# US 12,761,209 B2 · Devices and methods to induce adult type maturation of human pluripotent stem cell derived cardiomyocytes

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- Number: US 12,761,209 B2
- Document ID: US12761209B2
- Status: Granted
- Kind: B2
- Application number: 18/184,851
- Filed: 2023-03-16
- Published: 2023-09-21
- Granted: 2026-10-13
- Earliest claimed: 2022-03-18
- Assignees: University of Connecticut; The Regents of the University of California
- Applicants: University of Connecticut; The Regents of the University of California
- Inventors: Kshitiz; Junaid Afzal; Wenqiang Du; Yasir Suhail
- Web page: https://patentgrep.com/patent/US12761209B2

## CPC

- G01N 33/5082: {Supracellular entities, e.g. tissue, organisms}
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- C12N 2500/38: Vitamins
- C12N 2501/33: Insulin
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2513/00: 3D culture
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2535/10: Patterned coating
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed are constructs and methods to accelerate maturation of human pluripotent stem cell derived cardiomyocytes by maintaining them on a Cardiac Mimetic Matrix (CMM) substrate.

## Claims

### Claim 1

A construct, comprising:
(a) a patterned scaffold, wherein the patterned scaffold comprises a polymeric hydrogel substrate comprising a plurality of nanowrinkles, wherein the nanowrinkles comprise branched folds directionally aligned over a centimeter length scale, wherein the polymeric substrate has a viscoelasticity between about 15 kPa and about 100kPa;
(b) one or more cardiac matrix ligands conjugated to the patterned scaffold, wherein the one or more cardiac matrix ligands comprises 1, 2, 3, 4 or more of nephronectin (SEQ ID NO: 13), GRGDS (SEQ ID NO: 10), GFOGER (SEQ ID NO: 11), GFPGER (SEQ ID NO: 12) and/or other peptides containing one or more RGD motifs.

### Claim 2 (depends on 1)

The construct of claim 1, wherein the construct comprises nephronectin (SEQ ID NO: 13), RGD, and GFOGER (SEQ ID NO: 11) conjugated to the patterned scaffold.

### Claim 3 (depends on 2)

The construct of claim 2, wherein the nephronectin (SEQ ID NO: 13), RGD, and GFOGER (SEQ ID NO: 11) are present in about an equimolar ratio.

### Claim 4 (depends on 1)

The construct of claim 1, wherein the construct further comprises laminin conjugated to the patterned scaffold.

### Claim 5 (depends on 1)

The construct of claim 1, wherein the polymeric hydrogel substrate comprises aligned wrinkle ridges, wherein the aligned wrinkle ridges are arranged in one or more continuous and ordered patterns.

### Claim 6 (depends on 1)

The construct of claim 1, wherein a height of ridges ranges between about 10 nm and about 4 μm, and optionally the ridges are all of approximately the same height over the entire scaffold, or of approximately the same height in each discrete section of the scaffold.

### Claim 7 (depends on 1)

The construct of claim 1, wherein valley to valley distances and/or ridge peak to ridge peak distances of between about 400 nm and about 3 μm.

### Claim 8 (depends on 1)

The construct of claim 1, wherein the construct comprises fluorescent beads.

### Claim 9 (depends on 1)

The construct of claim 1, wherein the patterned scaffold comprises a polyacrylamide (PA) or polyethylene glycol (PEG) hydrogel, poly lactic-co-glycolic acid (PLGA), polyurethane (PUA), polyacrylate (PA) or their chemical branch derivatives.

### Claim 10 (depends on 1)

The construct of claim 1, wherein the patterned scaffold comprises a polyacrylamide (PA) hydrogel having a rigidity of between about 16-24 kPa.

### Claim 11 (depends on 10)

The construct of claim 10, wherein the polymeric hydrogel substrate binds to the one or more cardiac matrix ligands via covalent binding or functional group conjugation.

### Claim 12 (depends on 1)

The construct of claim 1, further comprising cardiomyocytes or precursors thereof seeded on the construct.

### Claim 13 (depends on 12)

The construct of claim 12, wherein the cardiomyocytes or precursors thereof comprise induced pluripotent stem cell (iPSC) derived cardiomyocytes, human cardiomyocytes or precursors thereof, and/or electrochemically connected cardiomyocytes.

### Claim 14 (depends on 1)

The construct of claim 1, further comprising cardiac fibroblasts or precursors thereof, endothelial cells, vascular smooth muscle cells and/or macrophages and/or other immune cells seeded on the construct.

### Claim 15 (depends on 1)

The construct of claim 1, wherein the polymeric hydrogel substrate is capable of shrinking or expanding to achieve a desired feature size ranging from 0.1 μm to 10 μm.

### Claim 16 (depends on 1)

The construct of claim 1, wherein the plurality of nanowrinkles comprises isotropic or non-aligned nanowrinkles capable of non-directional stretching, orthogonal stretching or circular stretching.