Midbrain dopamine (DA) neurons for engraftment

• US 10,711,243 B2
• Filed: 03/14/2019
• Issued: 07/14/2020
• 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:
  US16/353,546
• Publication Number:
  US 20190211306A1
• Time in Patent Office: 488 Days
• Field of Search: 435377
• 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 transforming growth factor beta (TGFβ

)/Activin-Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling, and exposing the cells to at least one activator of Sonic hedgehog (SHH) signaling and at least one glycogen synthesis kinase 3β

(GSK3β

) inhibitor that activates wingless (Wnt) signaling, wherein the cells are exposed to the at least one GSK3β

inhibitor three (3) days 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 to obtain a cell population comprising greater than about 40% differentiated cells expressing both forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A), and 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.

2. The method of claim 1, wherein the cells expressing FOXA2 and LMX1A are midbrain floor plate precursors.

3. The method of claim 1, wherein the cell population comprises at least about 50%, or at least about 70% cells expressing FOXA2 and LMX1A.

4. The method of claim 1, wherein the stem cells are differentiated into the cells expressing FOXA2 and LMX1A within about 11 days from the initial exposure the cells to the at least one inhibitor of TGFβ

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

5. 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).

6. The method of claim 1, wherein the at least one inhibitor of BMP signaling comprises LDN-193189.

7. The method of claim 1, wherein the modified N-terminal SHH comprises SHH C25II.

8. The method of claim 1, wherein the SAG comprises purmorphamine.

9. The method of claim 1, wherein the GSK3β

inhibitor comprises CHIR99021.

10. The method of claim 1, wherein:

(a) the initial exposure of the cells to the at least one activator 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, and/or

(b) the exposure of the cells to the at least one activator of SHH signaling is up to about 144 hours; and

/or

(c) the initial exposure of the cells to the at least one GSK3β

inhibitor is between about 24 hours and about 36 hours from the initial exposure of the cells to the at least one activator of SHH signaling.

11. The method of claim 1, further comprising exposing the cells to at least one activator of fibroblast growth factor (FGF) family signaling.

12. The method of claim 11, wherein the at least one activator of FGF family signaling comprises FGF8.

13. The method of claim 11, wherein the initial exposure of the cells to the at least one activator of FGF family 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, and/or the exposure of the cells to the at least one activator of FGF family signaling is up to about 144 hours.

14. The method of claim 1, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells (iPSCs), and engineered stem cells.

15. The method of claim 1, wherein the cells are exposed to the at least one GSK3β

inhibitor activator of Wnt 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.

16. The method of claim 1, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of LDN-193189, Noggin, dorsomorphin, and combinations thereof.

17. The method of claim 1, wherein the modified N-terminal SHE comprises two Isoleucines at the N-terminus.