Methods for neural conversion of human embryonic stem cells
First Claim
1. An in vitro method for differentiating pluripotent stem cells, comprising, exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling (first SMAD inhibitor) and (b) a second inhibitor of SMAD protein signaling (second SMAD inhibitor) to obtain a cell population comprising at least about 10% differentiated cells expressing paired box homeotic gene-6 (PAX6), wherein said first SMAD inhibitor comprises an inhibitor of bone morphogenetic protein (BMP) signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
- /Activin-Nodal signaling.
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Abstract
The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. Specifically, methods are provided for obtaining neural tissue, floor plate cells, and placode including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons, motorneurons, and sensory neurons. Further, neural plate tissue obtained using methods of the present inventions are contemplated for use in co-cultures with other tissues as inducers for shifting differentiation pathways, i.e. patterning.
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Citations
141 Claims
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1. An in vitro method for differentiating pluripotent stem cells, comprising, exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling (first SMAD inhibitor) and (b) a second inhibitor of SMAD protein signaling (second SMAD inhibitor) to obtain a cell population comprising at least about 10% differentiated cells expressing paired box homeotic gene-6 (PAX6), wherein said first SMAD inhibitor comprises an inhibitor of bone morphogenetic protein (BMP) signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
- /Activin-Nodal signaling.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. An in vitro method for differentiating pluripotent stem cells, comprising:
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exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor to obtain a cell population comprising at least about 10% differentiated cells expressing SIX1, wherein said exposing to said first SMAD inhibitor is discontinued after about two days or about three days from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor; and wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (22, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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23. An in vitro method for differentiating pluripotent stem cells, comprising:
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exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor to obtain a cell population comprising at least about 10% differentiated cells expressing CDX2, wherein said exposing to said first SMAD inhibitor is discontinued after about one day from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor; and wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (24, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
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25. An in vitro method for differentiating pluripotent stem cells, comprising:
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exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor; and
exposing said cells to (c) an activator of Sonic Hedgehog (SHH) signaling to obtain a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of SHH, FOXA2, Netrin-1, and F-Spondin,wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (26, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90)
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27. An in vitro method for differentiating pluripotent stem cells, comprising:
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exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor; and
exposing said cells to (c) an activator of SHH signaling and (d) one or more caudalizing factor to obtain a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of CORIN, LMX1B, EN1, NGN2, and NOV,wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (28, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113)
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29. An in vitro method for differentiating pluripotent stem cells, comprising exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor;
- and exposing said cells to (c) an activator of SHH signaling to obtain a cell population comprising at least about 10% differentiated cells expressing Tyrosine hydroxylase (TH),
wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (30, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130)
- and exposing said cells to (c) an activator of SHH signaling to obtain a cell population comprising at least about 10% differentiated cells expressing Tyrosine hydroxylase (TH),
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31. An in vitro method for differentiating pluripotent stem cells, comprising:
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exposing a plurality of pluripotent stem cells attached to a substrate to (a) a first SMAD inhibitor and (b) a second SMAD inhibitor and exposing said cells to a Brain-derived neurotrophic factor (BDNF), ascorbic acid, an activator of Sonic Hedgehog (SHH) signaling, and retinoic acid (RA) to obtain a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of ISL1 and HB9, wherein said first SMAD inhibitor comprises an inhibitor of BMP signaling, and said second SMAD inhibitor comprises an inhibitor of TGFβ
/Activin-Nodal signaling. - View Dependent Claims (32, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141)
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Specification