# US 10,478,458 B2 · In vitro pre-conditioned bone marrow-derived mesenchymal stem cells and uses thereof

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- Number: US 10,478,458 B2
- Document ID: US10478458B2
- Status: Granted
- Kind: B2
- Application number: 15/227,831
- Filed: 2016-08-03
- Published: 2017-04-06
- Granted: 2019-11-19
- Earliest claimed: 2015-08-03
- Assignees: The Johns Hopkins University; Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
- Applicants: The Johns Hopkins University
- Inventors: Xuhang Li; Xiaohua Hou; Rong Lin
- Web page: https://patentgrep.com/patent/US10478458B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- C12N 5/0619: {Neurons}
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- 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 2506/1353: from bone marrow mesenchymal stem cells (BM-MSC)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed is a composition including: an isolated in vitro pre-conditioned population of adult bone marrow derived mesenchymal stem cells (BMSCs), wherein the BMSCs express neuronal markers, and wherein the neuronal markers are PGP9.5, NSE, Tuj1, HuC/D and neuronal nitric oxide synthase (nNOS). Methods of preparing the BMSCs are also provided. In addition, the present disclosure is directed to a method of treating an enteric nervous system-related disorder including: administering to a subject in need thereof a pharmaceutical composition including the in vitro pre-conditioned BMSC population and a pharmaceutically acceptable carrier.

## Claims

### Claim 1

A composition comprising:
an isolated in vitro pre-conditioned population of adult bone marrow derived mesenchymal stem cells (BMSCs),
wherein at least 80% of the in vitro pre-conditioned BMSC population express PGP9.5 and NSE,
wherein at least 70% of the in vitro pre-conditioned BMSC population express Tuj1 and HuC/D,
wherein at least 75% of the in vitro pre-conditioned BMSC population express neuronal nitric oxide synthase (nNOS), and
wherein the BMSCs promote de novo regeneration of neurons not originated from the BMSCs when the BMSCs are administered to a subject.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the in vitro pre-conditioning comprises:
providing a population of mesenchymal stem cells from a bone marrow;
culturing the population of mesenchymal stem cells in a medium comprising a glial cell derived neurotrophic factor and a fetal gut culture medium.

### Claim 3 (depends on 1)

The composition of claim 1, wherein about 80% of the cells in the in vitro pre-conditioned BMSC population express PGP9.5, about 80% of the cells in the in vitro pre-conditioned BMSC population express NSE, about 75% of the cells in the in vitro pre-conditioned BMSC population express Tuj1, about 73% of the cells in the in vitro pre-conditioned BMSC population express HuC/D and about 75% of the in vitro pre-conditioned BMSC population express nNOS.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the in vitro pre-conditioned BMSCs are capable of maintaining a neuronal-like phenotype in vivo for at least 28 days.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the in vitro pre-conditioned BMSCs are human cells.

### Claim 6 (depends on 1)

The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the in vitro pre-conditioned BMSC population are labeled with a bio-imaging agent.

### Claim 8

A method of treating an enteric nervous system disorder comprising:
administering to a subject in need thereof a pharmaceutical composition comprising an isolated in vitro pre-conditioned population of adult bone marrow derived mesenchymal stem cells (BMSCs), wherein at least 80% of the in vitro pre-conditioned BMSC population express PGP9.5 and NSE,
wherein at least 70% of the in vitro pre-conditioned BMSC population express Tuj1 and HuC/D,
wherein at least 75% of the in vitro pre-conditioned BMSC population express neuronal nitric oxide synthase (nNOS), and wherein the BMSCs promote de novo regeneration of neurons not originated from the BMSCs when the BMSCs are administered to a subject
and a pharmaceutically acceptable carrier.

### Claim 9 (depends on 8)

The method of claim 8, wherein the enteric nervous system disorder is selected from the group consisting of a dysmotility syndrome, a diabetic gastroparesis, an intestinal pseudo-obstruction of motility, and neuronal loss in an enteric nervous system.

### Claim 10 (depends on 8)

The method of claim 8, wherein the dysmotility syndrome is selected from the group consisting of achalasia, gastro-esophageal reflux disease, delayed emptying of the stomach, abdominal pain, bloating, diarrhea and constipation.

### Claim 11 (depends on 10)

The method of claim 10, wherein the dysmotility syndrome is a congenital dysmotility syndrome.

### Claim 12

The method of claim ll, wherein the congenital dysmotility syndrome is Hirschsprung disease.

### Claim 13 (depends on 8)

The method of claim 8, wherein the pharmaceutical composition is administered into a gastrointenstinal tract of the subject.

### Claim 14 (depends on 8)

The method of claim 8, wherein the pharmaceutical composition is administered endoscopically to the subject.

### Claim 15 (depends on 8)

The method of claim 8, where the pharmaceutical composition is administered into a pylorus of the subject.

### Claim 16 (depends on 15)

The method of claim 15, wherein the pharmaceutical composition is administered into a submucosal layer of the pylorus.

### Claim 17

A method of preparing mesenchymal stem cells exhibiting a neuronal phenotype which method comprises:
isolating mesenchymal stem cells from an adult bone marrow;
culturing the mesenchymal stem cells in a medium comprising glial cell derived neurotrophic factor and a fetal gut culture medium to thereby obtain an isolated in vitro pre-conditioned population of adult bone marrow derived mesenchymal stem cells (BMSCs),
wherein at least 80% of the in vitro pre-conditioned BMSC population express PGP9.5 and NSE,
wherein at least 70% of the in vitro pre-conditioned BMSC population express Tuj1 and HuC/D,
wherein at least 75% of the in vitro pre-conditioned BMSC population express neuronal nitric oxide synthase (nNOS), and
wherein the BMSCs promote de novo regeneration of neurons not originated from the BMSCs when the BMSCs are administered to a subject.

### Claim 18 (depends on 17)

The method of claim 17, wherein the mesenchymal stem cells maintain the neuronal phenotype for at least 28 days in vivo.

### Claim 19 (depends on 17)

The method of claim 17, wherein about 80% of the cells in the in vitro pre-conditioned BMSC population express PGP9.5, about 80% of the cells in the in vitro pre-conditioned BMSC population express NSE, about 75% of the cells in the in vitro pre-conditioned BMSC population express Tuj1, about 73% of the cells in the in vitro pre-conditioned BMSC population express HuC/D and about 75% of the in vitro pre-conditioned BMSC population express nNOS.

### Claim 20 (depends on 17)

The method of claim 17, wherein the mesenchymal stem cells are human cells.