JPWO2020077204A5 - - Google Patents
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Description
本明細書において提供される任意の組成物または方法は、本明細書において提供され説明される他の組成物および方法のいずれかのうちの1つまたは複数と組み合わせることができる。
[本発明1001]
移植されたドナー細胞を複数回の間欠的ばく露でインターフェロンγ（IFNγ）に接触させ、それによって、移植されたドナー細胞の生残を向上させるかまたは移植されたドナー細胞の細胞死を低減する工程
を含む、移植されたドナー細胞の生残を向上させるかまたは移植されたドナー細胞の細胞死を低減する方法。
[本発明1002]
前記ドナー細胞がオルガノイド細胞、島細胞、島様オルガノイド細胞、β様島細胞である、本発明1001の方法。
[本発明1003]
Wnt4タンパク質またはWnt5aタンパク質を含む三次元マトリックス中で、多細胞島様オルガノイドを生成させるのに十分な時間、内分泌前駆細胞を培養する工程であって、該多細胞島様オルガノイドが、ベータ（β）細胞、アルファ（α）細胞、デルタ（δ）細胞、イプシロン（ε）細胞、および導管様細胞より選択される2つまたはそれ以上の細胞タイプを含み、該島様オルガノイドがグルコースに応答してインスリンを分泌する、該工程、ならびに
該島様オルガノイドを、インターフェロンγ（IFNγ）への複数回の間欠的ばく露に供し、それによって、該島様オルガノイドによる免疫チェックポイントタンパク質の持続的発現を誘導し、かつ免疫検出または自己免疫を該島様オルガノイドが回避することを可能にする工程
を含む、免疫検出または自己免疫を回避する島様オルガノイドを生成させる方法。
[本発明1004]
Wnt4タンパク質またはWnt5aタンパク質を含む三次元マトリックス中で、多細胞島様オルガノイドを生成させるのに十分な時間、免疫チェックポイントタンパク質を組換えにより発現する内分泌前駆細胞を培養する工程であって、該多細胞島様オルガノイドが、ベータ（β）細胞、アルファ（α）細胞、デルタ（δ）細胞、イプシロン（ε）細胞、および導管様細胞より選択される2つまたはそれ以上の細胞タイプを含み、該島様オルガノイドが、グルコースに応答してインスリンを分泌し、かつ免疫検出および自己免疫を回避する、該工程
を含む、免疫検出または自己免疫を回避する島様オルガノイドを生成させる方法。
[本発明1005]
前記三次元マトリックスがジェランガムを含む、本発明1003または本発明1004の方法。
[本発明1006]
前記三次元マトリックスが組換えヒトWnt4タンパク質を含む、本発明1003～1005のいずれかの方法。
[本発明1007]
前記免疫チェックポイントタンパク質の組換え発現が、該免疫チェックポイントタンパク質をコードするポリヌクレオチドを含有するベクターによる島様オルガノイド細胞の形質導入に起因する、本発明1004の方法。
[本発明1008]
前記免疫チェックポイントタンパク質が、免疫細胞で発現するコグネイトリガンドに結合し、該コグネイトリガンドが、プログラム細胞死タンパク質1（PD-1）;細胞傷害性Tリンパ球タンパク質4（CTLA-4）;リンパ球活性化遺伝子3タンパク質（LAG-3）;キラー細胞免疫グロブリン様受容体（KIR）;インドールアミン2,3-ジオキシゲナーゼ1（IDO1）;腫瘍壊死因子受容体スーパーファミリーメンバー9（4-1BB）;グルココルチコイド誘導性TNFRファミリー関連遺伝子（GITR）;T細胞免疫グロブリンドメインおよびムチンドメイン（TIM-3）;腫瘍壊死因子受容体スーパーファミリーメンバー4（OX40）;アデノシンA2A受容体（A2AR）;B7-H3;B7-H4;B7-1/B7-2;BTLA;T細胞活性化のVドメインIgサプレッサー（VISTA）;または前述のうちのいずれかの組合せより選択される、本発明1003～1007のいずれかの方法。
[本発明1009]
前記免疫チェックポイントタンパク質がプログラム死リガンド-1（PD-L1）である、本発明1003～1008のいずれかの方法。
[本発明1010]
前記細胞、島、オルガノイド、または島様オルガノイドが、少なくとも2日間の間に少なくとも2回、IFNγにばく露される、本発明1001～1003のいずれかの方法。
[本発明1011]
前記細胞、島、オルガノイド、または島様オルガノイドが、少なくとも3日間の間に少なくとも3回、IFNγにばく露される、本発明1001～1003のいずれかの方法。
[本発明1012]
前記細胞、島、オルガノイド、または島様オルガノイドが、少なくとも2日間の間に少なくとも2回、1時間超にわたってIFNγにばく露される、本発明1001～1003のいずれかの方法。
[本発明1013]
前記細胞、島、オルガノイド、または島様オルガノイドが、少なくとも3日間の間に少なくとも3回、1時間超にわたってIFNγにばく露される、本発明1001～1003のいずれかの方法。
[本発明1014]
前記細胞、島、オルガノイド、または島様オルガノイドが、少なくとも3日間の間に少なくとも3回、2時間にわたってIFNγにばく露される、本発明1013の方法。
[本発明1015]
前記内分泌前駆細胞が、誘導多能性幹細胞（iPSC）、胚性多能性幹細胞（ePSC）、および/または膵臓前駆細胞より選択される、本発明1003～1014のいずれかの方法。
[本発明1016]
前記内分泌前駆細胞が、ニューロゲニン3、neurod1、Nkx2.2、およびPax4バイオマーカーのうちの少なくとも1つのバイオマーカーを発現する、本発明1003～1015のいずれかの方法。
[本発明1017]
前記島様オルガノイドがヒト島様オルガノイド（HILO）である、本発明1002～1016のいずれかの方法。
[本発明1018]
前記島様オルガノイドが血管新生化される、本発明1017の方法。
[本発明1019]
前記島様オルガノイドが脂肪由来幹細胞および/または内皮細胞をさらに含む、本発明1002～1017のいずれかの方法。
[本発明1020]
前記脂肪由来幹細胞がヒト脂肪由来幹細胞（hADSC）であり、かつ/または前記内皮細胞がヒト臍帯静脈内皮細胞（HUVEC）である、本発明1019の方法。
[本発明1021]
前記島様オルガノイドが、KCl刺激インスリン分泌、GLP-1刺激インスリン分泌、ソマトスタチン分泌、グルカゴン分泌のうちの少なくとも1つをさらに呈する、本発明1002～1020のいずれかの方法。
[本発明1022]
前記島様オルガノイドが、NKX2-2、NEUROD1、RFX6、GCK、INS、NKX6-1、UCN3、MAFB、およびSYT4からなる群より選択されるβ細胞系列マーカーと、ARXα細胞系列マーカーとを発現する、本発明1002～1021のいずれかの方法。
[本発明1023]
前記三次元マトリックスが、ヒトWnt4タンパク質、組換えヒトWnt4タンパク質、ヒトWnt5タンパク質、または組換えヒトWnt5aタンパク質を含む、本発明1003または本発明1004の方法。
[本発明1024]
前記三次元マトリックスが組換えヒトWnt4タンパク質を含む、本発明1023の方法。
[本発明1025]
前記島様オルガノイドがエストロゲン関連受容体γ（ERRγ）の発現の増大を呈する、本発明1002～1024のいずれかの方法。
[本発明1026]
前記島様オルガノイドが、酸素消費速度（OCR）の増加と細胞酸性化速度（ECAR）の減少とを特徴とする増大した酸化的代謝を呈する、本発明1002～1025のいずれかの方法。
[本発明1027]
前記島様オルガノイドが、膵島オルガノイド、膵臓オルガノイド、肝臓オルガノイド、心臓オルガノイド、または腸オルガノイドである、本発明1002～1026のいずれかの方法。
[本発明1028]
前記島様オルガノイドがヒト膵島オルガノイドである、本発明1027の方法。
[本発明1029]
前記ドナー細胞が、心臓細胞、結腸細胞、腎臓細胞、肝臓細胞（肝細胞）、食道細胞、胃腸細胞、胃部（胃）細胞、肺細胞、膵臓細胞、膵臓β細胞、筋細胞、造血細胞、B細胞、T細胞、CD34＋造血細胞、キメラ抗原受容体-T細胞（CAR-T細胞）、骨髄細胞、ニューロン、神経細胞、網膜細胞、角膜細胞、脳細胞、ヒト皮膚細胞に由来するインスリン産生膵臓β細胞、卵巣細胞、子宮頸部細胞、精巣細胞、単核球、臍帯血（UCB）細胞、脂肪由来間葉系間質（幹）細胞、心臓幹細胞、結腸幹細胞、腎臓幹細胞、肝臓（肝細胞）幹細胞、胃腸幹細胞、胃部（胃）幹細胞、肺幹細胞、膵臓幹細胞、膵臓β幹細胞、筋幹細胞、造血幹細胞、T細胞幹細胞またはB細胞幹細胞、骨髄幹細胞、CD133＋幹細胞、CD34＋造血幹細胞、網膜幹細胞、神経幹細胞、間葉系幹細胞、臍帯間葉系幹細胞、外胚葉由来の神経細胞、外胚葉由来のドーパミン作動性神経細胞、角膜由来の細胞、正常ヒト角膜上皮細胞、不死化ドーパミン作動性ニューロン前駆細胞、内胚葉由来の肝臓細胞、中胚葉由来の筋細胞、骨髄細胞、腎臓細胞、および骨格筋細胞、または該細胞から生成されるもしくは該細胞を含有するオルガノイド;腸オルガノイド、肝オルガノイド、結腸オルガノイド、肝オルガノイド、腎臓オルガノイド、膀胱オルガノイド、卵巣オルガノイド、子宮頸部オルガノイド、神経オルガノイド、または肺臓（肺）オルガノイドより選択される、本発明1001または本発明1002の方法。
[本発明1030]
（a）Wnt4タンパク質またはWnt5aタンパク質を含む三次元マトリックス中で、多細胞ヒト島様オルガノイドを生成させるのに十分な時間、内分泌前駆細胞を培養する工程であって、該多細胞ヒト島様オルガノイドが、ベータ（β）細胞、アルファ（α）細胞、デルタ（δ）細胞、イプシロン（ε）細胞、および導管様細胞より選択される2つまたはそれ以上の細胞タイプを含み、該ヒト島様オルガノイドがグルコースに応答してインスリンを分泌する、該工程、
（b）工程（a）のHILOを、少なくとも48～72時間の全期間の間に2回または3回、各回1時間超にわたって、インターフェロンγ（IFNγ）と接触させる工程
を含む、免疫検出または自己免疫を回避するヒト島様オルガノイド（HILO）を生成させる方法であって、
IFNγとの接触時間同士の間はヒト島またはHILOがIFNγの非存在下で維持され、かつ工程（a）および工程（b）によって、該HILOにおける免疫チェックポイントタンパク質プログラム死リガンド-1（PD-L1）の持続的発現が誘導される、該方法。
[本発明1031]
工程（b）において、前記HILOを2時間にわたってIFNγと接触させる、本発明1030の方法。
[本発明1032]
前記HILOを、少なくとも48時間の間に2回、各回2時間にわたって、IFNγと接触させる、本発明1030または本発明1031の方法。
[本発明1033]
前記HILOを、少なくとも72時間の間に3回、各回2時間にわたって、IFNγと接触させる、本発明1030または本発明1031の方法。
[本発明1034]
前記内分泌前駆細胞が、誘導多能性幹細胞（iPSC）、胚性多能性幹細胞（ePSC）、および/または膵臓前駆細胞より選択される、本発明1030～1033のいずれかの方法。
[本発明1035]
前記内分泌前駆細胞が、ニューロゲニン3、neurod1、Nkx2.2、およびPax4バイオマーカーのうちの少なくとも1つのバイオマーカーを発現する、本発明1030～1034のいずれかの方法。
[本発明1036]
前記HILOが、血管新生化され、かつ酸素消費速度（OCR）の増加と細胞酸性化速度（ECAR）の減少とを特徴とする増大した酸化的代謝を呈する、本発明1030～1035のいずれかの方法。
[本発明1037]
IFNγが1～25ng/mlの量で使用される、本発明1001～1036のいずれかの方法。
[本発明1038]
IFNγが10ng/mlの量で使用される、本発明1037の方法。
[本発明1039]
前記島様オルガノイドまたはHILOにおけるPD-L1発現が7日超にわたって維持される、本発明1008～1036のいずれかの方法。
[本発明1040]
免疫チェックポイントタンパク質の持続的発現を有する、免疫保護された細胞、ヒト島様オルガノイドまたは膵島オルガノイドであって、
該オルガノイドが、本発明1001～1039のいずれかの方法によって産生される、該免疫保護された細胞、ヒト島様オルガノイドまたは膵島オルガノイド。
[本発明1041]
免疫チェックポイントタンパク質PD-L1の持続的発現を呈する、本発明1040のヒト島様オルガノイドまたは膵島オルガノイド。
[本発明1042]
Wnt4タンパク質またはWnt5タンパク質を含む三次元マトリックス中で培養された内分泌前駆細胞に由来し、かつ多系列細胞を含む、ヒト島様オルガノイド（HILO）であって、
該多系列細胞が、ベータ（β）細胞、アルファ（α）細胞、デルタ（δ）細胞、イプシロン（ε）細胞、および導管様細胞のうちの少なくとも2つを含み、該HILOが、血管新生化され、グルコース刺激インスリン分泌（GSIS）を呈し、かつ免疫チェックポイントタンパク質の持続的発現を呈する、該ヒト島様オルガノイド（HILO）。
[本発明1043]
膵島様オルガノイドまたは膵臓オルガノイドである、本発明1042のヒト島様オルガノイド（HILO）。
[本発明1044]
KCl刺激インスリン分泌またはグルコース刺激インスリン分泌をさらに呈する、本発明1042または本発明1043のヒト島様オルガノイド（HILO）。
[本発明1045]
前記三次元マトリックスがジェランガムを含む、本発明1042～1044のいずれかのヒト島様オルガノイド（HILO）。
[本発明1046]
前記三次元マトリックスが組換えヒトWnt4タンパク質を含む、本発明1042～1045のいずれかのヒト島様オルガノイド（HILO）。
[本発明1047]
前記三次元マトリックスが組換えヒトWnt5タンパク質を含む、本発明1042～1046のいずれかのヒト島様オルガノイド（HILO）。
[本発明1048]
前記内分泌前駆細胞が、誘導多能性幹細胞（iPSC）、胚性多能性幹細胞（ePSC）、および/または膵臓前駆細胞より選択される、本発明1042～1047のいずれかのヒト島様オルガノイド（HILO）。
[本発明1049]
前記内分泌前駆細胞が、ニューロゲニン3、neurod1、Nkx2.2、およびPax4バイオマーカーのうちの少なくとも1つのバイオマーカーを発現する、本発明1042～1048のいずれかのヒト島様オルガノイド（HILO）。
[本発明1050]
FLTP遺伝子およびESRRγ遺伝子を発現する、本発明1042～1049のいずれかのヒト島様オルガノイド（HILO）。
[本発明1051]
脂肪由来幹細胞および/または内皮細胞をさらに含む、本発明1042～1050のいずれかのヒト島様オルガノイド（HILO）。
[本発明1052]
前記脂肪由来幹細胞がヒト脂肪由来幹細胞（hADSC）であり、かつ/または前記内皮細胞がヒト臍帯静脈内皮細胞（HUVEC）である、本発明1051のヒト島様オルガノイド（HILO）。
[本発明1053]
KCl刺激インスリン分泌、GLP-1刺激インスリン分泌、ソマトスタチン分泌、またはグルカゴン分泌をさらに呈する、本発明1042～1052のいずれかのヒト島様オルガノイド（HILO）。
[本発明1054]
NKX2-2、NEUROD1、RFX6、GCK、INS、NKX6-1、UCN3、MAFB、およびSYT4からなる群より選択されるβ細胞系列マーカーと、ARXα細胞系列マーカーとを発現する、本発明1040～1053のいずれかのヒト島様オルガノイド（HILO）。
[本発明1055]
膵臓HILOが、Pdx1、MafA、Pax4、Pax6、NeuroD1、Nkx6-1、Gata6、およびFoxa2からなる群より選択されるβ細胞転写因子を発現する、本発明1040～1052のいずれかのヒト島様オルガノイド（HILO）。
[本発明1056]
前記免疫チェックポイントタンパク質が、免疫細胞で発現するコグネイトリガンドに結合し、該コグネイトリガンドが、プログラム細胞死タンパク質1（PD-1）;細胞傷害性Tリンパ球タンパク質4（CTLA-4）;リンパ球活性化遺伝子3タンパク質（LAG-3）;キラー細胞免疫グロブリン様受容体（KIR）;インドールアミン2,3-ジオキシゲナーゼ1（IDO1）;腫瘍壊死因子受容体スーパーファミリーメンバー9（4-1BB）;グルココルチコイド誘導性TNFRファミリー関連遺伝子（GITR）;T細胞免疫グロブリンドメインおよびムチンドメイン（TIM-3）;腫瘍壊死因子受容体スーパーファミリーメンバー4（OX40）;アデノシンA2A受容体（A2AR）;B7-H3;B7-H4;B7-1/B7-2;BTLA;T細胞活性化のVドメインIgサプレッサー（VISTA）;または前述のうちのいずれかの組合せより選択される、本発明1042～1055のいずれかのヒト島様オルガノイド（HILO）。
[本発明1057]
前記免疫チェックポイントタンパク質がプログラム死リガンド-1（PD-L1）である、本発明1042～1056のいずれかのヒト島様オルガノイド（HILO）。
[本発明1058]
前記ヒト島様オルガノイド、膵島オルガノイド、または本発明1042～1057のいずれかのHILOを移植または埋植されたヒト以外の生物。
[本発明1059]
哺乳動物である、本発明1058のヒト以外の生物。
[本発明1060]
マウスである、本発明1058のヒト以外の生物。
[本発明1061]
免疫保護された島様オルガノイドまたは膵島オルガノイドを対象に移植または埋植する工程を含む、該対象における膵臓疾患を処置する方法であって、
該島様オルガノイドまたは膵島オルガノイドが、β細胞、α細胞、δ細胞、ε細胞、導管様細胞、またはそれらの組合せを含み内分泌前駆細胞に由来する多系列細胞を含み、血管新生化され、グルコース刺激インスリン分泌（GSIS）を呈し、かつ免疫検出または自己免疫を回避するために免疫チェックポイントタンパク質の持続的発現を呈する、該方法。
[本発明1062]
免疫保護された島様オルガノイドまたは膵島オルガノイドを対象に移植または埋植する工程を含む、該対象における1型糖尿病を処置する方法であって、
該島様オルガノイドまたは膵島オルガノイドが、β細胞、α細胞、δ細胞、ε細胞、導管様細胞、またはそれらの組合せを含み内分泌前駆細胞に由来する多系列細胞を含み、血管新生化され、グルコース刺激インスリン分泌（GSIS）を呈し、かつ免疫検出または自己免疫を回避するために免疫チェックポイントタンパク質の持続的発現を呈する、該方法。
[本発明1063]
前記島様オルガノイドまたは膵島オルガノイドが、KCl刺激インスリン分泌、GLP-1刺激インスリン分泌、ソマトスタチン分泌、またはグルカゴン分泌をさらに呈する、本発明1061または本発明1062の方法。
[本発明1064]
前記島様オルガノイドまたは膵島オルガノイドが、NKX2-2、NEUROD1、RFX6、GCK、INS、NKX6-1、UCN3、MAFB、およびSYT4からなる群より選択されるβ細胞系列マーカーと、ARXα細胞系列マーカーとを発現する、本発明1061～1063のいずれかの方法。
[本発明1065]
前記内分泌前駆細胞が、誘導多能性幹細胞（iPSC）、胚性多能性幹細胞（ePSC）、および/または膵臓前駆細胞より選択される、本発明1061～1064のいずれかの方法。
[本発明1066]
前記内分泌前駆細胞が、ニューロゲニン3、neurod1、Nkx2.2、およびPax4バイオマーカーのうちの少なくとも1つのバイオマーカーを発現する、本発明1061～1065のいずれかの方法。
[本発明1067]
前記島様オルガノイドまたは膵島オルガノイドが、Pdx1、MafA、Pax4、Pax6、NeuroD1、Nkx6-1、Gata6、およびFoxa2からなる群より選択されるβ細胞転写因子を発現する、本発明1061～1066のいずれかの方法。
[本発明1068]
前記免疫チェックポイントタンパク質が、免疫細胞で発現するコグネイトリガンドに結合し、該コグネイトリガンドが、プログラム細胞死タンパク質1（PD-1）;細胞傷害性Tリンパ球タンパク質4（CTLA-4）;リンパ球活性化遺伝子3タンパク質（LAG-3）;キラー細胞免疫グロブリン様受容体（KIR）;インドールアミン2,3-ジオキシゲナーゼ1（IDO1）;腫瘍壊死因子受容体スーパーファミリーメンバー9（4-1BB）;グルココルチコイド誘導性TNFRファミリー関連遺伝子（GITR）;T細胞免疫グロブリンドメインおよびムチンドメイン（TIM-3）;腫瘍壊死因子受容体スーパーファミリーメンバー4（OX40）;アデノシンA2A受容体（A2AR）;B7-H3;B7-H4;B7-1/B7-2;BTLA;T細胞活性化のVドメインIgサプレッサー（VISTA）;または前述のいずれかの組合せより選択される、本発明1061～1067のいずれかの方法。
[本発明1069]
前記免疫チェックポイントタンパク質がプログラム死リガンド-1（PD-L1）である、本発明1061～1068のいずれかの方法。
[本発明1070]
前記島様オルガノイドまたは膵島オルガノイドが、本発明1001～1037のいずれかの方法によって産生される、本発明1061～1069のいずれかの方法。
[本発明1071]
前記島様オルガノイドまたは膵島オルガノイドが、本発明1040～1057のいずれかのオルガノイドである、本発明1061～1070のいずれかの方法。
[本発明1072]
免疫抑制物質が前記対象に投与される、本発明1061～1071のいずれかの方法。
[本発明1073]
前記対象がヒトである、本発明1061～1072のいずれかの方法。
[本発明1074]
前記膵臓疾患が1型糖尿病または2型糖尿病である、本発明1061または本発明1063～1073のいずれかの方法。
[本発明1075]
移植または埋植の後に生残しかつ細胞死が低減している細胞、島、またはオルガノイドを生成させる方法であって、
（a）インターフェロンγ（IFNγ）受容体を発現する細胞、島、またはオルガノイドを、インターフェロンγ（IFNγ）と、予め決められた時点において少なくとも0.5時間または少なくとも1時間接触させる工程、および
（b）約72時間または少なくとも約72時間の期間中に、工程（a）を少なくとも2回繰り返す工程
を含み、
IFNγとの接触時間同士の間は該細胞、島、またはオルガノイドがIFNγの非存在下で維持され、かつ工程（a）および工程（b）によって、該細胞、島、またはオルガノイドにおけるPD-L1の持続的発現が誘導される、
該方法。
[本発明1076]
工程（a）において、前記細胞、島、オルガノイド、または細胞を、少なくとも1時間、少なくとも2時間、または2時間超より選択される期間にわたって、IFNγと接触させる、本発明1075の方法。
[本発明1077]
工程（a）において、前記細胞、島、またはオルガノイドを、約2時間もしくは2時間または約12時間もしくは12時間より選択される期間にわたって、IFNγと接触させる、本発明1075または本発明1076の方法。
[本発明1078]
工程（a）が、工程（b）の少なくとも約72時間または少なくとも72時間の期間中に少なくとも3回繰り返され、各回が少なくとも約2時間にわたる、本発明1075～1077のいずれかの方法。
[本発明1079]
工程（a）と工程（b）の間で、IFNγの存在を除去するために前記細胞、島、またはオルガノイドが洗浄される、本発明1075～1078のいずれかの方法。
[本発明1080]
IFNγが1～25ng/mlの量で使用される、本発明1075～1079のいずれかの方法。
[本発明1081]
IFNγが10ng/mlの量で使用される、本発明1075～1079のいずれかの方法。
[本発明1082]
前記細胞、島、またはオルガノイドにおけるPD-L1発現が、工程（b）後に約7日超にわたって維持される、本発明1075～1081のいずれかの方法。
[本発明1083]
免疫検出または自己免疫を回避する細胞、島、またはオルガノイドおよびそれらの細胞を生成させる方法であって、
（a）インターフェロンγ（IFNγ）受容体を発現する細胞、島、またはオルガノイドおよびそれらの細胞を、少なくとも約24時間または少なくとも24時間の期間中の第1の時点において1時間超にわたって、約1ng/ml～25ng/mlの量のインターフェロンγ（IFNγ）と接触させる工程、ならびに
（b）該細胞、島、またはオルガノイドおよびそれらの細胞を、工程（a）後の後続する少なくとも約48時間の期間中の2つまたはそれ以上の追加の時点において、約0.5時間またはそれ以上を超える時間にわたって、約1ng/ml～25ng/mlの量のIFNγと接触させる工程
を含み、
IFNγと接触させること同士の間は、該細胞、島、またはオルガノイドを洗浄してIFNγの非存在下の培地中で休止させ、かつ工程（a）および工程（b）によって、該細胞、島、またはオルガノイドにおけるPD-L1の持続的発現が誘導される、
該方法。
[本発明1084]
工程（a）および工程（b）において、前記細胞、島、またはオルガノイドを、10ng/mlの量のIFNγと、少なくとも2時間にわたって接触させる、本発明1083の方法。
[本発明1085]
前記細胞、島、またはオルガノイドを、72時間の期間中の3つの時点において少なくとも約2時間にわたって、IFNγと接触させる、本発明1083または本発明1084の方法。
[本発明1086]
前記細胞、島、またはオルガノイドがヒトの細胞、島、またはオルガノイドである、本発明1001または本発明1075～1085のいずれかの方法。
[本発明1087]
前記オルガノイドがHILOまたはヒトHILOである、本発明1086の方法。
[本発明1088]
前記細胞が、心臓細胞、結腸細胞、腎臓細胞、膀胱細胞、肝臓細胞（肝細胞）、食道細胞、胃腸細胞、胃部（胃）細胞、肺細胞、卵巣細胞、子宮頸部細胞、子宮細胞、精巣細胞、膵臓細胞、膵臓β細胞、網膜細胞、角膜細胞、脳細胞、筋細胞、造血細胞、免疫細胞（B細胞、T細胞）、キメラ抗原受容体-T細胞（CAR-T細胞）、骨髄細胞、単核球、ニューロン、神経細胞、ヒト皮膚細胞に由来するインスリン産生膵臓β細胞、臍帯血（UCB）細胞、脂肪由来間葉系間質（幹）細胞、心臓幹細胞、結腸幹細胞、腎臓幹細胞、肝臓（肝細胞）幹細胞、胃腸幹細胞、胃部幹細胞、肺幹細胞、膵臓幹細胞、膵臓β幹細胞、筋幹細胞、造血幹細胞、免疫細胞（T細胞またはB細胞）幹細胞、骨髄幹細胞、CD133＋幹細胞、CD34＋造血細胞、CD34＋造血幹細胞、間葉系幹細胞、臍帯間葉系幹細胞、網膜幹細胞、神経幹細胞、外胚葉由来の神経細胞、不死化ドーパミン作動性ニューロン前駆細胞、および該細胞から生成されるまたは該細胞を含有するオルガノイドを含む、本発明1086の方法。
[本発明1089]
前記オルガノイドが、心臓オルガノイド、腸/胃腸オルガノイド、結腸オルガノイド、肝オルガノイド、腎臓オルガノイド、膀胱オルガノイド、卵巣オルガノイド、子宮頸部オルガノイド、神経オルガノイド、または肺臓（肺）オルガノイドを含む、本発明1086の方法。
[本発明1090]
前記島が、免疫系の細胞による破壊またはクリアランスから保護されたヒト死体島である、本発明1001、本発明1002、または本発明1075～1087のいずれかの方法。
[本発明1091]
その必要がある対象に、本発明1040～1057のいずれかの免疫保護された細胞、ヒト島様オルガノイドまたは膵島オルガノイドを投与する工程を含む、細胞移植の方法。
[本発明1092]
前記免疫保護された細胞、ヒト島様オルガノイドまたは膵島オルガノイドが、同系、自家、同種異系、または異種である、本発明1091の方法。
[本発明1093]
本発明1040～1057のいずれかの免疫保護された細胞、ヒト島様オルガノイドもしくは膵島オルガノイドまたは該免疫保護された細胞、ヒト島様オルガノイドもしくは膵島オルガノイドを含む薬学的に許容される組成物を含む、キット。
Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided and described herein.
[Invention 1001]
Implanted donor cells are contacted with interferon gamma (IFNγ) with multiple intermittent exposures, thereby improving the survival of the transplanted donor cells or reducing the cell death of the transplanted donor cells. process
A method of improving survival of transplanted donor cells or reducing cell death of transplanted donor cells comprising:
[Invention 1002]
1002. The method of invention 1001, wherein said donor cells are organoid cells, islet cells, islet-like organoid cells, beta-like islet cells.
[Invention 1003]
culturing endocrine progenitor cells in a three-dimensional matrix comprising Wnt4 protein or Wnt5a protein for a time sufficient to generate multicellular islet-like organoids, the multicellular islet-like organoids comprising beta (β) cells, comprising two or more cell types selected from alpha (α) cells, delta (δ) cells, epsilon (ε) cells, and duct-like cells, wherein said islet-like organoids respond to glucose by producing insulin and
subjecting the islet-like organoids to multiple intermittent exposures to interferon gamma (IFNγ), thereby inducing sustained expression of immune checkpoint proteins by the islet-like organoids and immunodetection or autoimmunity; Allowing the islet-like organoids to escape
A method of generating islet-like organoids that evade immune detection or autoimmunity, comprising:
[Invention 1004]
Culturing endocrine progenitor cells recombinantly expressing immune checkpoint proteins in a three-dimensional matrix comprising Wnt4 or Wnt5a protein for a time sufficient to generate multicellular islet-like organoids, the island-like organoids comprise two or more cell types selected from beta (β) cells, alpha (α) cells, delta (δ) cells, epsilon (ε) cells, and duct-like cells, said The islet-like organoids secrete insulin in response to glucose and evade immunodetection and autoimmunity.
A method of generating islet-like organoids that evade immune detection or autoimmunity, comprising:
[Invention 1005]
The method of Invention 1003 or Invention 1004, wherein said three-dimensional matrix comprises gellan gum.
[Invention 1006]
1005. The method of any of inventions 1003-1005, wherein said three-dimensional matrix comprises recombinant human Wnt4 protein.
[Invention 1007]
1005. The method of invention 1004, wherein recombinant expression of said immune checkpoint protein results from transduction of islet-like organoid cells with a vector containing a polynucleotide encoding said immune checkpoint protein.
[Invention 1008]
said immune checkpoint proteins bind to cognate ligands expressed on immune cells, said cognate ligands being programmed cell death protein 1 (PD-1); cytotoxic T lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG-3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3-dioxygenase 1 (IDO1); tumor necrosis factor receptor superfamily member 9 (4-1BB ); glucocorticoid-inducible TNFR family-related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4 (OX40); adenosine A2A receptor (A2AR); -H3; B7-H4; B7-1/B7-2; BTLA; V domain Ig suppressor of T cell activation (VISTA); either way.
[Invention 1009]
1008. The method of any of inventions 1003-1008, wherein said immune checkpoint protein is programmed death ligand-1 (PD-L1).
[Invention 1010]
1003. The method of any of inventions 1001-1003, wherein said cells, islets, organoids, or islet-like organoids are exposed to IFNγ at least twice over a period of at least two days.
[Invention 1011]
1003. The method of any of inventions 1001-1003, wherein said cells, islets, organoids, or islet-like organoids are exposed to IFNγ at least three times over a period of at least three days.
[Invention 1012]
1003. The method of any of inventions 1001-1003, wherein said cells, islets, organoids, or islet-like organoids are exposed to IFNγ for more than 1 hour at least twice during at least two days.
[Invention 1013]
1003. The method of any of inventions 1001-1003, wherein said cells, islets, organoids, or islet-like organoids are exposed to IFNγ for more than 1 hour at least 3 times over a period of at least 3 days.
[Invention 1014]
1013. The method of invention 1013, wherein said cells, islets, organoids, or islet-like organoids are exposed to IFNγ for 2 hours at least 3 times over a period of at least 3 days.
[Invention 1015]
1014. The method of any of the inventions 1003-1014, wherein said endocrine progenitor cells are selected from induced pluripotent stem cells (iPSC), embryonic pluripotent stem cells (ePSC), and/or pancreatic progenitor cells.
[Invention 1016]
1016. The method of any of inventions 1003-1015, wherein said endocrine progenitor cells express at least one biomarker of neurogenin 3, neurodl, Nkx2.2, and Pax4 biomarkers.
[Invention 1017]
The method of any of inventions 1002-1016, wherein said islet-like organoids are human islet-like organoids (HILO).
[Invention 1018]
1018. The method of the invention 1017, wherein said islet-like organoids are vascularized.
[Invention 1019]
1018. The method of any of inventions 1002-1017, wherein said islet-like organoids further comprise adipose-derived stem cells and/or endothelial cells.
[Invention 1020]
The method of the invention 1019, wherein said adipose-derived stem cells are human adipose-derived stem cells (hADSC) and/or said endothelial cells are human umbilical vein endothelial cells (HUVEC).
[Invention 1021]
1021. The method of any of the inventions 1002-1020, wherein said islet-like organoids further exhibit at least one of KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, glucagon secretion.
[Invention 1022]
said islet-like organoids express a beta cell lineage marker selected from the group consisting of NKX2-2, NEUROD1, RFX6, GCK, INS, NKX6-1, UCN3, MAFB, and SYT4, and an ARXα cell lineage marker; The method of any of the inventions 1002-1021.
[Invention 1023]
1004. The method of invention 1003 or invention 1004, wherein said three-dimensional matrix comprises human Wnt4 protein, recombinant human Wnt4 protein, human Wnt5 protein, or recombinant human Wnt5a protein.
[Invention 1024]
1023. The method of invention 1023, wherein said three-dimensional matrix comprises recombinant human Wnt4 protein.
[Invention 1025]
1025. The method of any of inventions 1002-1024, wherein said islet-like organoids exhibit increased expression of estrogen-related receptor gamma (ERRγ).
[Invention 1026]
1026. The method of any of inventions 1002-1025, wherein said islet-like organoids exhibit increased oxidative metabolism characterized by increased oxygen consumption rate (OCR) and decreased cellular acidification rate (ECAR).
[Invention 1027]
1027. The method of any of inventions 1002-1026, wherein said islet-like organoids are pancreatic islet organoids, pancreatic organoids, liver organoids, heart organoids, or intestinal organoids.
[Invention 1028]
1028. The method of invention 1027, wherein said islet-like organoids are human pancreatic islet organoids.
[Invention 1029]
The donor cells include heart cells, colon cells, kidney cells, liver cells (hepatocytes), esophageal cells, gastrointestinal cells, gastric (stomach) cells, lung cells, pancreatic cells, pancreatic β cells, muscle cells, hematopoietic cells, Insulin-producing pancreas derived from B cells, T cells, CD34+ hematopoietic cells, chimeric antigen receptor-T cells (CAR-T cells), myeloid cells, neurons, nerve cells, retinal cells, corneal cells, brain cells, human skin cells β cells, ovarian cells, cervical cells, testicular cells, mononuclear cells, umbilical cord blood (UCB) cells, adipose-derived mesenchymal stromal (stem) cells, cardiac stem cells, colonic stem cells, kidney stem cells, liver (hepatocytes) ) stem cells, gastrointestinal stem cells, gastric (stomach) stem cells, pulmonary stem cells, pancreatic stem cells, pancreatic β stem cells, muscle stem cells, hematopoietic stem cells, T-cell stem cells or B-cell stem cells, bone marrow stem cells, CD133+ stem cells, CD34+ hematopoietic stem cells, retinal stem cells, Neural stem cells, mesenchymal stem cells, umbilical cord mesenchymal stem cells, ectodermal-derived neurons, ectodermal-derived dopaminergic neurons, corneal-derived cells, normal human corneal epithelial cells, immortalized dopaminergic neuron progenitor cells , endoderm-derived liver cells, mesoderm-derived muscle cells, bone marrow cells, kidney cells, and skeletal muscle cells, or organoids produced from or containing such cells; intestinal organoids, liver organoids, colon organoids, The method of invention 1001 or invention 1002 selected from liver organoids, renal organoids, bladder organoids, ovarian organoids, cervical organoids, neural organoids, or lung (lung) organoids.
[Invention 1030]
(a) culturing endocrine progenitor cells in a three-dimensional matrix comprising Wnt4 or Wnt5a protein for a time sufficient to generate multicellular human islet-like organoids, wherein the multicellular human islet-like organoids are , beta (β) cells, alpha (α) cells, delta (δ) cells, epsilon (ε) cells, and duct-like cells, said human islet-like organoids comprising secreting insulin in response to glucose,
(b) contacting the HILO of step (a) with interferon gamma (IFNγ) two or three times for a total period of at least 48-72 hours, each time for more than 1 hour;
1. A method of generating human islet-like organoids (HILOs) that evade immunodetection or autoimmunity, comprising:
Human islets or HILO are maintained in the absence of IFNγ between contact times with IFNγ, and step (a) and step (b) induce immune checkpoint protein programmed death ligand-1 (PD-1) in said HILO. L1) sustained expression is induced.
[Invention 1031]
The method of invention 1030, wherein in step (b), said HILO is contacted with IFNγ for 2 hours.
[Invention 1032]
1032. The method of Invention 1030 or Invention 1031, wherein said HILO is contacted with IFNγ twice for at least 48 hours, each time for 2 hours.
[Invention 1033]
1032. The method of Invention 1030 or Invention 1031, wherein said HILO is contacted with IFNγ three times for at least 72 hours, each time for 2 hours.
[Invention 1034]
1034. The method of any of the inventions 1030-1033, wherein said endocrine progenitor cells are selected from induced pluripotent stem cells (iPSC), embryonic pluripotent stem cells (ePSC), and/or pancreatic progenitor cells.
[Invention 1035]
1034. The method of any of the inventions 1030-1034, wherein said endocrine progenitor cells express at least one biomarker of neurogenin 3, neurodl, Nkx2.2, and Pax4 biomarkers.
[Invention 1036]
1035. The method of any of invention 1030-1035, wherein said HILO is vascularized and exhibits increased oxidative metabolism characterized by increased oxygen consumption rate (OCR) and decreased cellular acidification rate (ECAR). Method.
[Invention 1037]
The method of any of inventions 1001-1036, wherein IFNγ is used in an amount of 1-25 ng/ml.
[Invention 1038]
The method of the invention 1037, wherein IFNγ is used in an amount of 10 ng/ml.
[Invention 1039]
The method of any of invention 1008-1036, wherein PD-L1 expression in said islet-like organoids or HILO is maintained for more than 7 days.
[Invention 1040]
Immunoprotected cells, human islet-like organoids or pancreatic islet organoids with sustained expression of immune checkpoint proteins,
Said immunoprotected cells, human islet-like organoids or pancreatic islet organoids, wherein said organoids are produced by the method of any of the inventions 1001-1039.
[Invention 1041]
Human islet-like or pancreatic islet organoids of the invention 1040 exhibiting sustained expression of the immune checkpoint protein PD-L1.
[Invention 1042]
Human islet-like organoids (HILOs) derived from endocrine progenitor cells cultured in a three-dimensional matrix comprising Wnt4 or Wnt5 proteins and containing multilineage cells,
said multi-lineage cells comprising at least two of beta (β) cells, alpha (α) cells, delta (δ) cells, epsilon (ε) cells, and duct-like cells; human islet-like organoids (HILO), which are isolated, exhibit glucose-stimulated insulin secretion (GSIS), and exhibit sustained expression of immune checkpoint proteins.
[Invention 1043]
A human islet-like organoid (HILO) of the invention 1042, which is an islet-like organoid or pancreatic organoid.
[Invention 1044]
A human islet-like organoid (HILO) of invention 1042 or invention 1043, further exhibiting KCl-stimulated insulin secretion or glucose-stimulated insulin secretion.
[Invention 1045]
The human islet-like organoid (HILO) of any of Inventions 1042-1044, wherein said three-dimensional matrix comprises gellan gum.
[Invention 1046]
The human islet-like organoid (HILO) of any of inventions 1042-1045, wherein said three-dimensional matrix comprises recombinant human Wnt4 protein.
[Invention 1047]
The human islet-like organoid (HILO) of any of inventions 1042-1046, wherein said three-dimensional matrix comprises recombinant human Wnt5 protein.
[Invention 1048]
The human islet-like organoid of any of the inventions 1042-1047, wherein said endocrine progenitor cells are selected from induced pluripotent stem cells (iPSC), embryonic pluripotent stem cells (ePSC), and/or pancreatic progenitor cells ( HILO).
[Invention 1049]
1048. The human islet-like organoid (HILO) of any of the inventions 1042-1048, wherein said endocrine progenitor cells express at least one biomarker among neurogenin 3, neurod1, Nkx2.2, and Pax4 biomarkers.
[Invention 1050]
The human islet-like organoid (HILO) of any of the inventions 1042-1049, expressing the FLTP gene and the ESRRγ gene.
[Invention 1051]
The human islet-like organoid (HILO) of any of the inventions 1042-1050, further comprising adipose-derived stem cells and/or endothelial cells.
[Invention 1052]
The human islet-like organoid (HILO) of the invention 1051, wherein said adipose-derived stem cells are human adipose-derived stem cells (hADSC) and/or said endothelial cells are human umbilical vein endothelial cells (HUVEC).
[Invention 1053]
The human islet-like organoid (HILO) of any of invention 1042-1052, further exhibiting KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, or glucagon secretion.
[Invention 1054]
NKX2-2, NEUROD1, RFX6, GCK, INS, NKX6-1, UCN3, MAFB, and SYT4, expressing a β cell lineage marker and an ARXα cell lineage marker of the present invention 1040-1053 Any human islet-like organoid (HILO).
[Invention 1055]
1052. The human islet-like organoid of any of the inventions 1040-1052, wherein the pancreatic HILO expresses a beta-cell transcription factor selected from the group consisting of Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2. (HILO).
[Invention 1056]
said immune checkpoint proteins bind to cognate ligands expressed on immune cells, said cognate ligands being programmed cell death protein 1 (PD-1); cytotoxic T lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG-3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3-dioxygenase 1 (IDO1); tumor necrosis factor receptor superfamily member 9 (4-1BB ); glucocorticoid-inducible TNFR family-related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4 (OX40); adenosine A2A receptor (A2AR); -H3; B7-H4; B7-1/B7-2; BTLA; V domain Ig suppressor of T cell activation (VISTA); Any human islet-like organoid (HILO).
[Invention 1057]
The human islet-like organoid (HILO) of any of the inventions 1042-1056, wherein said immune checkpoint protein is programmed death ligand-1 (PD-L1).
[Invention 1058]
A non-human organism transplanted or implanted with said human islet-like organoids, pancreatic islet organoids, or HILOs of any of the inventions 1042-1057.
[Invention 1059]
The non-human organism of the invention 1058, which is a mammal.
[Invention 1060]
The non-human organism of the invention 1058, which is a mouse.
[Invention 1061]
1. A method of treating pancreatic disease in a subject comprising transplanting or implanting immunoprotected islet-like or pancreatic islet organoids into the subject, comprising:
The islet-like or pancreatic islet organoids comprise multi-lineage cells derived from endocrine progenitor cells including beta-cells, alpha-cells, delta-cells, epsilon-cells, duct-like cells, or combinations thereof, are vascularized, and are glucose-stimulated. The method exhibits insulin secretion (GSIS) and persistent expression of immune checkpoint proteins to evade immunodetection or autoimmunity.
[Invention 1062]
1. A method of treating type 1 diabetes in a subject comprising transplanting or implanting immunoprotected islet-like or pancreatic islet organoids into the subject, comprising:
The islet-like or pancreatic islet organoids comprise multi-lineage cells derived from endocrine progenitor cells including beta-cells, alpha-cells, delta-cells, epsilon-cells, duct-like cells, or combinations thereof, are vascularized, and are glucose-stimulated. The method exhibits insulin secretion (GSIS) and persistent expression of immune checkpoint proteins to evade immunodetection or autoimmunity.
[Invention 1063]
The method of invention 1061 or invention 1062, wherein said islet-like or pancreatic islet organoids further exhibit KCl-stimulated insulin secretion, GLP-1-stimulated insulin secretion, somatostatin secretion, or glucagon secretion.
[Invention 1064]
wherein said islet-like or pancreatic islet organoids have a β cell lineage marker selected from the group consisting of NKX2-2, NEUROD1, RFX6, GCK, INS, NKX6-1, UCN3, MAFB, and SYT4, and an ARXα cell lineage marker. The method of any of the inventions 1061-1063, which expresses.
[Invention 1065]
1064. The method of any of the inventions 1061-1064, wherein said endocrine progenitor cells are selected from induced pluripotent stem cells (iPSC), embryonic pluripotent stem cells (ePSC), and/or pancreatic progenitor cells.
[Invention 1066]
1065. The method of any of the inventions 1061-1065, wherein said endocrine progenitor cells express at least one biomarker of neurogenin 3, neurodl, Nkx2.2, and Pax4 biomarkers.
[Invention 1067]
1066. Any of the inventions 1061-1066, wherein said islet-like or pancreatic islet organoids express a beta-cell transcription factor selected from the group consisting of Pdx1, MafA, Pax4, Pax6, NeuroD1, Nkx6-1, Gata6, and Foxa2. the method of.
[Invention 1068]
said immune checkpoint proteins bind to cognate ligands expressed on immune cells, said cognate ligands being programmed cell death protein 1 (PD-1); cytotoxic T lymphocyte protein 4 (CTLA-4); lymphocyte activation gene 3 protein (LAG-3); killer cell immunoglobulin-like receptor (KIR); indoleamine 2,3-dioxygenase 1 (IDO1); tumor necrosis factor receptor superfamily member 9 (4-1BB ); glucocorticoid-inducible TNFR family-related gene (GITR); T-cell immunoglobulin domain and mucin domain (TIM-3); tumor necrosis factor receptor superfamily member 4 (OX40); adenosine A2A receptor (A2AR); -H3; B7-H4; B7-1/B7-2; BTLA; V domain Ig suppressor of T cell activation (VISTA); the method of.
[Invention 1069]
The method of any of inventions 1061-1068, wherein said immune checkpoint protein is programmed death ligand-1 (PD-L1).
[Invention 1070]
The method of any of inventions 1061-1069, wherein said islet-like or pancreatic islet organoids are produced by any of the methods of inventions 1001-1037.
[Invention 1071]
The method of any of inventions 1061-1070, wherein said islet-like or pancreatic islet organoid is an organoid of any of inventions 1040-1057.
[Invention 1072]
The method of any of inventions 1061-1071, wherein an immunosuppressive substance is administered to said subject.
[Invention 1073]
The method of any of inventions 1061-1072, wherein said subject is a human.
[Invention 1074]
The method of Invention 1061 or any of Inventions 1063-1073, wherein said pancreatic disease is type 1 diabetes or type 2 diabetes.
[Invention 1075]
A method of producing cells, islets, or organoids that survive and have reduced cell death after transplantation or implantation, comprising:
(a) contacting interferon gamma (IFNγ) receptor-expressing cells, islets, or organoids with interferon gamma (IFNγ) at predetermined time points for at least 0.5 hours or at least 1 hour; and
(b) repeating step (a) at least twice during a period of about 72 hours or at least about 72 hours;
including
The cells, islets, or organoids are maintained in the absence of IFNγ between contact times with IFNγ, and step (a) and step (b) result in the reduction of PD-L1 in the cells, islets, or organoids. persistent expression is induced,
the method.
[Invention 1076]
1075. The method of invention 1075, wherein in step (a), said cells, islets, organoids, or cells are contacted with IFNγ for a period of time selected from at least 1 hour, at least 2 hours, or greater than 2 hours.
[Invention 1077]
1076. The method of invention 1075 or invention 1076, wherein in step (a) said cells, islets or organoids are contacted with IFNγ for a period of time selected from about 2 hours or 2 hours or about 12 hours or 12 hours.
[Invention 1078]
The method of any of Inventions 1075-1077, wherein step (a) is repeated at least 3 times during the period of at least about 72 hours or at least 72 hours of step (b), each time for at least about 2 hours.
[Invention 1079]
The method of any of inventions 1075-1078, wherein between steps (a) and (b) the cells, islets or organoids are washed to remove the presence of IFNγ.
[Invention 1080]
The method of any of inventions 1075-1079, wherein IFNγ is used in an amount of 1-25 ng/ml.
[Invention 1081]
The method of any of inventions 1075-1079, wherein IFNγ is used in an amount of 10 ng/ml.
[Invention 1082]
The method of any of inventions 1075-1081, wherein PD-L1 expression in said cells, islets, or organoids is maintained for more than about 7 days after step (b).
[Invention 1083]
1. A method of generating cells, islets, or organoids and cells thereof that evade immunodetection or autoimmunity, comprising:
(a) interferon gamma (IFNγ) receptor-expressing cells, islets, or organoids and those cells for at least about 24 hours or at a first time point during a period of at least 24 hours for more than 1 hour; contacting with an amount of interferon gamma (IFNγ) from ml to 25ng/ml, and
(b) treating the cells, islets, or organoids and their cells for about 0.5 hours or more at two or more additional time points during a subsequent period of at least about 48 hours after step (a); contacting with IFNγ in an amount of about 1 ng/ml to 25 ng/ml for more than a period of time
including
Between contacting with IFNγ, the cells, islets, or organoids are washed and allowed to rest in medium in the absence of IFNγ, and by steps (a) and (b), the cells, islets, or persistent expression of PD-L1 in organoids is induced,
the method.
[Invention 1084]
1083. The method of invention 1083, wherein in steps (a) and (b), said cells, islets or organoids are contacted with IFNγ in an amount of 10 ng/ml for at least 2 hours.
[Invention 1085]
The method of invention 1083 or invention 1084, wherein said cells, islets, or organoids are contacted with IFNγ for at least about 2 hours at 3 time points during a 72 hour period.
[Invention 1086]
The method of Invention 1001 or any of Inventions 1075-1085, wherein said cells, islets or organoids are human cells, islets or organoids.
[Invention 1087]
The method of invention 1086, wherein said organoid is HILO or human HILO.
[Invention 1088]
said cells are heart cells, colon cells, kidney cells, bladder cells, liver cells (hepatocytes), esophageal cells, gastrointestinal cells, gastric (stomach) cells, lung cells, ovarian cells, cervical cells, uterine cells, Testicular cells, pancreatic cells, pancreatic β cells, retinal cells, corneal cells, brain cells, muscle cells, hematopoietic cells, immune cells (B cells, T cells), chimeric antigen receptor-T cells (CAR-T cells), bone marrow cells, mononuclear cells, neurons, nerve cells, insulin-producing pancreatic beta cells derived from human skin cells, umbilical cord blood (UCB) cells, adipose-derived mesenchymal stromal (stem) cells, cardiac stem cells, colonic stem cells, kidney stem cells , liver (hepatocyte) stem cells, gastrointestinal stem cells, gastric stem cells, pulmonary stem cells, pancreatic stem cells, pancreatic β stem cells, muscle stem cells, hematopoietic stem cells, immune cell (T or B cell) stem cells, bone marrow stem cells, CD133+ stem cells, CD34+ hematopoietic cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, umbilical cord mesenchymal stem cells, retinal stem cells, neural stem cells, neuronal cells of ectodermal origin, immortalized dopaminergic neuronal progenitor cells, and cells generated from or comprising said cells The method of the invention 1086, comprising containing organoids.
[Invention 1089]
1086. The method of invention 1086, wherein said organoid comprises a heart organoid, an intestinal/gastrointestinal organoid, a colon organoid, a liver organoid, a kidney organoid, a bladder organoid, an ovarian organoid, a cervical organoid, a neuronal organoid, or a pulmonary (lung) organoid.
[Invention 1090]
The method of any of invention 1001, invention 1002, or inventions 1075-1087, wherein said islets are human cadaver islets protected from destruction or clearance by cells of the immune system.
[Invention 1091]
A method of cell transplantation comprising administering to a subject in need thereof the immunoprotected cells, human islet-like organoids or pancreatic islet organoids of any of the inventions 1040-1057.
[Invention 1092]
1091. The method of invention 1091, wherein said immunoprotected cells, human islet-like organoids or pancreatic islet organoids are syngeneic, autologous, allogeneic, or xenogeneic.
[Invention 1093]
immunoprotected cells, human islet-like organoids or pancreatic islet organoids of any of the inventions 1040-1057 or a pharmaceutically acceptable composition comprising said immunoprotected cells, human islet-like organoids or pancreatic islet organoids, kit.
Claims (49)
を含む、対象に投与された、移植された、もしくは埋植されたドナー細胞の生残を向上させるかまたは対象に投与された、移植された、もしくは埋植されたドナー細胞の細胞死を低減する方法。 Prior to administration, transplantation, or implantation, the donor cells are contacted with interferon gamma (IFNγ) in vitro or ex vivo with multiple intermittent exposures, thereby administering, transplanting , or implanting administering, transplanting or implanting to a subject, comprising enhancing survival of the administered donor cells or reducing cell death of the administered, transplanted or implanted donor cells. methods of enhancing survival of administered donor cells or reducing cell death of transplanted or implanted donor cells administered to a subject .
を含む、対象への投与もしくは移植、または対象における埋植の後に免疫検出または自己免疫を回避する、免疫保護された細胞、島、またはオルガノイドを生成させる方法。 subjecting an IFNγ receptor-expressing cell, islet, or organoid to multiple intermittent exposures to interferon-γ (IFNγ), wherein the multiple intermittent exposures to IFNγ are the cells, islets, or organoids; inducing sustained expression of immune checkpoint proteins by islets, or organoids, thereby allowing the cells, islets, or organoids to evade immune detection or autoimmunity . Methods of generating immunoprotected cells, islets, or organoids that evade immune detection or autoimmunity following administration or transplantation or implantation in a subject .
該オルガノイドが、請求項1～27のいずれか一項記載の方法によって産生される、該免疫保護された細胞、島またはオルガノイド。 Immunoprotected cells, islets or organoids with persistent expression of immune checkpoint proteins,
28. The immunoprotected cells, islets or organoids , wherein the organoids are produced by the method of any one of claims 1-27 .
該多系列細胞が、ベータ（β）細胞、アルファ（α）細胞、デルタ（δ）細胞、イプシロン（ε）細胞、および導管様細胞のうちの少なくとも2つを含み、該HILOが、血管新生化され、グルコース刺激インスリン分泌（GSIS）を呈し、かつ免疫チェックポイントタンパク質の持続的発現を呈する、該ヒト島様オルガノイド（HILO）。 Human islet-like organoids (HILOs) derived from endocrine progenitor cells cultured in a three-dimensional matrix comprising Wnt4 or Wnt5 proteins , or fragments thereof, and comprising multilineage cells, wherein
said multi-lineage cells comprising at least two of beta (β) cells, alpha (α) cells, delta (δ) cells, epsilon (ε) cells, and duct-like cells; human islet-like organoids (HILO), which are isolated, exhibit glucose-stimulated insulin secretion (GSIS), and exhibit sustained expression of immune checkpoint proteins.
該島様オルガノイドまたは膵島オルガノイドが、β細胞、α細胞、δ細胞、ε細胞、導管様細胞、またはそれらの組合せを含み内分泌前駆細胞に由来する多系列細胞を含み、血管新生化され、グルコース刺激インスリン分泌（GSIS）を呈し、かつ免疫検出または自己免疫を回避するために免疫チェックポイントタンパク質の持続的発現を呈する、該薬学的組成物。 A pharmaceutical composition for treating pancreatic disease in a subject comprising immunoprotected islet-like or pancreatic islet organoids, comprising :
The islet-like or pancreatic islet organoids comprise multi-lineage cells derived from endocrine progenitor cells including beta-cells, alpha-cells, delta-cells, epsilon-cells, duct-like cells, or combinations thereof, are vascularized, and are glucose-stimulated. Said pharmaceutical composition exhibiting insulin secretion (GSIS) and persistent expression of immune checkpoint proteins to avoid immune detection or autoimmunity.
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| KR102508156B1 (en) * | 2021-03-24 | 2023-03-09 | 연세대학교 산학협력단 | A Composition for Predicting Future Onset of Pancreatic Cancer |
| US20240207325A1 (en) * | 2021-04-12 | 2024-06-27 | Biocrine Ab | Pancreatic Islets as Protein Factories |
| CN114107196B (en) * | 2021-11-01 | 2024-06-11 | 浙江大学 | Method for differentiating natural killer cells by utilizing pluripotent stem cells based on single cell sequencing rational design and application of CSF1R inhibitor |
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