Midbrain dopamine (DA) neurons for engraftment

• US 10,280,398 B2
• Filed: 11/02/2012
• Issued: 05/07/2019
• Est. Priority Date: 11/04/2011
• Status: Active Grant

• Current Assignee: Memorial Sloan-Kettering Cancer Center
• Original Assignee: Memorial Sloan-Kettering Cancer Center
• Inventors: Studer, Lorenz, Shim, Jae-Won, Kriks, Sonja
• Primary Examiner(s): Bertoglio, Valarie E
• Application Number:
  US14/356,042
• Publication Number:
  US 20150010514A1
• Time in Patent Office: 2,377 Days
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

The present invention relates to the field of stem cell biology, in particular the lineage 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.

Claims

1. An in vitro method for differentiating pluripotent stem cells, comprising exposing a plurality of pluripotent stem cells to at least one inhibitor of TGFβ

/Activin-Nodal signaling, at least one inhibitor of bone morphogenetic protein (BMP) signaling, at least two activators of Sonic hedgehog (SHH) signaling comprising purmorphamine and SHH C25II, and at least one inhibitor of glycogen synthase kinase 3β

(GSK3β

) signaling that activates wingless (Wnt) signaling,

wherein the exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling and the at least one inhibitor of BMP signaling begins on day 0,

wherein said cells are exposed to the at least one inhibitor of GSK3β

signaling on the third (3^rd) day through the eleventh (11^th) day from the initial exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling and the at least one inhibitor of BMP signaling, and

wherein said cells are exposed to the at least one inhibitor of TGFβ

/Activin-Nodal signaling, the at least one inhibitor of BMP signaling, the at least two activators of SHH signaling, and the at least one inhibitor of GSK3β

signaling in amounts effective to produce 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).

2. The method of claim 1, wherein the initial exposure of the cells to the at least two activators of SHH signaling is between about 24 hours and about 36 hours from the initial exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling and the at least one inhibitor of BMP signaling.

3. The method of claim 1, wherein the exposure of the cells to the at least one inhibitor of GSK3β

signaling is initiated between about 24 hours and about 36 hours from the initial exposure of the cells to the at least two activators of SHE signaling.

4. The method of claim 1, wherein said cell population comprises at least about 40% cells expressing FOXA and LMX1A.

5. The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of human, nonhuman primate or rodent embryonic stem cells, induced pluripotent stem cells and engineered pluripotent stem cells.

6. The method of claim 1, wherein the at least one inhibitor of TGFβ

/Activin-Nodal signaling comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542).

7. The method of claim 1, wherein the at least one inhibitor of GSK3β

signaling comprises CHIR99021.

8. The method of claim 1, wherein the pluripotent stem cells:

(a) are differentiated into cells that express both FOXA2 and LMX1A within about 11 days from the initial exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling;

or

(b) are differentiated into dopamine neurons no later than 25 days from the initial exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling.

9. The method of claim 1, wherein

(a) the at least one inhibitor of TGFβ

/Activin-Nodal signaling and the at least one inhibitor of BMP signaling are selected from the group consisting of LDN-193189, SB431542, Noggin, dorsomorphin, and combinations of two or more of the foregoing; and

(b) the at least two activators of SHH signaling are selected from the group consisting of recombinant SHH, purified SHH, Smoothened agonists (SAG), and combinations of two or more of the foregoing.

10. The method of claim 1, wherein the differentiated cells expressing FOXA2 and LMX1A do not express at least one marker selected from the group consisting of PAX6, HES5, EMX2 and LHX2.

11. The method of claim 1, comprising exposing the cells to an effective concentration of fibroblast growth factor 8 (FGF8) for up to about 144 hours from the initial exposure of the cells to the at least one inhibitor of TGFβ

/Activin-Nodal signaling.

12. The method of claim 1, wherein said differentiated cells expressing FOXA2 and LMX1A are midbrain floor plate precursors.

13. The method of claim 1, wherein said cell population comprises at least about 50% differentiated cells expressing FOXA2 and LMX1A.

14. The method of claim 1, wherein said cell population comprises at least about 70% differentiated cells expressing FOXA2 and LMX1A.