JPWO2019157329A5 - - Google Patents
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- JPWO2019157329A5 JPWO2019157329A5 JP2020542885A JP2020542885A JPWO2019157329A5 JP WO2019157329 A5 JPWO2019157329 A5 JP WO2019157329A5 JP 2020542885 A JP2020542885 A JP 2020542885A JP 2020542885 A JP2020542885 A JP 2020542885A JP WO2019157329 A5 JPWO2019157329 A5 JP WO2019157329A5
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Description
本発明は以下を提供する。
［１］ 哺乳動物のインスリン分泌細胞の産生方法であって、
ａ．哺乳動物幹細胞を接着状態で培養することにより、前記哺乳動物幹細胞が自発的に三次元構造を形成することを可能にすることと、
ｂ．前記三次元構造を浮遊状態で培養することと、を含み、
前記培養ステップが、少なくとも２０日間のレチノイン酸およびシクロパミンへの曝露を含み、かつ前記三次元構造の前記幹細胞をＷｎｔ３Ａに曝露することを含まない、方法。
［２］ 前記哺乳動物幹細胞が、ヒト幹細胞である、上記［１］に記載の方法。
［３］ 前記哺乳動物幹細胞が、非ヒト霊長類幹細胞である、上記［１］に記載の方法。
［４］ 前記哺乳動物幹細胞が、細胞株に由来する、上記［１］に記載の方法。
［５］ インスリン分泌細胞の産生方法であって、
ａ．アクチビンＡおよびワートマニンを含む第１の培地内の接着性基質上で哺乳動物幹細胞を培養し、前記哺乳動物幹細胞がＷｎｔ３ａに曝露されないことと、
ｂ．レチノイン酸およびシクロパミンを含む少なくとも１つの追加の培地で前記細胞をさらに培養することと、
ｃ．前記細胞が三次元細胞構造を形成するときに、前記細胞を浮遊培養に移すことと、を含み、
前記細胞が、少なくとも２０日間、レチノイン酸およびシクロパミンに曝露される、方法。
［６］ 前記哺乳動物幹細胞が、前記接着性基質上で培養されるときに三次元構造を形成する、上記［５］に記載の方法。
［７］ 前記哺乳動物幹細胞が、ヒト幹細胞である、上記［５］に記載の方法。
［８］ 前記哺乳動物幹細胞が、非ヒト霊長類幹細胞である、上記［５］に記載の方法。
［９］ 哺乳動物のインスリン分泌細胞の産生方法であって、
ａ．内胚葉誘導因子を含む第１の培地で哺乳動物幹細胞を培養することにより、前記哺乳動物幹細胞を内胚葉細胞に分化させることと、
ｂ．内分泌誘導因子を含む第２の培地で（ａ）からの前記内胚葉細胞を培養することにより、前記内胚葉細胞を内分泌細胞に分化させることと、を含み、
前記哺乳動物幹細胞が、内胚葉細胞への分化前にケラチノサイト増殖因子（ＫＧＦ）に曝露されなかった、方法。
［１０］ 前記哺乳動物幹細胞が、ヒト幹細胞である、上記［９］に記載の方法。
［１１］ 前記哺乳動物幹細胞が、非ヒト霊長類幹細胞である、上記［９］に記載の方法。
［１２］ 前記内胚葉誘導因子が、アクチビンＡを含む、上記［９］～［１１］のいずれかに記載の方法。
［１３］ 前記第１の培地が、ワートマニンを含む、上記［９］～［１２］のいずれかに記載の方法。
［１４］ 前記第１の培地が、Ｗｎｔ３Ａを含まない、上記［９］～［１３］のいずれかに記載の方法。
［１５］ 前記細胞が、前記第１の培地で１～３日間培養される、上記［９］～［１４］のいずれかに記載の方法。
［１６］ 前記内分泌誘導因子が、レチノイン酸およびシロパミンを含む、上記［９］～［１５］のいずれかに記載の方法。
［１７］ 前記第２の培地が、ノギンを含む、上記［９］～［１６］のいずれかに記載の方法。
［１８］ 前記第２の培地が、ＫＧＦを含む、上記［９］～［１７］のいずれかに記載の方法。
［１９］ 前記細胞が、前記第２の培地で１～４日間培養される、上記［９］～［１８］のいずれかに記載の方法。
［２０］ ＫＧＦを含む第３の培地で前記内分泌細胞を培養することにより、前記内分泌細胞を膵臓始原細胞に分化させることをさらに含む、上記［９］～［１９］のいずれかに記載の方法。
［２１］ 前記第３の培地が、ノギンおよび上皮増殖因子（ＥＧＦ）を含む、上記［２０］に記載の方法。
［２２］ 前記第３の培地が、レチノイン酸およびシクロパミンを含む、上記［２０］または［２１］に記載の方法。
［２３］ 前記細胞が、前記第３の培地で１～４日間培養される、上記［２０］～［２２］のいずれかに記載の方法。
［２４］ ノギン、ＥＧＦ、γ－セクレターゼ阻害剤ＸＸＩ、およびＡｌｋ５ｉ ＩＩを含む第４の培地で前記膵臓始原細胞を培養することをさらに含む、上記［２０］に記載の方法。
［２５］ 前記第４の培地が、レチノイン酸およびシクロパミンを含む、上記［２４］に記載の方法。
［２６］ 前記細胞が、前記第４の培地で１～４日間培養される、上記［２４］または［２５］に記載の方法。
［２７］ Ａｌｋ５ｉ ＩＩおよびレチノイン酸を含む第５の培地で前記膵臓始原体を培養することをさらに含む、上記［２４］に記載の方法。
［２８］ 前記第５の培地が、シクロパミンを含む、上記［２７］に記載の方法。
［２９］ 前記細胞が、前記第５の培地で１～５日間培養される、上記［２７］または［２８］に記載の方法。
［３０］ Ａｌｋ５ｉ ＩＩ、ニコチンアミド、およびインスリン様増殖因子（ＩＧＦ）－Ｉを含む第６の培地で前記膵臓始原体を培養することをさらに含む、上記［２７］に記載の方法。
［３１］ 前記第６の培地が、レチノイン酸およびシクロパミンを含む、上記［３０］に記載の方法。
［３２］ 前記細胞が、前記第６の培地で１～９日間培養される、上記［３０］または［３１］に記載の方法。
［３３］ 前記哺乳動物幹細胞が、膵臓一次組織に由来する、上記［９］～［３２］のいずれかに記載の方法。
［３４］ 前記哺乳動物幹細胞が、ヒト胚性幹細胞である、上記［９］～［３２］のいずれかに記載の方法。
［３５］ 前記哺乳動物幹細胞が、人工多能性幹細胞である、上記［９］～［３２］のいずれかに記載の方法。
［３６］ 前記哺乳動物幹細胞が、多能性ではないリプログラム化された細胞である、上記［９］～［３２］のいずれかに記載の方法。
［３７］ 前記リプログラム化された細胞が、膵臓一次組織に由来する、上記［３６］に記載の方法。
［３８］ 前記リプログラム化された細胞が、リプログラミング遺伝子を、前記細胞のゲノムに前記リプログラミング遺伝子を組み込むことなく発現することによって、リプログラム化されている、上記［３６］または［３７］に記載の方法。
［３９］ 前記リプログラミング遺伝子が、少なくとも１つのエピソーム発現プラスミド上にコードされる、上記［３８］に記載の方法。
［４０］ 前記リプログラミング遺伝子が、Ｏｃｔ４、Ｓｏｘ２、Ｋｌｆ４、およびＬ－Ｍｙｃを含む、上記［３８］または［３９］に記載の方法。
［４１］ 前記細胞が、３０日以下の間培養される、上記［９］～［４０］のいずれかに記載の方法。
［４２］ インスリン分泌細胞の産生方法であって、
ａ．アクチビンＡおよびワートマニンを含む第１の培地でヒト幹細胞を培養することにより、前記ヒト幹細胞を内胚葉細胞に分化させ、前記ヒト幹細胞が内胚葉細胞への分化前にケラチノサイト増殖因子（ＫＧＦ）に曝露されなかったことと、
ｂ．レチノイン酸およびシクロパミンを含む第２の培地で（ａ）からの前記内胚葉細胞を培養することにより、前記内胚葉細胞を内分泌細胞に分化させることと、
ｃ．ＫＧＦ、ノギン、およびＥＧＦを含む第３の培地で（ｂ）からの前記内分泌細胞を培養することにより、前記内分泌細胞を膵臓始原細胞に分化させることと、
ｄ．ノギン、ＥＧＦ、γ－セクレターゼ阻害剤ＸＸＩ、およびＡｌｋ５ｉ ＩＩを含む第４の培地で（ｃ）からの前記膵臓始原細胞を培養することにより、前記膵臓始原細胞をインスリン産生細胞に分化させることと、を含む方法。
［４３］ 前記第２の培養培地が、ＫＧＦをさらに含む、上記［４２］に記載の方法。
［４４］ 前記第３および第４の培地が、レチノイン酸およびシクロパミンをさらに含む、上記［４２］または［４３］に記載の方法。
［４５］ Ａｌｋ５Ｉ ＩＩおよびレチノイン酸、ならびに任意選択的にシクロパミンを含む第５の培地で（ｄ）からの前記インスリン産生細胞をさらに培養する、上記［４２］～［４４］のいずれかに記載の方法。
［４６］ Ａｌｋ５ｉ ＩＩ、ニコチンアミン、ＩＧＦ－Ｉ、ならびに任意選択的にレチノイン酸およびシクロパミンを含む第６の培地で前記細胞をさらに培養することをさらに含む、上記［４５］に記載の方法。
［４７］ 総培養時間が、３０日未満である、上記［４２］～［４６］のいずれかに記載の方法。
［４８］ 前記ヒト幹細胞が、膵臓一次組織に由来する、上記［４２］～［４７］のいずれかに記載の方法。
［４９］ 糖尿病を治療するための細胞ベースの組成物であって、それを必要とするヒト対象への移植のための代理膵臓細胞の集団および好適な担体を含み、前記細胞の少なくとも６６％がインスリン産生膵臓細胞である、組成物。
［５０］ 前記代理膵臓細胞の少なくとも６６％が、ＮｅｕｒｏＤ１を発現する、上記［４９］に記載の細胞ベースの組成物。
［５１］ 前記代理膵臓細胞の少なくとも６８％が、Ｎｋｘ６．１を発現する、上記［４９］または［５０］に記載の細胞ベースの組成物。
［５２］ 上記［４９］～［５１］のいずれかに記載の細胞ベースの組成物であって、
前記インスリン産生膵臓細胞が、
ａ．内胚葉誘導因子を含む第１の培地内の接着性基質上でヒト幹細胞の集団を培養し、前記哺乳動物幹細胞はＷｎｔ３ａに曝露されないことと、
ｂ．レチノイン酸およびシクロパミンを含む少なくとも１つの追加の培地で前記細胞をさらに培養することと、
ｃ．前記細胞が三次元細胞構造を形成するときに、前記細胞を浮遊培養に移すことと、を含み、
前記細胞は、少なくとも２０日間、レチノイン酸およびシクロパミンに曝露され、前記細胞は、少なくとも２０日間、レチノイン酸およびシクロパミンに曝露される方法に従って誘導される、細胞ベースの組成物。
［５３］ 前記内胚葉誘導因子が、アクチビンＡおよびワートマニンを含む、上記［５２］に記載の細胞ベースの組成物。
［５４］ 前記ヒト幹細胞が、膵臓一次組織に由来する、上記［４９］～［５３］のいずれかに記載の細胞ベースの組成物。
［５５］ 前記ヒト幹細胞が、ヒト胚性幹細胞である、上記［４９］～［５３］のいずれかに記載の細胞ベースの組成物。
［５６］ 前記ヒト幹細胞が、人工多能性幹細胞である、上記［４９］～［５３］のいずれかに記載の細胞ベースの組成物。
［５７］ 前記ヒト幹細胞が、多能性ではないリプログラム化された細胞である、上記［４９］～［５３］のいずれかに記載の細胞ベースの組成物。
［５８］ 前記リプログラム化された細胞が、リプログラミング遺伝子を、前記細胞のゲノムに前記リプログラミング遺伝子を組み込むことなく発現することによって、リプログラム化されている、上記［５７］に記載の細胞ベースの組成物。
［５９］ 前記リプログラミング遺伝子が、少なくとも１つのエピソーム発現プラスミド上にコードされる、上記［５８］に記載の細胞ベースの組成物。
［６０］ 前記リプログラミング遺伝子が、Ｏｃｔ４、Ｓｏｘ２、Ｋｌｆ４、およびＬ－Ｍｙｃを含む、上記［５９］に記載の細胞ベースの組成物。
［６１］ 前記担体が、マクロカプセルを含む、上記［４９］～［６０］のいずれかに記載の細胞ベースの組成物。
［６２］ 前記マクロカプセルが、アルギン酸塩、硫酸セルロース、グルコマンナン、またはこれらの組み合わせを含む、上記［６１］に記載の細胞ベースの組成物。
［６３］ 内胚葉系譜に優先的に分化する未分化細胞を同定する方法であって、未分化細胞におけるＢＨＭＴ２およびＮＡＰ１Ｌ１の発現を評価することと、ＢＨＭＴ２の発現が対照細胞と比較して下方制御され、かつＮＡＰ１Ｌ１の発現が対照細胞と比較して上方制御される場合に、前記細胞を内胚葉系譜に分化する優先度を有するものとして同定することと、を含む方法。
［６４］ 前記未分化細胞におけるＣｏｘ７Ａ１およびＨＳＰＢ２の発現を評価することと、Ｃｏｘ７Ａ１およびＨＳＰＢ２の両方の発現が対照細胞と比較して下方制御される場合、前記細胞を内胚葉系譜に分化する優先度を有するものとして同定することと、をさらに含む、上記［６３］に記載の方法。
［６５］ 前記対照細胞が、前記内胚葉系譜への優先的分化を示さないまたは中胚葉系譜に分化することが実質的に不可能である、多能性細胞である、上記［６３］または［６４］に記載の方法。
［６６］ ＢＨＭＴ２の発現が、前記対照細胞に対して少なくとも２ｌｏｇ下方制御され、かつＮＡＰ１Ｌ１の発現が、前記対照細胞に対して少なくとも２ｌｏｇ上方制御される、上記［６３］に記載の方法。
［６７］ Ｃｏｘ７Ａ１およびＨＳＰＢ２の両方の発現が、前記対照細胞に対して少なくとも２ｌｏｇ下方制御される、上記［６６］に記載の方法。
［６８］ ＧＬＩＳ２、ＣＣＤＣ５８、ＭＴＸ３、およびＣ７ｏｒｆ２９の発現を評価することをさらに含む、上記［６３］または［６４］に記載の方法。
［６９］ ＧＬＩＳ２、ＣＣＤＣ５８、およびＭＴＸ３の発現が、前記対照細胞に対して上方制御され、かつＣ７ｏｒｆ２９の発現が、前記対照細胞に対して下方制御される、上記［６８］に記載の方法。
［７０］ 前記発現レベルは、Ｑ－ＰＣＲによって評価される、上記［６３］または［６４］に記載の方法。
［７１］ 前記発現レベルは、マイクロアレイ分析によって評価される、上記［６３］または［６４］に記載の方法。
以下の詳細な説明は、例示的かつ説明的であり、本発明のさらなる説明を提供することを意図する。
The present invention provides:
[1] A method for producing insulin-secreting cells in mammals.
a. By culturing mammalian stem cells in an adherent state, it is possible for the mammalian stem cells to spontaneously form a three-dimensional structure.
b. Including culturing the three-dimensional structure in a suspended state.
A method wherein the culture step comprises exposure to retinoic acid and cyclopamine for at least 20 days and does not include exposure of the stem cells of the three-dimensional structure to Wnt3A.
[2] The method according to the above [1], wherein the mammalian stem cell is a human stem cell.
[3] The method according to the above [1], wherein the mammalian stem cell is a non-human primate stem cell.
[4] The method according to the above [1], wherein the mammalian stem cells are derived from a cell line.
[5] A method for producing insulin-secreting cells.
a. Mammalian stem cells were cultured on an adhesive substrate in a first medium containing activin A and wortmanin so that the mammalian stem cells were not exposed to Wnt3a.
b. Further culturing the cells in at least one additional medium containing retinoic acid and cyclopamine,
c. Including transferring the cells to a suspension culture as the cells form a three-dimensional cell structure.
A method in which the cells are exposed to retinoic acid and cyclopamine for at least 20 days.
[6] The method according to [5] above, wherein the mammalian stem cells form a three-dimensional structure when cultured on the adhesive substrate.
[7] The method according to the above [5], wherein the mammalian stem cell is a human stem cell.
[8] The method according to [5] above, wherein the mammalian stem cell is a non-human primate stem cell.
[9] A method for producing insulin-secreting cells in mammals.
a. By culturing mammalian stem cells in a first medium containing an endoderm-inducing factor, the mammalian stem cells can be differentiated into endoderm cells.
b. It comprises differentiating the endoderm cells into endocrine cells by culturing the endoderm cells from (a) in a second medium containing an endocrine-inducing factor.
The method, wherein the mammalian stem cells were not exposed to keratinocyte growth factor (KGF) prior to differentiation into endoderm cells.
[10] The method according to [9] above, wherein the mammalian stem cell is a human stem cell.
[11] The method according to [9] above, wherein the mammalian stem cell is a non-human primate stem cell.
[12] The method according to any one of [9] to [11] above, wherein the endoderm-inducing factor contains activin A.
[13] The method according to any one of [9] to [12] above, wherein the first medium contains wortmanin.
[14] The method according to any one of [9] to [13] above, wherein the first medium does not contain Wnt3A.
[15] The method according to any one of [9] to [14] above, wherein the cells are cultured in the first medium for 1 to 3 days.
[16] The method according to any one of [9] to [15] above, wherein the endocrine inducing factor contains retinoic acid and siropamine.
[17] The method according to any one of [9] to [16] above, wherein the second medium contains nogin.
[18] The method according to any one of the above [9] to [17], wherein the second medium contains KGF.
[19] The method according to any one of [9] to [18] above, wherein the cells are cultured in the second medium for 1 to 4 days.
[20] The method according to any one of [9] to [19] above, further comprising differentiating the endocrine cells into pancreatic progenitor cells by culturing the endocrine cells in a third medium containing KGF. ..
[21] The method according to [20] above, wherein the third medium comprises nogin and epidermal growth factor (EGF).
[22] The method according to [20] or [21] above, wherein the third medium contains retinoic acid and cyclopamine.
[23] The method according to any one of [20] to [22] above, wherein the cells are cultured in the third medium for 1 to 4 days.
[24] The method of [20] above, further comprising culturing the pancreatic progenitor cells in a fourth medium containing nogin, EGF, γ-secretase inhibitor XXI, and Alk5i II.
[25] The method according to [24] above, wherein the fourth medium contains retinoic acid and cyclopamine.
[26] The method according to [24] or [25] above, wherein the cells are cultured in the fourth medium for 1 to 4 days.
[27] The method according to [24] above, further comprising culturing the pancreatic primordium in a fifth medium containing Alk5i II and retinoic acid.
[28] The method according to [27] above, wherein the fifth medium contains cyclopamine.
[29] The method according to [27] or [28] above, wherein the cells are cultured in the fifth medium for 1 to 5 days.
[30] The method of [27] above, further comprising culturing the pancreatic primordium in a sixth medium containing Alk5i II, nicotinamide, and insulin-like growth factor (IGF) -I.
[31] The method according to the above [30], wherein the sixth medium contains retinoic acid and cyclopamine.
[32] The method according to [30] or [31] above, wherein the cells are cultured in the sixth medium for 1 to 9 days.
[33] The method according to any one of [9] to [32] above, wherein the mammalian stem cells are derived from the primary pancreatic tissue.
[34] The method according to any one of [9] to [32] above, wherein the mammalian stem cell is a human embryonic stem cell.
[35] The method according to any one of [9] to [32] above, wherein the mammalian stem cell is an induced pluripotent stem cell.
[36] The method according to any one of [9] to [32] above, wherein the mammalian stem cell is a non-pluripotent reprogrammed cell.
[37] The method according to [36] above, wherein the reprogrammed cells are derived from the primary pancreatic tissue.
[38] The above [36] or [37], wherein the reprogrammed cell is reprogrammed by expressing the reprogramming gene without incorporating the reprogramming gene into the genome of the cell. The method described in.
[39] The method according to [38] above, wherein the reprogramming gene is encoded on at least one episome expression plasmid.
[40] The method according to [38] or [39] above, wherein the reprogramming gene comprises Oct4, Sox2, Klf4, and L-Myc.
[41] The method according to any one of [9] to [40] above, wherein the cells are cultured for 30 days or less.
[42] A method for producing insulin-secreting cells.
a. By culturing human stem cells in a first medium containing actibin A and wortmanin, the human stem cells are differentiated into endoderm cells, which are exposed to keratinocyte growth factor (KGF) prior to differentiation into endoderm cells. It wasn't done and
b. By culturing the endoderm cells from (a) in a second medium containing retinoic acid and cyclopamine, the endoderm cells can be differentiated into endocrine cells.
c. By culturing the endocrine cells from (b) in a third medium containing KGF, nogin, and EGF, the endocrine cells are differentiated into pancreatic progenitor cells.
d. Differentiating the pancreatic progenitor cells into insulin-producing cells by culturing the pancreatic progenitor cells from (c) in a fourth medium containing nogin, EGF, γ-secretase inhibitor XXI, and Alk5i II. How to include.
[43] The method according to [42] above, wherein the second culture medium further contains KGF.
[44] The method according to [42] or [43] above, wherein the third and fourth media further contain retinoic acid and cyclopamine.
[45] The above-mentioned [42] to [44], wherein the insulin-producing cells from (d) are further cultured in a fifth medium containing Alk5I II and retinoic acid, and optionally cyclopamine. Method.
[46] The method of [45] above, further comprising further culturing the cells in a sixth medium comprising Alk5i II, nicotine amine, IGF-I, and optionally retinoic acid and cyclopamine.
[47] The method according to any one of [42] to [46] above, wherein the total culture time is less than 30 days.
[48] The method according to any one of [42] to [47] above, wherein the human stem cells are derived from the primary pancreatic tissue.
[49] A cell-based composition for treating diabetes, comprising a population of surrogate pancreatic cells for transplantation into a human subject in need thereof and a suitable carrier, at least 66% of said cells. A composition that is an insulin-producing pancreatic cell.
[50] The cell-based composition according to [49] above, wherein at least 66% of the surrogate pancreatic cells express NeuroD1.
[51] The cell-based composition according to [49] or [50] above, wherein at least 68% of the surrogate pancreatic cells express Nkx6.1.
[52] The cell-based composition according to any one of [49] to [51] above.
The insulin-producing pancreatic cells
a. A population of human stem cells was cultured on an adhesive substrate in a first medium containing an endoderm-inducing factor, and the mammalian stem cells were not exposed to Wnt3a.
b. Further culturing the cells in at least one additional medium containing retinoic acid and cyclopamine,
c. Including transferring the cells to a suspension culture as the cells form a three-dimensional cell structure.
A cell-based composition in which the cells are exposed to retinoic acid and cyclopamine for at least 20 days, and the cells are induced according to a method exposed to retinoic acid and cyclopamine for at least 20 days.
[53] The cell-based composition according to [52] above, wherein the endoderm-inducing factor comprises activin A and wortmanin.
[54] The cell-based composition according to any one of [49] to [53] above, wherein the human stem cells are derived from the primary pancreatic tissue.
[55] The cell-based composition according to any one of [49] to [53] above, wherein the human stem cell is a human embryonic stem cell.
[56] The cell-based composition according to any one of [49] to [53] above, wherein the human stem cell is an induced pluripotent stem cell.
[57] The cell-based composition according to any one of [49] to [53] above, wherein the human stem cell is a non-pluripotent, reprogrammed cell.
[58] The cell according to [57] above, wherein the reprogrammed cell is reprogrammed by expressing the reprogramming gene without incorporating the reprogramming gene into the genome of the cell. Base composition.
[59] The cell-based composition according to [58] above, wherein the reprogramming gene is encoded on at least one episome expression plasmid.
[60] The cell-based composition according to [59] above, wherein the reprogramming gene comprises Oct4, Sox2, Klf4, and L-Myc.
[61] The cell-based composition according to any one of [49] to [60] above, wherein the carrier comprises macrocapsules.
[62] The cell-based composition according to [61] above, wherein the macrocapsules contain alginate, cellulose sulfate, glucomannan, or a combination thereof.
[63] A method for identifying undifferentiated cells that preferentially differentiate into the endoderm lineage, in which the expression of BHMT2 and NAP1L1 in undifferentiated cells is evaluated, and the expression of BHMT2 is downwardly regulated as compared with control cells. And when the expression of NAP1L1 is upregulated as compared to a control cell, the method comprising identifying the cell as having a priority to differentiate into an endoderm lineage.
[64] When the expression of Cox7A1 and HSPB2 in the undifferentiated cells is evaluated and the expression of both Cox7A1 and HSPB2 is downregulated as compared with the control cells, the priority for differentiating the cells into the endoderm lineage. The method according to [63] above, further comprising identifying as having.
[65] The control cell is a pluripotent cell that does not show preferential differentiation into the endoderm lineage or is substantially impossible to differentiate into the mesoderm lineage, [63] or [ 64].
[66] The method according to [63] above, wherein the expression of BHMT2 is down-regulated by at least 2 log with respect to the control cell, and the expression of NAP1L1 is down-regulated by at least 2 log with respect to the control cell.
[67] The method of [66] above, wherein expression of both Cox7A1 and HSPB2 is downregulated by at least 2 logs with respect to the control cell.
[68] The method according to [63] or [64] above, further comprising assessing the expression of GLIS2, CCDC58, MTX3, and C7orf29.
[69] The method according to [68] above, wherein the expression of GLIS2, CCDC58, and MTX3 is upregulated with respect to the control cell, and the expression of C7orf29 is downregulated with respect to the control cell.
[70] The method according to [63] or [64] above, wherein the expression level is evaluated by Q-PCR.
[71] The method according to [63] or [64] above, wherein the expression level is evaluated by microarray analysis.
The following detailed description is exemplary and descriptive and is intended to provide a further description of the invention.
Claims (15)
ａ．哺乳動物幹細胞を接着状態で培養することにより、前記哺乳動物幹細胞が自発的に三次元構造を形成することを可能にすることと、
ｂ．前記三次元構造を浮遊状態で培養することと、を含み、
前記培養ステップが、少なくとも２０日間のレチノイン酸およびシクロパミンへの曝露を含み、かつ前記三次元構造の前記幹細胞をＷｎｔ３Ａに曝露することを含まない、方法。 A method for producing insulin-secreting cells in mammals.
a. By culturing mammalian stem cells in an adherent state, it is possible for the mammalian stem cells to spontaneously form a three-dimensional structure.
b. Including culturing the three-dimensional structure in a suspended state.
A method wherein the culture step comprises exposure to retinoic acid and cyclopamine for at least 20 days and does not include exposure of the stem cells of the three-dimensional structure to Wnt3A.
ａ．アクチビンＡおよびワートマニンを含む第１の培地内の接着性基質上で哺乳動物幹細胞を培養し、前記哺乳動物幹細胞がＷｎｔ３ａに曝露されないことと、
ｂ．レチノイン酸およびシクロパミンを含む少なくとも１つの追加の培地で前記細胞をさらに培養することと、
ｃ．前記細胞が三次元細胞構造を形成するときに、前記細胞を浮遊培養に移すことと、を含み、
前記細胞が、少なくとも２０日間、レチノイン酸およびシクロパミンに曝露される、方法。 It is a method of producing insulin-secreting cells.
a. Mammalian stem cells were cultured on an adhesive substrate in a first medium containing activin A and wortmanin so that the mammalian stem cells were not exposed to Wnt3a.
b. Further culturing the cells in at least one additional medium containing retinoic acid and cyclopamine,
c. Including transferring the cells to a suspension culture as the cells form a three-dimensional cell structure.
A method in which the cells are exposed to retinoic acid and cyclopamine for at least 20 days.
ａ．アクチビンＡおよびワートマニンを含む第１の培地でヒト幹細胞を培養することにより、前記ヒト幹細胞を内胚葉細胞に分化させ、前記ヒト幹細胞が内胚葉細胞への分化前にケラチノサイト増殖因子（ＫＧＦ）に曝露されなかったことと、
ｂ．レチノイン酸およびシクロパミンを含む第２の培地で（ａ）からの前記内胚葉細胞を培養することにより、前記内胚葉細胞を内分泌細胞に分化させることと、
ｃ．ＫＧＦ、ノギン、およびＥＧＦを含む第３の培地で（ｂ）からの前記内分泌細胞を培養することにより、前記内分泌細胞を膵臓始原細胞に分化させることと、
ｄ．ノギン、ＥＧＦ、γ－セクレターゼ阻害剤ＸＸＩ、およびＡｌｋ５ｉ ＩＩを含む第４の培地で（ｃ）からの前記膵臓始原細胞を培養することにより、前記膵臓始原細胞をインスリン産生細胞に分化させることと、を含む方法。 It is a method of producing insulin-secreting cells.
a. By culturing human stem cells in a first medium containing actibin A and wortmanin, the human stem cells are differentiated into endoderm cells, which are exposed to keratinocyte growth factor (KGF) prior to differentiation into endoderm cells. It wasn't done and
b. By culturing the endoderm cells from (a) in a second medium containing retinoic acid and cyclopamine, the endoderm cells can be differentiated into endocrine cells.
c. By culturing the endocrine cells from (b) in a third medium containing KGF, nogin, and EGF, the endocrine cells are differentiated into pancreatic progenitor cells.
d. Differentiating the pancreatic progenitor cells into insulin-producing cells by culturing the pancreatic progenitor cells from (c) in a fourth medium containing nogin, EGF, γ-secretase inhibitor XXI, and Alk5i II. How to include.
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