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

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- Number: US 12,760,271 B2
- Document ID: US12760271B2
- Status: Granted
- Kind: B2
- Application number: 18/591,734
- Filed: 2024-02-29
- Published: 2024-08-08
- Granted: 2026-10-13
- Earliest claimed: 2011-11-04
- Assignees: Memorial Sloan Kettering Cancer Center
- Applicants: Memorial Sloan-Kettering Cancer Center
- Inventors: Lorenz Studer; Jae-Won Shim; Sonja Kriks
- Web page: https://patentgrep.com/patent/US12760271B2

## CPC

- C12N 5/0619: {Neurons}
- 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
- C12N 5/0623: {Stem cells}
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor)
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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.

## Claims

### Claim 1

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).

### Claim 2 (depends on 1)

The method of claim 1, further comprising subjecting the cell population to conditions favoring maturation of the differentiated cells into dopamine neurons, wherein the conditions comprise exposing the cell population to at least one of brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cell line-derived neurotrophic factor (GDNF), dibutyryl CAMP (dbcAMP), and transforming growth factor type β3 (TGFβ3).

### Claim 3 (depends on 2)

The method of claim 2, wherein the dopamine neurons express at least one marker selected from the group consisting of tyrosine hydroxylase (TH), orthodenticle homeobox 2 (OTX2), nuclear receptor related 1 protein (NURR1), neuron-specific class III beta-tubulin (Tuj1), Trefoil factor family 3 (TTF3), paired-like homeodomain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), and G-protein coupled, inwardly rectifying potassium channel (Kir3.2/GIRK2), CD142, DCSM1, CD63, CD99, and ALDH1.

### Claim 4 (depends on 3)

The method of claim 3, wherein the dopamine neurons are A9 subtype neurons.

### Claim 5 (depends on 3)

The method of claim 3, further comprising selecting a population of cells expressing at least one marker selected from the group consisting of Girk2, CD142, DCSM1, CD63, CD99, and ALDH1.

### Claim 6 (depends on 2)

The method of claim 2, wherein the cells are differentiated into the dopamine neurons no later than about 25 days from the initial induction of the inhibition of TGFβ/Activin-Nodal signaling and the inhibition of BMP signaling in the cells.

### Claim 7 (depends on 1)

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 pluripotent stem cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cell population comprises at least about 40% cells expressing FOXA2 and LMX1A.

### Claim 9 (depends on 8)

The method of claim 8, wherein the cell population comprises at least about 50% cells expressing FOXA2 and LMX1A.

### Claim 10 (depends on 8)

The method of claim 8, wherein the cell population comprises at least about 70% cells expressing FOXA2 and LMX1A.

### Claim 11 (depends on 1)

The method of claim 1, wherein the induction of inhibition of TGFβ/Activin-Nodal signaling comprises exposing the plurality of pluripotent cells to at least one inhibitor of TGFβ/Activin-Nodal signaling.

### Claim 12 (depends on 11)

The method of claim 11, wherein the at least one inhibitor of TGFβ/Activin-Nodal signaling comprises SB431542.

### Claim 13 (depends on 1)

The method of claim 1, wherein the induction of inhibition of BMP signaling comprises exposing the plurality of pluripotent cells to at least one inhibitor of BMP signaling.

### Claim 14 (depends on 13)

The method of claim 13, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of LDN-193189, Noggin, dorsomorphin, and combinations thereof.

### Claim 15 (depends on 1)

The method of claim 1, wherein the at least one activator of SHH signaling comprises purmorphamine.

### Claim 16 (depends on 1)

The method of claim 1, wherein the inhibition of GSK3β signaling comprises exposing the plurality of pluripotent cells to at least one inhibitor of GSK3β signaling.

### Claim 17 (depends on 16)

The method of claim 16, wherein the at least one inhibitor of GSK3β signaling comprises CHIR99021.

### Claim 18 (depends on 1)

The method of claim 1, wherein the pluripotent cells are differentiated into cells that express both FOXA2 and LMX1A within 11 days from the initial induction of the inhibition of TGFβ/Activin-Nodal signaling and the inhibition of BMP signaling.

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 1)

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.