JPWO2021203009A5 - - Google Patents
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- JPWO2021203009A5 JPWO2021203009A5 JP2022559927A JP2022559927A JPWO2021203009A5 JP WO2021203009 A5 JPWO2021203009 A5 JP WO2021203009A5 JP 2022559927 A JP2022559927 A JP 2022559927A JP 2022559927 A JP2022559927 A JP 2022559927A JP WO2021203009 A5 JPWO2021203009 A5 JP WO2021203009A5
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Description
様々な特許、特許出願、刊行物、製品説明、プロトコルおよび配列アクセッション番号が本出願全体にわたり引用され、その本開示は、あらゆる目的のためにその全体が参照により本明細書に組み込まれる。
本発明は、例えば、以下の項目を提供する。
(項目１)
幹細胞の分化を誘導するためのインビトロ方法であって、
前記幹細胞を、少なくとも１つのＳｍａｌｌ Ｍｏｔｈｅｒｓ Ａｇａｉｎｓｔ Ｄｅｃａｐｅｎｔａｐｌｅｇｉｃ（ＳＭＡＤ）シグナル伝達阻害剤、少なくとも１つのソニックヘッジホッグ（ＳＨＨ）シグナル伝達活性化剤、および少なくとも１つのウィングレス（Ｗｎｔ）シグナル伝達活性化剤に接触させる工程、ならびに
前記細胞を、少なくとも１つの線維芽細胞増殖因子（ＦＧＦ）シグナル伝達活性化剤および少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させて、中脳ドーパミンニューロンまたはその前駆体を示す少なくとも１つのマーカーを発現する分化細胞の集団を得る工程
を含む、方法。
(項目２)
前記細胞と前記少なくとも１つのＷｎｔシグナル伝達阻害剤との前記接触が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から少なくとも約５日で開始される、項目１に記載の方法。
(項目３)
前記細胞と前記少なくとも１つのＷｎｔシグナル伝達阻害剤との前記接触が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から約１５日以内に開始される、項目１または２に記載の方法。
(項目４)
前記細胞と前記少なくとも１つのＷｎｔシグナル伝達阻害剤との前記接触が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から約１０日で開始される、項目１～３のいずれか１項に記載の方法。
(項目５)
前記細胞と前記少なくとも１つのＷｎｔシグナル伝達阻害剤との前記接触が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から１０日、１１日、１２日、または１３日で開始される、項目１～４のいずれか１項に記載の方法。
(項目６)
前記細胞を、少なくとも約１日、前記少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させる、項目１～５のいずれか１項に記載の方法。
(項目７)
前記細胞を、最大約３０日間または最大約２５日間、前記少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させる、項目１～６のいずれか１項に記載の方法。
(項目８)
前記細胞を、約５日間、約１５日間、または約２０日間、前記少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させる、項目１～７のいずれか１項に記載の方法。
(項目９)
前記細胞を、４日間、５日間、６日間、７日間、１４日間、１５日間、１９日間または２０日間、前記少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させる、項目１～８のいずれか１項に記載の方法。
(項目１０)
前記細胞と前記少なくとも１つのＦＧＦシグナル伝達活性化剤との前記接触が、前記細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から少なくとも約５日または少なくとも約１０日で開始される、項目１～９のいずれか１項に記載の方法。
(項目１１)
前記細胞と前記少なくとも１つのＦＧＦシグナル伝達活性化剤との前記接触が、前記細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から約２０日以内または１８日以内に開始される、項目１～１０のいずれか１項に記載の方法。
(項目１２)
前記細胞と前記少なくとも１つのＦＧＦシグナル伝達活性化剤との前記接触が、前記細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から約１０日で開始される、項目１～１１のいずれか１項に記載の方法。
(項目１３)
前記細胞と前記少なくとも１つのＦＧＦシグナル伝達活性化剤との前記接触が、前記細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から１０日、１１日、１２日、または１３日で開始される、項目１～１２のいずれか１項に記載の方法。
(項目１４)
前記細胞を、少なくとも約１日および／もしくは最大約２０日間、少なくとも約３日間および／もしくは最大約１０日間、または少なくとも４日間および／もしくは最大７日間、前記少なくとも１つのＦＧＦシグナル伝達活性化剤と接触させる、項目１～１３のいずれか１項に記載の方法。
(項目１５)
前記細胞を、約５日間、前記少なくとも１つのＦＧＦシグナル伝達活性化剤と接触させる、項目１～１４のいずれか１項に記載の方法。
(項目１６)
前記細胞を、４日間、５日間、６日間、または７日間、前記少なくとも１つのＦＧＦシグナル伝達活性化剤と接触させる、項目１～１５のいずれか１項に記載の方法。
(項目１７)
前記細胞を、約５日間、前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤と接触させる、項目１～１６のいずれか１項に記載の方法。
(項目１８)
前記細胞を、６日間または７日間、前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤と接触させる、項目１～１７のいずれか１項に記載の方法。
(項目１９)
前記細胞を、約５日間、前記少なくとも１つのＳＨＨシグナル伝達活性化剤と接触させる、項目１～１８のいずれか１項に記載の方法。
(項目２０)
前記細胞を、６日間または７日間、前記少なくとも１つのＳＨＨシグナル伝達活性化剤と接触させる、項目１～１９のいずれか１項に記載の方法。
(項目２１)
前記細胞を、約１５日間、前記少なくとも１つのＷｎｔシグナル伝達活性化剤と接触させる、項目１～２０のいずれか１項に記載の方法。
(項目２２)
前記細胞を、１６日間または１７日間、前記少なくとも１つのＷｎｔシグナル伝達活性化剤と接触させる、項目１～２１のいずれか１項に記載の方法。
(項目２３)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤の濃度が、前記幹細胞とのその最初の接触から約４日増加する、項目１～２２のいずれか１項に記載の方法。
(項目２４)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤の前記濃度が、前記少なくとも１つのＷｎｔシグナル伝達活性化剤の初期濃度から約２００％～約１０００％増加する、項目２３に記載の方法。
(項目２５)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤の前記濃度が、前記少なくとも１つのＷｎｔシグナル伝達活性化剤の初期濃度から約５００％増加する、項目２３または２４に記載の方法。
(項目２６)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤の前記濃度が、約１μＭから約５μＭと約１０μＭとの間に増加する、項目２３～２５のいずれか１項に記載の方法。
(項目２７)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤の前記濃度が、約６μＭの濃度に増加する、項目２３～２６のいずれか１項に記載の方法。
(項目２８)
前記少なくとも１つのＷｎｔシグナル伝達阻害剤が、非古典的Ｗｎｔシグナル伝達および古典的Ｗｎｔシグナル伝達を阻害することができる、項目１～２７のいずれか１項に記載の方法。
(項目２９)
前記少なくとも１つのＷｎｔシグナル伝達阻害剤が、ＩＷＰ２、ＩＷＲ１－ｅｎｄｏ、ＸＡＶ９３９、ＩＷＰ－Ｏ１、Ｗｎｔ－Ｃ５９、ＩＷＰ－Ｌ６、およびＩＣＧ－００１、ならびにそれらの組み合わせからなる群から選択される、項目１～２８のいずれか１項に記載の方法。
(項目３０)
前記少なくとも１つのＷｎｔシグナル伝達阻害剤がＩＷＰ２を含む、項目１～２９のいずれか１項に記載の方法。
(項目３１)
前記少なくとも１つのＦＧＦシグナル伝達活性化剤が、ＦＧＦ１８、ＦＧＦ１７、ＦＧＦ８ａ、ＦＧＦ８ｂ、ＦＧＦ４、ＦＧＦ２、およびそれらの組み合わせからなる群から選択される、項目１～３０のいずれか１項に記載の方法。
(項目３２)
前記少なくとも１つのＦＧＦシグナル伝達活性化剤が、中脳の拡大を引き起こし、中脳遺伝子発現を上方制御することができる、項目１～３１のいずれか１項に記載の方法。
(項目３３)
前記少なくとも１つのＦＧＦシグナル伝達活性化剤が、ＦＧＦ１８、ＦＧＦ１７、ＦＧＦ８ａ、ＦＧＦ４、ＦＧＦ２、およびそれらの組み合わせからなる群から選択される、項目３２に記載の方法。
(項目３４)
前記少なくとも１つのＦＧＦシグナル伝達活性化剤がＦＧＦ１８を含む、項目３３に記載の方法。
(項目３５)
前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤が、ＴＧＦβ／Ａｃｔｉｖｉｎ－Ｎｏｄａｌシグナル伝達阻害剤、骨形成タンパク質（ＢＭＰ）シグナル伝達阻害剤、またはそれらの組み合わせを含む、項目１～３４のいずれか１項に記載の方法。
(項目３６)
前記少なくとも１つのＴＧＦβ／Ａｃｔｉｖｉｎ－Ｎｏｄａｌシグナル伝達阻害剤が、ＡＬＫ５の阻害剤を含む、項目３５に記載の方法。
(項目３７)
前記少なくとも１つのＴＧＦβ／Ａｃｔｉｖｉｎ－Ｎｏｄａｌシグナル伝達阻害剤が、ＳＢ４３１５４２、ＳＢ４３１５４２の誘導体、およびそれらの組み合わせからなる群から選択される、項目３５または３６に記載の方法。
(項目３８)
前記ＳＢ４３１５４２の誘導体がＡ８３－０１を含む、項目３７に記載の方法。
(項目３９)
前記少なくとも１つのＴＧＦβ／Ａｃｔｉｖｉｎ－Ｎｏｄａｌシグナル伝達阻害剤が、ＳＢ４３１５４２を含む、項目３５～３８のいずれか１項に記載の方法。
(項目４０)
前記少なくとも１つのＢＭＰシグナル伝達阻害剤が、ＬＤＮ１９３１８９、Ｎｏｇｇｉｎ、ドルソモルフィン、ＬＤＮ１９３１８９の誘導体、Ｎｏｇｇｉｎの誘導体、ドルソモルフィンの誘導体、およびそれらの組み合わせからなる群から選択される、項目３５に記載の方法。
(項目４１)
前記少なくとも１つのＢＭＰ阻害剤がＬＤＮ－１９３１８９を含む、項目３５または４０に記載の方法。
(項目４２)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤が、グリコーゲンシンターゼキナーゼ３β（ＧＳＫ３β）シグナル伝達阻害剤を含む、項目１～４１のいずれか１項に記載の方法。
(項目４３)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤が、ＣＨＩＲ９９０２１、ＣＨＩＲ９８０１４、ＡＭＢＭＰ塩酸塩、ＬＰ ９２２０５６、リチウム、デオキシコール酸、ＢＩＯ、ＳＢ－２１６７６３、Ｗｎｔ３Ａ、Ｗｎｔ１、Ｗｎｔ５ａ、それらの誘導体、およびそれらの組み合わせからなる群から選択される、項目１～４２のいずれか１項に記載の方法。
(項目４４)
前記少なくとも１つのＷｎｔシグナル伝達活性化剤がＣＨＩＲ９９０２１を含む、項目１～４３のいずれか１項に記載の方法。
(項目４５)
前記少なくとも１つのＳＨＨシグナル伝達活性化剤が、ＳＨＨタンパク質、Ｓｍｏｏｔｈｅｎｅｄアゴニスト（ＳＡＧ）、およびそれらの組み合わせからなる群から選択される、項目１～４４のいずれか１項に記載の方法。
(項目４６)
前記ＳＨＨタンパク質が、組換えＳＨＨ、修飾Ｎ末端ＳＨＨ、およびそれらの組み合わせからなる群から選択される、項目４５に記載の方法。
(項目４７)
前記修飾Ｎ末端ＳＨＨが、Ｎ末端に２つのイソロイシンを含む、項目４６に記載の方法。
(項目４８)
前記修飾Ｎ末端ＳＨＨが、非修飾Ｎ末端ＳＨＨと少なくとも約９０％の配列同一性を有する、項目４６または４７に記載の方法。
(項目４９)
前記非修飾Ｎ末端ＳＨＨが、非修飾マウスＮ末端ＳＨＨまたは非修飾ヒトＮ末端ＳＨＨである、項目４８に記載の方法。
(項目５０)
修飾Ｎ末端ＳＨＨがＳＨＨ Ｃ２５ＩＩを含む、項目４６～４９のいずれか１項に記載の方法。
(項目５１)
前記ＳＡＧがパルモルファミンを含む、項目４５に記載の方法。
(項目５２)
前記分化細胞の少なくとも約８０％が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から約１５日でＦＯＸＡ２およびＥＮ１を発現する、項目１～５１のいずれか１項に記載の方法。
(項目５３)
前記分化細胞の約８０％超または約９０％超が、前記幹細胞と前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤との最初の接触から１６日でＦＯＸＡ２およびＥＮ１を発現する、項目１～５２のいずれか１項に記載の方法。
(項目５４)
前記中脳ドーパミンニューロンまたはその前駆体を示す少なくとも１つのマーカーが、ＥＮ１、ＯＴＸ２、ＴＨ、ＮＵＲＲ１、ＦＯＸＡ２、ＰＩＴＸ３、ＬＭＸ１Ａ、ＬＭＯ３、ＳＮＣＡ、ＡＤＣＡＰ１、ＣＨＲＮＡ４、ＳＯＸ６、ＤＡＴ、ＶＭＡＴ２、ＷＮＴ１、ＧＩＲＫ２、およびそれらの組み合わせからなる群から選択される、項目１～５３のいずれか１項に記載の方法。
(項目５５)
前記分化細胞が、ＰＡＸ６、ＥＭＸ２、ＬＨＸ２、ＳＭＡ、ＳＩＸ１、ＰＩＴＸ２、ＳＩＭ１、ＰＯＵ４Ｆ１、ＰＨＯＸ２Ａ、ＢＡＲＨＬ１、ＢＡＲＨＬ２、ＧＢＸ２、ＨＯＸＡ１、ＨＯＸＡ２、ＨＯＸＢ１、ＨＯＸＢ２、ＰＯＵ５Ｆ１、ＮＡＮＯＧ、およびそれらの組み合わせからなる群から選択される少なくとも１つのマーカーを発現しない、項目１～５４のいずれか１項に記載の方法。
(項目５６)
少なくとも１つの陽性表面マーカーを発現し、少なくとも１つの陰性表面マーカーを発現しない細胞を単離することをさらに含む、項目１～５５のいずれか１項に記載の方法。
(項目５７)
前記少なくとも１つの陽性表面マーカーが、ＣＤ１７１、ＣＤ１８４、およびそれらの組み合わせからなる群から選択される、項目５６に記載の方法。
(項目５８)
前記少なくとも１つの陽性表面マーカーがＣＤ１８４を含む、項目５６または５７に記載の方法。
(項目５９)
前記少なくとも１つの陰性表面マーカーが、ＣＤ４９ｅ、ＣＤ９９、ＣＤ３４０、およびそれらの組み合わせから選択される、項目５６～５８のいずれか１項に記載の方法。
(項目６０)
前記少なくとも１つの陰性表面マーカーがＣＤ４９ｅを含む、項目５６～５９のいずれか１項に記載の方法。
(項目６１)
ＣＤ１８４を発現し、ＣＤ４９ｅを発現しない細胞を選別することを含む、項目５６～６０のいずれか１項に記載の方法。
(項目６２)
前記幹細胞が多能性幹細胞である、項目１～６１のいずれか１項に記載の方法。
(項目６３)
前記幹細胞が、非胚性幹細胞、胚性幹細胞、人工多能性幹細胞、およびそれらの組み合わせからなる群から選択される、項目１～６２のいずれか１項に記載の方法。
(項目６４)
前記幹細胞が、ヒト幹細胞、非ヒト霊長類幹細胞、または齧歯類幹細胞である、項目１～６３のいずれか１項に記載の方法。
(項目６５)
前記幹細胞がヒト幹細胞である、項目１～６４のいずれか１項に記載の方法。
(項目６６)
項目１～６５のいずれか１項に記載の方法によって得られるインビトロ分化細胞の細胞集団。
(項目６７)
項目６６に記載の細胞集団を含む組成物。
(項目６８)
薬学的に許容され得る担体をさらに含む医薬組成物である、項目６７に記載の組成物。
(項目６９)
中脳ドーパミンニューロンまたはその前駆体への幹細胞の分化を誘導するためのキットであって、
（ａ）少なくとも１つのＳＭＡＤシグナル伝達阻害剤、
（ｂ）少なくとも１つのＳＨＨシグナル伝達活性化剤、
（ｃ）少なくとも１つのＷｎｔシグナル伝達活性化剤、
（ｄ）少なくとも１つのＷｎｔシグナル伝達阻害剤、および
（ｅ）少なくとも１つのＦＧＦシグナル伝達活性化剤
を含む、キット。
(項目７０)
（ｆ）中脳ドーパミンニューロンまたはその前駆体を示す少なくとも１つのマーカーを発現する分化細胞の集団への幹細胞の分化を誘導するための説明書をさらに含む、項目６９に記載のキット。
(項目７１)
神経障害を有する対象における少なくとも１つの徴候を予防、モデリングおよび／または処置する方法であって、有効量の以下：
（ａ）項目６６に記載の細胞集団；または
（ｂ）項目６７もしくは６８に記載の組成物
の１つを前記対象に投与することを含む、方法。
(項目７２)
前記神経障害が、中脳ドーパミンニューロン機能の低下を特徴とする、項目７１に記載の方法。
(項目７３)
前記中脳ドーパミンニューロン機能の低下が年齢に関連する、項目７２に記載の方法。
(項目７４)
前記神経障害が、パーキンソニズム、パーキンソン病、ハンチントン病、アルツハイマー病、多発性硬化症、およびそれらの組み合わせからなる群から選択される、項目７１～７３のいずれか１項に記載の方法。
(項目７５)
前記神経障害が、パーキンソニズム、パーキンソン病、およびそれらの組み合わせからなる群から選択される、項目７１～７４のいずれか１項に記載の方法。
(項目７６)
神経障害の前記徴候が、振戦、運動緩慢、屈曲姿勢、姿勢不安定性、強直、嚥下障害および認知症からなる群から選択される、項目７１～７５のいずれか１項に記載の方法。
(項目７７)
対象において、神経障害を有する対象における少なくとも１つの徴候を予防、モデリングおよび／または処置する使用のための、項目６６に記載の細胞集団または項目６７もしくは６８に記載の組成物。
(項目７８)
前記神経障害が、中脳ドーパミンニューロン機能の低下を特徴とする、項目７７に記載の使用のための細胞集団または組成物。
(項目７９)
前記中脳ドーパミンニューロン機能の低下が年齢に関連する、項目７８に記載の使用のための細胞集団または組成物。
(項目８０)
前記神経障害が、パーキンソニズム、パーキンソン病、ハンチントン病、アルツハイマー病、多発性硬化症、およびそれらの組み合わせからなる群から選択される、項目７７～７９のいずれか１項に記載の使用のための細胞集団または組成物。
(項目８１)
前記神経障害が、パーキンソニズム、パーキンソン病、およびそれらの組み合わせからなる群から選択される、項目７７～８０のいずれか１項に記載の使用のための細胞集団または組成物。
(項目８２)
神経障害の前記徴候が、振戦、運動緩慢、屈曲姿勢、姿勢不安定性、強直、嚥下障害および認知症からなる群から選択される、項目７７～８１のいずれか１項に記載の使用のための細胞集団または組成物。
Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
The present invention provides, for example, the following items.
(Item 1)
1. An in vitro method for inducing differentiation of a stem cell, comprising:
contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and
contacting said cells with at least one activator of fibroblast growth factor (FGF) signaling and at least one inhibitor of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons or precursors thereof.
A method comprising:
(Item 2)
2. The method of claim 1, wherein said contacting of said cells with said at least one Wnt signaling inhibitor is initiated at least about 5 days after initial contacting of said stem cells with said at least one SMAD signaling inhibitor.
(Item 3)
3. The method of claim 1 or 2, wherein the contacting of the cells with the at least one Wnt signaling inhibitor is initiated within about 15 days of first contacting the stem cells with the at least one SMAD signaling inhibitor.
(Item 4)
4. The method of any one of items 1 to 3, wherein said contacting of said cells with said at least one Wnt signaling inhibitor is initiated about 10 days after initial contacting of said stem cells with said at least one SMAD signaling inhibitor.
(Item 5)
5. The method of any one of items 1 to 4, wherein said contacting of said cells with said at least one Wnt signaling inhibitor is initiated 10 days, 11 days, 12 days, or 13 days after initial contacting of said stem cells with said at least one SMAD signaling inhibitor.
(Item 6)
6. The method of any one of items 1 to 5, wherein said cells are contacted with said at least one inhibitor of Wnt signaling for at least about 1 day.
(Item 7)
7. The method of any one of items 1 to 6, wherein said cells are contacted with said at least one inhibitor of Wnt signaling for up to about 30 days or up to about 25 days.
(Item 8)
8. The method of any one of items 1 to 7, wherein said cells are contacted with said at least one inhibitor of Wnt signaling for about 5 days, about 15 days, or about 20 days.
(Item 9)
9. The method according to any one of items 1 to 8, wherein said cells are contacted with said at least one inhibitor of Wnt signalling for 4 days, 5 days, 6 days, 7 days, 14 days, 15 days, 19 days or 20 days.
(Item 10)
10. The method of any one of items 1 to 9, wherein said contacting of said cells with said at least one FGF signaling activator is initiated at least about 5 days or at least about 10 days after initial contacting of said cells with said at least one SMAD signaling inhibitor.
(Item 11)
11. The method of any one of items 1 to 10, wherein said contacting of said cells with said at least one FGF signaling activator is initiated within about 20 days or within 18 days of first contacting said cells with said at least one SMAD signaling inhibitor.
(Item 12)
12. The method of any one of items 1 to 11, wherein said contacting of said cells with said at least one FGF signaling activator is initiated about 10 days after initial contacting of said cells with said at least one SMAD signaling inhibitor.
(Item 13)
13. The method of any one of items 1 to 12, wherein said contacting of said cells with said at least one FGF signaling activator is initiated 10, 11, 12, or 13 days after initial contacting of said cells with said at least one SMAD signaling inhibitor.
(Item 14)
14. The method according to any one of items 1 to 13, wherein said cells are contacted with said at least one FGF signaling activator for at least about 1 day and/or up to about 20 days, at least about 3 days and/or up to about 10 days, or at least 4 days and/or up to 7 days.
(Item 15)
15. The method according to any one of items 1 to 14, wherein said cells are contacted with said at least one FGF signaling activator for about 5 days.
(Item 16)
16. The method according to any one of items 1 to 15, wherein said cells are contacted with said at least one FGF signaling activator for 4 days, 5 days, 6 days, or 7 days.
(Item 17)
17. The method of any one of items 1 to 16, wherein said cells are contacted with said at least one SMAD signaling inhibitor for about 5 days.
(Item 18)
18. The method of any one of items 1 to 17, wherein said cells are contacted with said at least one SMAD signaling inhibitor for 6 or 7 days.
(Item 19)
19. The method according to any of the preceding items, wherein said cells are contacted with said at least one SHH signalling activator for about 5 days.
(Item 20)
20. The method according to any of the preceding items, wherein said cells are contacted with said at least one SHH signalling activator for 6 or 7 days.
(Item 21)
21. The method according to any of items 1 to 20, wherein said cells are contacted with said at least one activator of Wnt signalling for about 15 days.
(Item 22)
22. The method according to any of the preceding items, wherein said cells are contacted with said at least one activator of Wnt signalling for 16 or 17 days.
(Item 23)
23. The method according to any one of items 1 to 22, wherein the concentration of said at least one activator of Wnt signalling is increased about 4 days from its first contact with said stem cells.
(Item 24)
24. The method of claim 23, wherein said concentration of said at least one Wnt signaling activator is increased by about 200% to about 1000% from an initial concentration of said at least one Wnt signaling activator.
(Item 25)
25. The method according to item 23 or 24, wherein said concentration of said at least one Wnt signalling activator is increased by about 500% from the initial concentration of said at least one Wnt signalling activator.
(Item 26)
26. The method according to any of items 23 to 25, wherein said concentration of said at least one Wnt signalling activator is increased from about 1 μM to between about 5 μM and about 10 μM.
(Item 27)
27. The method according to any of items 23 to 26, wherein said concentration of said at least one activator of Wnt signalling is increased to a concentration of about 6 μM.
(Item 28)
28. The method of any one of items 1 to 27, wherein the at least one Wnt signaling inhibitor is capable of inhibiting non-canonical Wnt signaling and canonical Wnt signaling.
(Item 29)
29. The method of any one of items 1 to 28, wherein the at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, XAV939, IWP-O1, Wnt-C59, IWP-L6, and ICG-001, and combinations thereof.
(Item 30)
30. The method of any one of items 1 to 29, wherein the at least one Wnt signaling inhibitor comprises IWP2.
(Item 31)
31. The method of any one of items 1 to 30, wherein the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF8b, FGF4, FGF2, and combinations thereof.
(Item 32)
32. The method of any one of items 1 to 31, wherein said at least one activator of FGF signaling is capable of causing midbrain enlargement and upregulating midbrain gene expression.
(Item 33)
33. The method of claim 32, wherein the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF4, FGF2, and combinations thereof.
(Item 34)
34. The method of claim 33, wherein the at least one FGF signaling activator comprises FGF18.
(Item 35)
35. The method of any one of items 1 to 34, wherein the at least one SMAD signaling inhibitor comprises a TGFβ/Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, or a combination thereof.
(Item 36)
36. The method of claim 35, wherein the at least one TGFβ/Activin-Nodal signaling inhibitor comprises an inhibitor of ALK5.
(Item 37)
37. The method of claim 35 or 36, wherein the at least one TGFβ/Activin-Nodal signaling inhibitor is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof.
(Item 38)
38. The method according to item 37, wherein the derivative of SB431542 comprises A83-01.
(Item 39)
39. The method of any one of items 35 to 38, wherein the at least one TGFβ/Activin-Nodal signaling inhibitor comprises SB431542.
(Item 40)
36. The method of claim 35, wherein the at least one BMP signaling inhibitor is selected from the group consisting of LDN193189, Noggin, Dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of Dorsomorphin, and combinations thereof.
(Item 41)
41. The method of claim 35 or 40, wherein the at least one BMP inhibitor comprises LDN-193189.
(Item 42)
42. The method of any one of items 1 to 41, wherein the at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor.
(Item 43)
43. The method of any one of items 1 to 42, wherein said at least one Wnt signaling activator is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wnt1, Wnt5a, derivatives thereof, and combinations thereof.
(Item 44)
44. The method of any one of items 1 to 43, wherein said at least one Wnt signaling activator comprises CHIR99021.
(Item 45)
45. The method according to any one of items 1 to 44, wherein the at least one SHH signalling activator is selected from the group consisting of SHH proteins, Smoothened agonists (SAGs), and combinations thereof.
(Item 46)
46. The method of claim 45, wherein the SHH protein is selected from the group consisting of recombinant SHH, modified N-terminal SHH, and combinations thereof.
(Item 47)
47. The method of claim 46, wherein the modified N-terminal SHH comprises two isoleucines at the N-terminus.
(Item 48)
48. The method of claim 46 or 47, wherein the modified N-terminal SHH has at least about 90% sequence identity with unmodified N-terminal SHH.
(Item 49)
49. The method of claim 48, wherein the unmodified N-terminal SHH is unmodified mouse N-terminal SHH or unmodified human N-terminal SHH.
(Item 50)
50. The method of any one of items 46 to 49, wherein the modified N-terminal SHH comprises SHH C25II.
(Item 51)
46. The method of claim 45, wherein the SAG comprises palmorfamine.
(Item 52)
52. The method of any one of items 1-51, wherein at least about 80% of the differentiated cells express FOXA2 and EN1 at about 15 days from initial contact of the stem cell with the at least one SMAD signaling inhibitor.
(Item 53)
53. The method of any one of items 1-52, wherein greater than about 80% or greater than about 90% of said differentiated cells express FOXA2 and EN1 16 days after initial contact of said stem cells with said at least one SMAD signaling inhibitor.
(Item 54)
54. The method of any one of items 1 to 53, wherein the at least one marker indicative of midbrain dopamine neurons or precursors thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.
(Item 55)
55. The method of any one of items 1-54, wherein the differentiated cells do not express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.
(Item 56)
56. The method of any one of items 1 to 55, further comprising isolating cells that express at least one positive surface marker and that do not express at least one negative surface marker.
(Item 57)
57. The method of claim 56, wherein the at least one positive surface marker is selected from the group consisting of CD171, CD184, and combinations thereof.
(Item 58)
58. The method of item 56 or 57, wherein the at least one positive surface marker comprises CD184.
(Item 59)
59. The method of any one of items 56 to 58, wherein the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof.
(Item 60)
60. The method of any one of items 56 to 59, wherein the at least one negative surface marker comprises CD49e.
(Item 61)
61. The method of any one of items 56 to 60, comprising selecting cells that express CD184 and do not express CD49e.
(Item 62)
62. The method of any one of items 1 to 61, wherein the stem cells are pluripotent stem cells.
(Item 63)
63. The method of any one of items 1 to 62, wherein the stem cells are selected from the group consisting of non-embryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.
(Item 64)
64. The method of any one of items 1 to 63, wherein the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells.
(Item 65)
65. The method of any one of items 1 to 64, wherein the stem cells are human stem cells.
(Item 66)
66. A cell population of in vitro differentiated cells obtainable by the method according to any one of items 1 to 65.
(Item 67)
A composition comprising the cell population described in item 66.
(Item 68)
70. The composition according to claim 67, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.
(Item 69)
A kit for inducing differentiation of stem cells into midbrain dopamine neurons or their precursors, comprising:
(a) at least one SMAD signaling inhibitor;
(b) at least one SHH signalling activator,
(c) at least one Wnt signaling activator;
(d) at least one Wnt signaling inhibitor, and
(e) at least one FGF signaling activator
Including the kit.
(Item 70)
(f) the kit of claim 69, further comprising instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons or their precursors.
(Item 71)
1. A method for preventing, modeling and/or treating at least one symptom in a subject having a neurological disorder, comprising administering to a subject an effective amount of:
(a) the cell population according to item 66; or
(b) The composition according to item 67 or 68.
The method comprises administering to the subject one of the following:
(Item 72)
72. The method of claim 71, wherein the neurological disorder is characterized by decreased midbrain dopamine neuron function.
(Item 73)
73. The method of claim 72, wherein the decline in midbrain dopamine neuron function is age-related.
(Item 74)
74. The method of any one of claims 71 to 73, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof.
(Item 75)
75. The method of any one of claims 71 to 74, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson's disease, and combinations thereof.
(Item 76)
76. The method of any one of items 71 to 75, wherein said symptoms of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, ankylosis, dysphagia and dementia.
(Item 77)
69. The cell population of item 66 or the composition of item 67 or 68 for use in preventing, modeling and/or treating at least one symptom in a subject having a neurological disorder.
(Item 78)
78. The cell population or composition for use according to item 77, wherein the neurological disorder is characterized by a decrease in midbrain dopamine neuron function.
(Item 79)
79. The cell population or composition for use according to item 78, wherein the decline in midbrain dopamine neuron function is age-related.
(Item 80)
80. The cell population or composition for use according to any one of items 77 to 79, wherein said neurological disorder is selected from the group consisting of Parkinsonism, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, and combinations thereof.
(Item 81)
81. The cell population or composition for use according to any one of items 77 to 80, wherein said neurological disorder is selected from the group consisting of Parkinsonism, Parkinson's disease, and combinations thereof.
(Item 82)
82. The cell population or composition for use according to any one of items 77 to 81, wherein said symptoms of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia and dementia.
Claims (42)
前記幹細胞を、少なくとも１つのＳｍａｌｌ Ｍｏｔｈｅｒｓ Ａｇａｉｎｓｔ Ｄｅｃａｐｅｎｔａｐｌｅｇｉｃ（ＳＭＡＤ）シグナル伝達阻害剤、少なくとも１つのソニックヘッジホッグ（ＳＨＨ）シグナル伝達活性化剤、および少なくとも１つのウィングレス（Ｗｎｔ）シグナル伝達活性化剤に接触させる工程、ならびに
前記細胞を、少なくとも１つの線維芽細胞増殖因子（ＦＧＦ）シグナル伝達活性化剤および少なくとも１つのＷｎｔシグナル伝達阻害剤と接触させて、中脳ドーパミンニューロンまたはその前駆体を示す少なくとも１つのマーカーを発現する分化細胞の集団を得る工程
を含む、方法。 1. An in vitro method for inducing differentiation of a stem cell, comprising:
contacting the stem cells with at least one Small Mothers Against Decapentaplegic (SMAD) signaling inhibitor, at least one Sonic Hedgehog (SHH) signaling activator, and at least one Wingless (Wnt) signaling activator; and contacting the cells with at least one Fibroblast Growth Factor (FGF) signaling activator and at least one Wnt signaling inhibitor to obtain a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons or precursors thereof.
（ｂ）前記少なくとも１つのＦＧＦシグナル伝達活性化剤が、ＦＧＦ１８、ＦＧＦ１７、ＦＧＦ８ａ、ＦＧＦ８ｂ、ＦＧＦ４、ＦＧＦ２、およびそれらの組み合わせからなる群から選択される、ならびに／または
（ｃ）前記少なくとも１つのＳＭＡＤシグナル伝達阻害剤が、ＴＧＦβ／Ａｃｔｉｖｉｎ－Ｎｏｄａｌシグナル伝達阻害剤、骨形成タンパク質（ＢＭＰ）シグナル伝達阻害剤、またはそれらの組み合わせを含む、ならびに／または
（ｄ）前記少なくとも１つのＷｎｔシグナル伝達活性化剤が、グリコーゲンシンターゼキナーゼ３β（ＧＳＫ３β）シグナル伝達阻害剤を含む、ならびに／または
（ｅ）前記少なくとも１つのＳＨＨシグナル伝達活性化剤が、ＳＨＨタンパク質、Ｓｍｏｏｔｈｅｎｅｄアゴニスト（ＳＡＧ）、組換えＳＨＨ、修飾Ｎ末端ＳＨＨ、ＳＨＨ Ｃ２５ＩＩ、パルモルファミン、およびそれらの組み合わせからなる群から選択される、
請求項１～２２のいずれか１項に記載の方法。 (a) the at least one Wnt signaling inhibitor is selected from the group consisting of IWP2, IWR1-endo, XAV939, IWP-O1, Wnt-C59, IWP-L6, and ICG-001, and combinations thereof; and/or
(b) the at least one FGF signaling activator is selected from the group consisting of FGF18, FGF17, FGF8a, FGF8b, FGF4, FGF2, and combinations thereof; and/or
(c) the at least one SMAD signaling inhibitor comprises a TGFβ/Activin-Nodal signaling inhibitor, a bone morphogenetic protein (BMP) signaling inhibitor, or a combination thereof; and/or
(d) the at least one Wnt signaling activator comprises a glycogen synthase kinase 3β (GSK3β) signaling inhibitor; and/or
(e) the at least one SHH signalling activator is selected from the group consisting of SHH protein, smoothened agonist (SAG), recombinant SHH, modified N-terminal SHH, SHH C25II, palmorfamine and combinations thereof;
The method according to any one of claims 1 to 22 .
（ａ）少なくとも１つのＳＭＡＤシグナル伝達阻害剤、
（ｂ）少なくとも１つのＳＨＨシグナル伝達活性化剤、
（ｃ）少なくとも１つのＷｎｔシグナル伝達活性化剤、
（ｄ）少なくとも１つのＷｎｔシグナル伝達阻害剤
（ｅ）少なくとも１つのＦＧＦシグナル伝達活性化剤、および
（ｆ）中脳ドーパミンニューロンまたはその前駆体を示す少なくとも１つのマーカーを発現する分化細胞の集団への幹細胞の分化を誘導するための説明書
を含む、キット。 A kit for inducing differentiation of stem cells into midbrain dopamine neurons or their precursors, comprising:
(a) at least one SMAD signaling inhibitor;
(b) at least one SHH signalling activator,
(c) at least one Wnt signaling activator;
(d) at least one Wnt signaling inhibitor.
(e) at least one FGF signaling activator , and
(f) instructions for inducing differentiation of the stem cells into a population of differentiated cells expressing at least one marker indicative of midbrain dopamine neurons or their precursors.
Including the kit.
42. The cell population or composition for use according to any one of claims 38 to 41 , wherein said symptoms of neurological disorder are selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia and dementia.
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