# US 2021/0340499 A1 · Production and Enrichment of Pancreatic Endocrine Progenitor Cells

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- Number: US 2021/0340499 A1
- Document ID: US20210340499A1
- Status: Published application
- Kind: A1
- Application number: 17/280,076
- Filed: 2019-09-25
- Published: 2021-11-04
- Earliest claimed: 2018-09-25
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Julie Beth Sneddon; Lauren E. Byrnes; Daniel M. Wong
- Web page: https://patentgrep.com/patent/US20210340499A1

## CPC

- C12N 5/0678: {Stem cells; Progenitor cells; Precursor cells}
- G01N 33/507: {Pancreatic cells}
- G01N 33/56966: {Animal cells}
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- C12N 2506/02: from embryonic cells
- G01N 2333/575: Hormones
- G01N 2333/62: Insulins
- G01N 2800/042: Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The disclosure provides methods for enriching for pancreatic endocrine progenitor cells, such as human pancreatic endocrine progenitor cells, including alpha cell progenitors, beta cell progenitors, delta cell progenitors, PP cell progenitors and epsilon cell progenitors. The disclosure provides mammalian, such as human, Fev+ pancreatic endocrine progenitor cells, including Fev+ alpha cell progenitors, Fev+ beta cell progenitors, Fev+ delta cell progenitors, Fev+ PP cell progenitors, and Fev+ epsilon cell progenitors. The disclosure further provides methods for producing or inducing such cells, including in vitro differentiation methods, and the cells so produced.

## Claims

### Claim 1

A method of enriching the pancreatic endocrine progenitor cell population in a cell sample comprising
(a) detecting cells in the sample expressing a pancreatic endocrine progenitor cell marker; and
(b) separating a pancreatic endocrine progenitor cell from at least one cell that does not express the pancreatic endocrine progenitor cell marker, thereby enriching the pancreatic endocrine progenitor cell population of the cell sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pancreatic endocrine progenitor cell is a human cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pancreatic endocrine progenitor cell is an alpha cell progenitor, a beta cell progenitor, a delta cell progenitor, a PP cell progenitor, or an epsilon cell progenitor.

### Claim 4 (depends on 3)

The method of claim 3, wherein the pancreatic endocrine progenitor cell marker is the E26 transformation-specific transcription factor Fev.

### Claim 5 (depends on 4)

The method of claim 4, wherein the pancreatic endocrine progenitor cell is a beta cell progenitor.

### Claim 6 (depends on 5)

The method of claim 5, wherein the Fev+ beta cell progenitor further comprises Gng12+, Tssc4+, Ece1+, Tmcm108+, Wipi1+, or Papss2+.

### Claim 7 (depends on 6)

The method of claim 6, wherein the beta cell progenitor is Fev+, Gng12+.

### Claim 8 (depends on 5)

The method of claim 5, wherein the Fev+ beta cell progenitor further comprises Pax4+, Chga+, Chgb+, Neurod1+, Runx1t1+, or Vim+.

### Claim 9 (depends on 5)

The method of claim 5 wherein the Fev+ beta cell progenitor does not express detectable Ngn3, Ins1 or Gcg.

### Claim 10 (depends on 9)

The method of claim 9, wherein the beta cell progenitor is Fev+, Ngn−.

### Claim 11 (depends on 10)

The method of claim 10, wherein the Fev+, Ngn− beta cell progenitor expresses a gene in the serotonin pathway, the insulin signaling pathway, sphingosine-1-phosphate signaling pathway, or Activating Transcription Factor-2.

### Claim 12 (depends on 5)

The method of claim 5, wherein the Fev+ beta cell progenitor further comprises Pdx1+ or Mafb+.

### Claim 13 (depends on 1)

The method of claim 1, wherein the at least one cell that does not express the pancreatic endocrine progenitor cell marker is a CD140+ mesenchyme cell.

### Claim 14 (depends on 5)

The method of claim 5, wherein the beta cell progenitor cell is a human cell.

### Claim 15

A method of producing a pancreatic endocrine progenitor cell comprising culturing a stem cell under conditions that induce differentiation of the stem cell into a pancreatic endocrine progenitor cell comprising the E26 transformation-specific transcription factor Fev.

### Claim 16 (depends on 15)

The method of claim 15, wherein the stem cell is an embryonic stem cell (ESC) or an inducible pluripotent stem cell (iPSC).

### Claim 17 (depends on 15)

The method of claim 15, wherein the pancreatic endocrine progenitor cell is an alpha cell progenitor, a beta cell progenitor, a delta cell progenitor, a PP cell progenitor, or an epsilon cell progenitor.

### Claim 18 (depends on 17)

The method of claim 17, wherein the pancreatic endocrine progenitor cell is a beta cell progenitor.

### Claim 19 (depends on 18)

The method of claim 18, wherein the Fev+ beta cell progenitor further comprises Gng12+, Tssc4+, Ece1+, Tmcm108+, Wipi1+, or Papss2+.

### Claim 20 (depends on 19)

The method of claim 19, wherein the beta cell progenitor is Fev+, Gng12+.

### Claim 21 (depends on 18)

The method of claim 18, wherein the Fev+ beta cell progenitor further comprises Pax4+, Chga+, Chgb+, Neurod1+, Runx1t1+, or Vim+.

### Claim 22 (depends on 18)

The method of claim 18 wherein the Fev+ beta cell progenitor does not express detectable Ngn3, Ins1 or Gcg.

### Claim 23 (depends on 22)

The method of claim 22, wherein the beta cell progenitor is Fev+, Ngn−.

### Claim 24 (depends on 23)

The method of claim 23, wherein the Fev+, Ngn− beta cell progenitor expresses a gene in the serotonin pathway, the insulin signaling pathway, sphingosine-1-phosphate signaling pathway, or Activating Transcription Factor-2.

### Claim 25 (depends on 18)

The method of claim 18, wherein the Fev+ beta cell progenitor further comprises Pdx1+ or Mafb+.

### Claim 26 (depends on 18)

The method of claim 18, wherein the Fev+ beta cell progenitor is a human cell.

### Claim 27 (depends on 17, 18)

An isolated Fev+ pancreatic endocrine progenitor cell produced according to claim 17 or claim 18.

### Claim 28

A method of inducing formation of a hormone-producing cell comprising contacting a progenitor of a hormone-producing cell with an effective amount of Fev to produce a hormone-producing cell.

### Claim 29 (depends on 28)

The method of claim 28, wherein the hormone-producing cell is a INS+ cell.

### Claim 30 (depends on 29)

The method of claim 29, wherein the hormone-producing progenitor cell is an ES4 cell.

### Claim 31 (depends on 28)

The method of claim 28, wherein the hormone-producing cell is a beta cell.

### Claim 32 (depends on 31)

The method of claim 31, wherein the hormone-producing progenitor cell is a beta-like cell.

### Claim 33 (depends on 28)

The method of claim 28, wherein the method is performed in vitro.

### Claim 34 (depends on 28)

The method of claim 28 further comprising removing a cell expressing at least one of PHOX2A, TLX2 or TBX2.

### Claim 35

A method of screening for a signaling compound that induces FEV+ progenitor cell replication comprising:
(a) contacting a FEV+ progenitor cell with a candidate compound;
(b) culturing the FEV+ progenitor cell under conditions suitable for cell proliferation;
(c) measuring the cell proliferation of the FEV+ progenitor cell in the presence or absence of the candidate compound; and
(d) identifying the compound as a signaling compound for FEV+ progenitor cell proliferation if the cell proliferation in the presence of the compound is greater than the cell proliferation in the absence of the compound.

### Claim 36 (depends on 35)

The method of claim 35 wherein the FEV+ progenitor cell is a FEV-MYC progenitor cell, a FEV-GFP progenitor cell, a FEV-KO progenitor cell, or a FEV-tNFGR progenitor cell.

### Claim 37

A method of screening for a signaling compound that enhances FEV+ progenitor cell differentiation into beta cells comprising:
(a) contacting FEV+ progenitor cells with a candidate compound;
(b) incubating the FEV+ progenitor cells under conditions suitable for cell differentiation;
(c) measuring the level of differentiation of the FEV+ progenitor cells to beta cells in the presence or absence of the candidate compound; and
(d) identifying the compound as a signaling compound for FEV+ progenitor cell differentiation into beta cells if the cell differentiation in the presence of the compound is greater than the cell differentiation in the absence of the compound.

### Claim 38 (depends on 37)

The method of claim 37 wherein the FEV+ progenitor cell is a FEV-MYC progenitor cell, a FEV-GFP progenitor cell, a FEV-KO progenitor cell, or a FEV-tNFGR progenitor cell.