# US 2014/0193373 A1 · Methods for establishing and improving the survival of a population of pancreatic progenitor or stem cells

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- Number: US 2014/0193373 A1
- Document ID: US20140193373A1
- Status: Published application
- Kind: A1
- Application number: 13/831,382
- Filed: 2013-03-14
- Published: 2014-07-10
- Earliest claimed: 2013-01-04
- Assignees: City of Hope; California Institute of Technology
- Applicants: City Of Hope
- Inventors: Hsun Ku
- Web page: https://patentgrep.com/patent/US20140193373A1

## CPC

- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- A61K 35/39: Pancreas; Islets of Langerhans (Langerhans cells of epidermis A61K35/36)
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- G01N 33/56966: {Animal cells}
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/335: Glucagon; Glucagon-like peptide [GLP]; Exendin
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/58: Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
- C12N 2533/52: Fibronectin; Laminin
- C12Q 2600/158: Expression markers
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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

In some embodiments, methods for improving the survival of pancreatic β-cell progenitors in culture are provided. Such methods may include contacting a population of pancreatic progenitor cells with an amino acid (aa) sequence comprising IKVAV (SEQ ID NO:1). In other embodiments, methods for (i) verifying the establishment of a population of pancreatic progenitor or stem cells and (ii) methods for generating or establishing a population of pancreatic endocrine progenitor cells in vitro are provided.

## Claims

### Claim 1

A method of improving the survival of pancreatic β-cell progenitors in culture comprising contacting a population of pancreatic progenitor cells with an amino acid sequence comprising IKVAV (SEQ ID NO:1), wherein the amino acid sequence improves survival of the population of pancreatic β-cell progenitors.

### Claim 2 (depends on 1)

The method of claim 1, wherein the population of pancreatic progenitor cells is derived from a population of adult pancreatic ductal cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amino acid supports differentiation of the pancreatic progenitor cells to pancreatic β-cell progenitors.

### Claim 4 (depends on 1)

The method of claim 1, wherein the amino acid sequence comprises SEQ ID NO:2.

### Claim 5 (depends on 1)

The method of claim 1, wherein the amino acid sequence is part of an artificial extracellular matrix protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the artificial extracellular matrix protein comprises an elastin-based sequence.

### Claim 7 (depends on 6)

The method of claim 6, wherein the elastin-based sequence comprises SEQ ID NO:3.

### Claim 8

A method of verifying the establishment of a population of pancreatic progenitor or stem cells comprising:
isolating a population of cells from a pancreatic tissue sample that express CD133 and Sox9 (CD133+/Sox9+ cells);
culturing at least one of the isolated CD133+/Sox9+ cells with an effective amount of R-Spondin 1 to establish at least one single-cell derived colony; and
verifying that the single-cell derived colony comprises a population of pancreatic progenitor or stem cells by detecting expression of one or more pancreatic progenitor cell markers in one or more colony cells.

### Claim 9 (depends on 8)

The method of claim 8, wherein the one or more pancreatic progenitor cell markers include Pdx1, Ngn3, Nkx6.1, Pax4, MafB, c-Met, DCAMKL1, DCAMKL2, or a combination thereof.

### Claim 10 (depends on 8)

The method of claim 8, wherein verifying that the one or more single-cell derived colonies comprise a population of pancreatic progenitor or stem cells is accomplished by RT-PCR or microarray.

### Claim 11

A method of establishing a population of pancreatic endocrine progenitor cells comprising
isolating a population of cells from a pancreatic tissue sample that express CD133 and Sox9 (CD133+/Sox9+ cells);
culturing at least one of the isolated CD133+/Sox9+ cells with an effective amount of R-Spondin 1 to establish at least one single-cell derived dense colony;
dissociating at least one single-cell derived dense colony to form a single-cell suspension;
culturing the single-cell suspension with an amino acid sequence comprising IKVAV (SEQ ID NO:1) in the absence of R-Spondin 1 to establish the population of pancreatic endocrine progenitor cells.

### Claim 12 (depends on 11)

The method of claim 11, further comprising dissociating and replating the single-cell derived dense colonies with an effective amount of R-Spondin 1 for one or more generations prior to forming a single-cell suspension.

### Claim 13 (depends on 11)

The method of claim 11, wherein the population of pancreatic endocrine progenitor cells are primarily pancreatic β-cell progenitor cells.

### Claim 14 (depends on 13)

The method of claim 13, further comprising selecting or verifying that the population of pancreatic progenitor endocrine cells comprise pancreatic β-cell progenitors by detecting expression of glucokinase, Pcsk1, Pcsk2 or a combination thereof.

### Claim 15 (depends on 13)

The method of claim 13, wherein the pancreatic β-cell progenitors secrete insulin in response to glucose.

### Claim 16 (depends on 13)

The method of claim 13, wherein the pancreatic β-cell progenitors are administered to a subject for treatment of diabetes.

### Claim 17 (depends on 11)

The method of claim 11, wherein the amino acid sequence comprises SEQ ID NO:2.

### Claim 18 (depends on 17)

The method of claim 17, wherein the amino acid sequence is part of an artificial extracellular matrix protein.

### Claim 19 (depends on 18)

The method of claim 18, wherein the artificial extracellular matrix protein comprises an elastin-based sequence.

### Claim 20 (depends on 19)

The method of claim 19, wherein the elastin-based sequence comprises SEQ ID NO:3.