＡ比として報告され得る。他の膵臓ホルモン（例えば、ソマトスタチンまたはグルカゴン）をコードするｍＲＮＡの発現は、同じ方法によって定量され得る。
【００４４】
（グルコース刺激インスリン分泌）
β細胞の重要な機能の１つは、グルコースレベルに従ってそのインスリン分泌を調整することである。代表的に、静的グルコース刺激（ＳＧＳ）アッセイは、増殖している付着膵臓細胞で行われて、これらの細胞が異なるグルコースレベルに応答してインスリンを分泌し得るか否かを同定し得る。細胞は一般に、ほぼコンフルエントになるまで適切な基材上で培養される。ＳＧＳ試験の３日前、培養培地は、同様の特徴だがインスリンを欠き、そして１ｇ／Ｌのグルコースのみを含む培地によって置換される。この培地は、毎日、３日間にわたって変更され、そしてＳＧＳ試験が４日目に行われる。
【００４５】
この試験の前に、培養培地は、グルコースおよびインスリンの分析のために収集され得る。この試験のために細胞を調製するために、細胞は、Ｄｕｌｂｅｃｃｏのリン酸緩衝化生理食塩水（ＤＰＢＳ）＋０．５％　ＢＳＡで２回洗浄され（各洗浄について５分間にわたってインキュベートされる）、次いでＤＰＢＳ単独で１回洗浄される（これもまた５分間にわたってインキュベートされる）。洗浄後、この細胞は、６０ｍｇ／ｄｌグルコースを含む、１０ｍｌ（１００ｍｍディッシュ中）または５ｍｌ（６０ｍｍディッシュ中）のＫｒｅｂｓ−Ｒｉｎｇｅｒｓ　ＳＧＳ溶液（ＫＲＢ−６０）を用いて、３７℃のインキュベーター中で３０分間にわたってインキュベートされる。次いで、このインキュベーションは繰り返される。
【００４６】
ＳＧＳアッセイを実施するために、細胞は、３ｍｌ（１００ｍｍディッシュ）または４ｍｌ（Ｔ７５フラスコ）または２ｍｌ（６０ｍｍディッシュ）ＫＲＢ−６０中で、３７℃で２０分間にわたってインキュベートされる。この培地は吸引され、そしてスピンされ、そしてＬＧ−１（低グルコース刺激工程）として、インスリンアッセイのために収集される。次いで、ＫＲＢ−４５０＋テオ（４５０ｍｇ／ｄｌグルコースおよび１０ｍＭテオフィリンを有するＫＲＢ）は、上記と同じ容量で添加され、そして細胞は、上記と同じ条件下で培養される。上清は、ＨＧ（高グルコース刺激）としてインスリンアッセイのために収集される。次いで、この細胞は、ＫＲＢ−６０と共に再度インキュベートされ、そしてこの培地はＬＧ−２として収集され、そして次のときにはＬＧ−３として収集される。この培地は、インスリン分析のために収集され、そしてインスリン含量が放射免疫アッセイ（ＲＩＡ）または他の適切なアッセイによって決定されるまで−２０℃で保存される。
【００４７】
ＳＧＳ試験の結果はしばしば、ＬＧ−１インスリン値によって割ったＨＧインスリン値として規定される、刺激指数として表現される。一般に、約２以上の刺激指数は、ＳＧＳアッセイにおいて陽性の結果であるとみなされるが、他の値（例えば、１．５、２．５、３．０、３．５など）を用いて特定の細胞集団を規定し得る。
【００４８】
制御された実験では、ウシ下垂体抽出物（ＢＰＥ）および組換えヒト成長ホルモンは、中間の分化の細胞からの所望のレベルのインスリン放出を維持することが見出された。
【００４９】
（ＩＩ．膵臓細胞を単離する方法）
本発明は、とりわけ、連続した複製を行い得るが、治療目的に適切な、より成熟した細胞へと分化するように誘導され得る、膵臓細胞の培養物を作製する方法を提供する。従って、本発明のいくつかの方法は、最初の工程として、膵臓からの細胞の単離を必要とする。膵臓から収集された細胞は、内分泌的分泌および外分泌的分泌をし得る分化した細胞を生じ得る、多様な集団である。これらの分化した細胞は、膵臓内分泌分子（例えば、インスリン、ソマトスタチン、グルカゴンおよび他の内分泌ホルモン）ならびに膵臓外分泌分子（例えば、アミラーゼ）を発現する。さらに、培養した細胞集団の一部は、培養において複製および増大し得る。本発明の中間細胞集団は、条件的幹細胞の分化、成熟内分泌細胞の脱分化、または他の膵臓細胞集団の分化形質転換によって、全てまたは部分的に生じ得る。以下に記載の培養方法は、種々の抽出条件および培養条件を活用して、膵臓細胞の種々の集団を作製する。
【００５０】
（ドナー供給源）
ドナー供給源は、培養膵臓細胞由来の１以上のドナー膵臓、または膵臓内分泌ホルモンおよび膵臓外分泌ホルモンを産生し得る細胞を生じ得る他の供給源であり得る。好ましい実施形態では、その後の培養のために分離された細胞は、１以上の寄贈された膵臓から得られる。本明細書中に記載される方法は、寄贈された膵臓の齢に依存しない。従って、胚から成体の齢にわたるドナーから単離された膵臓材料が用いられ得る。
【００５１】
別の実施形態では、膵臓細胞は、培養された供給源から単離される。例えば、「Ｍｉｃｒｏｅｎｃａｐｓｕｌａｔｉｏｎ　ｏｆ　ｃｅｌｌｓ」との発明の名称の、Ｓｏｏｎ−Ｓｈｉｏｎｇらに対する米国特許第５，７６２，９５９号の微小カプセル化方法に従って調製された細胞は、ドナー細胞の供給源として収集され得る。
【００５２】
中間細胞集団の作製のための出発材料はまた、本明細書中に開示される方法によって作製された、膵臓細胞のクローン集団を含む。従って、本発明の１つの実施形態では、本明細書中に開示された単離方法、分離方法および培養方法は、クローニングされた単一の膵臓細胞から誘導された中間の集団を生成するために、クローニング手順と結び付けられる。このような手順は、中間細胞集団のインビトロでの遺伝的操作のために特に適切である。
【００５３】
（膵臓細胞集団の単離）
一旦、膵臓がドナーから収集されたら、これは代表的に、種々の方法を用いて、培養するための個々の細胞または細胞の小さな群を生じるように処理される。１つのこのような方法は、回収された膵臓組織が酵素消化のために清浄および調製されることを要する。酵素処理は、回収された組織の実質が、より小さな単位の膵臓細胞材料へと解離されるように、結合組織を消化するために用いられる。収集された膵臓組織は、収集された器官の全体的構造から、膵臓細胞材料、部分構造および個々の膵臓細胞を分離するために１以上の酵素を用いて処理される。コラゲナーゼ、ＤＮＡｓｅ、Ｌｉｂｅｒａｓｅ調製物（米国特許第５，８３０，７４１号および同第５，７５３，４８５号を参照のこと）ならびに他の酵素は、本明細書中に開示される方法での使用が意図される。
【００５４】
単離された供給源材料は、１以上の所望の細胞集団を富化するためにさらに処理され得る。しかし、未分画膵臓組織もまた、培養のために一旦解離されたら、本発明の培養方法において、さらなる分離をすることなく直接用いられ得、そして中間細胞集団を生じる。１つの実施形態では、単離された膵臓細胞材料は、密度勾配（例えば、Ｎｙｃｏｄｅｎｚ、ＦｉｃｏｌｌまたはＰｅｒｃｏｌｌ）を通した遠心分離によって精製される。例えば、米国特許第５，７３９，０３３号に記載される勾配方法は、処理された膵臓材料について島を富化するための手段として用いられ得る。ドナー供給源から収集された細胞の混合物は代表的に、不均質であり、従って、細胞、細胞、δ細胞、管細胞、腺房細胞、条件的前駆細胞および他の膵臓細胞型を含む。
【００５５】
代表的な精製手順は、多数の層または界面への、単離された細胞材料の精製をもたらす。代表的に、２つの界面が形成される。上の界面は島が豊富であり、そして代表的に１０％〜１００％の島細胞を懸濁して含む。第二の界面は代表的に、島細胞、腺房細胞および管細胞を含む細胞の混合集団である。底の層はペレットであり、ペレットは、勾配の底に形成される。この層は代表的に、主に（＞８０％）腺房細胞を、いくつかの捕獲された島およびいくつかの管細胞を含む。管の樹成分は、さらなる操作のために別々に収集され得る。
【００５６】
さらなる操作のために選択される画分の細胞密度（ｃｅｌｌｕｌａｒ　ｃｏｎｓｔｉｔｕｅｎｃｙ）は、どの勾配画分が選択されるか、および各単離の最終結果に依存して変動する。島細胞が所望の細胞型である場合、単離された画分内に適切に富化された島細胞集団は、少なくとも１０％〜１００％の島細胞を含む。他の膵臓細胞型および濃度はまた、富化後に収集され得る。例えば、本明細書中に記載される培養方法は、用いられた精製勾配に依存して、第２の界面、ペレットまたは他の画分から単離された細胞について用いられ得る。
【００５７】
１つの実施形態では、中間膵臓細胞培養物は、島富化（上の）画分から作製される。しかし、さらに、それぞれ、島細胞、腺房細胞および管細胞の混合された細胞集団、または管樹成分、腺房細胞およびいくつかの捕獲された細胞の混合された細胞集団を代表的に含む、より不均質な第２の界面および底の層の画分もまた培養において用いられ得る。両方の層が、本明細書中に記載される中間段階の集団を生じ得る細胞を含むが、各層は、開示された方法について用いるための特定の利点を有し得る。例えば、島が富化された上の画分を用いることは、特定の利点を有し得る。この島が富化された画分は、より進んだ分化段階の、より多くの細胞を含み得、従って、ショック方法論において、より有効であり得、この方法論は、既存の中間段階集団を選択することによって起動し得る。あるいは、より不均質な第２の画分および第３の画分もまた、利点を有し得る。第２の（下の）界面は、内分泌発達のより初期の段階の、より多くの細胞を含み得、従って、離脱方法論によって即座の（ｉｍｍｅｄｉａｔｅ）集団を作製する際に、より効率的であり得、この方法論は、それほど分化していない前駆体由来の中間段階の細胞の発達を促進すると理論付けられる。しかし、本発明の方法は、これらの理論によって束縛されず、そして上の界面画分および下の界面画分の両方は、ショック方法論および離脱方法論のいずれについても、受容可能な結果を生じる。
【００５８】
（ＩＩＩ．高血清培地中での膵臓細胞の維持および増殖）
（Ａ．一般的細胞培養手順）
一旦膵臓細胞が入手および単離されたら、これらは、所望の中間段階集団の増殖を、または他の実施形態では、より成熟した細胞型の分化を選択する条件下で培養される。一般的細胞培養方法論は、Ｆｒｅｓｈｎｅｙ，Ｃｕｌｔｕｒｅ　ｏｆ　Ａｎｉｍａｌ　Ｃｅｌｌｓ：Ａ　Ｍａｎｕａｌ　ｏｆ　Ｂａｓｉｃ　Ｔｅｃｈｎｉｑｕｅ，第４版，Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ（２０００）に見出され得る。代表的に、膵臓細胞は、他の哺乳動物細胞に適切な条件下で、例えば、加湿インキュベーター中で３７℃で、５％　ＣＯ_２の雰囲気下で培養される。細胞は、当該分野で公知の種々の基材（例えば、負の表面電荷を有する、ホウケイ酸ガラス製のチューブ、ボトル、ディッシュ、クローニングリング、プラスチック製の組織培養チューブ、ディッシュ、フラスコ、マルチウェルプレート、増大した増殖表面積（ＧＳＡ）または食道ドップラーモニタ（Ｅｓｏｐｈａｇｅａｌ　Ｄｏｐｐｌｅｒ　Ｍｏｎｉｔｏｒ；ＥＤＭ）仕上げを有する容器、ＧＳＡを増大させるための複数の内部シートを有するフラスコ、Ｆｅｎｗａｌバッグおよび他の培養容器）で培養され得る。
【００５９】
一旦膵臓細胞材料が収集され、そして培養について選択されたら、または一旦集団がコンフルエントになり、そして新たな基材に移植されたら、細胞の集団は、培養のために適切な組織培養容器へと播種される。播種密度は、開示された方法を用いて培養される膵臓細胞の生存率に影響を有し得、そして特定の培養条件について最適な播種密度は、種々の密度の範囲で細胞を播種し、そして得られる細胞の生存率および増殖速度をモニタリングすることによって経験的に決定され得る。ある範囲の播種密度は、ホルモン分泌細胞を培養において産生する際に有効であることが示されている。代表的に、細胞濃度は、１枚の１００ｍｍ培養ディッシュあたり約１０^２細胞〜約１０^８細胞、例えば、１枚の１００ｍｍ培養ディッシュあたり１０^２細胞、１０^３細胞、１０^４細胞、１０^５細胞、１０^６細胞、１０^７細胞または１０^８細胞の範囲に及ぶが、より低い細胞濃度が、クローニング手順のために用いられ得る。他の培養容器についての細胞濃度は、種々の培養容器について相対基材表面積および／または培地気体交換表面積を計算することによって調整され得る。例えば、代表的な１００ｍｍ培養ディッシュは、５５平方センチメートルの基材表面積（Ｆｒｅｓｈｎｅｙ，前出を参照のこと）を有し、そして１枚のディッシュあたり１０，０００細胞の細胞濃度は、１平方センチメートルあたり約１８０細胞に相当し、一方、１枚のディッシュあたり１００，０００細胞の細胞濃度は、１平方センチメートルあたり約１，８００細胞に相当する。培養容器の表面積に関する細胞濃度は、培養表面積あたりの適切な培地容積（０．２ｍｌ／ｃｍ^２〜０．５ｍｌ／ｃｍ^２が静置培養について代表的な範囲である）を用いることによって、培地容積に関する細胞濃度に関連付けられ得る。１０倍の増大が生じたか否かを決定するために、細胞は、酵素消化によって取り出され、そして既知容積の流体中で顕微鏡下で計数される。細胞はまた、増殖および分化を促進する規定の細胞外マトリクス成分（例えば、フィブロネクチン、コラーゲンＩ、Ｅｎｇｅｌｂｒｅｔｈ−Ｈｏｌｍ−Ｓｗａｒｍマトリクスおよび好ましくはコラーゲンＩＶまたはラミニン）で予めコーティングした培養表面で増殖され得る。
【００６０】
標準的な細胞培養増殖技術は、本発明の実施のために適切である。細胞が、培養表面に付着して増殖する場合、この細胞は代表的に、８０％〜９０％コンフルエンスが達成されるまで単層として増殖し、この時点で、この細胞は、タンパク質分解消化によって表面から遊離され、そして新たな容器中での培養のために１：２または１：３に分けられる。この細胞のより高い希釈（一般に、１：４〜１：１０の範囲の間）もまた適切であるが、さらに低い細胞濃度は、クローニング手順において適切である。タンパク質分解酵素およびキレート剤の濃度は通常、細胞が無血清培地（例えば、０．０２５％トリプシンおよび０．５３ｍＭ　ＥＤＴＡ）中に継代される場合、より低くされる。培養培地は代表的に、毎週交換されるか、または培地のｐＨが新鮮な培地が必要とされることを示すときに交換される。
【００６１】
本発明の膵臓細胞は、種々の培地において培養され得る。本明細書中で記載されるように、特定の成分（特に血清）を含むかまたは欠く培地は、単離手順および増殖手順の特定の段階で好ましい。例えば、膵臓から新たに単離された細胞は、高血清培地中で維持されて、細胞が単離手順から回復するのを可能とし得る。逆に、低血清培地は、本明細書中に記載される中間段階集団の選択および増殖に有利に働く。最終的に、中間段階細胞の成熟は、特定の培地を用いる。従って、多数の培地処方は、本発明の実施において有用である。本明細書で開示される培地処方は、例示的な目的のためであり、そして培地の重要でない成分は、本明細書中に記載されるアッセイを用いて、細胞集団の複製または分化に対するバリエーションの影響をアッセイすることによって、単純に省略、置換、変更または添加され得る。例えば、Ｓｔｅｐｈａｎら，Ｅｎｄｏｃｒｉｎｏｌｏｇｙ　１４０：５８４１−５４（１９９９）を参照のこと。
【００６２】
培養培地は通常、基本培地を含み、基本培地は、無機塩、緩衝剤、アミノ酸、ビタミン、エネルギー源およびいくつかの場合、タンパク質代謝、核酸代謝、炭水化物代謝または脂質代謝に関与する有機中間体および前駆体の形態のさらなる栄養素を含む。基本培地としては、Ｆ１２、ＥａｇｌｅのＭＥＭ、Ｄｕｌｂｅｃｃｏの改変ＭＥＭ（ＤＭＥＭ）、ＲＰＭＩ　１６４０、Ｆ１２とＤＭＥＭとの１：１混合物などが挙げられる。Ｆｒｅｓｈｎｅｙ，前出を参照のこと。細胞の増殖を支持するために、基本培地には通常、増殖因子、他のタンパク質、ホルモンおよび微量元素の供給源が補充される。これらの補充物は、細胞の増殖、維持および／または分化を促進し、他の培地成分中の不純物または毒素を相殺し、そして基本培地中に欠けている微量元素を提供する。多くの培養培地では、血清は、これらの補充物の供給源である。血清は、種々の哺乳動物供給源（例えば、ヒト、ウシ、ヒツジ、ウマなど）から、および成体、若年期または胎仔の供給源から補充され得る。Ｆｒｅｓｈｎｅｙ，前出を参照のこと。胎仔ウシ血清は、通常用いられる補充物である。血清の濃度は、総培地溶液の百分率として、血清の容積に関して表され、そして代表的には、約０．１％〜約２５％、例えば、約０．１％、約０．２％、約０．５％、約１％、約２％、約３％、約４％、約５％、約６％、約７％、約８％、約９％、約１０％、約１５％、約２０％または約２５％の範囲にわたる。いくつかの適用では、基本培地には、血清というよりも、増殖因子、ホルモンおよび微量元素の規定のまたは半規定の混合物が補充される。血清置換補充物についての処方は、本明細書中に開示される；他のものは、当該分野で公知であるかまたは商業的供給源から入手可能である（Ｆｒｅｓｈｎｅｙ，前出を参照のこと）。いくつかの実施形態については、血清の濃度は、低くされるが除去されず、そして規定または半規定の補充混合物が規定培地に添加される。高濃度または低濃度の血清を含む培地についての好ましい適用は、本明細書中に記載される。
【００６３】
（Ｂ．高血清中の単離された細胞の維持および増殖）
ドナー膵臓から収集された細胞は通常、ドナーの死と単離手順の開始との間に、温かい虚血または冷たい虚血の期間を経ている。さらに、単離手順の間に、膵臓細胞は通常、タンパク質分解消化ならびに機械的ストレスおよび剪断ストレスに供される。特定の理論によって束縛されることを望まないが、これらの細胞によって経験された種々の外傷は、膵臓幹細胞集団（例えば、条件的前駆細胞）の増大をもたらす種々の細胞プロセスをアップレギュレートし得る。中間細胞集団は、単離または精製後に細胞を低血清培地中に直接配置することによって満足のいく効率で作製され得る。それにもかかわらず、単離手順の間に細胞によって経験された外傷は、細胞の生存および培養への適応に有害な影響を有し得るので、新鮮に単離された細胞を、高濃度の血清（例えば、＞４％）を含む安定化培地中で維持して、培養プロセスの効率を改善することがときどき所望される。この維持期間は、短くでもよい（例えば、一晩）。必要に応じて、細胞は、高血清培地中で長期の増殖期間にわたって維持され得る。
【００６４】
安定化のための高血清培地は代表的に、少なくとも４％の血清を含み、そしていくつかの実施形態では、より高濃度の血清（例えば、１０％または２０％）を含む。安定化または増殖のために用いられる培地は、多くの商業的供給源から入手可能な、そしてＭｏｏｒｅら，Ｊ　Ａｍ　Ｍｅｄ　Ａｓｓｏｃ　１９９：５１９−５２４（１９６７）によって記載される、基本培地（例えば、ＲＰＭＩ　１６４０）から誘導され得る。維持または増殖のための例示的な高血清培地としては、培地３（ＲＰＭＩ　１６４０＋１０ｍＭ　ＨＥＰＥＳ、２ｍＭグルタミン、５μＭ　ＺｎＳＯ_４および１０％ウシ胎児血清（ＦＢＳ））および培地７（ＲＰＭＩ　１６４０＋１０ｍＭ　ＨＥＰＥＳ、２ｍＭグルタミン、５μＭ　ＺｎＳＯ_４および２０％　ＦＢＳ）が挙げられる。高血清培地はまた、特定の容積の高血清培地（例えば、培地３または培地７）を特定の容積の無血清培地（例えば、ＳＭ９５、ＳＭ９６またはＳＭ９８（本明細書中に記載される））と混合して所望の血清濃度（例えば、４％〜９％）に達することによって誘導され得る。
【００６５】
収集後の安定化のために、細胞は、培養容器中で、高血清培地中で比較的高密度（例えば、７０ｍｌの培地７（２０％　ＦＢＳ）中で１０^９細胞）で便利に培養される。しかし、より低い細胞密度および血清濃度もまた用いられ得る。細胞は代表的に、比較的短時間（例えば、一晩）にわたって元の容器中で維持されて、収集手順からの回復が可能にされる。
【００６６】
維持期間後、細胞は、本明細書中に記載されるように、中間細胞集団の選択および増殖のために低血清培地へと移植され得る。必要に応じて、細胞は、高血清培地中で培養されて、混合細胞集団の増殖が可能にされ得る。代表的な実施形態では、維持培養からの細胞は、培地３（１０％　ＦＢＳ）、培地７（２０％　ＦＢＳ）または培地３と培地７との混合物（１５％　ＦＢＳ）を含む新たな培養容器中に再度播種される。細胞は代表的に、この培地中で７日間〜１０日間にわたって培養され、その間に、これらはコンフルエンスまで増殖し得る。一旦細胞がコンフルエンスに到達したら、これらは、本明細書中に記載される中間細胞集団の選択的増大のために低血清培地中へと継代され得る。
【００６７】
（ＩＶ．上皮細胞選択的培地中での培養による、中間分化段階の膵臓幹細胞の優先的増大）
一旦膵臓細胞が単離されたら、細胞は選択的培地へと移植されて、増殖している中間段階集団の出現が促進される。この選択的培地は、膵臓内分泌ホルモンを分泌する能力を保持する細胞の増殖、または高レベルの膵臓内分泌ホルモンを分泌する、より分化した細胞へと成熟する可能性を保持する細胞の増殖に好都合である。一般に、選択的培地は、線維芽細胞および間葉細胞を犠牲にして、上皮細胞または上皮様細胞の増殖に好都合であるが、純粋な上皮培養物は、本発明の方法における膵臓細胞の有利な使用のために必要とされるとは示されていない。代表的に、上皮選択的培地は、培養中での一定期間の増殖（例えば、各継代における集団の増大に依存して、２回、３回、４回または５回の継代）後に、ほぼ純粋な（例えば、＜１０％の線維芽細胞または間葉細胞）細胞の集団を生じる。
【００６８】
胚組織からの上皮細胞増殖に好都合なように用いられている１つの型の選択的培地は無血清培地である（例えば、Ｓｔｅｐｈａｎら，前出；ＰｅｅｈｌおよびＨａｍ，Ｉｎ　Ｖｉｔｒｏ　１６：５２６−４０（１９８０）を参照のこと）。成長ホルモンの供給源を含む、上皮特異的培地、そしてより好ましくは低血清培地が、成体哺乳動物から、膵臓細胞発達のマーカー（例えば、ＰＤＸ−１）を保持するが、適切な条件下でさらに分化して高レベルの膵臓内分泌ホルモンを発現し得る、増殖する膵臓細胞の別個の集団を選択するために用いられ得ることは、本発明の知見である。膵臓細胞の培養に適切な特定の上皮選択的培地は本明細書中に開示されるが、上皮細胞または上皮様細胞の優先的増大に好都合な、当該分野で公知の他の培地処方もまた用いられ得る。
【００６９】
上皮選択的低血清培地への移植は、高血清培地中での一定期間の維持（「離脱（ｗｅａｎｉｎｇ）」）後に、または単離および分離手順後に細胞を選択的低血清培地中へ直接移植すること（「ショック」）によって、達成され得る。いずれの方法論も、本明細書中の実施例に示すように、所望の中間細胞集団の作製のために適切である。
【００７０】
ウシ下垂体抽出物［ＢＰＥ］および組換えヒト成長ホルモンを用いる制御された実験では、下垂体抽出物が、ＢＰＥが存在しないものと比較して、増殖速度において有益な増大を与えることが決定された。組換え成長ホルモンは、ＢＰＥに伴った増殖速度を有意に回復し得た。
【００７１】
（Ａ．選択的低血清培地の調製）
この状況で用いられる場合、［低血清培地」とは、約１％未満の血清を有する培地をいう。従って、無血清培地は、一種の低血清培地である。０％と１％との間の血清、例えば、約０．１％、０．２％、０．３％、０．４％、０．５％、０．６％、０．７％、０．８％または０．９％の血清の濃度を有する培地は、適切な濃度の血清を無血清培地へと添加することによってか、または無血清培地と血清含有培地とを混合して所望の濃度の血清を達成するかのいずれかによって調製され得る。
【００７２】
完全無血清培地は、基本培地（例えば、ＳＭ９６または１：１　Ｆ１２／ＤＭＥＭ）に増殖因子、他のタンパク質、ホルモンおよび微量元素の混合物（これは、血清によって提供される生物学的機能を代理する）を補充することによって調製される。無血清培地の利点は、補充混合物の組成が、所望でない細胞（例えば、腺房細胞または結合組織細胞（例えば、線維芽細胞））の増殖を防止しながら、所望の細胞集団（例えば、中間細胞集団）の増殖を促進するために、容易に操作され得ることである。補充混合物はまた、より成熟した細胞への幹細胞集団の分化を促進するために、または高速度の増殖を維持するために幹細胞集団の分化を妨害するために、操作され得る。
【００７３】
（１．成長ホルモン）
成長ホルモン（ＧＨ）を含む低血清上皮選択培養培地が、中間分化の有益な膵臓細胞集団の出現を促進することは、本発明の知見である。特定の理論によって束縛されることを望まないが、細胞増殖を支持する、血清中に通常見出されるマイトジェン性物質をＧＨが代理し得るが、血清は、それほど望ましくない細胞集団（例えば、線維芽細胞および間葉細胞）の過剰増殖を促進する他のマイトジェン性因子を含むことが仮定される。それゆえ、ＧＨを含有する補充混合物での血清の置換は、中間の分化状態の細胞集団の増殖を選択する。無血清培地中でのＧＨの機能は、本発明の代替実施形態では他の補充成分で置換され得るが、天然抽出物中の、または組換えタンパク質としての、ＧＨの容易な入手可能によって、ＧＨ含有培地は、本明細書中に開示された方法について適切な上皮選択培地となる。
【００７４】
成長ホルモン（ソマトトロピンとしても公知）は、下垂体前葉において合成され、正常な身体の成長および乳汁分泌を促進し、そして細胞代謝の種々の局面に影響を与える、ポリペプチド性ホルモンである。ＧＨは、細胞に直接的影響、ならびにＩＧＦ−Ｉおよび類似の分子によって媒介される間接的影響の両方を有する；インタクトな膵臓では、島細胞の増殖は、ＧＨならびに相同なホルモンプロラクチンおよびラクトゲンの発現に結び付けられている（例えば、Ｎｉｅｌｓｅｎら，Ｊ　Ｍｏｌ　Ｍｅｄ　７７（１）：６２−６（１９９９）を参照のこと）。ヒトでは、成熟ＧＨは、１９１アミノ酸残基を含み、そして２２ｋＤａの分子量を示す。しかし、通常観察されるジスルフィドダイマーに加えて、ヒトＧＨの一部（残基１〜４３および４４〜１９１）から作製される２つのペプチドが血清中に検出されており、そして成体島組織に対して別個の効果を有する（Ｌｅｗｉｓら，Ｅｎｄｏｃｒ　Ｊ　４７増補：Ｓ１−８（２０００）を参照のこと）。ヒトＧＨの種々の天然に存在する、誘導体、改変体、代謝産物および操作された誘導体が公知であり、これらとしては、グリコシド化ＧＨ、メチオニルＧＨ、２０ｋＤａ　ＧＨ、アセチル化ＧＨ、タンパク質分解切断ＧＨ、脱アミド（ｄｅｓａｍｉｄｏ）ＧＨ、スルホキシドＧＨおよび短縮形態のＧＨが挙げられる。
【００７５】
ＧＨは、ホルモンの保存されたファミリーのメンバーであり、このファミリーは、ヒトにおけるＧＨ−Ｖ１およびＧＨ−Ｖ２、コリオマンモトロピンおよびプロラクチン、ならびに他の脊椎動物由来のタンパク質（例えば、齧歯類の胎盤性ラクトゲンＩおよびＩＩならびに他のウシおよびヒツジのラクトゲン、マウスのプロリフェリン（ｐｒｏｌｉｆｅｒｉｎ）Ｉ、ＩＩおよびＩＩＩ、ならびにプロリフェリン関連タンパク質、ウシプロラクチン関連タンパク質Ｉ、ＩＩおよびＩＩＩ、ラットプロラクチン様タンパク質ＡおよびＢ、ならびに種々の魚由来のソマトラクチンを含む。このファミリーのメンバーは、以下のコンセンサス配列によって特徴付けられる：Ｃ−ｘ−［ＳＴ］−ｘ（２）−［ＬＩＶＭＦＹ］−ｘ−［ＬＩＶＭＳＴＡ］−Ｐ−ｘ（５）−［ＴＡＬＩＶ］−ｘ（７）−［ＬＩＶＭＦＹ］−ｘ（６）−［ＬＩＶＭＦＹ］−ｘ（２）−［ＳＴＡ］−ＷまたはＣ−［ＬＩＶＭＦＹ］−ｘ（２）−Ｄ−［ＬＩＶＭＦＹＳＴＡ］−ｘ（５）−［ＬＩＶＭＦＹ］−ｘ（２）−［ＬＩＶＭＦＹＴ］−ｘ（２）−Ｃ。
【００７６】
本発明の実施のために適切な成長ホルモンは、種々の天然供給源および人工供給源から入手され得る。同種のＧＨをしばしば必要とするＧＨの治療的用途とは対照的に、霊長類種、哺乳動物種または脊椎動物種の範囲由来のＧＨは、膵臓細胞の培養のための低血清培地の処方において用いられ得る。成長ホルモンの便利な供給源は、ウシ下垂体抽出物（ＢＰＥ）であり、これは、天然のＧＨの豊富な供給源である。ＢＰＥ（７５μｇ／ｍｌタンパク質）は、培養培地中に約０．１μｌ／ｍｌ〜約１００μｌ／ｍｌ、好ましくは０．５μｌ／ｍｌ〜５０μｌ／ｍｌ、そして最も好ましくは５μｌ／ｍｌまたは３７．５ｍｇ／ｌで含まれ得る。他の種から入手可能な下垂体抽出物（例えば、ブタ、ヒツジなど）もまた、同様の濃度で用いられ得る。下垂体抽出物中に存在する他の因子は、その効果を増強し得るが、満足のいく結果がまた、精製したＧＨを用いて、および組換えＧＨを用いて達成され得る。組換えウシＧＨおよび組換えヒトＧＨは、広範に入手可能であり、そしてＧＨ活性の適切な供給源である。組換えＧＨは、培養培地に、０．０１ｍｇ／ｌと１００ｍｇ／ｌとの間、好ましくは０．１ｍｇ／ｌと１０ｍｇ／ｌとの間、より好ましくは約０．２ｍｇ／ｌ、約０．５ｍｇ／ｌ、約０．７５ｍｇ／ｌ、約１ｍｇ／ｌ、約１．２５ｍｇ／ｌ、約２ｍｇ／ｌまたは５ｍｇ／ｌ、そして最も好ましくは約１．２５ｍｇ／Ｌで添加され得、ここで、１ｍｇの組換えタンパク質は、３ＩＵのＧＨにほぼ等価である。
【００７７】
（２．他の補充物）
完全無血清培地のために基本培地へと添加される代表的成分としては、以下が挙げられる：組換えヒトインスリン（０．１μｇ／ｍｌ〜１００μｇ／ｍｌ）、トランスフェリン（０．１μｇ／ｍｌ〜１００μｇ／ｍｌ）、上皮増殖因子（０．１ｎｇ／ｍｌ〜１００ｎｇ／ｍｌ）、エタノールアミン（０．１μｇ／ｍｌ〜１００μｇ／ｍｌ）、アプロチニン（０．１μｇ／ｍｌ〜１００μｇ／ｍｌ）、グルコース（０．１ｍｇ／ｍｌ〜１００ｍｇ／ｍｌ）、ホスホエタノールアミン（０．１μＭ〜１００μＭ）、トリヨードチロニン（０．１ｐＭ〜１００ｐＭ）、セレン（０．１ｎＭ〜１００ｎＭ）、ヒドロコルチゾン（０．０１μＭ〜１００μＭ）、プロゲステロン（０．１ｎＭ〜１０ｎＭ）、フォルスコリン（０．１μＭ〜１００μＭ）、ヒレグリン（０．１ｎＭ〜１００ｎＭ）およびウシ下垂体抽出物（０．１μｇ／ｍｌ〜５００μｇ／ｍｌ）。細胞増殖を支持するために全ての補充成分が必要とされるわけではない；特定の補充物についての最適な濃度または必要性は、単一成分を省略するかまたはその濃度を低減し、そして細胞増殖に対する影響を観察することによって経験的に決定され得る（Ｓｔｅｐｈａｎら，前出を参照のこと）。
【００７８】
一般に、補充成分は、同じ生物学的特性を有する天然産物または合成産物によって置換され得る。例えば、トリヨードチロニン、ヒドロコルチゾンおよびプロゲステロンは全て、同じ細胞内レセプター（甲状腺レセプター、グルココルチコイドレセプターおよびプロゲステロンレセプター）を活性化することが公知の天然または合成のホルモンによって置換され得る。インスリンおよびＥＧＦは代表的に、組換えＤＮＡ方法論によって産生されるヒトタンパク質であるが、天然の供給源から精製されたポリペプチドによって、他の種由来のポリペプチドによって、またはインスリンレセプターおよびＥＧＦレセプターの他のアゴニストによって、置換され得る。ＧＨは、いくつかの場合、ＧＨレセプターの他のアンタゴニストによって置換され得る。同様に、ＥｒｂＢ３レセプターのリガンドであるヒレグリンは、ヒレグリンアイソフォームならびに他のＥｒｂＢ３アゴニスト（例えば、ＮＲＧ２、ＮＲＧ３およびＮＲＧ４、感覚ニューロンおよび運動ニューロン由来因子、ニューレスチン（ｎｅｕｒｅｓｔｉｎ）ならびにＥｂｐ−１、ヒレグリンα、ヒレグリンβ、ヒレグリンγ、ニューレグリン−１およびニューレグリン−２（ＮＲＧ−１α；ＮＲＧ−１β、ＮＲＧ−２αおよびＮＲＧ−２β））によって置換され得る。
【００７９】
例示的な無血清培地としては、基本培地ＳＭ９６および完全培地ＳＭ９５（これは、以下の表に示されるように補充されたＳＭ９６からなる）が挙げられる。ＳＭ９８は、Ｓｔｅｐｈａｎら，前出によって記載された培地補充物１４Ｆの改変物が補充された、１：１のＦ１２／ＤＭＥＭからなる。ＳＭ９８は、１４Ｆよりも少ないヒレグリン（８ｎｇ／ｍｌに対して１ｎｇ／ｍｌ）を含む。従って、ＳＭ９８は、以下が補充された、１：１のＦ１２／ＤＭＥＭからなる：組換えヒトインスリン、１０μｇ／ｍｌ；トランスフェリン、１０μｇ／ｍｌ；上皮増殖因子、１０ｎｇ／ｍｌ；エタノールアミン、６１ｎｇ／ｍｌ；アプロチニン、２５μｇ／ｍｌ；グルコース、５ｍｇ／ｍｌ；ホスホエタノールアミン、１４１ｎｇ／ｍｌ；トリヨードチロニン、３．３６５ｐｇ／ｍｌ；セレン、４．３２５ｎｇ／ｍｌ；ヒドロコルチゾン、１８１ｎｇ／ｍｌ；プロゲステロン、３．１５ｎｇ／ｍｌ；フォルスコリン、４１０ｎｇ／ｍｌ；ヒレグリン、１ｎｇ／ｍｌ；およびウシ下垂体抽出物、７５μｇ／ｍｌ。ＳＭ９５およびＳＭ９８中のＥＧＨおよびヒレグリンの例示的供給源は、組換えヒトＥＧＦ（Ｓｉｇｍａ　Ｅ９６４４）およびヒトヒレグリン−１のＥＧＦドメイン（アミノ酸１７６〜２４６）（Ｒ＆Ｄ　ｓｙｓｔｅｍｓ　３９６−ＨＢ／ＣＦ）である。
【００８０】
【表５】

【００８１】
【表６】

【００８２】
【表７】

（Ｂ．低血清培地への細胞の移植）
本明細書中に記載される場合、低血清培地へと膵臓細胞の培養物を移植することは、中間分化状態を有する、規定された細胞集団の選択を促進する。この細胞集団は、継代培養された場合増殖し続けるが、膵臓マーカー（例えば、ＰＤＸ−１）の高い発現レベルを維持する。刺激されていない、この集団は、比較的低レベルの膵臓内分泌ホルモン（例えば、インスリン）を分泌するが、本発明の方法に従って成熟して、高分泌細胞が得られ得る。低血清培地へと膵臓細胞の培養物を移植するために、細胞は、高血清培地から低血清培地へと離脱され得るか、または単離後に低血清培地中に直接配置され得る。ＳＭ９５およびＳＭ９８のような培地は、適切な低血清培地であるが、ＳＭ９５は、膵臓細胞の成熟の際にわずかに改善されたインスリン分泌を生じる。
【００８３】
離脱手順において、細胞は、高血清培地中で一定期間にわたって維持され、次いで、中間細胞集団の選択的増殖のために低血清培地へと移植される。この維持期間は、本明細書中上記のように、短くてもよい（例えば、一晩）。あるいは、細胞は、低血清培地への移植前に高血清培地中で増殖され得る。従って、１つの実施形態では、膵臓細胞は、単離され、精製され、次いで１０％添加された血清を有する培地（例えば、培地３）を含む培養容器中にプレーティングされる。細胞は、ほぼコンフルエントになるまで増殖させられ、次いで中間集団の選択のために低血清培地または無血清培地（例えば、ＳＭ９５またはＳＭ９８）へと継代される。得られる細胞集団は、免疫組織化学染色によって調べた場合、９０％より多くがＰＤＸ−１陽性であり、そして１：１００と１０００：１との間のインスリン：アクチンｍＲＮＡ比を維持する。
【００８４】
離脱手順の別の実施形態では、培地中の血清の濃度は、細胞を継代培養する際に毎回、または培地を除去して再度配置する際に毎回、培地を順次低下する濃度の血清で置換することによって、徐々に低下される。例えば、この培養手順の１つの実施形態では、単離された膵臓細胞は、培地７（２０％　ＦＢＳ）中で一晩、最初に維持され、次いで培地３（１０％　ＦＢＳ）中で７〜１０日間にわたって増殖される。ほぼコンフルエンスに達したら、細胞は、４％血清を含む培地（６０％　ＳＭ９５および４０％培地３）を有する新たな容器へと継代培養される。さらに、ほぼコンフルエントになるまでの増殖後、細胞は、血清を含まない培地（１００％　ＳＭ９５）を有する新たな容器へと継代培養される。細胞は、増殖し続け、そしてその後の継代の際に、純粋なＳＭ９５培地中に継代培養される。
【００８５】
離脱手順の各工程の間の血清の濃度は重要ではない；従って、細胞を１０％血清中での増殖から０％血清へと徐々に離脱する場合、連続した培地変更は、約９％、８％、７％、６％、５％、４％、３％、２％または１％の血清を含み得る。このような中間濃度の血清を有する培地は、培地３（１０％　ＦＢＳ）とＳＭ９５またはＳＭ９８（０％　ＦＢＳ）とを適切な比で混合することによって調製され得る。一般に、細胞を新たな培地に継代するかまたは細胞に新たな培地を与える場合、以前の培地からのいくらかの残りの血清が、新たな培地中へと持ち越され得る。従って、培養培地中の血清の百分率は、添加された新たな培地中の血清の濃度と正確には等しくないかもしれない。しかし、通常の細胞培養プロトコルでは、ごく少量の培地が持ち越され、続いて培地の交換を続けることによって希釈される。いずれにせよ、持ち越された残りの血清の存在は、本発明の方法を妨害しない。所望の場合、培養培地中の血清の量は、特定の百分率の血清を有することが既知の培地と問題の培養培地との間で血清特異的成分（例えば、アルブミン、グロブリン、フェチュインなどの免疫アッセイ）の濃度を比較することによって決定され得る。
【００８６】
膵臓細胞の培養物はまた、高血清培地中で細胞を維持することなく、１回の工程で、低血清培地中へと移植され得る。この「ショック」方法論では、細胞は通常、低血清培地を有する培養容器中に、単離手順後直ちにプレーティングされる。このような実施形態では、単離された膵臓細胞の最初の継代は、１％未満の血清を有する培地（例えば、ＳＭ９５またはＳＭ９８）中で実施される。
【００８７】
ショック方法論の代替の実施形態では、細胞は、１％と４％との間の血清、例えば、約１．３３％、１．５％、１．６６％、２％、２．３３％、２．５％、２．６６％、３％、３．３３％、３．５％または３．６６％の血清を含む培地中に最初に継代される。細胞が密度勾配によって精製された場合、これらは一般に、血清を含む培地中で洗浄または回収され得る。細胞が低血清培地中で洗浄されない場合、細胞の最初の継代のための培養培地は、単離手順からの中間濃度の血清を含む。例示的な実施形態では、密度勾配界面からの細胞は、１容積の培地７（２０％血清）中に収集される。この容積に、５容積の無血清培地（例えば、ＳＭ９５）が添加されて、３．３３％の最終血清濃度が得られる。次いで、細胞は、この培地中で１回の継代にわたって増殖される。その後の継代または培地変更は低血清培地を用いて実施され、その結果、細胞が１％以下の血清を含む培地中で増殖されて、中間細胞集団（すなわち、中間の分化の細胞）が選択されることとなる。
【００８８】
ショック方法論または離脱方法論のいずれかを用いると、低血清培地中での膵臓細胞の培養物は、適切な培養条件下で増殖し続け、膵臓内分泌細胞に対して特異的な特定のマーカーを発現するが、成熟島集団とは異なる、中間細胞集団の出現を促進する。本発明の１つの実施形態では、この中間細胞集団は、膵臓内分泌マーカーＰＤＸ−１のその高発現（代表的に、少なくとも９０％の細胞はＰＤＸ−１について陽性に染まり、そしてＰＤＸ−１の染色は、細胞の核に濃縮する傾向がある）によって、およびそのインスリンｍＲＮＡ発現パターンによって区別される。この実施形態では、中間集団の大部分の細胞は、インスリン発現について陽性であるが、この集団中のインスリン遺伝子発現の平均レベルは比較的低い（代表的に、インスリン：アクチンｍＲＮＡ比は、１：１０と１００：１との間である）。いくつかの実施形態では、ショック方法論によって産生される中間細胞集団は、グルコース負荷に対して陽性の応答を維持し、ＳＧＳ試験によってアッセイされた場合、２以上の刺激指数を生じる。他の実施形態では、離脱方法論によって産生された中間集団は、さらに成熟するまでＳＧＳアッセイにおいて負のスコアとなる。
【００８９】
本発明の方法によって産生された中間細胞集団は代表的に、増殖する能力および高分泌内分泌細胞へとさらに分化する能力の両方を保持する。増殖能力は一般に、第１の密度で播種された培養物が、第２の密度へと増大する能力によって評価される；例えば、１平方センチメートルあたり１８０細胞でプレーティングされた細胞は、１回の継代で１ｍｌあたり１，８００細胞へと増大し得る。増殖と継代とのサイクルの繰り返しによって、単離された膵臓細胞の出発集団は、内分泌マーカー（例えば、ＰＤＸ−１およびインスリンｍＲＮＡ発現）を保持し、かつ成熟した高分泌内分泌細胞へと分化する能力を保持しながら、約１０，０００倍以上（例えば、約１００倍、５００倍、１０００倍、５０００倍、１０，０００倍、５０，０００倍、１００，０００倍、５００，０００倍または１，０００，０００倍）増大し得る。
【００９０】
この中間段階の集団の細胞は、さらに操作されることなく、膵臓内分泌機能の回復または増強のために哺乳動物中に移植され得る。中間集団の細胞はまた、島細胞の発達およびホメオスタシス（例えば、薬物スクリーニング、島形態形成または自己免疫応答）についてのモデル系として用いられ得る。しかし、本発明の１つの実施形態では、中間段階の集団由来の細胞は、培養中にさらに成熟して、高レベルの膵臓内分泌ホルモンを分泌する細胞を産生する。
【００９１】
（Ｖ．高インスリン産生凝集物への中間細胞の成熟）
適切な培養条件下では、本発明の中間段階の膵臓細胞は、高レベルの膵臓内分泌ホルモンを発現する別の型の細胞へと成熟する。代表的に、このプロセスは、この細胞に、凝集物または偽島として公知の細胞クラスター（これは、膵臓中の成熟した島に特有の形態学的特徴および免疫組織化学的特徴のいくつかを獲得する）を形成させることを含む。膵臓細胞の増殖によって予め馴化された基材が、凝集物発達の一貫性の増強を提供し、そしてより本物の島の形態を生じることは本発明の知見である。
【００９２】
この手順のための好ましい出発材料は、本発明の方法によって産生された中間段階の細胞の培養物であるが、ＰＤＸ−１陽性条件的幹細胞を含む他の細胞集団が用いられ得る。中間段階の細胞集団をコンフルエンスになるまで単に増殖させた場合、細胞の中心または凝集物が出現し得る。凝集物の形成はまた、本発明の特定の方法によって生成される細胞に特異的な処理方法によって誘導され得る（例えば、ショック方法論によって生成される中間集団は、コラゲナーゼでの処理によって凝集物を形成するように誘導され得る）。しかし、一貫した凝集物形成は、細胞が、膵臓細胞を培養するために以前に用いられた基材上で培養される場合、増強される。特定の理論によって束縛されることを望まないが、中間段階の細胞の増殖は、特定の細胞外マトリクス分子の蓄積または細胞外マトリクスの特定の空間的配置を刺激し得る。細胞を除去して新たな細胞をこのマトリクスに播種した場合、このマトリクスは、インビボでの島と類似した形態学的特性および生化学的特性を有する凝集物への細胞の分化を促進する。
【００９３】
代表的実施形態では、膵臓細胞が単離され、そして本発明の方法に従って増殖される。次いで、細胞は、基材上に継代培養され、その間に、これらは、単層としてコンフルエントになるまで増殖する。一旦細胞が単層を形成したら（最初の継代培養物の播種密度に依存して、代表的には約５日間）、これらは、トリプシン処理によって除去される。低下させた濃度のトリプシン（代表的には、標準的細胞培養技術において用いられる濃度の１／２または１／４）が、マトリクスの大規模な分解を防ぐために好ましい。あるいは、細胞の単層は、界面活性剤を用いて基材を抽出することによって除去され得、界面活性剤は、細胞を除去するが、分泌されたマトリックスを残す（Ｇｏｓｐｏｄａｒｏｗｉｃｚら，Ｐｒｏｃ　Ｎａｔｌ　Ａｃａｄ　Ｓｃｉ　ＵＳＡ　７７：４０９４−８（１９８０）を参照のこと）。
【００９４】
凝集物形成を誘導するために、膵臓細胞は、この予め馴化された基材上で培養される。便利には、基材上で予め増殖され除去された細胞は、分けられ、そして馴化基材に再度播種され得る。しかし、基材を馴化させる培養物と基材に播種される培養物とが同じ培養物である必要はない。従って、１つの細胞培養物は、基材を馴化させるために基材上で増殖され、この細胞は除去され、そして別の培養物由来の細胞は馴化基材上に播種されて凝集物形成を促進し得る。馴化細胞は、その後培養される細胞と同じまたは異なるドナーまたは種に由来し得る。
【００９５】
上記のように予め馴化された基材上で増殖された細胞は、代表的な単層としてよりもむしろ、細胞凝集物として増殖する。これらの凝集物は、約５０〜約５０００の細胞を含み、そして直径が約５０ミクロン〜約３００ミクロンである。切片化し、そして免疫組織化学染色によって調べた場合、これらの凝集物は、特徴的形態を提示する。１細胞の厚みでかつ管マーカーサイトケラチン１９について陽性に染まる、連続した被膜層は、サイトケラチン１９染色を一般に欠く内部細胞塊を取り囲む。内部塊は代表的に、膵臓内分泌ホルモン、ＫＳ１／４およびＰＤＸ−１について陽性に染まる細胞を含む。特定の理論によって束縛されることを望まないが、この形態は、外被膜を含む管様幹細胞が内分泌前駆体細胞を生じ、これが内部細胞塊内で、より成熟した内分泌細胞へと分化する機構を示唆する。従って、本発明の方法によって産生された細胞凝集物は、インビボで内分泌膵臓細胞を生じる発達プロセスを模倣する。従って、これらの凝集物は哺乳動物中に移植されて、膵臓内分泌機能を増強または回復し得る。
【００９６】
哺乳動物への移植およびその後の内分泌機能のモニタリングは、島移植のために通常用いられる方法に従って実施され得る；例えば、Ｒｙａｎら，Ｄｉａｂｅｔｅｓ　５０：７１０−１９（２００１）；Ｐｅｃｋら，Ａｎｎ　Ｍｅｄ　３３：１８６−９２（２００１）；Ｓｈａｐｉｒｏら，Ｎ　Ｅｎｇｌ　Ｊ　Ｍｅｄ　３４３（４）：２３０−８（２０００）；Ｃａｒｌｓｓｏｎら，Ｕｐｓ　Ｊ　Ｍｅｄ　Ｓｃｉ　１０５（２）：１０７−２３（２０００）およびＫｕｈｔｒｅｉｂｅｒ，ＷＭ，Ｃｅｌｌ　Ｅｎｃａｐｓｕｌａｔｉｏｎ　Ｔｅｃｈｎｏｌｏｇｙ　ａｎｄ　Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｂｉｒｋｈａｕｓｅｒ，Ｂｏｓｔｏｎ，１９９９を参照のこと。好ましい移植部位としては、肝臓および腎臓皮膜が挙げられる。移植された凝集物に対する免疫反応に問題がある場合、種々のアプローチを用いて、移植された凝集物の免疫認識を妨害し得る。これらとしては、以下が挙げられる：免疫細胞による接近を妨害するための凝集物のカプセル化（米国特許第５，７６２，９５９、同第５，５５０，１７８号および同第５，５７８，３１４号を参照のこと）、凝集物形成の前に細胞を遺伝子操作して、宿主によって認識される抗原を除去すること（米国特許第６，００１，６４７号を参照のこと）および免疫抑制レジメンでの宿主の処理（Ｒｙａｎら，Ｄｉａｂｅｔｅｓ　５０：７１０−１９（２００１）を参照のこと）。
【００９７】
（実施例）
以下の実施例は、本願発明を例示するために提供されるが、本願発明を限定するためには提供されない。
【００９８】
（実施例１：離脱によるＰＤＸ−１陽性の中間細胞集団の作製）
ＨＤ３５７は、５７歳の女性白人器官ドナーであった。膵臓組織を、機械的破壊およびＨＢＳＳ中のＬｉｂｅｒａｓｅ（１．５ｍｇ／ｍｌ）を用いた消化によって解離させた。解離した細胞を、米国特許第５，７３９，０３３号に記載されるように、ＰＩＰＳの溶液中での三層密度勾配分離（ＵＷ溶液中でのＮｙｃｏｄｅｎｚ（Ｎｙｃｏｍｅｄ　ＡＳ，Ｎｏｒｗａｙ））によって分画した。４０ｍｌの１．１１４ｇ／ｍｌ　ＰＩＰＳ溶液中の、２．６ｍｌの解離膵臓組織の底部層、８０ｍｌの１．０９０ｇ／ｍｌ　ＰＩＰＳの中間層および７０ｍｌ　ＲＰＭＩ　１６４０＋２％　ＦＢＳの上部層を積層することによって、密度勾配を２５０ｍｌ遠心管中に準備した。遠心分離を、１，５００ＲＰＭで、ブレーキなしで６分間、０５，ＡＲＣローターを備えたＳｏｒｖａｌｌ　ＲＣ−３Ｃ　Ｐｌｕｓを用いて実施した。以下の３つの別々の組織集団が、勾配分離後に収集された：上部層と第２層との間の上（島が富化される）界面、中間層と底部層との間の下界面、およびペレット画分。収集された細胞画分を培地７（第ＩＩＩ節Ｂ）で洗浄して勾配溶液を除去し、その後培養した。上部界面からの総島収量は、１２８，０００ＩＥＱ（精製後細胞の検鏡によって決定され、そして１５０μｍの標準的島に対する島容積によって正規化された島当量）であり、純度は７９％であった。
【００９９】
下の界面由来の約５％の純度の２０，０００ＥＱの膵臓細胞を、３５ｍｌの培地７（２０％　ＦＢＳ）を有するＴ１６２培養フラスコ中で一晩培養した（Ｐ０）。１日目に、この培地を、継代培養（第ＩＩＩＢ節）を行わずに培地３（１０％　ＦＢＳ）に交換した。９日目に、この培地を、継代培養を行わずに１／３　ＳＭ９８および２／３培地３（合計６．６７％　ＦＢＳ）に交換した。１３日目に、この細胞をＰ１に継代し、そしてＳＭ９８（０％　ＦＢＳ）を有する１００ｍｍ組織培養ディッシュ中で培養した。細胞をさらに７回の継代（Ｐ２〜Ｐ８）にわたって継代した。継代時に、細胞の一部を、免疫細胞化学分析のためにチャンバスライド上で培養した。
【０１００】
染色プロセスを、スライドのチャンバ部分を除去して開始した。細胞を、カルシウム／マグネシウムを有するＤＰＢＳ（Ｄｕｌｂｅｃｃｏのリン酸緩衝化生理食塩水）で３分間にわたって３回、非常に穏やかにリンスした。次いで、この細胞を、室温で、ＤＰＢＳ中の氷冷４％パラホルムアルデヒド溶液中で１０分間にわたって固定し、続いて、カルシウム／マグネシウムを有するＤＰＢＳ中で各々３分間、３回リンスした。細胞を、ブロッキング溶液（ＴＢＳ／３％　ＢＳＡ／１％正常ヤギ血清）中で１時間にわたって室温でインキュベートした。次いで、細胞を、１０分間〜１５分間にわたってブロッキング溶液中の０．３％　ＴｒｉｔｏｎＸ−１００を用いて透過化した。適切な希釈でブロッキング溶液中に１０００：１希釈されたウサギ抗ＰＤＸ−１一次抗体（Ｓｃｒｉｐｐｓ製）を細胞に添加し、そして４℃にて一晩インキュベートした。ＰＤＸ−１抗体を、Ｌｅｏｎａｒｄ　Ｊ．ら，Ｍｏｌ．Ｅｎｄｏｃｒｉｎｏｌ．，１９９３年１０月７日，（１０）１２７５−８３の教示に従って作製し得る。
【０１０１】
一次抗体とのインキュベーション後、細胞を、カルシウム／マグネシウムを含まないＤＰＢＳ中で３分間にわたってリンスした。さらに２回のリンス後、細胞をブロッキング溶液中で３０分間にわたって室温で再度ブロックした。二次抗体（ブロッキング溶液中に２００：１希釈されたヤギ抗ウサギＩｇＧ−ローダミン結合体）をこの細胞に添加し、そして暗所にて１時間〜２時間にわたって室温でインキュベートした。
【０１０２】
インキュベーション後、細胞を、カルシウムもマグネシウムも含まないＤＰＢＳ中で３回、それぞれ３分間にわたってリンスした。核を、ＤＰＢＳ中のＨｏｅｃｈｓｔ色素（１０，０００：１）で５分間にわたって対比染色し、続いてＤＰＢＳ中で１回リンスした。過剰な流体を、ペーパータオルで軽くたたいて除去した。スライドに３〜４滴の水性マウント培地をマウントし、そしてカバースライドで覆った。スライドを暗所にて２時間にわたって室温で乾燥させ、そして縁部をマニキュア液でシールした。スライドを暗所にて４℃で保存した後に、Ｏｌｙｍｐｕｓ　ＡＸ　７０蛍光顕微鏡で検鏡した。本質的に１００％のＨＤ３５７　Ｐ８細胞がＰＤＸ−１について陽性に染まった。
【０１０３】
（実施例２：ショックによる、ＰＤＸ−１陽性の中間細胞集団の作製）
ヒトドナー番号３６９（ＨＤ３６９）は、４８歳のラテンアメリカ系男性であり、体重が８０ｋｇであり、そしてＣＭＶ以外は、大部分のウイルス学試験について陰性であった。温かい虚血時間は存在せず、冷たい虚血時間は１５時間であった。膵臓は太っていたが、明らかな損傷は存在しなかった。主な膵臓管にカニューレ挿入し、そして試験の間に見出された明らかな漏出は存在しなかった。酵素の注入は満足がいくものであった。静的消化は１９分間かかり、そしてチャンバ消化は１８分間持続した。この消化によって、精製前に約２７０，０００　ＩＥＱが得られた。冷たい虚血の長さに起因して、島のほぼ３分の１は、不明確であり、インタクトな被膜を有さなかった。
【０１０４】
３層の密度勾配遠心分離を、実施例１の手順に従って実施した。いくつかの島がフラグメントへと解離して見出され、そして上の界面における島の純度は５４．３％であり、最終的な島の収量は約１４９，０００ＩＥＱであった。
【０１０５】
上の界面由来の約６，０００ＩＥＱ（純度約５４％）を８ｍｌの培地７（２０％　ＦＢＳ）中に収集し、そして２ｍｌのアリコート３つおよび０．５ｍｌのアリコート３つに分けた。これらの画分を、処理した非コーティングプラスチック組織培養ディッシュ中で培養した。細胞を、３つの群に分配した。群１は、以下からなっていた：ａ）２ｍｌの培地７および１０ｍｌの培地ＳＭ９５（合計３．３３％のＦＢＳ）中の１，５００ＩＥＱ（これを、１００ｍｍ培養ディッシュに播種した）、ならびにｂ）０．５ｍｌの培地７および３ｍｌの培地ＳＭ９５（合計２．８６％のＦＢＳ）中の４００ＩＥＱ（これを、６０ｍｍ培養ディッシュに播種した）。群２は以下からなっていた：ａ）２ｍｌの培地７および１０ｍｌの培地ＳＭ９６（合計３．３３％のＦＢＳ）中の１，５００ＩＥＱ（これを、１００ｍｍ培養ディッシュ中に播種した）、ならびにｂ）０．５ｍｌの培地７および３ｍｌの培地ＳＭ９６（合計２．８６％のＦＢＳ）中の４００ＩＥＱ（これを、６０ｍｍ培養ディッシュ中に播種した）。群３は以下からなっていた：ａ）２ｍｌの培地７および１０ｍｌの培地ＳＭ９８（合計３．３３％のＦＢＳ）中の１，５００ＩＥＱ（これを、１００ｍｍ培養ディッシュ中に播種した）、ならびにｂ）０．５ｍｌの培地７および３ｍｌの培地ＳＭ９８中の４００ＩＥＱ（これを、６０ｍｍ培養ディッシュ中に播種した）（合計２．８６％のＦＢＳ）。このディッシュを、３７℃で、５％　ＣＯ_２の存在下で１４日間にわたって、継代１回目までインキュベートした。培地を１週間に２回、無血清培地に交換した。
【０１０６】
継代１回目（１４日目）に、細胞を、トリプシン／ＥＤＴＡを約１０分間用いて培養ディッシュの底部から解離し、次いで１０％　ＦＢＳ　ＨＢＳＳ培地を用いて洗浄した。各群において、１００ｍｍディッシュおよび６０ｍｍディッシュの両方からの細胞を合わせ、そして３枚の１００ｍｍディッシュ中に播種した。
【０１０７】
継代２回目（２０日目）に、細胞をトリプシン処理し、そして１対２の比で継代培養した。ＳＭ９５およびＳＭ９８中の細胞は、増殖し、そして１週間以内にコンフルエントになった。ＳＭ９５中での６回の継代後、細胞を固定し、そして実施例１に記載した通りにＰＤＸ−１発現について染色した。ほぼ１００％の細胞は、ＰＤＸ−１について陽性に染まった。
【０１０８】
（実施例３：中間細胞集団のインスリン発現）
ＰＤＸ−１陽性の中間細胞集団を、ヒトドナーから、実施例２のショック方法に従って作製した、そして無血清培地中で繁殖させた。継代１回目から継代６回目までの細胞を、インスリンおよびアクチンのｍＲＮＡの発現についてアッセイした。
【０１０９】
ＲＮＡを、ＲＮｅａｓｙミニキット（ＱＩＡＧＥＮ　＃７４１０４）を用いて製造業者の指示に従って単離した。手短には、溶解緩衝液（１０ｃｍプレートあたり６５０μｌ）をこの細胞に添加し、使い捨て細胞スクレーパー（Ｆｉｓｈｅｒ＃０８７７３２）を用いて収集し、次いでＱＩＡｓｈｒｅｄｄｅｒ（ＱＩＡＧＥＮ＃７９６５４）を用いて破壊した。総ＲＮＡを溶解産物から単離し、次いでＲｉｂｏＧｒｅｅｎ　ＲＮＡ　Ｑｕａｎｔｉｔａｔｉｏｎアッセイ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ＃Ｒ−１１４９０）を用いて定量した。ＲＮＡサンプルを、ｃＤＮＡ合成まで−８０℃で凍結保存した。各サンプルの二連のアリコート（各々０．５μｇ）を、２０ｐｍｏｌｅのオリゴ（ｄＴ）_１６を用い、Ｏｍｎｉｓｃｒｉｐｔ　ＲＴキット（ＱＩＡＧＥＮ＃２０５１１１）を製造業者の指示に従って用いて逆転写し、次いで各ｃＤＮＡサンプルを、ＴＥ緩衝液（ｐＨ８．０）を用いて１００μｌになるように希釈し、そして−２０℃にて保存した。リアルタイムＰＣＲを、２μｌの各ｃＤＮＡサンプルおよび示したプライマーを用いて、Ｒｏｃｈｅ　Ｍｏｌｅｃｕｌａｒ　ＬｉｇｈｔＣｙｃｌｅｒで実施した。アクチンおよびインスリンを、ハイブリッドプローブプロトコルおよびＤＮＡ　Ｍａｓｔｅｒ　Ｈｙｂｒｉｄｉｚａｔｉｏｎミックス（Ｒｏｃｈｅ＃２１５８８２５）を製造業者の指示に従って用いて測定した。ＰＣＲを、並行して増幅された各産物の検量線との比較によって定量した。
【０１１０】
【表８】

この結果は、平均のインスリン対アクチンｍＲＮＡ比が、継代１回目から継代６回目まで、２１１と１．９９との間の範囲にあることを示した。
【０１１１】
【表９】

ショック方法によって作製された中間集団はまた、グルコース負荷に応答してインスリンを分泌する能力についてアッセイされた。示した培養物からのほぼコンフルエントなプレートを、刺激グルコース分泌試験（ＳＧＳ）を用いてインスリン分泌についてアッセイした。
【０１１２】
【表１０】

（実施例４：低血清培地中での増殖によるＰＤＸ−１陽性の中間細胞の選択）
本実施例は、低血清培地中での増殖による中間段階の集団の選択を例示する。単離した膵臓細胞を、血清含有培地条件から無血清培養培地条件へと移行させた。このことは、インスリン発現膵臓培養物をもたらす、内分泌前駆細胞の所望の集団を富化および選択するために役立つ。膵臓細胞を、実施例１の通りにドナー膵臓から単離した。島が富化された上の界面層Ａおよび下の界面Ｂならびにペレット画分を、血清含有培地（２０％ウシ胎仔血清を有する培地７）中で最初に増殖させた（継代０回目、ＰＯ）。一晩のインキュベーション後、培養物を、より少ない血清を含む培地（１０％ウシ胎児血清を有する培地３）に切り替え、そして７日間〜１０日間培養した。この期間の最後に、ほぼコンフルエントな培養物をトリプシン処理（トリプシン／ＥＤＴＡ）し、そして選択のための規定された無血清培地（ＳＭ９８またはＳＭ９５）中に、１：３の比で継代した（Ｐ１）。培養物に毎週補給し、そして８０％〜９０％のコンフルエンスに達したときに１：２の比で継代した。
【０１１３】
細胞培養前の層Ｂ中の細胞集団は不均質であり、そして腺房細胞（アミラーゼ陽性）、いくつかの成熟した島（インスリン、グルカゴン、ソマトスタチンに陽性の細胞）、およびいくつかの管成分（サイトケラチン１９）から構成されていた（表１を参照のこと）。増殖プロセスおよび選択プロセスに供された、富化された集団は、これらのマーカーのタンパク質および遺伝子の発現パターンにおいて多くの変化を提示した（表２および表３）。継代０回目（Ｐ０）には、細胞集団は依然として不均質であり、そして有意な割合（２０％〜５０％）がアミラーゼ陽性であり、サイトケラチン１９陽性（８５％〜９５％）であり、そしてほんの１％〜３％の細胞がインスリンについての陽性染色を示した。これらの予め離脱された初期インスリン陽性培養細胞は小さく、インスリンについて陽性に染まる、多くの小さな細胞質顆粒を含み、ＰＤＸ−１（膵臓ホメオボックスドメインタンパク質−１）について強く染まる小さな丸い核を有し、そしてサイトケラチン１９についての細胞質染色を示さなかった；それゆえ、新たに富化された膵臓材料中に存在する成熟した成体島から誘導されたようであった。この小さなインスリン陽性細胞集団は、細胞を継代して血清含有培地から無血清培地へと移行させるにつれて減少し、そして継代４回目（Ｐ４）までにもはや検出可能でなかった。継代２回目〜継代４回目（Ｐ２〜Ｐ４）では、第２の異なるインスリン陽性集団が検出され得た（総細胞の２％〜４０％）。離脱細胞のこの集団は、インスリンについて陽性に染まる楕円形の核および核周囲細胞質顆粒を有し、そしてＰＤＸ−１陽性核を有する、より大きな細胞からなっていた（継代６回目である図１を参照のこと）。サイトケラチン１９および／または核増殖抗原Ｋｉ６７の同時発現は、有意な割合のこれらの細胞において観察された。大きなインスリン陽性細胞は、継代４回目〜継代８回目（Ｐ４〜Ｐ８）では、細胞集団の１０％〜６０％を構成していた。インスリン陽性細胞の百分率は離脱プロセスの間に上昇するが、総インスリンＲＮＡ発現レベルは減少した。このことは、初期インスリン陽性細胞集団と比較して、大きなインスリン陽性細胞中の、１細胞あたりの、より低いインスリンＲＮＡ発現を示す。離脱されても、血清含有培地から無血清培地（ＳＭ９８またはＳＭ９５）へと移行されてもいない細胞は、検出可能なレベルのインスリンを発現せず、核というよりも細胞質にある傾向のある弱いＰＤＸ−１発現を示し、そしてより大きな割合（離脱集団における＜１０％と比較して、＞３０％）の離脱されていないこれらの細胞は、腺房マーカーアミラーゼを発現し続けた。増殖した膵臓細胞の選択の間のホルモン遺伝子発現レベルにおける変化をまた、定量的ＲＴ−ＰＣＲによってモニタリングした（表３）。
【０１１４】
【表１】

データをパラフィン切片から得て、そして示されたマーカーを発現する総細胞の百分率として示す。
【０１１５】
【表２】

データを、示されたマーカーを発現する総細胞の百分率として示す。
【０１１６】
【表３】

（実施例５：インスリン分泌凝集物を形成する中間細胞の成熟）
ドナー膵臓ＨＤ３８１の消化から得られたヒト膵臓組織を、培地７（２０％　ＦＢＳ）中で２日間にわたって培養した。培地を、３日間にわたって培地３（１０％　ＦＢＳ）へと交換した。この時間の間に、ほとんど単層として増殖した細胞集団が発達した（継代０回目）。この培養物を、ＳＭ　９８（０％　ＦＢＳ）培地中の新たな組織培養プレート中に５日間にわたって継代培養して、内分泌前駆細胞を選択した（継代１回目）。ほぼ全ての細胞がこの培養物において単層として増殖した。
【０１１７】
継代１回目の培養物を、新たなプレートへと継代培養し、ここで、この培養物は、単層として増殖し続けた（継代２回目）。継代１回目の培養物をまた、リサイクルしたプレート中に継代培養し、このプレートから、継代培養のためにトリプシン処理によって継代１回目の細胞を除去した。リサイクルしたプレートでは、細胞は単層として増殖するのを停止した。むしろ、細胞は、約５０ミクロンと約３００ミクロンとの間の寸法を有する、約５０細胞から約５０００細胞の凝集物を形成した。単層増殖物の４回の連続した継代からの細胞を、リサイクルしたプレートに継代培養した。各場合において、新たなプレートで培養した細胞は、単層として繁殖し、一方、リサイクルしたプレートで培養した細胞は細胞凝集物を形成した。
【０１１８】
細胞凝集物のサンプルを、手で拾い、４％ホルマリン中で固定し、そしてパラフィンブロック中に包埋した。スライドのための切片を約６μｍの厚さに切断した。パラフィン除去（ｄｅｐａｒａｆｆｉｎｉｚａｔｉｏｎ）のために、スライドを以下の試薬中に逐次浸漬した：１０分間にわたって２回、キシレン；１分間にわたって３回、１００％エタノール；１分間にわたって１回、９５％エタノール；１分間にわたって１回、７０％エタノール。スライドを、０．３％過酸化水素メタノール溶液中に３０分間にわたって室温で浸漬して、内因性ペルオキシダーゼ活性を阻害した。スライドを、０．０１クエン酸緩衝液（ｐＨ６．０）中で３０分間にわたって煮沸して、特定のエピトープを回復させた。スライドをＰＢＳで各々５分間、３回リンスし、そして湿潤チャンバ中で室温で、３０分間〜６０分間にわたって、ＰＢＳ中の１０％正常ヤギ血清中でブロックした。ブロッキング溶液中に調製した、Ｄａｋｏ　Ｃｏｒｐｏｒａｔｉｏｎ（Ｃａｔ．Ｎｏ．Ｍ０８８８）からの一次（ＣＫ−１９特異的）抗体をサンプルに添加し、そして湿潤チャンバ中で室温で６０分間にわたってインキュベートした。スライドを、ＰＢＳで３回、５分間にわたって洗浄し、そしてブロッキング溶液中に調製された二次抗体（ビオチン化抗ＩｇＧ）と共に、湿潤チャンバ中で室温で３０分間にわたってインキュベートした。スライドを、ＰＢＳで５分間にわたって３回リンスし、そしてアビジン−西洋ワサビペルオキシド（ａｖｉｄｉｎ−ｈｏｒｓｅｒａｄｉｓｈ　ｐｅｒｏｘｉｄｅ）またはＡＢＣ複合体（Ｄａｋｏ）と共に室温で３０分間にわたってインキュベートした。スライドを、ＰＢＳで５分間にわたって３回リンスし、そしてビアミノベンジジン発色溶液とともに１０分間にわたってインキュベートした。蒸留水での２回の洗浄後、スライドをＭａｙｅｒのＨｅｍａｔｏｘｙｌｉｎ中に５分間にわたって浸漬し、次いで、水が無色になりかつ核が青くなるまで、流れる水道水中でリンスし続けた。スライドを、以下の試薬中に順次浸漬することによって脱水した：７０％エタノール、１分間；９５％エタノール、１分間；１００％エタノール、１分間、３回；キシレン、一晩。スライドをマウント培地中にマウントし、そして４℃で保存した。
【０１１９】
膵臓マーカーおよびホルモンに対する抗体のパネルでの染色は、凝集物が、ＣＫ−１９について大部分陰性の細胞の内部塊を取り囲む、１層の厚さのＣＫ−１９陽性細胞の被膜からなることを示した（被膜がこの処理によって部分的に撹乱されている、図２を参照のこと。内部塊の細胞は不均質に染まったが、内部細胞塊内には、膵臓内分泌ホルモン（インスリン、ソマトスタチンおよびグルカゴン）ならびにＫＳ１／４およびＰＤＸ−１について陽性に染まる細胞が見出され得る。ＣＫ−１９陽性被膜細胞のうちの約５０％もまたＰＤＸ−１陽性であった。
【０１２０】
単層培養物のインスリンレベルおよび種々の継代において凝集した細胞のインスリンレベルを比較した。細胞を高塩緩衝液で溶解し、そして溶解産物中のインスリンの量を、^１２５Ｉ標識インスリンを用いる競合放射免疫アッセイによって決定した。値を、Ｈｏｅｃｈｓｔ　３３２５８の存在下での蛍光によって測定した溶解産物の総ＤＮＡ含量に対して正規化した。新たに単離された膵臓島のインスリン含量を、比較のために決定した。継代３回目の凝集物についての比較を示す：
【０１２１】
【表１１】

本明細書中に記載される実施例および実施形態は、例示の目的のためのみであること、そしてそれらを考慮した種々の改変または変更が当業者に示唆され、そして本出願および添付の特許請求の範囲の趣旨および範囲内に含まれるべきであることが理解される。本明細書中に引用される全ての刊行物、特許および特許出願は、その全体が全ての目的のために本明細書中に参考として援用される。
【図面の簡単な説明】
【図１】
図１（ａおよびｂ）は、ＰＤＸ−１陽性染色を示す、培養膵臓細胞の２枚のマイクロ写真である。（ａ）は、非特異的抗体を用いたコントロールであり、そして（ｂ）は、ＰＤＸ−１特異的抗体で染色した細胞である。
【図２】
図２（ａおよびｂ）は、ＣＫ−１９陽性細胞の被膜を示す、培養膵臓細胞の凝集物の２枚のマイクロ写真である。（ａ）は、非特異的抗体を用いたコントロールであり、そして（ｂ）は、ＣＫ−１９特異的抗体で染色した細胞である。
【図３】
図３は配列表を示す。[0001]
(Citation of related application)
This application is based on U.S. Provisional Application No. 60 / 215,634 (filed on June 30, 2000) and U.S. Provisional Application No. 60 / 246,306 (filed on November 11, 2000) and U.S. Provisional Application No. Unassigned) (filed May 17, 2001).
[0002]
(Reference to rights to inventions made under federally supported research and development)
Not applicable.
[0003]
(Refer to the "Sequence Listing" filed on the compact disc, table or appendix to the computer program listing)
Not applicable.
[0004]
(Field of the Invention)
The present invention relates to the finding that there is an intermediate differentiation stage pancreatic stem cell that can be grown in a stable manner in successive serial passages while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture and can be induced to a late developmental stage. The present invention further provides culture techniques for selecting cells at these intermediate differentiation stages and for selectively removing early or late stage pancreatic cells.
[0005]
(Background of the Invention)
The mammalian pancreas develops from the embryonic foregut. As the embryo grows, the tubing develops by branching morphogenesis. After fusion of the ventral and dorsal primordia, new organs grow and mature into two connected structures (the exocrine and endocrine systems). Most of the pancreas is composed of acinar cells that produce digestive enzymes. The endocrine system includes β cells, which produce insulin, α cells, which produce glucagon, and δ cells, which produce somatostatin. Endocrine cells are organized into clusters called islets.
[0006]
Animal studies have shown at least two mechanisms of β-cell formation: neoplastic and mature β-cell replication from ductal precursor cells. Replication of differentiated β cells is maintained after birth until adulthood. Beta cell replication is accelerated, for example, by increased demand for insulin as a result of high glucose infusion, partial pancreatectomy, and during pregnancy. Under these conditions, the β-cell mass grows rapidly through both cell hypertrophy (expansion of the volume of individual cells) and hyperplasia (increase in the number of β cells).
[0007]
In type I, insulin-dependent diabetes mellitus (IDDM), there is a clear decrease in the number of β cells due to an autoimmune attack on the β cells. Eisenbarth, N .; Eng. J. Med. 314: 1360-1368 (1986). Treatment for Type I diabetes may include increasing the number of β-cells in a subject suffering from Type I diabetes. Bonner-Weir, Endocrin. 141: 1926-1929 (2000).
[0008]
Another treatment for diabetes using islet cells involves transplanting pancreatic tissue from an immunocompatible donor into a transplant recipient. Typically, transplant recipients are required to prevent rejection of transplanted organs receiving immunosuppressive drug treatment. A recently developed immunosuppressant regimen has improved the outcome of clinical islet transplantation in humans. Although this technique remains experimental, this much simpler surgical procedure plays an important role in the treatment of diabetes if islet cell transplantation can perform the same function as whole organ pancreas transplantation.
[0009]
Although transplantation of human islets shows promise as a powerful treatment for diabetes, there are a number of obstacles that currently limit the usefulness of this procedure. One significant obstacle is the inability to produce enough islet cells for use in this procedure. This procedure, currently used to obtain islets for transplantation, typically involves the isolation of pancreatic tissue, enzymatic digestion of pancreatic tissue to release individual cells from surrounding cells, and the use of gradient centrifugation purification techniques including. Gradient centrifugation purification techniques are well known in the art and are performed by many islet transplant centers. Unfortunately, the yield of islets from a single pancreas treated using standard procedures is usually insufficient for transplantation. Therefore, alternatives to this procedure are sought and developed. Although the use of fetal or xenograft tissue has been developed, ethical concerns, availability of source materials, and concerns about immunorejection or xenobiotic pathogens complicate such approaches.
[0010]
To date, the ability to isolate, culture and expand pancreatic cells for use in transplantation to treat pancreatic endocrine disease has remained elusive. Although islets and islet cells can be isolated from pancreatic tissue, this isolated material, if not properly stored, remains viable for only a short period of time and can only perform endocrine functions for only a short period of time. is there. Various approaches have been reported for isolating pancreatic stem cells and inducing differentiation in vitro (Peck et al., Ann Med 33: 186-192 (2001); Bonner-Weir et al., Proc Natl Acad Sci USA 14: 7999-8004 (2000); U.S. Patent Nos. 6,001,647; 5,928,942; 5,888,916; PCT publications WO 00/78929 and WO 00/47721. That). However, the previous methodology was subject to some limitations. Expansion of the pancreatic cell population after isolation generally required a period of growth in serum-containing media (see, for example, US Pat. No. 5,888,916). This raises cost and safety issues. Furthermore, while satisfactory cell growth is achieved by such methods, the resulting cell population may not retain markers of pancreatic cell differentiation, nor may retain the ability to produce insulin, and Often, they cannot consistently differentiate into viable and high hormone producing cells.
[0011]
Serum-free selective media (see Stephan et al., Endocrinology 140: 5841-54 (1999)) that can promote the growth of epithelial cell populations compared to less desirable cell types, Offer the possibility to overcome. Serum-free culture conditions have been reported for the culture of pancreatic cells isolated from adult tissue. See Bonner-Weir et al., Supra; WO 00/78929. However, these procedures are not entirely satisfactory. The period of culture in serum-containing media, which requires special culture media, is still essential, and the transfer of cells to serum-free media for differentiation renders them incapable of growing. What is needed is a general method of isolating and culturing pancreatic cell material that consistently yields cells that can grow in vitro while retaining the potential to produce pancreatic hormones. Such cell populations can reverse the diabetic state after transplantation, and can serve as a source for pancreatic endocrine hormones in vitro and provide a model system for studying pancreatic development and disease. The present invention fulfills these and other needs.
[0012]
(Summary of the Invention)
The present invention provides methods and compositions for culturing pancreatic cells in vitro. In one aspect, the invention provides a method of preparing a cell culture of growing pancreatic cells, comprising the steps of isolating growing pancreatic cells, growth hormone and serum having less than 1% of total volume. Transplanting the cells into an epithelial selective culture medium comprising: and culturing the cells through at least one passage in culture. This method yields a cell population that can be expanded from about 180 cells per square centimeter to about 1,800 cells per square centimeter, which is characterized by: at least 90% of the cells have the transcription factor PDX-1 Is positive for / IPF-1 and the insulin: actin mRNA ratio of this population is between 1: 100 and 1000: 1. In certain embodiments of the invention, at least 95%, 98%, 99% or 100% of the cells stain positive for PDX-1. In one embodiment of the invention, the cells may be expanded from about 90 cells per square centimeter, and may be expanded to about 36,000 cells per square centimeter. In another embodiment, the insulin: actin mRNA ratio is between 1:10 and 100: 1. In some embodiments of the invention, the insulin: actin mRNA ratio is an unstimulated level of insulin mRNA.
[0013]
In one embodiment of the invention, before transferring the cells to a culture medium containing less than 1% serum, the cells are maintained in a medium containing serum between 1% and 4% of the volume of the medium. Is done. In another embodiment, the cells are maintained in a medium containing at least 4% serum by volume. In some embodiments, the maintenance period is less than 24 hours, while in other embodiments, the maintenance period is many days, such as any number of days between 1 and 14 days. It is. In some embodiments of the present invention, transplantation of cells from a medium containing more than 1% serum or more than 4% serum continuously transfers the cells to a medium containing less and less serum. It is done gradually while transplanting. In other embodiments, transplantation of cells from a medium containing more than 1% serum or more than 4% serum is achieved in one step by one medium change or several passages Is done. In one embodiment of the invention, the isolated pancreatic cells have a mixture of a PDX-1 positive and a PDX-1 negative phenotype and are cultured in a medium containing less than 1% serum. Proliferation selectively expands PDX-1 positive cells.
[0014]
In another aspect, the invention provides a method of maturing pancreatic cells to more differentiated cells that express high levels of endocrine hormones. The method comprises the steps of culturing pancreatic cells on a substrate to acclimate the substrate, removing the cells from the substrate and replating pancreatic cells on the substrate, including the inner cell mass. Producing an aggregate of pancreatic cells comprising an encapsulating coat of cytokeratin-19 positive cells, wherein the aggregate comprises 50-5000 cells and comprises between 50 and 300 microns Having a diameter. In one embodiment, at least one cell of the inner cell mass stains positive for a marker of endocrine development selected from the group consisting of PDX-1, insulin, glucagon, somatostatin and KS1 / 4. In other embodiments, a higher percentage of the inner cell mass (eg, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells). , Stains positive for markers of endocrine development. In one embodiment, at least one culturing step to produce aggregates is performed in a medium containing growth hormone and less than 1% of serum by volume. In another embodiment, the starting culture of pancreatic cells is at least 90% PDX-1 positive and has an insulin: actin mRNA ratio of between 1: 100 and 1000: 1.
[0015]
In another aspect, the invention provides a method for providing a mammal with pancreatic endocrine function, comprising the steps of culturing pancreatic cells on a substrate, removing the cells from the substrate. Reseeding pancreatic cells on the substrate, forming the cells into an aggregate having an encapsulating coat of ck-19-positive cells and an inner cell mass, and transplanting the aggregate into a mammal. And providing a pancreatic endocrine function.
[0016]
In yet another aspect, the invention retains the property that at least 90% of the cells stain positive for PDX-1 and that the population has an insulin: actin mRNA ratio between 1: 100 and 1000: 1. While growing a culture of growing pancreatic cells having the ability to grow from a first culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter to about 1,800 cells per square centimeter. provide.
[0017]
In another aspect, the present invention provides an aggregate of cultured pancreatic cells, wherein the aggregate comprises a surrounding capsule and an inner cell mass of ck-19 positive cells, wherein the aggregate comprises 50-5000. Pancreatic cells and has a diameter between about 50 and 300 microns.
[0018]
(Definition)
“Aggregate” in the context of cells refers to a three-dimensional structure.
[0019]
"CK-19" is cytokeratin 19, a 40 Kd acidic keratin.
[0020]
"Insulin: actin mRNA ratio" is measured by band density using a gel scanner or by real-time PCR using different labels for insulin and actin (see Example 3). This is the average across the population of cells.
[0021]
"Transplanting" is the implantation or placement of cells into a recipient. This includes encapsulated cells and non-encapsulated cells, for example, in an alginate matrix. The cells can be placed subcutaneously, intramuscularly, in the portal vein, or intraperitoneally by methods known in the art.
[0022]
`` Passage '' of cells that grow as a monolayer attached to the surface is usually the transition of a seeded culture vessel from a partially confluent state to a confluent state, at which point the cells are removed from the culture vessel. Refers to a transfer that is removed and reseeded into the culture vessel at a lower density. However, cells can be passaged before reaching confluence. Passaging typically results in an expansion of the cell population as the cells grow to confluence. The expansion of the cell population depends on the initial seeding density, but is typically a 1-10, 1-5, 1-3 or 1-2 fold expansion. Thus, passaging generally requires that the cells be able to divide more than once in culture.
[0023]
A "population" of cells refers to a plurality of cells obtained by a particular isolation or culture procedure. Although the selection process of the present invention results in a population having relatively homogeneous properties, the population of cells can be heterogeneous when assayed for marker expression or other phenotype. The characteristics of a cell population are generally determined by the percentage of individual cells having a particular characteristic (eg, the percentage of cells that stain positive for a particular marker) or the bulk average of the characteristic as measured for the entire population (eg, from the cell population). Amount of mRNA in the lysate generated).
[0024]
"90% PDX-1 positive" refers to statistical sampling of randomly selected cells. Standard immunochemical techniques are used, and positively stained cells are visually counted under a microscope. Percentages are determined by comparison to properly controlled samples (ie, preparing identical cells and using antibodies of similar isotype but not specific for PDX-1).
[0025]
"Serum" refers to material obtained from blood other than blood cells. Serum is typically obtained by coagulating blood cells or physically separating blood cells by centrifugation and fibrin removal. As used herein, serum can be functionally defined by its biological activity: serum generally supports the growth of mammalian cells in culture when added to a culture medium. Serum can be obtained from various species (eg, humans, cows, sheep, horses, pigs, rabbits, chickens, etc.) and from various stages of development (eg, fetal, young or adult). In certain embodiments, “serum” also refers to fractionated serum or a serum replacement or replacement product obtained from another source (eg, Select Soytone (Becton Dickinson)) or other commercially available product Say. Such serum equivalents can be completely or partially defined.
[0026]
"Mantle" refers to the membrane of cells that surrounds the inner cell mass in three dimensions.
[0027]
(Detailed description)
The present invention relates to the finding that there is an intermediate differentiation stage pancreatic stem cell that can be grown in a stable manner in successive serial passages while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture and can be induced into a late developmental stage, ie, prototypical islet cells. In the past, attempts to isolate and culture pancreatic prototypic cells for use in artificial insulin-producing organs have failed. This is because late stage pancreatic cells are not cultured well, and overgrowth of early stage cells results in a cell culture that does not produce insulin.
[0028]
These earlier methods of culturing pancreatic prototypic cells have partially failed. This is because these culture conditions did not select cells at the appropriate differentiation stage. It is a finding of the present invention that early stage prototypic cells from pancreatic tissue overgrow in culture media containing high concentrations of serum. By culturing pancreatic tissue in a medium that selects for the growth of epithelial cells, an advantageous subpopulation of intermediately differentiated cells that can be passaged in culture but retain the ability to secrete endocrine hormones is selected. An example of such an epithelial selective medium is a low serum medium or a serum-free medium, and especially a low serum medium containing a source of growth hormone (GH).
[0029]
The addition of growth hormone to serum-free, epithelial selective media has at least two advantages. First, it enhances the growth rate of the desired intermediate cells, and more importantly, it increases the glucose-stimulated response of the cell culture.
[0030]
(I. Phenotypic assay)
In practicing the methods of the present invention, it is useful to assay the phenotype of pancreatic cells at a particular stage of culture. Since the expression of a particular protein correlates with the identity or differentiation status of the cell, the cells can be analyzed for the expression of the marker gene or marker protein to assess their identity or differentiation status. For example, in freshly isolated pancreatic tissue, expression of amylase identifies the cells as exocrine acinar cells, while expression of insulin identifies the cells as endocrine islet cells. Similarly, islet cells at an early stage of differentiation are usually positive for cytokeratin ck-19, while mature islet cells show less expression of ck-19.
[0031]
Phenotypic characteristics can be assayed in a cell-by-cell manner or as a population average. The assay format depends on the specific requirements and methodology of the assay technology. Thus, assays for marker expression by immunohistochemistry performed on fixed sections or suspension cells by FACS analysis measure the frequency and intensity for a given marker expressed by individual cells. On the other hand, it may be desirable to measure a property such as the average of insulin to actin mRNA expression ratio for the entire cell population. In such cases, assays are typically performed by collecting mRNA from the cell pool and measuring the total amount of insulin and actin messages. Many phenotypic characteristics can be assayed based on either cells or populations. For example, insulin expression can be assayed either by staining individual cells for the presence of insulin in secretory granules, or by lysing the cell pool and assaying for total insulin protein. Similarly, mRNA levels can be measured on a cell population by lysing the cells and collecting the mRNA, or on an individual cell basis by in situ hybridization.
[0032]
(Cell differentiation marker)
There are a number of cell markers that can be used to identify a pancreatic cell population. Isolated and cultured donor cells begin to display various phenotypic and genotypic indicators of differentiated pancreatic cells. Changes in these indicators or markers, whether after the initial growth phase or immediately after isolation and purification, are believed to be a response to the shift of pancreatic cells to a serum-free environment. Examples of phenotypic and genotypic indicators include various molecular markers present in a regulated (eg, either up-regulated or down-regulated) conditional progenitor cell population. Can be These molecular markers include ck-19, which is postulated to be a marker for pancreatic conditional stem cells.
[0033]
Typically, mammalian stem cells undergo a number of stages of development as they mature to their final developmental endpoint. The stage of development can often be determined by identifying markers that are present or absent from the developing cell. Because human endocrine cells develop in a similar manner, the methods disclosed herein provide various markers for identifying cells as they transition from a stem cell-like phenotype to a pseudo-islet phenotype. Intended for use.
[0034]
Expression of markers induced to proliferate or differentiate by the methods of the invention retains some similarity to the order of marker expression in normal human pancreas development. Very early in development, primordial epithelial cells express PDX-1, an early cell marker that is a homeodomain nuclear factor. As the cells develop, they begin to sprout and form tubes. These cells express cytokeratin 19, a marker for epithelial duct cells, and transiently express PDX-1 to become developmentally endocrine cells. As these cells develop, they acquire the ability to express insulin, somatostatin or glucagon. The final differentiated cells can only express one and become α cells (glucagon), β cells (insulin) and δ cells (somatostatin). The intermediate cell population of the present invention is considered to be at an underdeveloped stage of development and retains the ability to proliferate and the potential to differentiate into mature endocrine cells. Regardless of whether the cells are actual examples of precursors in the developmental pathway or are simply the result of in vitro manipulation, the intermediate stage cells obtained by the methods of the present invention can proliferate, and It can express endocrine hormones and therefore has the potential to be used to correct deficiencies in any type of islet cells.
[0035]
Markers of interest are molecules expressed in the pancreas in a temporal and tissue-specific pattern (see Hollingsworth, Ann NY Acad Sci 880: 38-49 (1999)). These molecular markers fall into three general categories: transcription factors, notch pathway markers, and intermediate filament markers. Examples of transcription factor markers include PDX-1, NeuroD, Nkx-6.1, Isl-1, Pax-6, Pax-4, Ngn-3 and HES-1. Examples of notch markers include Notch1, Notch2, Notch3, Notch4, Jagged1, Jagged2, Dll1, and RBPjk. Examples of intermediate filament markers include ck-19 and Nestin.
[0036]
Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art and include: quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots and in situ hybridization (see, eg, Current Protocols in Molecular Biology (ed. Ausubel et al., Eds. 2001) and immunoassays (eg, immunohistochemical analysis of sectioned material), Western blotting, and For markers accessible in intact cells, see Flow Cytometry Analysis (FACS) (eg, Harlow and Lane, Using Antibodies: A Laboratory). Manual, New York: Refer to the Cold Spring Harbor Laboratory Press (1998)). Conventional histochemical markers of endocrine cell differentiation can also be used. Cells to be examined by immunohistochemistry can be cultured on glass chamber slides for microscopy. Alternatively, cells grown in conventional tissue culture can be manually removed from the culture and embedded in paraffin for sectioning. The PDX-1 antibody is described in Leonard J. et al. Et al., Mol. Endocrinol. , Oct. 7, 1993, (10) 1275-83.
[0037]
Cell differentiation markers vary and can be detected by conventional immunohistochemistry. The commonly applicable protocols follow.
[0038]
The staining process begins by removing the chamber portion of the slide. Cells were rinsed very gently in buffer and fixed in paraformaldehyde solution. The cells are then incubated at room temperature in a blocking solution containing normal serum. Cells were permeabilized with a non-ionic detergent in a blocking solution. Primary antibodies, as listed below, are prepared in blocking solutions at appropriate dilutions, added to the cells, and incubated. Following incubation with the primary antibody, cells were rinsed in buffer and blocked again in blocking solution.
[0039]
The secondary antibody, prepared in the blocking solution at the appropriate dilution, is added to the cells and incubated in the dark. After incubation, the cells were rinsed and nuclei were counterstained with Hoechst dye. Excess fluid is removed and the slide is mounted and covered with a cover slide. Slides are dried and stored in the dark.
[0040]
Alternatively, the cells can be prepared using the ABC method for immunocytochemistry. Briefly, the cells are embedded in paraffin and slides with paraffin sections are dried at 37 ° C. overnight. The cells are deparaffinized and immersed in a methanolic hydrogen peroxide solution to inhibit endogenous peroxidase activity. Slides are boiled in 0.01 citrate buffer (pH 6.0) for 30 minutes to recover specific epitopes. Slides were rinsed with buffer and blocked with normal serum at room temperature in a humid chamber.
[0041]
Primary antibodies prepared in blocking solution (Table A lists commonly used primary and secondary antibodies) are added to the sample and incubated in a humid chamber. The slide is washed and incubated with the secondary antibody prepared in a blocking solution. The slides were rinsed again with buffer and incubated with avidin-horseradish peroxidase reagent or ABC conjugate from a commercial kit (eg, Dako Corporation). The slides are rinsed again and incubated in a gold wrap with a diaminobenzidine color developing solution; urea hydrogen peroxide. After washing with distilled water, the slides are immersed in Mayer's Hematoxylin for 5 minutes, then the slides are kept in flowing tap water until the water becomes colorless and the nuclei turn blue. Slides are dehydrated and mounted for observation (Table A lists commonly used primary and secondary antibodies).
[0042]
[Table 4]

(Insulin mRNA expression)
One marker that can be used to characterize the identity, differentiation or maturity of pancreatic cells is the level of insulin mRNA. For example, an intermediate cell population of the present invention exhibits expression of insulin mRNA within a defined range. Methods for quantifying insulin mRNA include Northern blot, nuclease protection and primer extension. In one embodiment, RNA is extracted from a population of cultured cells and the amount of proinsulin message is measured by quantitative reverse transcription PCR. After reverse transcription, the insulin cDNA is specifically and quantitatively amplified from the sample using primers that hybridize to the insulin cDNA sequence and amplification conditions where the amount of amplification product is related to the amount of mRNA present in the sample. (See, for example, Zhou et al., J Biol Chem 272: 25648-51 (1997)). The kinetic quantification procedure is preferred due to the accuracy with which the starting mRNA levels can be determined.
[0043]
Frequently, the amount of insulin mRNA is normalized to mRNA that is constitutively expressed (eg, actin), and actin is specifically amplified from the same RNA sample using actin-specific primers. Thus, the expression level of insulin mRNA can be reported as the ratio of insulin mRNA amplification product to actin mRNA amplification product, or simply as the insulin: actin mRNA ratio. Expression of mRNA encoding other pancreatic hormones such as somatostatin or glucagon can be quantified by the same method.
[0044]
(Glucose stimulated insulin secretion)
One of the important functions of beta cells is to regulate their insulin secretion according to glucose levels. Typically, a static glucose stimulation (SGS) assay can be performed on growing adherent pancreatic cells to identify whether these cells can secrete insulin in response to different glucose levels. Cells are generally cultured on a suitable substrate until near confluency. Three days before the SGS test, the culture medium is replaced by a medium with similar characteristics but lacking insulin and containing only 1 g / L glucose. The medium is changed daily for three days, and the SGS test is performed on day four.
[0045]
Prior to this test, the culture medium may be collected for glucose and insulin analysis. To prepare cells for this test, cells are washed twice with Dulbecco's phosphate buffered saline (DPBS) + 0.5% BSA (incubated for 5 minutes for each wash), then Wash once with DPBS alone (also incubated for 5 minutes). After washing, the cells were incubated with 10 ml (in a 100 mm dish) or 5 ml (in a 60 mm dish) of Krebs-Ringers SGS solution (KRB-60) containing 60 mg / dl glucose for 30 minutes in a 37 ° C. incubator. Incubate over This incubation is then repeated.
[0046]
To perform the SGS assay, cells are incubated in 3 ml (100 mm dish) or 4 ml (T75 flask) or 2 ml (60 mm dish) KRB-60 for 20 minutes at 37 ° C. The medium is aspirated and spun and collected as an LG-1 (low glucose stimulation step) for the insulin assay. Then, KRB-450 + theo (KRB with 450 mg / dl glucose and 10 mM theophylline) is added in the same volume as above, and the cells are cultured under the same conditions as above. The supernatant is collected for insulin assay as HG (high glucose stimulation). The cells are then re-incubated with KRB-60, and the medium is collected as LG-2 and the next time as LG-3. This medium is collected for insulin analysis and stored at -20 <0> C until the insulin content is determined by radioimmunoassay (RIA) or other suitable assay.
[0047]
The results of the SGS test are often expressed as a stimulation index, defined as the HG insulin value divided by the LG-1 insulin value. Generally, a stimulation index of about 2 or greater is considered a positive result in the SGS assay, but is identified using other values (eg, 1.5, 2.5, 3.0, 3.5, etc.). May be defined.
[0048]
In controlled experiments, bovine pituitary extract (BPE) and recombinant human growth hormone were found to maintain desired levels of insulin release from cells of intermediate differentiation.
[0049]
(II. Method for isolating pancreatic cells)
The invention provides, inter alia, a method of producing a culture of pancreatic cells that can undergo continuous replication but can be induced to differentiate into more mature cells suitable for therapeutic purposes. Thus, some methods of the invention require, as an initial step, the isolation of cells from the pancreas. Cells collected from the pancreas are a diverse population that can give rise to differentiated cells capable of endocrine and exocrine secretion. These differentiated cells express pancreatic endocrine molecules (eg, insulin, somatostatin, glucagon and other endocrine hormones) and pancreatic exocrine molecules (eg, amylase). In addition, a portion of the cultured cell population can replicate and expand in culture. Intermediate cell populations of the present invention may result, in whole or in part, from conditional stem cell differentiation, dedifferentiation of mature endocrine cells, or differentiation transformation of other pancreatic cell populations. The culture methods described below utilize various extraction and culture conditions to produce various populations of pancreatic cells.
[0050]
(Donor source)
The donor source can be one or more donor pancreas from cultured pancreatic cells, or other source that can produce cells capable of producing pancreatic endocrine hormones and pancreatic exocrine hormones. In a preferred embodiment, cells isolated for subsequent culture are obtained from one or more donated pancreas. The methods described herein do not depend on the age of the donated pancreas. Thus, pancreatic material isolated from donors of adult to adult age can be used.
[0051]
In another embodiment, the pancreatic cells are isolated from a cultured source. For example, cells prepared according to the microencapsulation method of U.S. Patent No. 5,762,959 to Soon-Shing et al., Entitled "Microencapsulation of cells," can be collected as a source of donor cells.
[0052]
Starting materials for the production of the intermediate cell population also include a clonal population of pancreatic cells produced by the methods disclosed herein. Thus, in one embodiment of the present invention, the isolation, isolation and culture methods disclosed herein are used to generate an intermediate population derived from a single cloned pancreatic cell. , Associated with the cloning procedure. Such a procedure is particularly suitable for in vitro genetic manipulation of an intermediate cell population.
[0053]
(Isolation of pancreatic cell population)
Once the pancreas has been collected from a donor, it is typically processed using a variety of methods to yield individual cells or small groups of cells for culture. One such method requires that the recovered pancreatic tissue be cleaned and prepared for enzymatic digestion. Enzymatic treatment is used to digest connective tissue such that the recovered parenchyma is dissociated into smaller units of pancreatic cell material. The collected pancreatic tissue is treated with one or more enzymes to separate pancreatic cell material, substructures and individual pancreatic cells from the overall structure of the collected organ. Collagenase, DNAse, Liberase preparations (see U.S. Patent Nos. 5,830,741 and 5,753,485) and other enzymes are useful for use in the methods disclosed herein. Is intended.
[0054]
The isolated source material can be further processed to enrich for one or more desired cell populations. However, unfractionated pancreatic tissue, once dissociated for culture, can also be used directly in the culture methods of the present invention without further separation and yield an intermediate cell population. In one embodiment, the isolated pancreatic cell material is purified by centrifugation through a density gradient (eg, Nycodenz, Ficoll or Percoll). For example, the gradient method described in US Pat. No. 5,739,033 can be used as a means to enrich for islets for processed pancreatic material. The mixture of cells collected from the donor source is typically heterogeneous and thus contains cells, cells, delta cells, duct cells, acinar cells, conditional progenitor cells and other pancreatic cell types.
[0055]
Typical purification procedures result in the purification of the isolated cellular material into multiple layers or interfaces. Typically, two interfaces are formed. The upper interface is rich in islets and typically contains 10-100% islet cells in suspension. The second interface is typically a mixed population of cells, including islet cells, acinar cells, and duct cells. The bottom layer is a pellet, and the pellet is formed at the bottom of the gradient. This layer typically contains predominantly (> 80%) acinar cells, some captured islets and some duct cells. The tube tree components may be collected separately for further manipulation.
[0056]
The cellular concentration of the fractions selected for further manipulation will vary depending on which gradient fractions are selected and the end result of each isolation. If the islet cells are of the desired cell type, the islet cell population suitably enriched in the isolated fraction contains at least 10% to 100% islet cells. Other pancreatic cell types and concentrations can also be collected after enrichment. For example, the culture methods described herein can be used on cells isolated from a second interface, pellet, or other fraction, depending on the purification gradient used.
[0057]
In one embodiment, the intermediate pancreatic cell culture is made from the islet-enriched (top) fraction. However, it further typically comprises, respectively, a mixed cell population of islet cells, acinar cells and duct cells, or a mixed cell population of ductal tree components, acinar cells and some captured cells, A more heterogeneous second interface and bottom layer fraction can also be used in the culture. Although both layers contain cells that can give rise to the intermediate populations described herein, each layer can have certain advantages for use with the disclosed methods. For example, using an islet-enriched upper fraction may have certain advantages. This islet-enriched fraction may contain more cells at a more advanced differentiation stage and, therefore, may be more effective in shock methodology, which selects for an existing interstage population Can be activated by Alternatively, the more heterogeneous second and third fractions may also have advantages. The second (lower) interface may include more cells at an earlier stage of endocrine development, and may therefore be more efficient in creating an immediate population by withdrawal methodology. It is theorized that this methodology promotes the development of intermediate stage cells from less differentiated precursors. However, the method of the present invention is not bound by these theories, and both the upper and lower interface fractions produce acceptable results for both shock and release methodology.
[0058]
III. Maintenance and growth of pancreatic cells in high serum medium
(A. General cell culture procedure)
Once the pancreatic cells are obtained and isolated, they are cultured under conditions that select for the expansion of the desired intermediate stage population, or, in another embodiment, the differentiation of more mature cell types. General cell culture methodologies can be found in Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th edition, John Wiley & Sons (2000). Typically, pancreatic cells are grown under conditions appropriate for other mammalian cells, for example, at 37 ° C. in a humidified incubator at 5% CO 2. ₂ Cultured under an atmosphere of Cells can be prepared from various substrates known in the art (eg, borosilicate glass tubes, bottles, dishes, cloning rings, plastic tissue culture tubes, dishes, flasks, multiwell plates with a negative surface charge). , Vessels with increased growth surface area (GSA) or Esophageal Doppler Monitor (EDM) finish, flasks with multiple inner sheets to increase GSA, Fenwal bags and other culture vessels) .
[0059]
Once the pancreatic cell material is collected and selected for culture, or once the population is confluent and transplanted to a new substrate, the population of cells is seeded into a suitable tissue culture vessel for culture. Is done. Seeding density can have an effect on the viability of pancreatic cells cultured using the disclosed methods, and the optimal seeding density for a particular culture condition is to seed the cells in a range of different densities, and It can be determined empirically by monitoring the viability and growth rate of the resulting cells. A range of seeding densities has been shown to be effective in producing hormone-secreting cells in culture. Typically, the cell concentration is about 10 per 100 mm culture dish. ² Cells to about 10 ⁸ Cells, for example, 10 per 100 mm culture dish ² Cells, 10 ³ Cells, 10 ⁴ Cells, 10 ⁵ Cells, 10 ⁶ Cells, 10 ⁷ Cells or 10 ⁸ Although lower in cell range, lower cell concentrations can be used for cloning procedures. Cell concentrations for other culture vessels can be adjusted by calculating relative substrate surface area and / or medium gas exchange surface area for various culture vessels. For example, a typical 100 mm culture dish has a substrate surface area of 55 square centimeters (Freshney, supra) and a cell concentration of 10,000 cells per dish is approximately 180 cells per square centimeter. A cell concentration of 100,000 cells per dish corresponds to about 1,800 cells per square centimeter. The cell concentration for the surface area of the culture vessel is determined by the appropriate medium volume per culture surface area (0.2 ml / cm ² ~ 0.5ml / cm ² Are typical ranges for static cultures) can be linked to the cell concentration with respect to the medium volume. To determine if a 10-fold increase has occurred, cells are removed by enzymatic digestion and counted under a microscope in a known volume of fluid. Cells can also be grown on culture surfaces pre-coated with defined extracellular matrix components that promote growth and differentiation, such as fibronectin, collagen I, Engelbreth-Holm-Swarm matrix and preferably collagen IV or laminin.
[0060]
Standard cell culture propagation techniques are suitable for practicing the present invention. If the cells grow by attaching to the culture surface, they typically grow as a monolayer until 80% -90% confluence is achieved, at which point the cells are brought to the surface by proteolytic digestion. And split 1: 2 or 1: 3 for cultivation in new vessels. Higher dilutions of the cells (generally in the range of 1: 4 to 1:10) are also appropriate, but lower cell concentrations are appropriate in the cloning procedure. Proteolytic enzyme and chelator concentrations are usually lower when cells are passaged in serum-free medium (eg, 0.025% trypsin and 0.53 mM EDTA). The culture medium is typically changed weekly or when the pH of the medium indicates that fresh medium is needed.
[0061]
The pancreatic cells of the present invention can be cultured in various media. As described herein, media containing or lacking certain components (especially serum) are preferred at certain stages of the isolation and growth procedures. For example, freshly isolated cells from the pancreas may be maintained in a high serum medium to allow the cells to recover from the isolation procedure. Conversely, a low serum medium favors the selection and expansion of the interstage population described herein. Finally, maturation of the intermediate stage cells uses a particular medium. Accordingly, a number of media formulations are useful in the practice of the present invention. The media formulations disclosed herein are for illustrative purposes, and non-essential components of the media may be modified using the assays described herein to replicate or differentiate cell populations. By assaying for the effects, they can simply be omitted, replaced, modified or added. See, for example, Stephan et al., Endocrinology 140: 5841-54 (1999).
[0062]
The culture medium usually comprises a basal medium, which comprises inorganic salts, buffers, amino acids, vitamins, energy sources and, in some cases, organic intermediates involved in protein, nucleic acid, carbohydrate or lipid metabolism and Contains additional nutrients in precursor form. Examples of the basic medium include F12, Eagle's MEM, Dulbecco's modified MEM (DMEM), RPMI 1640, and a 1: 1 mixture of F12 and DMEM. See Freshney, supra. To support cell growth, the basal medium is usually supplemented with sources of growth factors, other proteins, hormones and trace elements. These supplements promote cell growth, maintenance and / or differentiation, counteract impurities or toxins in other media components, and provide the missing trace elements in the basal media. In many culture media, serum is the source of these supplements. Serum can be replenished from a variety of mammalian sources, such as humans, cows, sheep, horses, and the like, and from adult, young or fetal sources. See Freshney, supra. Fetal calf serum is a commonly used supplement. The concentration of serum is expressed in terms of serum volume as a percentage of the total media solution, and is typically from about 0.1% to about 25%, for example, about 0.1%, about 0.2%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about It ranges from 20% or about 25%. In some applications, the basal medium is supplemented with a defined or semi-defined mixture of growth factors, hormones and trace elements, rather than serum. Formulations for serum replacement supplements are disclosed herein; others are known in the art or are available from commercial sources (see Freshney, supra). . For some embodiments, the concentration of serum is reduced but not removed, and a defined or semi-defined supplement mixture is added to the defined medium. Preferred applications for media containing high or low concentrations of serum are described herein.
[0063]
(B. Maintenance and growth of isolated cells in high serum)
Cells collected from the donor pancreas typically undergo a period of warm or cold ischemia between the death of the donor and the start of the isolation procedure. Furthermore, during the isolation procedure, pancreatic cells are usually subjected to proteolytic digestion and mechanical and shear stress. While not wishing to be bound by any particular theory, the various traumas experienced by these cells can up-regulate various cellular processes that result in an expansion of the pancreatic stem cell population (eg, conditional progenitor cells) . Intermediate cell populations can be produced with satisfactory efficiency by placing the cells directly in low serum medium after isolation or purification. Nevertheless, freshly isolated cells are treated with high concentrations of serum since trauma experienced by the cells during the isolation procedure can have a detrimental effect on cell survival and adaptation to culture. It is sometimes desirable to maintain in a stabilized medium containing (eg,> 4%) to improve the efficiency of the culture process. This maintenance period can be short (eg, overnight). If desired, the cells can be maintained in a high serum medium for an extended period of growth.
[0064]
High serum media for stabilization typically includes at least 4% serum, and in some embodiments, includes higher concentrations of serum (eg, 10% or 20%). Media used for stabilization or growth are available from a number of commercial sources and are described in Moore et al., J. Am Med Assoc 199: 519-524 (1967), including basal media (eg, RPMI). 1640). An exemplary high serum medium for maintenance or growth includes medium 3 (RPMI 1640 + 10 mM HEPES, 2 mM glutamine, 5 μM ZnSO ₄ And 10% fetal bovine serum (FBS)) and medium 7 (RPMI 1640 + 10 mM HEPES, 2 mM glutamine, 5 μM ZnSO ₄ And 20% FBS). A high serum medium also includes a specific volume of high serum medium (eg, Medium 3 or Medium 7) with a specific volume of serum-free medium (eg, SM95, SM96 or SM98 (described herein)). It can be induced by mixing to reach the desired serum concentration (eg, 4-9%).
[0065]
For post-harvest stabilization, cells are grown in culture vessels in high serum medium at a relatively high density (eg, 70 ml of Medium 7 (20% FBS) for 10 days). ⁹ Cells). However, lower cell densities and serum concentrations can also be used. Cells are typically maintained in their original containers for a relatively short period of time (eg, overnight) to allow recovery from the collection procedure.
[0066]
After a maintenance period, the cells can be transplanted into low serum medium for selection and expansion of an intermediate cell population, as described herein. If necessary, the cells can be cultured in a high serum medium to allow expansion of the mixed cell population. In an exemplary embodiment, the cells from the maintenance culture are in a new culture vessel containing Medium 3 (10% FBS), Medium 7 (20% FBS) or a mixture of Medium 3 and Medium 7 (15% FBS). Is re-sown. Cells are typically cultured in this medium for 7-10 days, during which they can grow to confluence. Once the cells reach confluence, they can be passaged into low serum medium for selective expansion of the intermediate cell population described herein.
[0067]
(IV. Preferential expansion of pancreatic stem cells at intermediate differentiation stages by culture in epithelial cell selective medium)
Once the pancreatic cells have been isolated, the cells are transplanted into a selective medium to facilitate the appearance of a growing interstage population. This selective medium favors the growth of cells that retain the ability to secrete pancreatic endocrine hormones or that retain the potential to mature into more differentiated cells that secrete high levels of pancreatic endocrine hormones. is there. In general, selective media favors the growth of epithelial cells or epithelial-like cells at the expense of fibroblasts and mesenchymal cells, while pure epithelial cultures favor the pancreatic cells in the methods of the invention. It is not shown to be required for use. Typically, epithelial selective media are grown after a period of growth in culture (eg, 2, 3, 4, or 5 passages, depending on the population growth at each passage). Produces a population of cells that are nearly pure (eg, <10% fibroblasts or mesenchymal cells).
[0068]
One type of selective medium that has been favorably used for epithelial cell growth from embryonic tissue is a serum-free medium (eg, Stephan et al., Supra; Peehl and Ham, In Vitro 16: 526-40 ( 1980)). An epithelial-specific medium, and more preferably a low-serum medium, containing a source of growth hormone, retains a marker of pancreatic cell development (eg, PDX-1) from an adult mammal, but under appropriate conditions. It is an observation of the present invention that it can be used to select a distinct population of proliferating pancreatic cells that can differentiate to express high levels of pancreatic endocrine hormones. Although certain epithelial selective media suitable for culturing pancreatic cells are disclosed herein, other media formulations known in the art that favor the preferential expansion of epithelial cells or epithelial-like cells may also be used. Can be
[0069]
Transplantation into epithelial-selective low-serum media involves transplanting cells directly into selective low-serum media after a period of maintenance in high-serum media ("weaning"), or following isolation and separation procedures. Things ("shock") can be achieved. Either methodology is suitable for generating the desired intermediate cell population, as shown in the examples herein.
[0070]
In controlled experiments using bovine pituitary extract [BPE] and recombinant human growth hormone, it was determined that pituitary extract provided a beneficial increase in growth rate compared to the absence of BPE. Was. Recombinant growth hormone was able to significantly restore the growth rate associated with BPE.
[0071]
(A. Preparation of selective low serum medium)
As used in this context, "low serum medium" refers to a medium having less than about 1% serum. Therefore, a serum-free medium is a kind of low serum medium. Serum between 0% and 1%, for example, about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0% The medium having a serum concentration of 0.8% or 0.9% can be obtained by adding an appropriate concentration of serum to the serum-free medium or by mixing the serum-free medium with the serum-containing medium to obtain the desired concentration. To achieve the desired serum.
[0072]
Complete serum-free medium is a mixture of growth factors, other proteins, hormones and trace elements in a basal medium (eg, SM96 or 1: 1 F12 / DMEM), which surrogate the biological functions provided by serum. ). The advantage of a serum-free medium is that the composition of the supplementation mixture prevents the growth of unwanted cells (eg, acinar cells or connective tissue cells (eg, fibroblasts)) while maintaining the desired cell population (eg, intermediate cells). Population) can be easily manipulated to promote growth. The replenishment mixture can also be manipulated to promote differentiation of the stem cell population into more mature cells, or to prevent differentiation of the stem cell population to maintain a high rate of growth.
[0073]
(1. growth hormone)
It is an observation of the present invention that low serum epithelial selective culture medium containing growth hormone (GH) promotes the emergence of a beneficial pancreatic cell population for intermediate differentiation. While not wishing to be bound by any particular theory, it is possible that GH can survive mitogenic substances normally found in serum that support cell growth, but that serum is less desirable in cell populations such as fibroblasts. And other mitogenic factors that promote hyperproliferation of mesenchymal cells). Therefore, replacement of serum with a supplementation mixture containing GH selects for expansion of an intermediately differentiated cell population. Although the function of GH in serum-free media can be replaced by other supplementary components in alternative embodiments of the invention, the ready availability of GH in natural extracts or as a recombinant protein makes GH The containing medium will be a suitable epithelial selection medium for the methods disclosed herein.
[0074]
Growth hormone (also known as somatotropin) is a polypeptide hormone synthesized in the anterior pituitary gland, promoting normal body growth and lactation, and affecting various aspects of cellular metabolism. GH has both a direct effect on cells and an indirect effect mediated by IGF-I and similar molecules; in the intact pancreas, proliferation of islet cells depends on the expression of GH and the homologous hormones prolactin and lactogen. (See, e.g., Nielsen et al., J Mol Med 77 (1): 62-6 (1999)). In humans, mature GH contains 191 amino acid residues and exhibits a molecular weight of 22 kDa. However, in addition to the normally observed disulfide dimer, two peptides made from a portion of human GH (residues 1-43 and 44-191) have been detected in serum, and are compared to adult islet tissue. (See Lewis et al., Endocr J 47 Augmentation: S1-8 (2000)). Various naturally occurring derivatives, variants, metabolites and engineered derivatives of human GH are known, including glycosidated GH, methionyl GH, 20 kDa GH, acetylated GH, proteolytically cleaved GH, Deamido GH, sulfoxide GH and truncated forms of GH are included.
[0075]
GH is a member of a conserved family of hormones, which include GH-V1 and GH-V2 in humans, choliomanmotropins and prolactin, and other vertebrate-derived proteins (eg, rodent placenta). Sex lactogens I and II and other bovine and ovine lactogens, mouse proliferin I, II and III, and proliferin-related proteins, bovine prolactin-related proteins I, II and III, rat prolactin-like proteins A and B, and Including somatolactin from various fishes, members of this family are characterized by the following consensus sequence: Cx- [ST] -x (2)-[LIVMFY] -x- [LIVMSTA]- -X (5)-[TALIV] -x (7)-[LIVMFY] -x (6)-[LIVMFY] -x (2)-[STA] -W or C- [LIVMFY] -x (2)- D- [LIVMFYSTA] -x (5)-[LIVMFY] -x (2)-[LIVMFYT] -x (2) -C.
[0076]
Suitable growth hormones for the practice of the present invention can be obtained from a variety of natural and artificial sources. In contrast to the therapeutic uses of GH, which often require allogeneic GH, GH from a range of primate, mammalian or vertebrate species is useful in formulating low serum media for the culture of pancreatic cells. Can be used. A convenient source of growth hormone is bovine pituitary extract (BPE), which is a rich source of natural GH. BPE (75 μg / ml protein) is present in the culture medium at about 0.1 μl / ml to about 100 μl / ml, preferably 0.5 μl / ml to 50 μl / ml, and most preferably 5 μl / ml or 37.5 mg / ml. May be included. Pituitary extracts available from other species (eg, pigs, sheep, etc.) can also be used at similar concentrations. Other factors present in the pituitary extract may enhance its effect, but satisfactory results may also be achieved with purified GH and with recombinant GH. Recombinant bovine GH and recombinant human GH are widely available and are suitable sources of GH activity. Recombinant GH is present in the culture medium between 0.01 mg / l and 100 mg / l, preferably between 0.1 mg / l and 10 mg / l, more preferably about 0.2 mg / l, about 0.2 mg / l. Can be added at 5 mg / l, about 0.75 mg / l, about 1 mg / l, about 1.25 mg / l, about 2 mg / l or 5 mg / l, and most preferably about 1.25 mg / l, 1 mg of recombinant protein is approximately equivalent to 3 IU of GH.
[0077]
(2. Other supplements)
Representative components added to the basal medium for a complete serum-free medium include: recombinant human insulin (0.1 μg / ml-100 μg / ml), transferrin (0.1 μg / ml-100 μg) / Ml), epidermal growth factor (0.1 ng / ml to 100 ng / ml), ethanolamine (0.1 μg / ml to 100 μg / ml), aprotinin (0.1 μg / ml to 100 μg / ml), glucose (0. 1 mg / ml to 100 mg / ml), phosphoethanolamine (0.1 μM to 100 μM), triiodothyronine (0.1 pM to 100 pM), selenium (0.1 nM to 100 nM), hydrocortisone (0.01 μM to 100 μM), Progesterone (0.1 nM to 10 nM), forskolin (0.1 μM to 100 μM), heregulin (0.1 nM to 100 nM) and bovine pituitary extract (0.1 μg / ml to 500 μg / ml). Not all supplementary components are required to support cell growth; the optimal concentration or need for a particular supplement is to omit or reduce the concentration of a single component, and It can be determined empirically by observing the effect on growth (see Stephan et al., Supra).
[0078]
Generally, the supplemental components can be replaced by natural or synthetic products having the same biological properties. For example, triiodothyronine, hydrocortisone and progesterone can all be replaced by natural or synthetic hormones known to activate the same intracellular receptors (thyroid, glucocorticoid and progesterone receptors). Insulin and EGF are typically human proteins produced by recombinant DNA methodology, but by polypeptides purified from natural sources, by polypeptides from other species, or by insulin and EGF receptors. It can be replaced by another agonist. GH can be replaced in some cases by other antagonists of the GH receptor. Similarly, heregulin, a ligand for the ErbB3 receptor, is a component of heregulin isoforms and other ErbB3 agonists (eg, NRG2, NRG3 and NRG4, sensory and motor neuron-derived factors, neurestin and Ebp-1, heregulin α, heregulin β, heregulin γ, neuregulin-1 and neuregulin-2 (NRG-1α; NRG-1β, NRG-2α and NRG-2β).
[0079]
Exemplary serum-free media include basal medium SM96 and complete medium SM95, which consists of SM96 supplemented as shown in the table below. SM98 consists of 1: 1 F12 / DMEM supplemented with a modification of the medium supplement 14F described by Stephan et al., Supra. SM98 contains less than 14F heregulin (1 ng / ml vs. 8 ng / ml). Thus, SM98 consists of 1: 1 F12 / DMEM supplemented with: recombinant human insulin, 10 μg / ml; transferrin, 10 μg / ml; epidermal growth factor, 10 ng / ml; ethanolamine, 61 ng / ml. Aprotinin, 25 μg / ml; glucose, 5 mg / ml; phosphoethanolamine, 141 ng / ml; triiodothyronine, 3.365 pg / ml; selenium, 4.325 ng / ml; hydrocortisone, 181 ng / ml; Forskolin, 410 ng / ml; heregulin, 1 ng / ml; and bovine pituitary extract, 75 μg / ml. Exemplary sources of EGH and heregulin in SM95 and SM98 are recombinant human EGF (Sigma E9644) and the EGF domain of human heregulin-1 (amino acids 176-246) (R & D systems 396-HB / CF).
[0080]
[Table 5]

[0081]
[Table 6]

[0082]
[Table 7]

(B. Transplantation of cells into low serum medium)
As described herein, transplanting a culture of pancreatic cells into a low serum medium facilitates the selection of defined cell populations with an intermediate differentiation state. This cell population continues to grow when subcultured, but maintains high expression levels of pancreatic markers (eg, PDX-1). This unstimulated population secretes relatively low levels of pancreatic endocrine hormones, such as insulin, but can be matured according to the methods of the invention to obtain hypersecreting cells. To transplant a culture of pancreatic cells into low serum medium, cells can be detached from high serum medium to low serum medium, or can be placed directly into low serum medium after isolation. Media such as SM95 and SM98 are suitable low serum media, but SM95 results in slightly improved insulin secretion during pancreatic cell maturation.
[0083]
In the detachment procedure, cells are maintained in high serum medium for a period of time, and then are transplanted into low serum medium for selective expansion of the intermediate cell population. This maintenance period may be short (eg, overnight), as described herein above. Alternatively, cells can be grown in high serum medium before transplantation into low serum medium. Thus, in one embodiment, pancreatic cells are isolated, purified, and then plated in a culture vessel containing a medium with 10% added serum (eg, Medium 3). Cells are grown to near confluence and then passaged to low or serum-free media (eg, SM95 or SM98) for selection of intermediate populations. The resulting cell population, when examined by immunohistochemical staining, is greater than 90% PDX-1 positive and maintains an insulin: actin mRNA ratio between 1: 100 and 1000: 1.
[0084]
In another embodiment of the withdrawal procedure, the concentration of serum in the medium is replaced with successively decreasing concentrations of serum each time the cells are subcultured, or each time the medium is removed and replaced. By doing so, it is gradually reduced. For example, in one embodiment of this culture procedure, the isolated pancreatic cells are first maintained overnight in Medium 7 (20% FBS), then 7-10 in Medium 3 (10% FBS). Propagated for days. When near confluence, the cells are subcultured into new vessels with media containing 4% serum (60% SM95 and 40% media 3). In addition, after growth to near confluence, the cells are subcultured into new vessels with serum-free medium (100% SM95). The cells continue to grow and, upon subsequent passage, are subcultured in pure SM95 medium.
[0085]
The concentration of serum during each step of the withdrawal procedure is not critical; therefore, when cells are gradually withdrawn from growth in 10% serum to 0% serum, a continuous medium change is about 9%, 8%. %, 7%, 6%, 5%, 4%, 3%, 2% or 1% serum. A medium having such an intermediate concentration of serum can be prepared by mixing Medium 3 (10% FBS) with SM95 or SM98 (0% FBS) in a suitable ratio. Generally, when cells are passaged to new media or cells are given new media, some residual serum from the previous media may be carried over into the new media. Thus, the percentage of serum in the culture medium may not be exactly equal to the concentration of serum in the fresh medium added. However, in a typical cell culture protocol, only a small amount of medium is carried over and subsequently diluted by continuing to change the medium. In any case, the presence of residual serum carried over will not interfere with the method of the invention. If desired, the amount of serum in the culture medium can be determined by comparing serum-specific components (eg, immunoassays such as albumin, globulin, fetuin, etc.) between the medium known to have a certain percentage of serum and the culture medium in question. ) Can be determined by comparing the concentrations.
[0086]
Pancreatic cell cultures can also be transplanted into low serum medium in a single step without maintaining the cells in high serum medium. In this "shock" methodology, cells are usually plated immediately after the isolation procedure in culture vessels with low serum medium. In such embodiments, the first passage of the isolated pancreatic cells is performed in a medium with less than 1% serum (eg, SM95 or SM98).
[0087]
In an alternative embodiment of the shock methodology, the cells are between 1% and 4% serum, eg, about 1.33%, 1.5%, 1.66%, 2%, 2.33%, 2%. It is first passaged in medium containing 0.5%, 2.66%, 3%, 3.33%, 3.5% or 3.66% serum. If the cells have been purified by density gradient, they can generally be washed or recovered in a medium containing serum. If the cells are not washed in low serum medium, the culture medium for the first passage of cells will contain intermediate concentrations of serum from the isolation procedure. In an exemplary embodiment, cells from the density gradient interface are collected in one volume of Medium 7 (20% serum). To this volume, 5 volumes of serum-free medium (eg, SM95) is added to give a final serum concentration of 3.33%. The cells are then grown in this medium for one passage. Subsequent passages or media changes are performed using low serum media, such that cells are grown in media containing 1% or less serum and an intermediate cell population (ie, cells of intermediate differentiation) are selected. Will be done.
[0088]
Using either shock or withdrawal methodology, cultures of pancreatic cells in low serum medium continue to grow under appropriate culture conditions and express specific markers specific for pancreatic endocrine cells However, it promotes the emergence of an intermediate cell population that differs from the mature islet population. In one embodiment of the invention, this intermediate cell population is characterized by its high expression of the pancreatic endocrine marker PDX-1 (typically at least 90% of the cells stain positive for PDX-1 and stain for PDX-1) Tend to concentrate in the nucleus of cells) and by their insulin mRNA expression patterns. In this embodiment, the majority of cells in the intermediate population are positive for insulin expression, but the average level of insulin gene expression in this population is relatively low (typically, the insulin: actin mRNA ratio is 1: 1). Between 10 and 100: 1). In some embodiments, the population of intermediate cells produced by the shock methodology maintains a positive response to the glucose load and produces a stimulation index of 2 or more when assayed by the SGS test. In other embodiments, the intermediate population produced by the withdrawal methodology scores negatively in the SGS assay until further maturity.
[0089]
Intermediate cell populations produced by the methods of the invention typically retain both the ability to proliferate and to further differentiate into highly secretory endocrine cells. Proliferative capacity is generally assessed by the ability of a culture seeded at a first density to grow to a second density; for example, cells plated at 180 cells per square centimeter are used for one passage. Can grow to 1,800 cells per ml per liter. Through repeated cycles of proliferation and passage, the starting population of isolated pancreatic cells retains endocrine markers (eg, PDX-1 and insulin mRNA expression) and differentiates into mature, high secreting endocrine cells. While maintaining the ability, about 10,000 times or more (for example, about 100 times, 500 times, 1000 times, 5000 times, 10,000 times, 50,000 times, 100,000 times, 500,000 times or 1, (Million times).
[0090]
The cells of this intermediate stage population can be transplanted into a mammal for restoring or enhancing pancreatic endocrine function without further manipulation. The cells of the intermediate population can also be used as a model system for islet cell development and homeostasis (eg, drug screening, islet morphogenesis or autoimmune response). However, in one embodiment of the invention, cells from the intermediate stage population become more mature in culture to produce cells that secrete high levels of pancreatic endocrine hormones.
[0091]
V. Maturation of intermediate cells into high insulin producing aggregates
Under appropriate culture conditions, the intermediate stage pancreatic cells of the present invention mature into another type of cell that expresses high levels of pancreatic endocrine hormones. Typically, this process causes the cells to acquire some of the morphological and immunohistochemical features unique to mature islets in the pancreas, known as aggregates or pseudo islets To form). It is an observation of the present invention that substrates preconditioned by proliferation of pancreatic cells provide enhanced consistency of aggregate development and result in more authentic islet morphology.
[0092]
The preferred starting material for this procedure is a culture of intermediate stage cells produced by the method of the invention, although other cell populations, including PDX-1 positive conditional stem cells, can be used. If the intermediate cell population is simply grown to confluence, cell centers or aggregates may appear. Aggregate formation can also be induced by cell-specific processing methods generated by certain methods of the invention (eg, an intermediate population generated by shock methodology forms aggregates upon treatment with collagenase). Can be induced to do so). However, consistent aggregate formation is enhanced when cells are cultured on substrates previously used to culture pancreatic cells. While not wishing to be bound by any particular theory, the proliferation of cells in an intermediate stage can stimulate the accumulation of certain extracellular matrix molecules or a particular spatial arrangement of the extracellular matrix. When cells are removed and new cells are seeded on the matrix, the matrix promotes the differentiation of the cells into aggregates with morphological and biochemical properties similar to islets in vivo.
[0093]
In an exemplary embodiment, pancreatic cells are isolated and expanded according to the methods of the invention. The cells are then subcultured onto a substrate, during which they grow to confluence as a monolayer. Once the cells have formed a monolayer (typically about 5 days, depending on the seeding density of the first subculture), they are removed by trypsinization. Reduced concentrations of trypsin (typically ま た は or の of those used in standard cell culture techniques) are preferred to prevent extensive degradation of the matrix. Alternatively, a monolayer of cells can be removed by extracting the substrate with a detergent, which removes the cells but leaves a secreted matrix (Gospodarowicz et al., Proc Natl Acad Sci. ScL USA 77: 4094-8 (1980)).
[0094]
Pancreatic cells are cultured on this pre-conditioned substrate to induce aggregate formation. Conveniently, cells that have been previously grown and removed on the substrate can be separated and replated on a conditioned substrate. However, the culture for acclimating the substrate and the culture inoculated on the substrate need not be the same culture. Thus, one cell culture is grown on a substrate to acclimate the substrate, the cells are removed, and cells from another culture are seeded on the conditioned substrate to allow for aggregate formation. Can promote. The conditioned cells can be from the same or a different donor or species as the cells that are subsequently cultured.
[0095]
Cells grown on substrates previously conditioned as described above grow as cell aggregates rather than as a representative monolayer. These aggregates contain about 50 to about 5000 cells and are about 50 microns to about 300 microns in diameter. These aggregates exhibit characteristic morphology when sectioned and examined by immunohistochemical staining. A continuous coating layer, one cell thick and staining positive for the tube marker cytokeratin 19, surrounds the inner cell mass, which generally lacks cytokeratin 19 staining. The inner mass typically contains cells that stain positive for pancreatic endocrine hormones, KS1 / 4 and PDX-1. While not wishing to be bound by any particular theory, this morphology provides a mechanism by which tube-like stem cells, including the outer capsule, give rise to endocrine precursor cells, which differentiate within the inner cell mass into more mature endocrine cells. Suggest. Thus, the cell aggregates produced by the methods of the present invention mimic the developmental process that gives rise to endocrine pancreatic cells in vivo. Thus, these aggregates can be transplanted into mammals to enhance or restore pancreatic endocrine function.
[0096]
Transplantation into a mammal and subsequent monitoring of endocrine function can be performed according to methods commonly used for islet transplantation; for example, Ryan et al., Diabetes 50: 710-19 (2001); Peck et al., Ann Med 33: 186-92 (2001); Shapiro et al., N Engl J Med 343 (4): 230-8 (2000); Carlsson et al., Ups J Med Sci 105 (2): 107-23 (2000) and Kuhtreiber, WM, Cell. See Encapsulation Technology and Therapeutics, Birkuuser, Boston, 1999. Preferred implantation sites include liver and kidney capsules. If there is a problem with the immune response to the transplanted aggregate, various approaches may be used to interfere with immune recognition of the transplanted aggregate. These include: Encapsulation of aggregates to prevent access by immune cells (U.S. Patent Nos. 5,762,959, 5,550,178 and 5,578,314). See, US Pat. No. 6,001,647) and genetically engineering cells prior to aggregate formation to remove antigens recognized by the host (see US Pat. No. 6,001,647). Treatment of the host (see Ryan et al., Diabetes 50: 710-19 (2001)).
[0097]
(Example)
The following examples are provided to illustrate the present invention, but not to limit the present invention.
[0098]
(Example 1: Production of PDX-1 positive intermediate cell population by withdrawal)
HD357 was a 57-year-old female Caucasian organ donor. Pancreatic tissue was dissociated by mechanical disruption and digestion with Liberase (1.5 mg / ml) in HBSS. Dissociated cells were fractionated by trilayer density gradient separation in solution of PIPS (Nycodenz in UW solution (Nycomed AS, Norway)) as described in US Pat. No. 5,739,033. . By laminating the bottom layer of 2.6 ml of dissociated pancreatic tissue, the middle layer of 80 ml of 1.090 g / ml PIPS and the top layer of 70 ml RPMI 1640 + 2% FBS in 40 ml of 1.114 g / ml PIPS solution, The gradient was set up in a 250 ml centrifuge tube. Centrifugation was performed at 1,500 RPM for 6 minutes without brake using a 05, Sorvall RC-3C Plus equipped with an ARC rotor. Three separate tissue populations were collected after gradient separation: the upper (island-enriched) interface between the top and second layers, the lower interface between the middle and bottom layers, And pellet fraction. The collected cell fraction was washed with medium 7 (Section IIIB) to remove the gradient solution and then cultured. The total islet yield from the upper interface was 128,000 IEQ (islet equivalent as determined by microscopic examination of cells after purification and normalized by islet volume relative to a 150 μm standard islet) with a purity of 79%. .
[0099]
Approximately 5% pure 20,000 EQ pancreatic cells from the lower interface were cultured overnight in a T162 culture flask with 35 ml of Medium 7 (20% FBS) (P0). On day 1, the medium was changed to medium 3 (10% FBS) without subculture (Section IIIB). On day 9, the medium was changed to 1/3 SM98 and 2/3 medium 3 (6.67% FBS total) without subculture. On day 13, the cells were passaged to P1 and cultured in a 100 mm tissue culture dish with SM98 (0% FBS). Cells were passaged for a further seven passages (P2-P8). At passage, some of the cells were cultured on chamber slides for immunocytochemical analysis.
[0100]
The staining process was started by removing the chamber portion of the slide. Cells were very gently rinsed three times for 3 minutes in DPBS with calcium / magnesium (Dulbecco's phosphate buffered saline). The cells were then fixed in an ice-cold 4% paraformaldehyde solution in DPBS at room temperature for 10 minutes, followed by three rinses in DPBS with calcium / magnesium for 3 minutes each. Cells were incubated for 1 hour at room temperature in blocking solution (TBS / 3% BSA / 1% normal goat serum). The cells were then permeabilized with 0.3% Triton X-100 in blocking solution for 10-15 minutes. Rabbit anti-PDX-1 primary antibody (from Scripps) diluted 1000: 1 in blocking solution at the appropriate dilution was added to the cells and incubated overnight at 4 ° C. The PDX-1 antibody was purchased from Leonard J. et al. Et al., Mol. Endocrinol. , Oct. 7, 1993, (10) 1275-83.
[0101]
After incubation with the primary antibody, cells were rinsed in calcium / magnesium free DPBS for 3 minutes. After two more rinses, the cells were re-blocked in the blocking solution for 30 minutes at room temperature. A secondary antibody (goat anti-rabbit IgG-rhodamine conjugate diluted 200: 1 in blocking solution) was added to the cells and incubated for 1-2 hours at room temperature in the dark.
[0102]
After incubation, cells were rinsed three times in DPBS without calcium and magnesium, each for 3 minutes. Nuclei were counterstained with Hoechst dye (10,000: 1) in DPBS for 5 minutes, followed by a single rinse in DPBS. Excess fluid was removed by tapping with a paper towel. Slides were mounted with 3-4 drops of aqueous mounting medium and covered with cover slides. Slides were dried at room temperature in the dark for 2 hours and edges were sealed with nail varnish. After storing the slides at 4 ° C. in the dark, they were examined with an Olympus AX 70 fluorescence microscope. Essentially 100% of the HD357 P8 cells stained positive for PDX-1.
[0103]
(Example 2: Production of PDX-1 positive intermediate cell population by shock)
Human donor number 369 (HD369) was a 48 year old Latin American male, weighing 80 kg, and was negative for most virological tests except for CMV. There was no warm ischemia time and the cold ischemia time was 15 hours. The pancreas was fat, but there was no obvious damage. The main pancreatic duct was cannulated and there were no apparent leaks found during the study. Injection of the enzyme was satisfactory. Static digestion took 19 minutes and chamber digestion lasted 18 minutes. This digest provided about 270,000 IEQ before purification. Due to the length of cold ischemia, almost one third of the islets were ambiguous and did not have an intact capsule.
[0104]
Three-layer density gradient centrifugation was performed according to the procedure of Example 1. Several islets were found dissociated into fragments, and the purity of the islets at the upper interface was 54.3%, with a final islet yield of about 149,000 IEQ.
[0105]
About 6,000 IEQ (about 54% pure) from the upper interface was collected in 8 ml of Medium 7 (20% FBS) and split into three 2 ml aliquots and three 0.5 ml aliquots. These fractions were cultured in treated uncoated plastic tissue culture dishes. Cells were divided into three groups. Group 1 consisted of: a) 1,500 IEQ in 2 ml of Medium 7 and 10 ml of Medium SM95 (3.33% FBS total), which were seeded on a 100 mm culture dish, and b). 400 IEQ in 0.5 ml of Medium 7 and 3 ml of Medium SM95 (2.86% FBS total), which were seeded in a 60 mm culture dish. Group 2 consisted of: a) 1,500 IEQ in 2 ml of Medium 7 and 10 ml of Medium SM96 (3.33% FBS total), which were seeded in a 100 mm culture dish, and b). 400 IEQ in 0.5 ml of medium 7 and 3 ml of medium SM96 (2.86% total FBS), which were seeded in a 60 mm culture dish. Group 3 consisted of: a) 1,500 IEQ in 2 ml of medium 7 and 10 ml of medium SM98 (3.33% FBS total), which were seeded in a 100 mm culture dish, and b). 400 IEQ (inoculated in a 60 mm culture dish) in 0.5 ml of Medium 7 and 3 ml of Medium SM98 (total 2.86% FBS). This dish is placed at 37 ° C. with 5% CO 2 ₂ Was incubated up to the first passage for 14 days in the presence of The medium was changed to a serum-free medium twice a week.
[0106]
At the first passage (day 14), the cells were dissociated from the bottom of the culture dish using trypsin / EDTA for about 10 minutes and then washed with 10% FBS HBSS medium. In each group, cells from both 100 mm and 60 mm dishes were combined and seeded into three 100 mm dishes.
[0107]
At the second passage (day 20), cells were trypsinized and subcultured at a ratio of 1: 2. Cells in SM95 and SM98 grew and became confluent within one week. After six passages in SM95, cells were fixed and stained for PDX-1 expression as described in Example 1. Nearly 100% of the cells stained positive for PDX-1.
[0108]
(Example 3: Insulin expression of intermediate cell population)
A PDX-1 positive intermediate cell population was generated from a human donor according to the shock method of Example 2 and propagated in serum-free medium. Cells from passage 1 to passage 6 were assayed for insulin and actin mRNA expression.
[0109]
RNA was isolated using the RNeasy mini kit (QIAGEN # 74104) according to the manufacturer's instructions. Briefly, lysis buffer (650 μl per 10 cm plate) was added to the cells, harvested using a disposable cell scraper (Fisher # 087732), and then disrupted using a QIAshredder (QIAGEN # 79654). Total RNA was isolated from the lysate and then quantified using the RiboGreen RNA Quantitation assay (Molecular Probes # R-11490). RNA samples were stored frozen at -80 ° C until cDNA synthesis. Duplicate aliquots (0.5 μg each) of each sample were transferred to 20 pmole oligo (dT) ₁₆ Using the Omniscript RT kit (QIAGEN # 205111) according to the manufacturer's instructions, then dilute each cDNA sample to 100 μl with TE buffer (pH 8.0) and -20 Stored at ° C. Real-time PCR was performed on a Roche Molecular LightCycler using 2 μl of each cDNA sample and the indicated primers. Actin and insulin were measured using a hybrid probe protocol and a DNA Master Hybridization mix (Roche # 2158825) according to the manufacturer's instructions. PCR was quantified by comparison to a standard curve of each product amplified in parallel.
[0110]
[Table 8]

The results indicated that the average insulin to actin mRNA ratio ranged from 211 to 1.99 from passage 1 to passage 6.
[0111]
[Table 9]

Intermediate populations generated by the shock method were also assayed for their ability to secrete insulin in response to a glucose load. Nearly confluent plates from the indicated cultures were assayed for insulin secretion using the stimulated glucose secretion test (SGS).
[0112]
[Table 10]

(Example 4: Selection of PDX-1 positive intermediate cells by growth in a low serum medium)
This example illustrates the selection of an intermediate stage population by growth in low serum medium. The isolated pancreatic cells were transferred from serum-containing medium conditions to serum-free culture medium conditions. This helps to enrich and select for the desired population of endocrine precursor cells, resulting in an insulin-expressing pancreatic culture. Pancreatic cells were isolated from donor pancreas as in Example 1. The islet-enriched upper and lower interface layers A and B and the pellet fraction were first grown in serum-containing medium (medium 7 with 20% fetal calf serum) (passage 0, PO ). After overnight incubation, the culture was switched to medium with less serum (medium 3 with 10% fetal bovine serum) and cultured for 7-10 days. At the end of this period, nearly confluent cultures were trypsinized (trypsin / EDTA) and passaged in a defined serum-free medium (SM98 or SM95) for selection at a ratio of 1: 3 ( P1). Cultures were supplemented weekly and passaged at a ratio of 1: 2 when reaching 80% -90% confluence.
[0113]
The cell population in layer B before cell culture is heterogeneous and acinar cells (amylase positive), some mature islets (cells positive for insulin, glucagon, somatostatin), and some tube components ( Cytokeratin 19) (see Table 1). The enriched population that was subjected to the expansion and selection processes displayed many changes in the protein and gene expression patterns of these markers (Tables 2 and 3). At passage 0 (P0), the cell population is still heterogeneous and a significant proportion (20% -50%) are amylase positive, cytokeratin 19 positive (85% -95%), And only 1% to 3% of cells showed positive staining for insulin. These pre- detached primary insulin-positive cells are small, contain many small cytoplasmic granules that stain positive for insulin, have small round nuclei that stain strongly for PDX-1 (pancreatic homeobox domain protein-1), And did not show cytoplasmic staining for cytokeratin 19; therefore, it appeared to be derived from mature adult islets present in freshly enriched pancreatic material. This small insulin-positive cell population diminished as cells were passaged from serum-containing medium to serum-free medium and was no longer detectable by the fourth passage (P4). From passage 2 to passage 4 (P2 to P4), a second different insulin positive population could be detected (2% -40% of total cells). This population of detached cells had oval nuclei and perinuclear cytoplasmic granules that stained positive for insulin, and consisted of larger cells with PDX-1 positive nuclei (FIG. 1 at passage 6). checking). Co-expression of cytokeratin 19 and / or the nuclear proliferation antigen Ki67 was observed in a significant proportion of these cells. Large insulin-positive cells constituted 10% to 60% of the cell population from passage 4 to passage 8 (P4-P8). Although the percentage of insulin positive cells increased during the withdrawal process, total insulin RNA expression levels decreased. This indicates lower insulin RNA expression per cell in large insulin positive cells compared to the initial insulin positive cell population. Cells that have not been detached and have not been transferred from serum-containing media to serum-free media (SM98 or SM95) do not express detectable levels of insulin and are weak PDXs that tend to be in the cytoplasm rather than the nucleus. These cells that exhibited -1 expression and a greater percentage (> 30% compared to <10% in the withdrawn population) continued to express the acinar marker amylase. Changes in hormone gene expression levels during selection of expanded pancreatic cells were also monitored by quantitative RT-PCR (Table 3).
[0114]
[Table 1]

Data were obtained from paraffin sections and are shown as percentage of total cells expressing the indicated marker.
[0115]
[Table 2]

Data are shown as percentage of total cells expressing the indicated marker.
[0116]
[Table 3]

Example 5: Maturation of intermediate cells forming insulin secretory aggregates
Human pancreatic tissue from digestion of the donor pancreas HD381 was cultured in Medium 7 (20% FBS) for 2 days. The medium was changed to Medium 3 (10% FBS) for 3 days. During this time, a population of cells that grew almost as a monolayer developed (passage 0). This culture was subcultured in fresh tissue culture plates in SM 98 (0% FBS) medium for 5 days to select for endocrine precursor cells (passage 1). Almost all cells grew as a monolayer in this culture.
[0117]
The first passage culture was subcultured to a new plate, where the culture continued to grow as a monolayer (second passage). The first passage culture was also subcultured into recycled plates from which the first passage cells were removed by trypsinization for subculture. In the recycled plate, the cells stopped growing as a monolayer. Rather, the cells formed aggregates of about 50 to about 5000 cells, with dimensions between about 50 and about 300 microns. Cells from four consecutive passages of the monolayer growth were subcultured on recycled plates. In each case, cells cultured on fresh plates grew as monolayers, while cells cultured on recycled plates formed cell aggregates.
[0118]
Samples of cell aggregates were picked by hand, fixed in 4% formalin, and embedded in paraffin blocks. Sections for slides were cut to a thickness of about 6 μm. For deparaffinization, slides were sequentially immersed in the following reagents: 2 times over 10 minutes, xylene; 3 times over 1 minute, 100% ethanol; 1 time over 1 minute, 95% ethanol; 1 minute 70% ethanol once over. Slides were immersed in a 0.3% hydrogen peroxide solution in methanol for 30 minutes at room temperature to inhibit endogenous peroxidase activity. Slides were boiled in 0.01 citrate buffer (pH 6.0) for 30 minutes to restore specific epitopes. Slides were rinsed three times for 5 minutes each with PBS and blocked in 10% normal goat serum in PBS for 30-60 minutes at room temperature in a humid chamber. Primary (CK-19-specific) antibodies from Dako Corporation (Cat. No. M0888), prepared in blocking solution, were added to the samples and incubated in a humid chamber at room temperature for 60 minutes. The slides were washed three times with PBS for 5 minutes and incubated with a secondary antibody (biotinylated anti-IgG) prepared in blocking solution for 30 minutes at room temperature in a humid chamber. Slides were rinsed three times with PBS for 5 minutes and incubated with avidin-horseradish peroxide or ABC complex (Dako) for 30 minutes at room temperature. The slides were rinsed three times for 5 minutes with PBS and incubated with the biaminobenzidine color development solution for 10 minutes. After two washes with distilled water, the slides were immersed in Mayer's Hematoxylin for 5 minutes and then rinsed in running tap water until the water became colorless and the nuclei turned blue. Slides were dehydrated by immersing sequentially in the following reagents: 70% ethanol, 1 minute; 95% ethanol, 1 minute; 100% ethanol, 1 minute, 3 times; xylene, overnight. Slides were mounted in mounting media and stored at 4 ° C.
[0119]
Staining with a panel of antibodies to pancreatic markers and hormones indicated that the aggregates consisted of a single layer of CK-19-positive cell coating surrounding the inner mass of cells mostly negative for CK-19. (See FIG. 2, in which the capsule is partially disturbed by this treatment. The cells of the inner mass stained heterogeneously, but the inner cell mass contained pancreatic endocrine hormones (insulin, somatostatin and glucagon). ) And cells staining positive for KS1 / 4 and PDX-1 About 50% of the CK-19 positive capsule cells were also PDX-1 positive.
[0120]
The insulin levels of the monolayer culture and of the aggregated cells at various passages were compared. The cells are lysed in a high salt buffer and the amount of insulin in the lysate is determined ¹²⁵ Determined by competitive radioimmunoassay using I-labeled insulin. Values were normalized to the total DNA content of the lysate as measured by fluorescence in the presence of Hoechst 33258. The insulin content of freshly isolated pancreatic islets was determined for comparison. A comparison for the third passage aggregate is shown:
[0121]
[Table 11]

The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations in light of them will be suggested to those skilled in the art, and the present application and the appended claims Should be included within the spirit and scope of the scope. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[Brief description of the drawings]
FIG.
FIG. 1 (a and b) are two microphotographs of cultured pancreatic cells showing PDX-1 positive staining. (A) is a control using a non-specific antibody, and (b) is a cell stained with a PDX-1 specific antibody.
FIG. 2
2 (a and b) are two microphotographs of aggregates of cultured pancreatic cells showing the coating of CK-19 positive cells. (A) is a control using a non-specific antibody, and (b) is a cell stained with a CK-19-specific antibody.
FIG. 3
FIG. 3 shows the sequence listing.

Claims (16)

Approximately 180 cells per square centimeter from the first culture vessel to the second vessel while retaining the property of having 90% PDX-1 positive and having an insulin: actin mRNA ratio of between 1: 100 and 1000: 1. A method of preparing a cell culture of growing pancreatic cells having the ability to grow to about 1,800 cells per square centimeter when passaged at an initial concentration of, comprising the following steps:
(A) isolating growing pancreatic cells;
(B) transplanting the cells into a culture medium containing growth hormone and having a total serum volume of 1% or less, having an insulin: actin mRNA ratio of between 1: 100 and 1000: 1 and PDX Selectively growing cells that are -1 positive; and (c) passing the cells to 90% PDX-1 positive, and between 1: 100 and 1000: 1 insulin: actin mRNA. Having the ability to pass from the first culture vessel to the second vessel at an initial concentration of about 180 cells per square centimeter to increase to about 1,800 cells per square centimeter while retaining the property of having a ratio. Providing a cell culture of growing pancreatic cells. After the cells are maintained in a first medium containing between 1% and 4% serum of the total volume of the first medium, the cells are transferred to a second medium containing growth hormone and 1% or less serum of the total volume. The method of claim 1, wherein the method is implanted. The cells are maintained in a first medium containing more than 4% of serum in the total volume of the first medium, and then transplanted into a second medium containing growth hormone and less than 1% of serum in total volume; The method of claim 1. The cells are progressively reduced in amount of serum from a first medium containing more than about 4% serum of the first medium to a second medium containing growth hormone and less than 1% serum. 4. The method of claim 3, wherein the cells are transplanted by serial passage into a medium having the cells. 2. The method of claim 1, wherein the isolated growing pancreatic cells have a mixture of a PDX-1 positive phenotype and a PDX-1 negative phenotype. The method of claim 1, wherein the cell culture has an insulin: actin mRNA ratio between 1:10 and 100: 1. A method for producing an aggregate of cultured pancreatic cells comprising an encapsulating coat of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells, and comprises 50 microns Having a diameter of between 300 microns, the method comprises the following steps:
(A) culturing pancreatic cells on a substrate;
(B) removing the cells from the substrate;
(C) replating PDX-1 positive pancreatic cells on the substrate produced in step (b); and (d) culturing the cells of step (c) on the substrate of step (c) Providing an aggregate of cultured pancreatic cells comprising a coating around the ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and comprises 50 microns Having a diameter of between 1 and 300 microns. 8. The method of claim 7, wherein the culturing of step (a) or step (c) is performed in a medium containing growth hormone and less than 1% total volume of serum. A method for providing a pancreatic endocrine function to a mammal, comprising the following steps:
A method comprising: (a) producing an aggregate of cultured pancreatic cells by the method of claim 7; and (b) transplanting the aggregate into the mammal. 10. The method of claim 9, wherein the cells are passaged in a medium containing between 1 mg and 2 mg of recombinant growth hormone per liter. 2. The method of claim 1, wherein said cells are passaged in a medium containing recombinant human growth hormone. 2. The method of claim 1, wherein the cells are passaged in a medium containing epidermal growth factor. A culture of growing pancreatic cells produced by the method of claim 1. Approximately 180 cells per square centimeter from the first culture vessel to the second vessel while retaining the property of having 90% ΔPDX-1 positive and having an insulin: actin mRNA ratio of between 1: 100 and 1000: 1. A culture of proliferating pancreatic cells having the ability to grow to about 1,800 cells per square centimeter at an initial concentration of. An aggregate of pancreatic cells produced by the method of claim 7. An aggregate of cultured pancreatic cells, comprising an encapsulating coat of CK-19-positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells, and comprises 50 and 300 microns. Agglomerates having a diameter between

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