# US 8,465,975 B2 · Human late stage motor neuron progenitor cells produced from pluripotent stem cells

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- Number: US 8,465,975 B2
- Document ID: US8465975B2
- Status: Granted
- Kind: B2
- Application number: 13/324,956
- Filed: 2011-12-13
- Published: 2012-05-03
- Granted: 2013-06-18
- Earliest claimed: 2008-03-24
- Assignees: California Stem Cell, Inc.
- Applicants: Aleksandra Jovanovic Poole
- Inventors: Aleksandra Jovanovic Poole
- Web page: https://patentgrep.com/patent/US8465975B2

## CPC

- C12N 5/0623: {Stem cells}
- A61P 25/00: Drugs for disorders of the nervous system
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Motor neuron progenitor (MNP) cells and populations of MNP cells, are provided, in particular, populations of human late stage MNP cells having a purity of greater than about 65% late stage MNP cells and high-purity populations of MNP cells having greater than 95% viable cells, as well as method of making and using the same, including deriving late stage MNP cells from pluripotent embryonic stem cells, producing high-purity populations of late stage MNP cells, producing populations of viable MNP cells, transporting viable MNP cells, and transplanting MNP cells.

## Claims

### Claim 1

A method of generating a high-purity population of late stage motor neuron progenitor cells, wherein the population has a purity of greater than about 65% late stage motor neuron progenitor cells, comprising:
(a) neuralizing a culture of pluripotent stem cells by culturing pluripotent stem cells in a growth media that contains beta fibroblast growth factor (bFGF) and that lacks bone morphogenic protein (BMP), and that is supplemented with one or more of albumin, a fatty acid, a thyroid hormone, a vitamin, a trace mineral, insulin and transferrin;
(b) ventralizing the neutralized cell population of step (a) comprising culturing the cell population under non-adherent conditions in a motor neuron media supplemented with retinoic acid and beta fibroblast growth factor (bFGF) for at least about five days,
(c) incubating the ventralized cell population of step (b) comprising culturing the cell population in growth media with bFGF without retinoic acid to promote formation of neurospheres,
(d) selecting the neurospheres;
(e) expanding neurospheres on an adherent substrate to form a layer of cells comprising a population of cells comprising greater than about 65% late stage motor neuron progenitor cells, wherein said late stage motor neuron cells express markers of the motor neuron HB9, Ach, Isl1, Lhx3, and Lim1, but lack a functional calcium channel.

### Claim 2 (depends on 1)

The method of claim 1, wherein the growth media is a serum-free media.

### Claim 3 (depends on 1)

The method of claim 1, wherein the motor neuron media is a serum-free media.

### Claim 4 (depends on 1)

The method of claim 1, wherein selecting the neurospheres comprises gravitational sedimentation of the neurospheres.

### Claim 5 (depends on 1)

The method of claim 1, wherein the population of late stage motor neuron progenitor cells comprises cells characterized by the expression of at least one of a neurofilament marker or a neuronal nuclei specific protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the at least one of a neurofilament marker is at least one of Tuj1, MAP2, and SM132.

### Claim 7 (depends on 6)

The method of claim 6, wherein the neuronal nuclei specific protein is NeuN.

### Claim 8 (depends on 3)

The method of claim 3, wherein the serum-free growth media comprises non-essential amino acids, dipeptide L-glutamine, pyruvate, human albumin, ethanolamine, insulin, transferring, selenite, and T3 thyroid hormone.

### Claim 9 (depends on 3)

The method of claim 3, wherein the serum-free motor neuron media comprises B27 supplement with vitamin A, magnesium chloride, and dipeptide L-glutamine.

### Claim 10 (depends on 3)

The method of claim 3, wherein the serum-free motor neuron media is supplemented with retinoic acid at a concentration of between about 5 μM to about 20 uM.

### Claim 11 (depends on 1)

The method of claim 1, wherein the bFGF is at a concentration of between about 2 ng/mL to about 20 ng/mL.

### Claim 12 (depends on 1)

The method of claim 1, wherein the layer of cells comprising a population of cells having greater than about 65% late stage motor neuron progenitor cells, has a purity of between 65% and 100% late stage motor neuron progenitor cells.

### Claim 13 (depends on 1)

The method of claim 1, wherein the contaminant population in the layer of cells comprising a population of cells having greater than about 65% late stage motor neuron progenitor cells, is less than about 35% astrocytes.

### Claim 14 (depends on 1)

The method of claim 1, wherein the contaminant population in the layer of cells comprising a population of cells having greater than about 65% late stage motor neuron progenitor cells, is between 0% and about 35% astrocytes.

### Claim 15 (depends on 1)

The method of claim 1, wherein the cells do not express one or more of OCT4, GFAP, SMA, or AFP.

### Claim 16 (depends on 1)

An isolated population of late stage motor neuron progenitor cells prepared by the method of claim 1, wherein the population of cells comprises greater than about 65% late stage motor neuron progenitor cells and the late stage motor neuron progenitor cells express markers of the motor neuron HB9, Ach, Isl1, Lhx3, and Lim1, but lack a functional calcium channel.