US 12,760,271 B2 · Midbrain dopamine (DA) neurons for engraftment

Abstract

The present invention relates to the field of stem cell biology, in particular the linage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC) in addition to nonembryonic human induced pluripotent stem cells (hiPSC), somatic stem cells, stem cells from patients with a disease, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and/or hiPSC into floor plate midbrain progenitor cells and then further into large populations of midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons using novel culture conditions. The midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons made using the methods of the present invention are further contemplated for various uses including, but not limited to, use in in vitro drug discovery assays, neurology research, and as a therapeutic to reverse disease of, or damage to, a lack of dopamine neurons in a patient. Further, compositions and methods are provided for differentiating midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons from human pluripotent stem cells for use in disease modeling, in particular Parkinson's disease. Additionally, authentic DA neurons are enriched for markers, such as CD142, and A9 type neuronal cells.

Claims20

Dependent claims

An in vitro method for differentiating pluripotent stem cells, comprising:

inducing the inhibition of TGFβ/Activin-Nodal signaling;
inducing the inhibition of bone morphogenetic protein (BMP) signaling;
inducing the activation of Sonic hedgehog (SHH) signaling;
and
inducing the inhibition of glycogen synthase kinase 3β (GSK3β) signaling that activates wingless (Wnt) signaling in the cells,
wherein the induction of activation of SHH signaling comprises exposing the plurality of pluripotent cells to at least one activator of SHH signaling, wherein the at least one activator of SHH signaling is selected from the group consisting of:
a Smoothened agonist (SAG);
a modified N-terminal SHH;
and a SHH protein comprising a N-terminal fragment and a C-terminal fragment;
wherein the inhibition of GSK3β signaling is induced in the cells three (3) days from the initial induction of:
(i) the inhibition of TGFβ/Activin-Nodal signaling;
and (ii) the inhibition of BMP signaling;
to thereby obtain a cell population comprising at least about 10% differentiated cells expressing both forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A).

10 more elements

Dependencies are inferred from the claim text.

Midbrain dopamine (DA) neurons for engraftment

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Midbrain dopamine (DA) neurons for engraftment

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Abstract

Claims20