# US 10,767,164 B2 · Microenvironments for self-assembly of islet organoids from stem cells differentiation

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,767,164 B2
- Document ID: US10767164B2
- Status: Granted
- Kind: B2
- Application number: 15/841,004
- Filed: 2017-12-13
- Published: 2018-10-04
- Granted: 2020-09-08
- Earliest claimed: 2017-03-30
- Assignees: The Research Foundation for The State University of New York
- Applicants: The Research Foundation for The State University of New York
- Inventors: Sha Jin; Kaiming Ye; Huanjing Bi
- Web page: https://patentgrep.com/patent/US10767164B2

## CPC

- C12N 5/0677: {Three-dimensional culture, tissue culture or organ culture; Encapsulated cells}
- A61L 27/14: Macromolecular materials
- A61L 27/3633: {Extracellular matrix [ECM]}
- A61L 27/3695: {characterised by the function or physical properties of the final product, where no specific conditions are defined to achieve this (A61L27/3687, A61L27/3691 take precedence)}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61L 2430/34: for soft tissue reconstruction
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)

## Abstract

Human pluripotent stem cells (hPSCs) are promising cell source to produce therapeutic endocrine cells for diabetes treatment. A gel solution made by decellularized tissue-specific extracellular matrix (dpECM) significantly promotes three-dimensional (3D) islet-like organogenesis during induced hPSC differentiation into endocrine lineages. Islet organoids are self-organized even in a two-dimensional (2D) culture mode. Cells derived from hPSCs differentiated on such ECM coated substrates exhibit similar cellular composition to native pancreatic islets. These cells express islet signature markers insulin, PDX-1, C-peptide, MafA, glucagon, somatostatin, and pancreatic polypeptide, and secrete more insulin in response to glucose level compared to a traditional matrix substrate (Matrigel). The dpECM facilitates generating more C-peptide+/glucagon− cells rather than C-peptide+/glucagon+ cells. Remarkably, dpECM also facilitated intra-organoid vascularity by generating endothelial cells and pericytes. Furthermore, dpECM niches also induced intra-organoid microvascularization during pancreatic differentiation.

## Claims

### Claim 1

A synthetic pancreatic organoid, comprising:
at least one cross-linked collagen-containing surface, coated with osmotically processed acellular tissue-specific matrix extracted from a mammalian organ without use of detergent, and cell growth factors comprising collagen, mucopolysaccharides, and basement membrane factors; and
stem cells cultured and induced into differentiation on the surface, having at least two differentiated pancreatic cell types.

### Claim 2 (depends on 1)

The synthetic pancreatic organoid according to claim 1,
wherein the osmotically processed acellular tissue-specific matrix comprises a pancreas tissue-specific matrix, and
wherein the at least two differentiated pancreatic cell types comprise:
about 50-70% pancreatic beta cells,
about 20-30% pancreatic alpha cells,
about 10% pancreatic delta cells, and
less than 5% pancreatic polypeptide cells.

### Claim 3 (depends on 2)

The synthetic pancreatic organoid according to claim 2, wherein the pancreatic beta cells, pancreatic alpha cells, pancreatic delta cells, and pancreatic polypeptide cells are derived from human pluripotent stem cells.

### Claim 4 (depends on 3)

The synthetic pancreatic organoid according to claim 3, wherein the pancreas tissue-specific matrix is derived from a non-human mammal.

### Claim 5 (depends on 1)

The synthetic pancreatic organoid according to claim 1, wherein the acellular tissue-specific matrix is osmotically processed for cellular removal by cycles of different osmotic tension.

### Claim 6 (depends on 1)

The synthetic pancreatic organoid according to claim 1, wherein the acellular tissue-specific matrix has a residual DNA content of less than 1% of the DNA of the tissue from which it is derived.

### Claim 7

A synthetic pancreatic organoid, comprising:
a cross-linked collagen-containing surface,
a coating on the cross-linked collagen-containing surface, comprising an osmotically processed acellular tissue-specific matrix, extracted from a mammalian pancreas, and cell growth factors comprising collagen, mucopolysaccharides, and basement membrane factors, and
a plurality of differentiated pancreatic cell types, derived from stem cells cultured and induced into differentiation on the surface.

### Claim 8 (depends on 7)

The synthetic pancreatic organoid according to claim 7, wherein the plurality of differentiated cell types are induced to differentiate by a process comprising sequentially incubating the stem cells in:
RPMI 1640 containing B27, 50 ng/ml activin A and 0.5-1 mM sodium butyrate,
RPMI 1640 containing B27, 250 μM ascorbic acid, 50 ng/ml keratinocyte growth factor, 50 ng/ml Noggin, 1 μM retinoic acid, 300 nM (−)-indolactam V, and 100 nM LDN193189,
DME/F12 containing B27, 1 μM RA, 200 nM LDN, 300 nM ILV, 1 μM 3,3′,5-Triiodo-L-thyronine sodium salt, 10 μM ALK5 inhibitor II, and 10 μg/ml heparin, supplemented with glucose to a final concentration of 20 mM,
RPMI 1640 containing B27, 1 μM T3, 10 μM ALKi, 1 mM N-acetyl cysteine, 0.5 μM R428, 10 μM trolox, 100 nM γ-secretase inhibitor XX, 10 μM zinc sulfate, 10 mM nicotinamide, and 10 μg/ml HP, supplemented with glucose to a final concentration of 20 mM glucose, and
CMRL supplement containing 10% fetal bovine serum, 1 μM T3, 10 μM ALKi, 0.5 μM R428, and 10 mM Nic.