# US 11,299,710 B2 · Brown adipocyte progenitors in human skeletal muscle

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- Number: US 11,299,710 B2
- Document ID: US11299710B2
- Status: Granted
- Kind: B2
- Application number: 16/409,926
- Filed: 2019-05-13
- Published: 2020-11-05
- Granted: 2022-04-12
- Earliest claimed: 2011-11-10
- Assignees: Energesis Pharmaceuticals, Inc.
- Applicants: Energesis Pharmaceuticals, Inc.
- Inventors: Olivier D. Boss; Mihaela Crisan; Jean-Paul Giacobino
- Web page: https://patentgrep.com/patent/US11299710B2

## CPC

- C12N 5/0653: {Adipocytes; Adipose tissue}
- A61K 35/35: Fat tissue; Adipocytes; Stromal cells; Connective tissues (adipose-derived stem cells A61K35/28; collagen A61K38/39)
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- G01N 33/56966: {Animal cells}
- C12N 2501/01: Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
- 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 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/40: Regulators of development
- G01N 2333/70503: Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N 2333/70589: CD45
- G01N 2333/70596: Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

This invention relates to brown adipose tissue (BAT) progenitor cells and methods for isolating BAT progenitor cells from skeletal muscle. BAT progenitor cell surface markers and medium and agents for inducing cell differentiation into brown adipocytes are also provided. In some embodiments, the BAT progenitor cell expresses a first cell surface marker associated with endothelial cells, the first cell surface marker being detectable in an antibody based assay using a first antibody. In addition, the BAT progenitor cell can be substantially free of a second cell surface marker associated with endothelial cells, the second cell surface marker being substantially undetectable in said antibody based assay using a second antibody. The BAT progenitor cell can also be substantially free of additional cell surface markers.

## Claims

### Claim 1

A method for identifying an agent that induces differentiation of a brown adipocyte progenitor cell into a brown adipocyte, comprising:
contacting a population of skeletal muscle cells with a first and a second antibody that are specific for CD34 and CD31, respectively;
selecting a brown adipocyte progenitor cell from the population of skeletal muscle cells on the basis of expression of CD34 and CD31, wherein said brown adipocyte progenitor cell is positive for CD34 and negative for CD31;
contacting the selected brown adipocyte progenitor cell with an agent that induces differentiation brown adipocyte progenitor cell into a brown adipocyte; and
determining in the brown adipocyte progenitor cell the presence of an indicator of differentiation into a brown adipocyte induced by said agent.

### Claim 2 (depends on 1)

The method of claim 1, wherein the indicator of differentiation is an increase in one or more of the following: expression of uncoupling protein-1 (UCP1) protein or mRNA, expression of fatty acid-binding protein-4 (FABP4) (aP2) protein or mRNA, expression of peroxisome-proliferator-activated receptor γ2 (PPARγ2) protein or mRNA, expression of mitochondrial transcription factor A (mtTFA) protein or mRNA, expression of PPARγ coactivator-1α (PGC-1α) protein or mRNA, uncoupled respiration, respiration rate, metabolic rate, glucose utilization rate, fatty acid oxidation rate, and a combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD146 and the selected brown adipocyte progenitor cell is negative for CD146.

### Claim 4 (depends on 1)

The method of claim 1, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD45 and the selected brown adipocyte progenitor cell is negative for CD45.

### Claim 5 (depends on 1)

The method of claim 1, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD56 and the selected brown adipocyte progenitor cell is negative for CD56.

### Claim 6 (depends on 1)

The method of claim 1, wherein the agent is one or more of the following: a peroxisome-proliferator-activated receptor (PPAR)γ activator, modulator or inhibitor; a PPARγ activator or modulator; a PPARγ activator or modulator; a dual PPARγ and PPARγ activator or modulator; a pan-PPAR (α, δ, γ) activator or modulator; a phosphodiesterase (PDE)4 inhibitor; a PDE7 inhibitor; a nuclear receptor-interacting protein 1 (NRIP1) (RIP 140) inhibitor, a phosphatase and tensin homolog (PTEN) inhibitor; an α1-adrenergic full or partial agonist; a retinoid receptor α (RXRα) activator or modulator; a PPARγ coactivator (PGC)-1α activator; a PGC-1β inhibitor or activator; adiponectin or an activator of adiponectin receptor AdipoR1 and/or AdipoR2; a nitric oxide synthase (NOS) inhibitor or activator; a Rho kinase-ROCK inhibitor; brain-derived neurotrophic factor (BDNF); a monoamine oxidase (MAO) A inhibitor and/or an MAO B inhibitor; an activator of SRC proto-oncoprotein; an inhibitor of epidermal growth factor receptor (EGFR); an inhibitor of fatty acid amide hydrolase (FAAH); an inhibitor of mitogen-activated protein kinase (MAPK) 1 or 2 or 4 or 5 or 7 or 8; an inhibitor of cyclin-dependent kinase 9 (CDK9); a TGR5 agonist; an adenosine monophosphate activated protein kinase (AMPK) activator; bone morphogenetic protein 7 (BMP-7); an mammalian target of rapamycin (mTOR) inhibitor; an adenylate cyclase activator; or combinations of any of the foregoing.

### Claim 7 (depends on 1)

The method of claim 1, wherein the indicator of differentiation is an increase in one or both of uncoupling protein-1 (UCP1) protein and UCP1 mRNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the brown adipocyte progenitor cell is capable of expansion and passage in a culture.

### Claim 9 (depends on 1)

The method of claim 1, wherein the population of skeletal muscle cells is obtained from one or more of fetal, juvenile, and adult human skeletal muscle.

### Claim 10

A method for identifying an agent that induces differentiation of a brown adipocyte progenitor cell into a brown adipocyte, comprising:
contacting a population of skeletal muscle cells with a first and a second antibody that are specific for CD34 and CD31, respectively;
selecting a plurality of brown adipocyte progenitor cells from the population of skeletal muscle cells on the basis of expression of CD34 and CD31, wherein said brown adipocyte progenitor cell is positive for CD34 and negative for CD31;
contacting the selected brown adipocyte progenitor cell with an agent that induces differentiation brown adipocyte progenitor cell into a brown adipocyte; and
determining in the plurality of brown adipocyte progenitor cells the presence of an indicator of differentiation into a brown adipocyte induced by said agent.

### Claim 11 (depends on 10)

The method of claim 10, wherein the indicator of differentiation is an increase in one or both of uncoupling protein-1 (UCP1) protein and UCP1 mRNA in the plurality of brown adipocyte progenitor cells.

### Claim 12 (depends on 10)

The method of claim 10, wherein the agent is one or more of the following: a PPARγ activator, modulator or inhibitor; a PPARα activator or modulator; a PPARδ activator or modulator; a dual PPARα and PPARδ activator or modulator; a pan-PPAR (α, δ, γ) activator or modulator; a PDE4 inhibitor; a PDE7 inhibitor; a NRIP1 (RIP 140) inhibitor, a PTEN inhibitor; an α1-adrenergic full or partial agonist; an RXRα activator or modulator; a PGC-1α activator; a PGC-1β inhibitor or activator; adiponectin or an activator of adiponectin receptor AdipoR1 and/or AdipoR2; an NOS inhibitor or activator; a Rho kinase-ROCK inhibitor; BDNF; a monoamine oxidase (MAO) A inhibitor and/or an MAO B inhibitor; an activator of SRC proto-oncoprotein; an inhibitor of EGFR; an inhibitor of FAAH; an inhibitor of MAPK 1 or 2 or 4 or 5 or 7 or 8; an inhibitor of CDK9; a TGR5 agonist; an AMPK activator; BMP-7; an mTOR inhibitor; an adenylate cyclase activator; or combinations of any of the foregoing.

### Claim 13 (depends on 10)

The method of claim 10, wherein the brown adipocyte progenitor cell is capable of expansion and passage in a culture.

### Claim 14 (depends on 10)

The method of claim 10, wherein the population of skeletal muscle cells is obtained from one or more of fetal, juvenile, and adult human skeletal muscle.

### Claim 15 (depends on 10)

The method of claim 10, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD146 and the selected brown adipocyte progenitor cells are negative for CD146.

### Claim 16 (depends on 10)

The method of claim 10, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD45 and the selected brown adipocyte progenitor cells are negative for CD45.

### Claim 17 (depends on 10)

The method of claim 10, wherein the population of skeletal muscle cells is further contacted with an antibody specific for CD56 and the selected brown adipocyte progenitor cells are negative for CD56.