Muscle cell patches and uses thereof

Abstract

To provide a contractile cell construct that includes a contractile cell adhered on a surface of a three-dimensional scaffold (3DFCS) including a fibroblast or its precursor cell, and a method for using the construct in order to treat a disease.SOLUTION: In one embodiment, the present invention discloses a method for preparing a contractile construct including: (a) disseminating an immature contractile cell onto a surface of a three-dimensional scaffold (3DFCS) including a fibroblast in order to generate the contractile construct; and (b) culturing the contractile construct under a condition of accelerating differentiation of the immature contractile cell into a mature contractile cell forming streak lines. In another embodiment, the present invention discloses a method for treating a disorder characterized by malfunction of a contractile cell which includes bringing a construct of any embodiment or a combination of embodiments of the present invention in contact with a patient having a contractile cell-based disorder in an amount effective for treating the disorder.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 61 / 888,882, filed October 9, 2013, which is incorporated by reference in its entirety.

[0002] Statement of Government Rights This invention was made with Government support under Grant No. 1-101-BX001406-01A1 awarded by the VA. [Background technology]

[0003] New therapies are needed for patients with chronic heart failure (CHF), the number one hospital discharge diagnosis for patients aged 65 years or older in the United States, as well as associated ischemic and nonischemic heart disease. The prevalence of heart failure is over 5 million, with an annual incidence of 550,000 patients. Heart failure is more deadly than cancer, accidents, and combined strokes, and costs more than $2.3 billion annually. Once patients develop symptoms of NY class III or IV heart failure, their mortality approaches 50% within 2 years without heart transplantation. The latest approach to treat CHF is to inject stem and / or progenitor cells directly into the heart, using several different cell types. However, results of recent clinical trials using such injection methods have been generally disappointing. Summary of the Invention

[0004] In one aspect, the present invention provides a method for preparing a contraction construct, comprising: (a) seeding immature contractile cells onto the surface of a fibroblast-containing three-dimensional scaffold (3DFCS) to generate a contractile construct; (b) culturing the contractile construct under conditions that promote differentiation of immature contractile cells into mature contractile cells that form striations; The present invention provides a method comprising:

[0005] In one embodiment, the immature contractile cells are immature cardiomyocytes and the mature contractile cells are mature cardiomyocytes. In another embodiment, the immature contractile cells are immature smooth muscle cells or skeletal muscle cells and the mature contractile cells are mature smooth muscle cells or skeletal muscle cells. In another embodiment, the contractile construct is implanted after culturing into a subject in need thereof. The construct may be implanted before the initiation of cell contraction and / or patch-level contraction, and in another embodiment, the contractile construct is implanted after the initiation of patch-level contraction.

[0006] In another aspect, the present invention provides a construct comprising contractile cells or precursors thereof attached to the surface of a fibroblast-containing three-dimensional scaffold (3DFCS), the construct being capable of spontaneous synchronous contraction across the surface of the 3DFCS, and the contractile cells being capable of contracting at least 1.3×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 and present on the surface of the 3DFCS at a ratio of about 1:15 to about 6:1 with the fibroblasts on the 3DFCS. In one embodiment, the contractile cells include a combination of progenitor contractile cells and mature contractile cells. In another embodiment, the progenitor contractile cells and mature contractile cells are present on the surface of the construct at a ratio of about 1:2 to about 2:1. In another embodiment, the contractile cells include immature cardiomyocytes, mature cardiomyocytes, or a combination thereof. In another embodiment, the immature contractile cells are immature smooth muscle cells or immature skeletal muscle cells, and the mature contractile cells are mature smooth muscle cells or mature skeletal muscle cells. In another embodiment, the contractile cells form striations on the construct.

[0007] In a further aspect, the invention provides a method for treating a disorder characterized by dysfunction of contractile cells, comprising contacting a patient having a contractile cell system disorder with a construct of any embodiment or combination of embodiments of the invention in an amount effective to treat the disorder. In one embodiment, the contractile cells comprise immature cardiomyocytes, mature cardiomyocytes, or a combination thereof, and the method comprises contacting the heart of a subject suffering from such a disorder with a construct in an amount effective to treat the disorder, the disorder being ischemia-induced heart failure, chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy, dilated cardiomyopathy (DCM), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling, insufficient blood flow, impaired cardiac function ... Symptoms may include, but are not limited to, adaptive left ventricular remodeling, decreased left ventricular function, left heart failure, right heart failure, posterior failure, anterior failure, systolic dysfunction, diastolic dysfunction, increased or decreased systemic vascular resistance, low output heart failure, high output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cold extremities at rest, exercise intolerance, fatigue, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, lateral shift of the apical beat, gallop rhythm, heart murmur, parasternal pulse, and pleural effusion.

[0008] In one embodiment, the construct is attached to the epicardium of the subject. In another embodiment, the construct is non-contracted when in contact with the epicardium or is contracted when in contact with the epicardium. In a further embodiment, the treatment comprises one or more of the following heart failure parameters, including but not limited to: improved left ventricular function, lowered left ventricular end diastolic pressure (EDP), improved myocardial perfusion, remodeling of the heart wall by cardiomyocytes, reversal of maladaptive left ventricular remodeling in CHF subjects, improved diastolic function such as passive filling, active filling of the left ventricle, ventricular chamber compliance, and increased E' (mm / sec), decreased E / E', increased LV dP / dt (mmHg / sec) and decreased Tau (milliseconds). In another embodiment, the cardiomyocytes on the construct are electrically coupled to the patient's native myocardium.

[0009] In further embodiments, the contractile cells comprise immature skeletal muscle cells, immature smooth muscle cells, mature skeletal muscle cells, mature smooth muscle cells, or combinations thereof, and the method comprises treating any disorder that can benefit from augmentation, repair, or restoration of skeletal and / or smooth muscle tissue by contacting a patient having the disorder with the construct in an amount effective to treat the disorder.

[0010] In another aspect, the present invention provides a method for drug screening, comprising contacting a structure of any embodiment or combination of embodiments of the present invention with a compound of interest and determining the effect of the compound on one or more characteristics of the structure.In one embodiment, the method comprises culturing the structure under conditions that promote the contraction of the structure before contacting the structure with the compound of interest.In another embodiment, the effect of the compound on one or more of contraction displacement, contraction rate, contraction synchrony, and contraction velocity is determined. [Brief description of the drawings]

[0011] [Figure 1-1] Electrical activation mapping was performed in neonatal cardiomyocyte (NCM)-3D fibroblast constructs (3DFC) in tissue culture 5 days after co-culture using a custom designed multi-electrode array (MEA) with 18 recording sites spaced 500 μm apart (A). Recordings were made from 10 electrodes, with each recording site numbered sequentially as channels 1-10 (B). Electrical activation of the patch was shown at 7 s intervals, demonstrating consistent beat-to-beat activation displaying peak lateral conduction voltages for each individual channel (C). Amplitudes show that all channels overlapped during the beat-to-beat sequence (D) and during unitary activation (E). Amplitudes were recorded as 0.03-0.42 and -0.13--0.75 mV (D and E). [Figure 1-2]Electrical activation mapping was performed in neonatal cardiomyocyte (NCM)-3D fibroblast constructs (3DFC) in tissue culture 5 days after co-culture using a custom designed multi-electrode array (MEA) with 18 recording sites spaced 500 μm apart (A). Recordings were made from 10 electrodes, with each recording site numbered sequentially as channels 1-10 (B). Electrical activation of the patch was shown at 7 s intervals, demonstrating consistent beat-to-beat activation displaying peak lateral conduction voltages for each individual channel (C). Amplitudes show that all channels overlapped during the beat-to-beat sequence (D) and during unitary activation (E). Amplitudes were recorded as 0.03-0.42 and -0.13--0.75 mV (D and E). [Diagram 2] a) Paced activation maps in a chronic heart failure (CHF) rat with a seeded patch for the region of interest indicated by the black box. b) Electrocardiogram taken from the epicardial surface during introduction of the pacing electrode at location "P" showing successful capture. c) Provides data for 9 different activation maps for activation time compiled over 72 contractions at 32 locations. Multiple maps created show consistency in the measurements. [Diagram 3] Induced pluripotent stem cell derived cardiomyocytes (stained red) were seeded onto the fibroblast constructs and co-cultured. The vicryl fibers can be observed as a net-like mesh weave. The crimson fluorescence is the embedded fibroblasts. These cells were seeded locally and do not penetrate the patch or the embedded fibroblasts. The patch started spontaneous, synchronous contractions immediately after seeding. The cells were randomly seeded using centrifugal force. [Figure 4] When seeded on fibroblast patches, induced pluripotent stem cell-derived cardiomyocytes produced a force response. Data are from fibroblast patches seeded with 2 x 106 cells each (1.2 x 106 cells / cm2) on day 5 of culture. This indicates that iPSC-derived cardiomyocytes align and contract in unison, potentially aiding in the resulting improved function. [Diagram 5]Trichrome staining shows LV cross-sections 3 weeks after patch implantation. Corresponding asterisks and boxes indicate areas of higher magnification. Arrows indicate bands of myocytes expressing RFP as shown in the fluorescent image on the right. The epicardium (EPI) and endocardium (END) are labeled for orientation. Positive red fluorescent positive cells suggest survival of human iPSC-dCMs, as indicated by the presence of RFP expression (red). Nuclei of tissues and constructs are labeled with DAPI (blue). As expected, human iPSC-dCMs remain localized 3 weeks after implantation. [Figure 6] Trichrome staining of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on day 2 (A) versus day 6 (B) in standard tissue culture. All cells stain positive (red / purple) for muscle in both 2- and 6-day cultures. After 6 days in culture, hiPSC-CMs expanded. When seeded on fibroblast patches, hiPSC-CMs remain small in size on day 2 (C and E), but by day 6 (D and F) they have developed into an intact layer with clearly defined striations, suggesting that the fibroblast patches provide structural support to allow hiPSC-CMs to mature in vitro. [Figure 7] Trichrome stained left ventricular cross sections of infarcted but untreated chronic heart failure (CHF) controls at 6 weeks (A and B), and CHF+ human induced pluripotent stem cell-derived cardiomyocyte patch (hiPSC-CM) at 6 weeks after coronary artery ligation (3 weeks after transplantation) (D and E). Hearts were excised, the right ventricle removed, and cut into 5 μm transverse sections along the midpoint of the ventricle. Healthy myocardium is represented in magenta, collagen / scar in blue, and red blood cells as small red dots. Box insets represent areas of higher magnification. Transplantation of hiPSC-CM patches increases LV wall thickness (D) and results in the retention and / or generation of myocardium (D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] All references cited are incorporated herein by reference in their entirety. Unless otherwise indicated, techniques utilized within this application may be found in any of several known references, such as Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Techniques (Methods in Enzymology, Vol. 185, D. Goeddel (Ed.), 1991, Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutschcer (Ed.), (1990) Academic Press); PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990. Academic Press, San Diego, CA), Animal Cell Culture: A Manual of Basic Techniques, 2nd Edition (RI Freshney. 1987. Liss Co., New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray (Ed.), The Humana Press, Clifton, NJ), and Ambion's 1998 Catalog (Ambion, Austin, TX).

[0013] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless otherwise stated.

[0014] As used herein, the term "about" means + / - 5% of the stated parameter.

[0015] All embodiments of any aspect of the invention can be used in combination unless the context clearly dictates otherwise.

[0016] In a first aspect, the present invention provides a construct comprising contractile cells attached to the surface of a fibroblast-containing three-dimensional scaffold (3DFCS), the construct being capable of spontaneous synchronous contraction across the surface of the 3DFCS, the contractile cells being present in a concentration of 1.3×10 5cells / cm 2 ~2.95×10 6 cells / cm 2 The cells are seeded onto the surface of the construct at a density of about 1:15 to about 6:1 with the fibroblasts on the 3DFCS.

[0017] The constructs of the invention can be used for the treatments and drug screening described herein, and are demonstrated in the Examples to provide functional benefits when implanted in rodent models of congestive heart failure, and to be electrically stable when implanted.

[0018] As used herein, a "three-dimensional fibroblast construct" is a construct comprising fibroblasts grown on a three-dimensional substrate comprising a biocompatible, non-biological material, formed into a three-dimensional structure with gaps bridged by cells within the construct. It is understood that the 3DFC may comprise cell types, in addition to the appropriate fibroblasts, as needed for a given purpose. For example, the 3DFC may comprise other stromal cells, including but not limited to endothelial cells. See, for example, U.S. Patent Application Publication No. 2009 / 0269316 and U.S. Patent No. 4,963,489, both of which are incorporated herein by reference in their entireties.

[0019] The fibroblasts and other cells may be fetal or adult in origin and may be derived from convenient sources such as skin, cardiac muscle, smooth muscle, skeletal muscle, liver, pancreas, brain, adipose tissue (fat). Such tissues and / or organs may be obtained by appropriate biopsy or at autopsy. In an alternative embodiment for all aspects of the invention, the fibroblasts and other cells are human cells. In an alternative embodiment for all aspects of the invention, the 3DFC is a matrix-embedded human skin construct of neonatal dermal fibroblasts cultured in vitro on a bioabsorbable mesh to generate metabolically active, viable tissue. These fibroblasts proliferate throughout the mesh and secrete a wide variety of growth factors and cytokines, including human dermal collagen, fibronectin, and glycosaminoglycans (GAGs), which are themselves embedded in the self-produced dermal matrix. In culture, fibroblasts produce the angiogenic growth factors: VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), bFGF (basic fibroblast growth factor), and angiopoietin-1 (see, e.g., J. Anat. (2006) 209, pp 527-532).

[0020] Any suitable 3D FCS can be used, including, but not limited to, any and all scaffolds, such as woven, bonded, spun, printed, degradable, non-degradable, allogeneic, autograft, xenograft, synthetic, biological, degradable, non-degradable, porous, which may include one or more of the following: woven, bonded, spun, printed, degradable, non-degradable, allogeneic, autograft, xenograft, micropores (evenly spaced, unevenly spaced, various sizes), extracellular matrix, etc.

[0021] The three-dimensional support framework may be of any material and / or shape that (a) allows cells to adhere (or can be modified to allow cells to adhere) and b) allows cells to grow within two or more layers. Several different materials may be used to form the framework, including, but not limited to, nylon (polyamide), Dacron (polyester), polystyrene, polypropylene, polyacrylate, polyvinyl compounds (e.g., polyvinyl chloride, PVC), polycarbonate, polytetrafluoroethylene (PTFE; TEFLON), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), catgut suture, cellulose, gelatin, dextran, and the like. Any of these materials may be woven into a mesh to form the three-dimensional framework. Certain materials, such as nylon, polystyrene, and the like, are poor substrates for cell attachment. When these materials are used as the three-dimensional support framework, it is desirable to pretreat the framework prior to inoculation of fibroblasts and other stromal cells to enhance attachment to the framework. For example, nylon screens can be treated with 0.1 M acetic acid and incubated in polylysine, fetal bovine serum, and / or collagen prior to seeding with fibroblasts and other stromal cells to coat the nylon. Polystyrene can be similarly treated with sulfuric acid.

[0022] When the 3DFC is directly implanted in vivo, it may be preferable to use biodegradable materials such as PGA, catgut suture material, collagen, polylactic acid, or hyaluronic acid. For example, these materials may be woven into a three-dimensional framework such as a collagen sponge or collagen gel. When the cultures are to be maintained for long periods or cryopreserved, non-degradable materials such as nylon, Dacron, polystyrene, polyacrylate, polyvinyl, Teflon, cotton, etc. may be preferred. A convenient nylon mesh that can be used according to the present invention is a nylon filtration mesh (#3-210 / 36, Tetko, Inc., NY) with an average pore size of 140 μm and an average fiber diameter of 90 μm for the nylon fibers.

[0023] Any suitable contractile cells can be used, including, but not limited to, smooth muscle cells, skeletal muscle cells, and cardiac muscle cells, or combinations thereof.

[0024] Contractile cells can be derived from any source, including, but not limited to, fetal tissue, neonatal tissue, adult tissue from stem cell population, progenitor cell population, embryonic cell or somatic cell reprogrammed via induced pluripotent stem cell (iPSC) by virus, mRNA, episomal vector, etc. Contractile cells can be fully mature contractile cells or immature cells of a specific contractile cell pathway, or combinations thereof. These cells can be derived from any suitable organism, such as primate cells, such as rodent or human cells. These cells can be derived from male or female subjects, or cells from male and female subjects can be combined.

[0025] In one alternative embodiment, the 3DFC comprises a patch and the cells are seeded on top of the patch. In this embodiment, the bottom of the patch can be attached to a surface of interest, such as a heart.

[0026] In one embodiment, the contractile cells are present on the surface of the 3DFCS in a ratio of about 1:10 to about 4:1 relative to the fibroblasts. In another embodiment, the contractile cells are present on the surface of the construct in a ratio of about 1:3 to about 1.2:1 relative to the fibroblasts. In various further embodiments, the contractile cells of any embodiment or combination of embodiments are present in a ratio of about 4:20 to about 1.2:1, about 1:4 to about 1.2:1, about 6:20 to about 1.2:1, about 7:20 to about 1.2:1, about 2:5 to about 1.2:1, about 9:20 to about 1.2:1, about 1:2 to about 1.2:1, about 11: The fluorophore is present on the surface of the construct in a ratio of about 20 to about 1.2:1, about 3:5 to about 1.2:1, about 13:20 to about 1.2:1, about 7:10 to about 1.2:1, about 3:4 to about 1.2:1, about 4:5 to about 1.2:1, about 17:20 to about 1.2:1, about 9:10 to about 1.2:1, about 19:20 to about 1.2:1, and about 1:1 to about 1.2:1.

[0027] In one embodiment, the contracting cells are 2×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 In another embodiment, the contracting cells are seeded onto the surface of the construct at a density of 2×10 6 cells / cm 2 ~2.5×10 6 cells / cm 2 In various further embodiments, the contracting cells are seeded onto the surface of the construct at a density of 2×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 ;5×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1.5×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1.3×10 5 cells / cm 2~2.5×10 6 cells / cm 2 ; or 1.3 × 10 5 cells / cm 2 ~2×10 6 cells / cm 2 are seeded onto the surface of the construct at a density of 100 μg / ml.

[0028] In further embodiments, the contractile cells include a combination of immature and mature contractile cells. In one such embodiment, the immature and mature contractile cells are present on the construct surface in a ratio of about 1:2 to about 2:1. In other embodiments, the ratio is about 1:1 to about 2:1, or about 1:1 to about 1:2.

[0029] In a further embodiment, the contractile cells are engineered to reduce or eliminate expression of CD40 and / or HLA. This embodiment provides cells selected for a diminished immune profile that allows for better retention of transplanted cells in a host particularly suitable for allogeneic transplantation.

[0030] In further embodiments, the contractile cells are derived from induced pluripotent stem cells (iPSCs). In a non-limiting embodiment, mature contractile cells may be generated on the construct using the methods of the invention described herein.

[0031] In one embodiment, the contractile cells comprise immature cardiomyocytes.

[0032] As used herein, "immature cardiomyocytes" lack observable sarcomeres. In various embodiments, compared to "mature cardiomyocytes," they have one or more of the following characteristics: · Morphologically small cell size; · Decreased myofibril density; ·Electrophysiologically suppressed / reduced action potential amplitude; Decreased gene and / or protein expression of MYH7 (beta myosin heavy chain), MYH6 (alpha myosin heavy chain), SCN5A, GJA1 (connexin 43), HCN4 (hyperpolarization-activated K+ channel), KCNJ2 (inwardly rectifying potassium ion channel), SERCA2a (sarco / endoplasmic reticulum ATPase), alpha actinin, cardiac troponin I (cTnI), and cardiac troponin T (cTnT).

[0033] In another embodiment, the contractile cells comprise mature cardiomyocytes. As used herein, "mature cardiomyocytes" have observable sarcomeres. In various embodiments, compared to "mature cardiomyocytes," immature cardiomyocytes have one or more of the following characteristics: · Morphologically small cell size; · Decreased myofibril density; ·Electrophysiologically suppressed / reduced action potential amplitude; Decreased gene and / or protein expression of MYH7 (beta myosin heavy chain), MYH6 (alpha myosin heavy chain), SCN5A, GJA1 (connexin 43), HCN4 (hyperpolarization-activated K+ channel), KCNJ2 (inwardly rectifying potassium ion channel), SERCA2a (sarco / endoplasmic reticulum ATPase), alpha actinin, cardiac troponin I (cTnI), and cardiac troponin T (cTnT).

[0034] By showing maturation of immature cardiomyocytes (such as those derived from iPSCs) on the construct, the inventors have demonstrated that the construct provides a unique and supportive environment that promotes the survival and maturation of contracting cells and is therefore effective for in vivo administration of cells.

[0035] In one embodiment, the immature and / or mature cardiomyocytes are 1.3×10 5 cells / cm 2 ~2.7×10 6 cells / cm 2In another embodiment, the immature and / or mature cardiomyocytes are seeded on the surface of the construct at a density of 1.2×10 to 1.5×10, and the contractile cells are present on the surface of the 3DFCS at a ratio of about 1:7 to about 3:1 with the fibroblasts on the 3DFCS. 6 cells / cm 2 ~2.3×10 6 cells / cm 2 In various embodiments, the constructs are seeded on the surface of the construct at a total density of 2.9×10 cardiomyocytes for therapeutic use. 5 cells / cm 2 , 1.2×10 6 cells / cm 2 or 2.3 × 10 6 cells / cm 2 in the dosage range.

[0036] In various embodiments, cardiomyocytes are present on the surface of the 3DFCS in a ratio of about 1.5:1 to 1:1.7; 1:1 to 3:1; 1:15 to 3.5:1; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1 together with fibroblasts on the 3DFCS.

[0037] The cardiomyocyte population may be 100% mature cardiomyocytes or 100% immature cardiomyocytes, 50% mature cardiomyocytes and 50% immature cardiomyocytes, or any suitable ratio thereof.

[0038] In another embodiment, the contractile cells comprise smooth muscle cells. In one such embodiment, the smooth muscle cells comprise 1.2×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 and the smooth muscle cells are present on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS. In various embodiments, the smooth muscle cells are present on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 2.5:1 to 6:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1 with the fibroblasts on the 3DFCS.

[0039] In various further embodiments, the smooth muscle cells are 1.3×10 5 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.2×10 6 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.3×10 5 cells / cm 2 ~1.2×10 6 cells / cm 2 ; or 1.0 × 10 6 cells / cm 2 ~1.2×10 6 cells / cm 2 In another embodiment, smooth muscle cells are seeded onto the surface of the construct at a density of 1.0×10 6 cells / cm 2 ~1.2×10 6 cells / cm 2 and the smooth muscle cells are present on the surface of the 3DFCS at a ratio of about 1:1.7 to about 1.5:1 with the fibroblasts on the 3DFCS.

[0040] In a further embodiment, the contractile cells comprise skeletal muscle cells. In one such embodiment, the skeletal muscle cells comprise 1.3×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 and the skeletal muscle cells are present on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS. In various embodiments, the skeletal muscle cells are present on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1 with the fibroblasts on the 3DFCS. In various further embodiments, the skeletal muscle cells are present on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1. 5 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.2×10 6 cells / cm 2~2.94×10 6 cells / cm 2 ;1.3×10 5 cells / cm 2 ~1.2×10 6 cells / cm 2 ; or 1.0 × 10 6 cells / cm 2 ~1.2×10 6 cells / cm 2 In another embodiment, the skeletal muscle cells are seeded onto the surface of the construct at a density of 1.0×10 5 cells / cm 2 ~1.2.0×10 6 cells / cm 2 The skeletal muscle cells are seeded onto the surface of the construct at a density of about 1:1.7 to about 1.5:1 with the fibroblasts on the 3DFCS.

[0041] In one embodiment, the contractile cells of any embodiment or combination of embodiments, particularly for the cardiomyocyte and skeletal muscle embodiments of the construct, form striations on the construct, in these embodiments, the contractile cells form repeating sarcomeres that can be visualized by microscopy.

[0042] The constructs of any embodiment may include contractile cells engineered to express any biological agent, gene activation, cell scaffolding, extracellular matrix for muscle repair, etc., where engineering may include pretreatment, preloading, overexpression, general drug elution properties, where biological agents may include proteins, amino acid derivatives, polypeptide hormones, steroids, mRNA, DNA, cytokines, growth factors, cell or scaffold associated receptors (endogenous or modified), enzymes, enzyme precursors, viral agents, antimicrobial agents, etc., or any combination of the above.

[0043] Exemplary such compounds include, but are not limited to, one or more of thymosin beta-4 (TB4), akt murine thymoma viral oncogene homolog (ΑΚΤ1), stromal cell-derived factor-1 alpha (SDF-1), genes that promote angiogenesis, and hepatocyte growth factor (HGF).

[0044] The constructs of the present invention may further include the incorporation of any biological agents, gene activation, cell scaffolding, extracellular matrix, or established blood vessels capable of surgical or biological attachment to the native vasculature.

[0045] In another aspect, the present invention provides a method for treating a disorder characterized by the dysfunction of contractile cells, comprising contacting a patient with a contractile cell system disorder with a construct of any embodiment or combination of embodiments of the present invention in an amount effective to treat the disorder. The inventors have demonstrated that the construct provides a unique and supportive environment that promotes the survival and maturation of contractile cells by showing the maturation of immature cardiomyocytes (such as those derived from iPSCs) on the construct, and is therefore effective for in vivo administration of cells. The construct provides functional benefits when transplanted into a rodent model of congestive heart failure, and is demonstrated in the examples to be electrically stable when transplanted. The inventors have also demonstrated that cardiac patches of human iPSC-derived cardiomyocytes increase mRNA expression levels of angiopoietin 1 (ANG-1), connexin 43 (Cx43), and vascular endothelial growth factor (VEGF) after transplantation into left ventricular heart tissue.

[0046] The constructs of the present invention may be implanted by any surgical means (open cavity, minimally invasive, robotic, catheter, etc.) and may be implanted / embedded in place through the use of sutures, adhesives, cell adhesion, polarization (magnetic), etc. The constructs may be manufactured and cryopreserved prior to use.

[0047] In one embodiment, the contractile cells comprise immature cardiomyocytes, mature cardiomyocytes, or a combination thereof, and the method comprises contacting a heart of a subject suffering from such a disorder with an amount of the construct effective to treat the disorder. In this embodiment, the disorder includes ischemia-induced heart failure, chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy, dilated cardiomyopathy (DCM), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling, maladaptive left ventricular remodeling, reduced left ventricular function, left heart failure, right heart failure, posterior failure, anterior failure, contractile heart failure, or ventricular failure. Symptoms of pulmonary circulation abnormalities may include, but are not limited to, pulmonary circulation abnormalities, pulmonary edema ...

[0048] Thus, the method of the present invention utilizes 3DFC as a delivery system for cell-based therapy, using the heart as its own bioreactor to support the engraftment / growth of cells seeded on the 3DFC. In contrast to isolated cell injection, the method of the present invention can cover the majority of the myocardium, i.e., the amount of damaged myocardium required for therapy, thus addressing one criticism of why cell injection seems to work better in rodents than in humans. Also, cells seeded on the 3DFC are not cleared into the circulation as seen with isolated cell injection.

[0049] In an alternative embodiment that can be combined with any other embodiment herein, the subject is a mammal, most preferably a human. In a further alternative embodiment that can be combined with any other embodiment herein, the subject is a human. In another alternative embodiment, the immature cardiomyocytes, mature cardiomyocytes, or a combination thereof are obtained from the subject.

[0050] As used herein, "CHF" is a chronic (as opposed to rapid onset) impairment of the heart's ability to supply sufficient blood to meet the body's needs. CHF can be caused by, but is distinct from, cardiac arrest, myocardial infarction, and cardiomyopathies. In one alternative embodiment, the subject suffers from congestive heart failure. In various further alternative embodiments that can be combined with any other embodiment herein, the subject's heart failure includes left heart failure, right heart failure, backward failure (increased venous back pressure), forward failure (failure to provide adequate arterial perfusion), systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low output heart failure, and high output heart failure. In various further alternative embodiments that can be combined with any other embodiment herein, the subject's CHF can be any of class I-IV, more preferably class III or IV, according to the New York Heart Association functional classification. Class I: No limitation experienced in any activity; no symptoms from usual activities. Class II: There is only mild limitation in activity; patient is comfortable at rest or with mild exertion. Class III: There is significant limitation in any activity; the patient is comfortable only at rest. Class IV: Any physical activity causes discomfort and symptoms occur at rest.

[0051] In further alternative embodiments that can be combined with any other embodiment herein, the subject is diagnosed with CHF according to the New York Heart Association functional classification. In further alternative embodiments that can be combined with any other embodiment herein, the subject is further characterized by one or more of the following: hypertension, obesity, smoking, diabetes, valvular heart disease, and ischemic heart disease.

[0052] As used herein, "treat" or "treatment" refers to achieving one or more of the following: (a) reducing the severity of the disorder (e.g., treating a class IV subject to improve the condition to class III for CHF subjects); (b) limiting or preventing the onset of symptoms characteristic of the disorder; (c) inhibiting the worsening of symptoms characteristic of the disorder; (d) limiting or preventing the recurrence of symptoms in patients who previously exhibited symptoms of the disorder. Signs characteristic of CHF include, but are not limited to, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling (e.g., left ventricular remodeling), reduced left ventricular function, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cold extremities at rest, exercise intolerance, fatigue, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, lateral shift of the apical beat, galloping rhythm, heart murmur, parasternal pulse, and pleural effusion.

[0053] In various embodiments, treatment includes one or more of the following heart failure parameters including, but not limited to, improved left ventricular function, reduced left ventricular end diastolic pressure (EDP), improved myocardial perfusion, remodeling of the heart wall by cardiomyocytes, reversal of maladaptive left ventricular remodeling in CHF subjects, improved diastolic function such as passive filling, active filling of the left ventricle, ventricular chamber compliance, and increased E' (mm / sec), decreased E / E', increased LV dP / dt (mmHg / sec), and decreased Tau (milliseconds).

[0054] In one embodiment, the constructs described herein are used to promote healing of ischemic cardiac tissue. The ability of the constructs to promote healing of ischemic tissue depends in part on the severity of ischemia. As will be appreciated by those skilled in the art, the severity of ischemia depends in part on the length of time the tissue is deprived of oxygen. Among such activities is the reduction or prevention of remodeling of ischemic tissue. As used herein, "remodeling" refers to the presence of one or more of the following: (1) progressive thinning of ischemic tissue, (2) a decrease in the number or vascularity of blood vessels supplying the ischemic tissue, and / or (3) occlusion of one or more of the blood vessels supplying the ischemic tissue, and, if the ischemic tissue includes muscle tissue, (4) a decrease in the contractile ability of the muscle tissue. Without treatment, remodeling typically leads to a weakening of the ischemic tissue, which can no longer function at the same level as the corresponding healthy tissue. Cardiovascular ischemia is generally a direct consequence of coronary artery disease, typically caused by the rupture of atherosclerotic plaques in the coronary arteries, leading to the formation of thrombi that can occlude or obstruct the coronary arteries, thereby depriving the downstream myocardium of oxygen. Prolonged ischemia can lead to cell death or necrosis, and the area of ​​dead tissue is commonly referred to as an infarct.

[0055] In some embodiments, the candidate subjects for the methods described herein are patients with stable angina and reversible myocardial ischemia. Stable angina is characterized by constrictive chest pain that occurs during exertion or stress and is relieved by rest or sublingual nitroglycerin. Coronary angiography of patients with stable angina typically reveals 50-70% blockage of at least one coronary artery. Stable angina is typically diagnosed by evaluation of clinical symptoms and ECG changes. Patients with stable angina may have transient ST segment abnormalities, but the sensitivity and specificity of these changes associated with stable angina is low.

[0056] In some embodiments, the candidates for the methods described herein are patients with unstable angina and reversible myocardial ischemia. Unstable angina is characterized by constricting chest pain at rest that is relieved by sublingual nitroglycerin. Angina chest pain is generally relieved by sublingual nitroglycerin, and the pain usually subsides within 30 minutes. There are three classes of severity of unstable angina: Class I, characterized as new-onset, severe or accelerated angina; Class II, subacute angina at rest that is characterized by increased severity, duration, or nitroglycerin requirement; Class III, acute angina at rest. Unstable angina represents a clinical condition between stable angina and acute myocardial infarction (AMI), and is believed to be primarily due to the progression of severity and extent of atherosclerosis, coronary artery spasm, or hemorrhage to non-occlusive plaque with thrombotic occlusion. Coronary angiography in patients with unstable angina usually reveals more than 90% blockage of at least one coronary artery, such that oxygen delivery cannot meet even baseline myocardial oxygen demand. Slow growth of stable atherosclerotic plaque or rupture of unstable atherosclerotic plaque, accompanied by thrombus formation, can cause unstable angina. Both of these can result in significant coronary artery stenosis. Unstable angina is usually associated with atherosclerotic plaque rupture, platelet activation, and thrombus formation. Unstable angina is usually diagnosed by clinical symptoms, ECG changes, and changes in cardiac markers.

[0057] In some embodiments, candidates for the methods described herein are human patients with left ventricular dysfunction and reversible myocardial ischemia who have undergone coronary artery bypass graft (CABG) surgery, have at least one transplantable coronary vessel, at least one coronary vessel, and are unable to undergo bypass or percutaneous coronary intervention.

[0058] In some embodiments, application of the construct to the ischemic tissue increases the number of blood vessels present in the ischemic tissue as measured using laser Doppler imaging (see, e.g., Newton et al., 2002, J Foot Ankle Surg, 41(4):233-7). In some embodiments, the number of blood vessels increases by 1%, 2%, 5%, in other embodiments, the number of blood vessels increases by 10%, 15%, 20%, or even 25%, 30%, 40%, 50%, and in some embodiments, the number of blood vessels increases even further, although intermediate values ​​are acceptable.

[0059] In some embodiments, application of the construct to ischemic cardiac tissue increases the ejection fraction. In a healthy heart, the ejection fraction is about 65-95 percent. In a heart containing ischemic tissue, the ejection fraction is about 20-40 percent in some embodiments. Thus, in some embodiments, treatment with the construct results in an absolute improvement in the ejection fraction of 0.5-1 percent compared to the ejection fraction prior to treatment. In other embodiments, treatment with the construct results in an absolute improvement in the ejection fraction of more than 1 percent. In some embodiments, the absolute improvement in the ejection fraction is 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or more compared to the ejection fraction prior to treatment. For example, if the ejection fraction before treatment was 40%, in these embodiments an ejection fraction of 41% to 59% or greater is observed after treatment. In yet other embodiments, treatment with the construct improves the ejection fraction by more than 10% compared to the ejection fraction before treatment.

[0060] In some embodiments, application of the construct to ischemic cardiac tissue increases one or more of cardiac output (CO) (up to 55% or more increase compared to pre-treatment state), left ventricular end-diastolic volume index (LVEDVI), left ventricular end-systolic volume index (LVESVI) and systolic wall thickening (SWT). These parameters are measured by standard clinical diagnostic methods in the art, including, for example, nuclear scans such as radionuclide ventriculography (RNV) or multi-gate acquisition (MUGA), as well as x-rays.

[0061] In some embodiments, application of the construct to ischemic cardiac tissue results in a demonstrable improvement in the blood levels of one or more protein markers used clinically as indicators of cardiac injury, such as creatine kinase (CK), serum glutamic oxaloacetic transaminase (SGOT), lactate dehydrogenase (LDH), which can be used to diagnose myocardial injury (see, e.g., U.S. Patent Application Publication No. 2005 / 0142613), troponin I, and troponin T (see, e.g., U.S. Patent Application Publication No. 2005 / 0021234). In yet other embodiments, modifications affecting the N-terminus of albumin can be measured (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0142613, 2005 / 0021234, and 2005 / 0004485; the disclosures of which are incorporated herein by reference in their entireties).

[0062] Additionally, the constructs can be used with therapeutic devices used to treat cardiac disease, including heart pumps, endovascular stents, endovascular stent grafts, left ventricular assist devices (LVADs), biventricular cardiac pacemakers, artificial hearts, and augmented external counterpulsation (EECP).

[0063] In further alternative embodiments that may be combined with any other embodiment herein, the treatment generates new cardiomyocytes and new blood vessels in the subject. In further alternative embodiments that may be combined with any other embodiment herein, the treatment improves left ventricular function, reduces end diastolic pressure (EDP) (up to 50-60% or more reduction compared to pre-treatment state), myocardial perfusion, remodeling of the anterior cardiac wall by cardiomyocytes, and / or reverses maladaptive left ventricular remodeling in the subject.

[0064] In one alternative, non-limiting embodiment, where a synchronously pulsating construct is placed on the heart to assist the contraction of the left ventricle, beneficial treatment can be demonstrated by an improvement in the ejection fraction. In a further alternative, non-limiting embodiment, a non-pulsating construct is placed on the heart and then begins to spontaneously pulsate on the heart to assist the contraction of the heart.

[0065] The construct can be contacted with the heart in any suitable manner that promotes adhesion. The construct may be attached to various locations on the heart, including the epicardium, myocardium, and endocardium, most preferably the epicardium. Attachment means include, but are not limited to, direct adhesion between the construct and the cardiac tissue, biological adhesives, sutures, synthetic adhesives, laser dyes, or hydrogels. Some commercially available hemostatic agents and sealants include SURGICAL® (oxidized cellulose), ACTIFOAM® (collagen), FIBRX® (light-activated fibrin sealant), BOHEAL® (fibrin sealant), FIBROCAPS® (dry powder fibrin sealant), polysaccharide polymer p-GlcNAc (SYVEC® patch; Marine Polymer Technologies), Polymer 27CK (Protein Polymer Tech). Medical devices and instruments are also known for preparing autologous fibrin sealant from 120 ml of the patient's blood in an operating room in an hour and a half (e.g., Vivostat System).

[0066] In an alternative embodiment of the present invention using direct adhesion, the construct is placed directly on the heart, and the product adheres by natural cell adhesion.In a further alternative embodiment, the construct is adhered to the heart using a surgical adhesive, preferably a biological adhesive such as fibrin adhesive.The use of fibrin adhesive as a surgical adhesive is known.Fibrin adhesive compositions are known (see, for example, U.S. Patent Nos. 4,414,971, 4,627,879 and 5,290,552), and the obtained fibrin can be autologous (see, for example, U.S. Patent No. 5,643,192). The adhesive compositions may contain additional components, such as liposomes containing one or more agents or drugs (see, e.g., U.S. Pat. Nos. 4,359,049 and 5,605,541), and may be introduced by injection (see, e.g., U.S. Pat. No. 4,874,368) or by spraying (see, e.g., U.S. Pat. Nos. 5,368,563 and 5,759,171). Kits for applying fibrin adhesive compositions are also available (see, e.g., U.S. Pat. No. 5,318,524).

[0067] In another embodiment, the laser dye is applied to the heart, construct, or both and activated using a laser of a wavelength suitable for adhering to the tissue. In an alternative embodiment, the laser dye has an activation frequency in a range that does not alter the function or integrity of the tissue. For example, 800 nm light passes through the tissue and red blood cells. A laser wavelength that passes through the tissue may be used using indocyanine green (ICG) as the laser dye. A solution of 5 mg / ml ICG is applied onto the surface of the three-dimensional stromal tissue (or target site), and the ICG binds to the collagen of the tissue. Activating the laser dye using a 5 ms pulse from an emitting laser with a peak intensity near 800 nm denatures the collagen and fuses the elastin of the adjacent tissue to the modified surface.

[0068] In another embodiment, the construct is attached to the heart using hydrogel. Several natural and synthetic polymeric materials are sufficient to form suitable hydrogel compositions. For example, polysaccharides, such as alginates, can be crosslinked with divalent cations, and polyphosphazenes and polyacrylates can be ionically crosslinked or crosslinked by ultraviolet polymerization (US Pat. No. 5,709,854). Alternatively, synthetic surgical adhesives such as 2-octyl cyanoacrylate (DERMABOND™, Ethicon, Somerville, NJ) can be used to attach the three-dimensional stromal tissue.

[0069] In an alternative embodiment of the invention, the construct is secured to the heart using one or more sutures, including, but not limited to, 5-O, 6-O and 7-O prolene sutures (Ethicon catalog numbers 8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyglactin or other suitable non-biodegradable or biodegradable suture materials. When suturing, a double ended needle is typically used, although this is not required.

[0070] In another embodiment, 3DFC is grown in a bioreactor system (e.g., U.S. Patent Nos. 5,763,267 and 5,843,766) where the framework is slightly larger than the tissue engineered final product. The final product includes an edge, end, flap or tab of the scaffold material that is used as a site for application of biological / synthetic adhesives, laser dyes or hydrogels. In an alternative embodiment, the scaffold fabric may be used as an adhesive material for suturing or microsuturing.

[0071] The phrase "effective amount" as used herein means an amount of construct effective to treat a disorder as described herein. As will be apparent to one of skill in the art, the method includes the use of one or more of the described constructs to treat a disorder characterized by dysfunction of cardiomyocytes. In one embodiment, the method includes contacting the heart with an amount of one or more constructs effective to cover one or more ischemic areas of the heart, preferably all ischemic areas of the heart. The construct is used in an amount effective to promote tissue healing and / or revascularization of weakened or damaged cardiac tissue in an individual diagnosed with a disorder characterized by dysfunction of cardiomyocytes. The amount of construct administered will depend, in part, on the severity of the disorder, whether the construct is used as an injectable composition (see US Patent Publication No. 20060154365, which is incorporated herein by reference in its entirety), the concentration of various growth factors and / or Wnt proteins present, the number of viable cells containing the construct, and / or the accessibility to the cardiac tissue(s) to be treated. Determining an effective dosage is well within the skill of one of skill in the art. A suitable animal model, such as the dog model described in US Patent Application Publication No. 20060292125 (hereby incorporated by reference in its entirety), can be used for dose-efficacy testing on specific tissues of the heart.

[0072] As used herein, "dose" refers to the number of adhesive pieces of the construct that are applied to the heart of an individual diagnosed with congestive heart failure. A typical adhesive piece of the construct is approximately 35 cm. 2 As will be appreciated by those of skill in the art, the absolute dimensions of the adhesive coupon can vary, so long as it contains a sufficient number of cells to promote healing of weakened or damaged cardiac tissue in an individual diagnosed with a disorder characterized by myocardial dysfunction. Thus, adhesive coupons suitable for use in the methods described herein may be 15 cm or larger. 2 ~50cm 2 The size range may be as follows:

[0073] Applying more than one adhesive piece of construct can increase the area of ​​the heart treatable by the methods described herein. For example, in an embodiment using two adhesive pieces of construct, the treatable area is approximately doubled in size. In an embodiment using three adhesive pieces of construct, the treatable area is approximately tripled in size. In an embodiment using four adhesive pieces of construct, the treatable area is approximately quadrupled in size. In an embodiment using five adhesive pieces of construct, the treatable area is approximately five times larger, i.e., 35 cm2. 2 ~175cm 2 It is.

[0074] In some embodiments, an adhesive strip of one of the constructs is attached to an area of ​​the heart of an individual diagnosed with a disorder characterized by dysfunction of cardiomyocytes.

[0075] In other embodiments, two adhesive pieces of the construct are attached to an area of ​​the heart of an individual diagnosed with a disorder characterized by dysfunction of cardiomyocytes.

[0076] In other embodiments, three adhesive pieces of the construct are attached to an area of ​​the heart of an individual diagnosed with a disorder characterized by dysfunction of cardiomyocytes.

[0077] In other embodiments, four, five, or more pieces of the construct are attached to an area of ​​the heart of an individual diagnosed with a disorder characterized by dysfunction of cardiomyocytes.

[0078] In embodiments in which two or more adhesive pieces of the construct are administered, the proximity of one adhesive piece to another can be adjusted depending, in part, on the severity of the disorder characterized by cardiomyocyte dysfunction, the extent of the area to be treated, and / or the accessibility of the cardiac tissue(s) to be treated. For example, in some embodiments, the pieces of 3DFC can be placed directly adjacent to each other such that one or more ends of the first piece contact one or more ends of the second piece. In other embodiments, the pieces can be adhered to the cardiac tissue such that the ends of one piece do not touch the ends of the other piece. In these embodiments, the pieces can be separated from each other by an appropriate distance based on the anatomical condition and / or disease condition exhibited by the subject. Determining the proximity of one piece to another piece is within the ordinary skill of one of ordinary skill in the art and can be tested, if necessary, using an appropriate animal model, such as the canine model described in US Patent Application Publication No. 20060292125.

[0079] In embodiments involving multiple pieces of the construct, some or all of the pieces may be attached to the same or different regions of the heart.

[0080] In embodiments involving multiple pieces of the construct, the pieces are attached to the heart simultaneously or in parallel.

[0081] In some embodiments, pieces of the construct are administered over time. The number and interval of administration depends in part on the severity of the disorder, whether 3DFC is used as an injectable composition (see US Patent Publication No. 20060154365, which is incorporated by reference in its entirety), the concentration of various growth factors and / or Wnt proteins present, the number of viable cells containing 3DFC, and / or the accessibility of the cardiac tissue to be treated. The determination of the number of administrations and the duration between successive applications is well within the skill of the artisan, and can be tested, if necessary, using a suitable animal model, such as the dog model described in US Patent Publication No. 20060292125.

[0082] In a further alternative embodiment, the one or more constructs are in contact with the left ventricle, hi a further alternative embodiment, the one or more constructs cover the entire heart.

[0083] In embodiments that include multiple pieces of the construct, some or all of the pieces can be adhered to an area that includes the heart. In other embodiments, one or more of the pieces of the construct can be adhered to an area that does not include damaged myocardium. For example, in some embodiments, a first piece can be adhered to an area that includes ischemic tissue, and a second piece can be adhered to an adjacent area that does not include ischemic tissue. In these embodiments, the adjacent area can include damaged or defective tissue. As used herein, "damaged" or "defective" tissue refers to an abnormal state of tissue that can be caused by internal and / or external events, including but not limited to events that initiate ischemic tissue. Other events that can result in ischemic, damaged or defective tissue include disease, surgery, environmental exposure, injury, aging, and / or combinations thereof.

[0084] In embodiments involving multiple pieces of cultured three-dimensional tissue, the pieces of the construct can be attached to the ischemic tissue simultaneously or in parallel.

[0085] Constructs can either contract (at the cell level, the patch (i.e., construct) level, or both) or not upon contact with the epicardium. Construct contraction is described in two ways: 1) cell contraction and 2) patch-level contraction. In cell-level contraction, the seeded contractile cells contract with synchronous, spontaneous characteristics, but are unable to move the 3DFC and require a microscope for visualization. Patch-level contraction occurs after the cells are organized and aligned, resulting in movement or contraction of the entire patch at a gross level and does not require any microscope for visualization.

[0086] In one embodiment, the cardiomyocytes on the construct are electrically coupled to the patient's native myocardium, which serves to improve electrical activity within the heart, including but not limited to, maintaining the recipient in normal sinus rhythm without inducing arrhythmias, including but not limited to, ventricular tachycardia and ventricular fibrillation.

[0087] The method may further include systemic administration to the subject of cytokines, including but not limited to insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and stromal cell-derived factor alpha (SDF-Lα).

[0088] The methods and compositions described herein can be used in combination with conventional treatments, such as administration of various medicines and surgery. For example, in some embodiments, the cultured three-dimensional tissue is administered with one or more medicines used to treat disorders characterized by dysfunction of cardiomyocytes. Medicines suitable for use in the methods described herein include angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril, lisinopril, and captopril), angiotensin II (A-II) receptor blockers (e.g., losartan and valsartan), diuretics (e.g., bumetanide, furosemide, and spironolactone), digoxin, beta-blockers, and nesiritide.

[0089] Additionally, the constructs can be used in conjunction with other options used to treat disorders characterized by dysfunction of cardiomyocytes, including heart pumps, also referred to as left ventricular assist devices (LVADs), biventricular cardiac pacemakers, cardiac wrap procedures, artificial hearts and augmented external counterpulsation (EECP), and cardiac wrap procedures (see, e.g., U.S. Pat. Nos. 6,425,856, 6,085,754, 6,572,533, and 6,730,016, the contents of which are incorporated herein by reference).

[0090] In some embodiments, the construct is used in combination with a cardiac wrap procedure. In these embodiments, a flexible pouch or jacket is used to deliver and / or attach the construct, and the construct can be placed or embedded in the pouch before being placed on the damaged or weakened cardiac tissue. In other embodiments, the pouch and the 3DFC can be bonded together. For example, the pouch and the construct can be bonded together using a retractable sewing assembly. In other embodiments, the construct can be configured to include threads useful for bonding the framework to the pouch. U.S. Patent Nos. 6,416,459, 5,702,343, 6,077,218, 6,126,590, 6,155,972, 6,241,654, 6,425,856, 6,230,714, 6,241,654, 6,155,972, 6,293,906, the contents of which are incorporated herein by reference. Nos. 6,425,856, 6,085,754, 6,572,533 and 6,730,016, and U.S. Patent Application Publication Nos. 2003 / 0229265 and 2003 / 0229261, describe various embodiments of pouches and jackets, e.g., cardiac restraint devices, that can be used to deliver and / or attach the constructs.

[0091] In some embodiments, in addition to the construct, other devices are attached to the pouch, such as defibrillation electrodes, tension indicators that indicate when the jacket is adjusted to the desired degree of tension on the heart, and are used in the methods and compositions described herein.See, for example, U.S. Patent Nos. 6,169,922 and 6,174,279, the contents of which are incorporated herein by reference.

[0092] Several methods can be used to measure changes in cardiac function in subjects before and after the attachment of the construct. For example, echocardiograms can be used to determine the heart's ability to pump. The percentage of blood that is pumped out of the left ventricle with each heartbeat is called the ejection fraction. In a healthy heart, the ejection fraction is about 60 percent. In individuals with chronic heart failure caused by the inability of the left ventricle to contract vigorously, i.e., systolic heart failure, the ejection fraction is usually less than 40 percent. Depending on the severity and cause of heart failure, the ejection fraction typically ranges from less than 40 percent to 15 percent or less. Echocardiograms can also be used to distinguish between systolic heart failure, in which the pumping function is normal but the heart is stiff, and diastolic heart failure.

[0093] In some embodiments, echocardiograms are used to compare the ejection fraction before and after treatment with the construct. In certain embodiments, treatment with the construct improves the ejection fraction by 3-5 percent. In other embodiments, treatment with the construct improves the ejection fraction by 5-10 percent. In still other embodiments, treatment with the construct improves the ejection fraction by more than 10 percent.

[0094] Nuclear scans such as radionuclide ventriculography (RNV) or multiple gated acquisition (MUGA) scans can be used to determine how much blood the heart pumps with each beat. These tests are done using a small amount of dye injected into an individual's veins. A special camera is used to detect the radioactive material flowing through the heart. Other tests include x-rays and blood tests. Chest x-rays can be used to determine the size of the heart and whether fluid has built up in the lungs. Blood tests can be used to check brain natriuretic peptide (BNP), a specific indicator of congestive heart failure. BNP is secreted in high levels by the heart when it is overworked. Therefore, changes in BNP levels in the blood can be used to monitor the effectiveness of a treatment plan.

[0095] In a further aspect, the present invention provides a kit for treating CHF comprising a suitable construct as disclosed above and means for adhering said construct to the heart or organ, which may include any such adhesive means as described above, for example a surgical adhesive composition, a hydrogel, or a pre-attached microsuture prolene needle.

[0096] In another embodiment, the contractile cells include immature skeletal muscle cells, immature smooth muscle cells, mature skeletal muscle cells, mature smooth muscle cells, or combinations thereof. Although the method has been demonstrated using cardiomyocytes, they are exemplary of the full range of contractile cells that can be used to provide an effective drug screening system to evaluate whether drug candidates function in vivo. In this aspect, the method includes the treatment of any disorder that can benefit from the enhancement, repair, or restoration of skeletal and / or smooth muscle tissue, and can include contacting a patient having the disorder with the construct in an amount effective to treat the disorder. Exemplary such disorders include, but are not limited to, neuromuscular diseases, degenerative diseases, inflammatory diseases, autoimmune muscle diseases, and / or any form of injury, such as, but not limited to, trauma, including vascular disorders (peripheral arterial disease, atherosclerosis, aneurysms, etc.), respiratory diseases (chronic obstructive pulmonary disease, diaphragmatic hernia, hemidiaphragmatic hernia (which may include Bohodalek hernia or congenital diaphragmatic hernia), diaphragmatic atony, etc.), hernias (groin, abdominal, semilunar, umbilical, Bohodalek, esophageal hiatus, Morgagni, etc.), any form of injury may include sports injuries, burns, post-traumatic wounds, war injuries, muscle fatigue, etc., which may result from blunt and / or penetrating trauma, etc., or any combination of such.

[0097] In another aspect, the present invention provides a method for drug screening comprising contacting a construct of any embodiment or combination of embodiments of the present invention with a compound of interest and determining the effect of the compound on one or more properties of the construct.

[0098] In this aspect, the construct of the present invention can be used for drug screening. The inventors have demonstrated maturation of immature cardiomyocytes (such as those derived from iPSCs) on the construct. Thus, the construct of the present invention provides tissue-like development and signaling. This is important because drug development companies want to test drugs on the most mature cells possible. Current iPSC (immature) cardiomyocytes do not show complete maturation. Our data shows that iPSC cardiomyocytes mature (i.e., into mature cardiomyocytes) when cultured on the construct of the present invention, in contrast to standard culture of iPSC cardiomyocytes. In one embodiment, the method of this aspect is used with the cardiomyocyte construct of the present invention. Although the method has been demonstrated with cardiomyocytes, these are examples of a complete range of contractile cells that can be used to provide an effective drug screening system to evaluate whether drug candidates function in vivo.

[0099] In this aspect, the method may include culturing the construct under conditions to promote contraction of the construct prior to contacting the construct with a compound of interest. In this embodiment, the patch is cultured until cell and / or patch-level contraction (preferably patch-level contraction) occurs, and then the drug is added. Patch contraction (displacement, contraction rate / peak frequency, synchrony, rate, velocity, action potential, beat / contraction pattern, etc.) is recorded and analyzed. In one embodiment, the contracting cells may have an inherent genetic defect, such as long QT. Appropriate culturing techniques can be determined by one of skill in the art based on this disclosure and the intended purpose of the assay being performed.

[0100] In a further aspect, the present invention provides a method for preparing a contraction construct, comprising the steps of: (a) seeding immature contractile cells onto the surface of a fibroblast-containing three-dimensional scaffold (3DFCS) to generate a contractile construct; (b) culturing the contractile construct under conditions that promote differentiation of immature contractile cells into mature contractile cells that form striations; The present invention provides a method comprising:

[0101] The inventors have surprisingly discovered that fibroblast-containing constructs enhance / promote maturation of immature contractile cells into more mature cells as defined morphologically or via gene or protein expression, thus greatly facilitating the preparation of contractile constructs that can be used, for example, for implantation of therapeutic preparations or drug screening assays as described herein.

[0102] In one embodiment, the immature contractile cells are immature cardiomyocytes and the mature contractile cells are mature cardiomyocytes, as defined herein. In another embodiment, the immature contractile cells are immature smooth muscle cells and the mature contractile cells are mature smooth muscle cells. In a further embodiment, the immature contractile cells are immature skeletal muscle cells and the mature contractile cells are mature skeletal muscle cells.

[0103] In one embodiment, the contractile cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to about 6:1, or about 1:10 to about 4:1 with respect to the fibroblasts. In another embodiment, the contractile cells are seeded on the surface of the construct at a ratio of about 1:3 to about 1.2:1 with respect to the fibroblasts. In various further embodiments, the contractile cells of any embodiment or combination of embodiments are seeded at a ratio of about 4:20 to about 1.2:1, about 1:4 to about 1.2:1, about 6:20 to about 1.2:1, about 7:20 to about 1.2:1, about 2:5 to about 1.2:1, about 9:20 to about 1.2:1, about 1:2 to about 1.2:1, about 11:1 to about 1.2:1, about 12:1 to about 1.2:1, about 13:1 to about 1.2:1, about 14:1 to about 1.2:1, about 15:1 to about 1.2:1, about 16:1 to about 1.2:1, about 17:1 to about 1.2:1, about 18:1 to about 1.2:1, about 19:1 to about 1.2:1, about 20:1 to about 1.2:1, about 21:1 to about 1.2:1, about 22:1 to about 1.2:1, about 23:1 to about 1.2:1, about 24:1 to about 1.2:1, about 25:1 to about 1.2:1, about 26:1 to about 1.2:1, about 27:1 to about 1.2:1, about 28:1 to about 1.2:1, about 29:1 to about 1.2:1 The cells are seeded onto the surface of the construct at a ratio of about 1:1 to about 1.2:1, about 13:20 to about 1.2:1, about 7:10 to about 1.2:1, about 3:4 to about 1.2:1, about 4:5 to about 1.2:1, about 17:20 to about 1.2:1, about 9:10 to about 1.2:1, about 19:20 to about 1.2:1, or about 1:1 to about 1.2:1.

[0104] In one embodiment, the contracting cells are 1.3×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 or 2×10 5 cells / cm 2 ~2.95×10 6 cells / cm2 In another embodiment, the contracting cells are seeded onto the surface of the construct at a density of 2×10 6 cells / cm 2 ~2.5×10 6 cells / cm 2 In various further embodiments, the contracting cells are seeded onto the surface of the construct at a density of 2×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 ;5×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1.5×10 6 cells / cm 2 ~2.95×10 6 cells / cm 2 ;1.3×10 5 cells / cm 2 ~2.5×10 6 cells / cm 2 ; or 1.3 × 10 5 cells / cm 2 ~2×10 6 cells / cm 2 are seeded onto the surface of the construct at a density of 100 μg / ml.

[0105] In further embodiments, the contractile cells include a combination of immature and mature contractile cells. In one such embodiment, the immature and mature contractile cells are present on the construct surface in a ratio of about 1:2 to about 2:1. In other embodiments, the ratio is about 1:1 to about 2:1; or about 1:1 to about 1:2.

[0106] In one embodiment, the contracting cells comprise immature cardiomyocytes. In another embodiment, the contracting cells comprise mature cardiomyocytes. In one embodiment, the immature and / or mature cardiomyocytes comprise 1.3×10 5 cells / cm 2 ~2.7×10 6 cells / cm 2In another embodiment, immature and / or mature cardiomyocytes are seeded on the surface of the construct at a density of 2.9×10, and contractile cells are seeded on the surface of the 3DFCS at a ratio of about 1:7 to about 3:1 with fibroblasts on the 3DFCS. 5 cells / cm 2 ~2.3×10 6 cells / cm 2 In various embodiments, the constructs are seeded with cardiomyocytes for therapeutic use at a total density of 2.9×10 5 cells / cm 2 , 1.2×10 6 cells / cm 2 or 2.3 × 10 6 cells / cm 2 The cardiomyocyte population can be 100% mature cardiomyocytes or 100% immature cardiomyocytes, 50% mature cardiomyocytes and 50% immature cardiomyocytes, or any suitable ratio thereof.

[0107] In another embodiment, the contractile cells comprise smooth muscle cells. In one such embodiment, the smooth muscle cells comprise 1.3×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 and the contractile cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS. In various embodiments, the smooth muscle cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1 with the fibroblasts on the 3DFCS.

[0108] In various further embodiments, the smooth muscle cells are 1.3×10 5 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.2×10 6 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.3×10 5cells / cm 2 ~1.2×10 6 cells / cm 2 ; or 1.0 × 10 6 cells / cm 2 ~1.2×10 6 cells / cm 2 In another embodiment, smooth muscle cells are seeded onto the surface of the construct at a density of 1.0×10 6 cells / cm 2 ~1.2×10 6 cells / cm 2 and the smooth muscle cells are present on the surface of the 3DFCS at a ratio of about 1:1.7 to about 1.5:1 with the fibroblasts on the 3DFCS.

[0109] In a further embodiment, the contractile cells comprise skeletal muscle cells. In one such embodiment, the skeletal muscle cells comprise 1.3×10 5 cells / cm 2 ~2.95×10 6 cells / cm 2 and the skeletal muscle cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS. In various embodiments, the skeletal muscle cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to about 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1 with the fibroblasts on the 3DFCS. In various further embodiments, the skeletal muscle cells are seeded on the surface of the 3DFCS at a ratio of about 1:15 to 3.5:1 with the fibroblasts on the 3DFCS; 1:15 to 1.7:1; 1:6 to 3.5:1; 1.6 to 1.5:1; or 1:1.7 to 1.5:1. 5 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.2×10 6 cells / cm 2 ~2.94×10 6 cells / cm 2 ;1.3×10 5 cells / cm 2 ~1.2×10 6 cells / cm 2 ; or 1.0 × 10 6 cells / cm 2 ~1.2×10 6 cells / cm 2In another embodiment, the skeletal muscle cells are seeded onto the surface of the construct at a density of 1.0×10 5 cells / cm 2 ~1.2×10 6 cells / cm 2 The skeletal muscle cells are seeded onto the surface of the construct at a density of about 1:1.7 to about 1.5:1 with the fibroblasts on the 3DFCS.

[0110] Appropriate culture conditions can be determined by one skilled in the art, so long as immature cardiomyocytes are cultured on the 3DFC. Any useful medium may be used, including, but not limited to, DMEM-LG supplemented with fetal bovine serum (5-15%, preferably 10%) and other appropriate factors (including, but not limited to, sodium bicarbonate and antibiotics).

[0111] The contact between the cultured 3DFC and the contracting cells seeded on the 3DFC can be performed under any suitable conditions that facilitate the application of a force that brings the cells into contact with the 3DFC. In one embodiment, when the 3DFC is placed in a medium and the cells are introduced into the suspension, the volume of the cell suspension is about twice the volume of the medium in which the 3DFC is placed. In one alternative embodiment that can be combined with any other embodiment herein, the contact occurs at about 37°C. The cell density and the ratio of fibroblasts on the 3DFC are as described herein.

[0112] In one embodiment, each 3DFC to be seeded is placed in the well so that it covers the bottom of the well and lies horizontally. Subjecting the cells in the cell suspension to a force that brings the cells into contact with the 3DFC may include the use of any suitable force, including but not limited to centrifugal force and electric force generated by an electric field, or a combination of such forces. In an alternative embodiment, centrifugal force is used. The centrifugal force applied depends on various factors, such as the cell type to be seeded on the 3DFC. In one alternative embodiment, which can be combined with any other embodiment herein, the constructs are centrifuged at 1200 rpm to 1600 rpm for 2 to 10 minutes. In an alternative embodiment, all 3DFC constructs to be seeded are placed in a horizontal arrangement (rather than vertical) in the well, so that each well rotates at the same radius.

[0113] In one embodiment, the medium is xenobiotic-free. For example, the constructs can be maintained at 37° C. and 5% CO2. Medium can be changed every 10-48 hours, although 24 hours is preferred. Seeding and culture is performed using any tissue culture-tested 35 mm 2 , 65mm 2 , 100mm 2Such as "open top" culture dishes or well plates in formats such as 96, 24, or 6. The plates / dishes can be low or high adhesion. Contractile cells can be seeded and co-cultured on the 3DFC, making a distinction between cryopreserved and freshly isolated (from tissue) or tissue culture preparations. Cryopreserved cells, freshly isolated cells from tissue or viable tissue culture cells can be seeded directly on the 3DFC by any suitable technique. Patches can be cultured for any suitable period of time, as best suits the intended use of the construct. In one embodiment, the construct is used for transplantation and the culture is for 14 to 240 hours, while in various further embodiments the culture is for 14 to 120 hours, 14 to 36 hours, 14 to 48 hours, 14 to 22 hours, 24 to 240 hours, 24 to 120 hours, 24 to 72 hours, 24 to 48 hours, 48 ​​to 240 hours, 48 ​​to 120 hours, 48 ​​to 72, 14 to 22 hours, 18 to 22 hours, less than 48 hours, less than 24 hours, less than 20 hours, less than 16 hours, or less than 14 hours prior to transplantation.

[0114] In a further embodiment, the method further comprises implanting the contraction construct into a subject in need thereof. In one embodiment, the construct does not exhibit cell-level or patch-level contraction upon implantation. In this embodiment, implantation occurs after cells have attached onto the construct but before either cell-level or patch-level contraction begins, so that the heart promotes cell placement and binding and limits arrhythmia. In another embodiment, the construct is implanted after cell-level and / or construct-level contraction is exhibited.

[0115] Seeded patches typically initiate cell-level contractions across the surface of the patch within 48 hours, and these contractions evolve into full "patch" contractions where the underlying 3DFC can be visually observed to contract (by video) after approximately 72 hours. However, certain cell sources (such as cryopreserved cells) may require additional culture time before cell-level contraction can be detected. Constructs can be cultured for approximately 10 days, as the vicryl mesh begins to hydrolyze and disintegrate.

[0116] When implanted, the method of the invention can be carried out similarly to that disclosed herein. For example, when the immature contractile cells are immature cardiomyocytes and the mature contractile cells are mature cardiomyocytes, implantation includes contacting the heart of a subject suffering from such a disorder with an effective amount of a contractile construct to treat the disorder. In this embodiment, the disorder includes ischemia-induced heart failure, chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy, dilated cardiomyopathy (DCM), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling, maladaptive left ventricular remodeling, reduced left ventricular function, left heart failure, right heart failure, posterior failure, anterior failure, contractile These may include, but are not limited to, functional impairment, diastolic dysfunction, increased or decreased systemic vascular resistance, low output heart failure, high output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cold extremities at rest, exercise intolerance, fatigue, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, lateral shift of apical beat, gallop rhythm, heart murmur, parasternal pulse, and pleural effusion. All other embodiments of the therapy as disclosed above may be used in this aspect of the invention.

[0117] In another embodiment, the method further comprises contacting the contractile construct with a compound of interest and determining the effect of the compound on one or more properties of the construct. This embodiment of drug screening is described above, and all embodiments disclosed therein can be used with this embodiment. For example, the method may comprise culturing the construct under conditions that promote the contraction of the construct before contacting the compound of interest with the construct. In another embodiment, the effect of the compound on one or more of contractile displacement, contractile rate, contractile synchrony, and contractile velocity is determined.

[0118] Working Example Patch manufacturing Seeding method Briefly, centrifugal force is applied to cells in suspension. Pushing / forcing the cells onto the surface of a three-dimensional fibroblast construct (3DFC) allows for a random but uniform distribution of the cells. Contractile forces are generated once the base construct (3DFC) provides support as well as the appropriate requirements for cell implantation and positioning. The final "product" is a degradable mesh embedded with fibroblasts and seeded with a contractile cell population, in this preparation iPSC-derived ("immature") cardiomyocytes.

[0119] Seeding density The seeding density of induced human pluripotent stem cells (hiPSCs) was 0.3 × 10 6 cells / cm 2 ~2.4×10 6 cells / cm 2 The range is 1.2×10 6 cells / cm 2 is the ideal.

[0120] Cell-to-cell ratio The starting material for the cardiac patch is a 3DFC comprising a synthetic vicryl mesh embedded with human dermal fibroblasts. The fibroblasts exhibit angiogenesis and therefore provide trophic support to the seeded iPSC-derived cardiomyocyte population following transplantation into the heart. Our data show that cell ratios (iPSC-derived cardiomyocytes to dermal fibroblasts) can range from 3:20 to 1.2:1, with 1.2:2 being ideal.

[0121] We developed a method to electrically map the heart to examine the stability and attachment of the implanted patch. Electrical activation mapping was performed on rat neonatal cardiomyocytes (NCM)-3DFC in tissue culture 5 days after co-culture (maintained at 37°C, 5% CO2 with 10% FBS in DMEM). Medium was changed every 24 h and a custom designed multi-electrode array (MEA) was used to record at 18 recording sites spaced 500 μm apart (Figure 1A). Recordings were made from 10 electrodes and each recording site was numbered sequentially as channels 1-10 (Figure 1B). Electrical activation of the patch was shown at 7 s intervals and showed consistent beat-to-beat activation displaying peak lateral conduction voltages for each individual channel (Figure 1C). Amplitudes show that all channels overlapped during the beat-to-beat sequence (Figure 1D) and during unitary activation (Figure 1E). The amplitudes were recorded as 0.03 to 0.42 and -0.13 to -0.75 mV (Figure 1D and E). These results demonstrate that NCM-3DFCs are electrically stable (Figure 1) and unlikely to induce arrhythmias when implanted.

[0122] The inventors have evaluated human iPSCs seeded on fibroblast patches, which show a trend towards improvement in terms of R-wave amplitude. The inventors performed a full functional study with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) implanted at 18±4 hours after patch creation. Echocardiography was performed using images in the parasternal short and long axis to evaluate the anterior, lateral, anterolateral, inferior and posterior walls using a dedicated rodent echocardiography system (Vevo2100) at 3 and 6 weeks after intervention, and LV systolic and diastolic function was assessed, i.e., to define mitral inflow patterns, using M-mode for LV function analysis, and tissue Doppler for quantification of myocardial tissue movement during diastole (anterior LV wall). The data are shown in Figure 1.

[0123] [Table 1]

[0124] The data show that hiPSC-CM-3DFCs improve LV function at 3 weeks post-transplantation by increasing EF by 13%, tissue Doppler parameters E' by 23% and E' / a' by 33% (p<0.05), and decreasing EDP by 47%, Tau by 18% and E / e' by 23% (p<0.05). Passive pressure-volume relationships show a leftward shift of the pressure axis toward normal with hiPSC-CM-3DFC patch treatment. These data support the improvement shown in LV passive filling and chamber stiffness as well as hemodynamics in terms of reduced operational LVEDP. No functional improvement was observed with transplantation of 3DFCs alone.

[0125] Electrical coupling was performed and assessed by peak voltage amplitude and conduction velocity (Figure 3). Human iPSCs seeded on 3DFC showed a trend of improvement in terms of voltage (Figure 2). To evaluate voltage, a) pacing activation maps were created in a chronic heart failure (CHF) rat model with seeded patches for the region of interest indicated by the black box (Figure 2a). b) An electrocardiogram taken from the epicardial surface during introduction of the pacing electrode at location "P" shows successful capture (Figure 2b). We provide data for 9 different activation maps for activation time compiled over 72 contractions at 32 locations (Figure 2c). The multiple maps created showed consistency in measurements of activation time (msec) and amplitude (mV). These results regarding improvement in R-wave amplitude and voltage are important as they indicate that cells seeded on the patch are excitable and electromechanically coupled with cardiac tissue.

[0126] Patches seeded with human iPSC-derived cardiomyocytes beat spontaneously with forces generated in a synchronized fashion, can be electrically paced, and are easily implanted.

[0127] Human induced pluripotent stem cell derived cardiomyocytes (stained red) were seeded onto the fibroblast constructs and co-cultured (Figure 3). The vicryl fibers can be observed as a net-like mesh weave. The crimson fluorescence is the embedded fibroblasts. These cells were seeded locally and do not penetrate the patch or the embedded fibroblasts. These patches started spontaneous, synchronous contractions immediately after seeding. The cells were randomly seeded using centrifugal force.

[0128] Patch contraction is described in two ways: 1) cell contraction and 2) patch-level contraction. In cell-level contraction, the seeded contracting cells are synchronous and contracting with spontaneous characteristics, but are unable to move the 3DFC and a microscope is required for visualization. Patch-level contraction occurs after the cells are organized and aligned, resulting in movement or contraction of the entire patch at a gross level and does not require any microscope for visualization. Seeded patches initiate cell-level contractions across the surface of the patch within 48 hours, and these contractions develop into complete "patch" contraction where the underlying 3DFC can be observed visually contracting after approximately 72 hours. Patches can be cultured for approximately 10 days as the vicryl mesh begins to hydrolyze and disintegrate. After 10 days of culture, these patches lose structural integrity.

[0129] As shown in Figure 4, human induced pluripotent stem cell-derived cardiomyocytes generated a force response when seeded on a fibroblast patch. The data show that after 5 days in culture, 2 x 10 6 cells (1.2×10 6 cells / cm 2) from fibroblast patches seeded with iPSCs. Force measurements were performed using a miniature intact fiber testing device (Aurora Scientific - Model 801C) with thermal control and perfusion capabilities. Patches were fabricated as described and cultured for 1-6 days. Patches were then trimmed into approximately 2 mm x 17 mm sections and attached to force transducers. Both the transducer well and perfusion fluid were maintained at 37 °C during force experiments. Resting tension was achieved prior to acquiring force generation. Force generation indicates that iPSC-derived cardiomyocytes are aligned and contract in unison, which could potentially aid in the resulting functional improvement.

[0130] Pathophysiologically ischemia-induced CHF is manifested by anterior and anterolateral thinning as well as dilation of the LV as a compensatory measure to maintain cardiac output. These regions are relatively devoid of viable cardiomyocytes due to the ischemic nature of the tissue, resulting in impaired myocardial contractility and performance. Therapeutic strategies such as cell-based tissue engineering relevant to CHF may involve cell replacement by exogenous, endogenous, or a combination thereof to reconstitute the infarcted region with viable cardiomyocytes. As shown in Figure 5, hiPSC-CM-3DFCs help facilitate the exchange of cardiomyocytes into the infarcted region.

[0131] Maturation of cardiomyocytes for therapy or various in vitro assays may be important. Therapeutically, cardiomyocyte maturation may help promote force generation and better restore LV function while providing a larger tissue and therefore representative physiological in vitro assay. Maturation of hiPSCs was assessed after culture on fibroblast constructs (3DFC) (Figure 6). Trichrome staining of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) at day 2 (A) versus day 6 (B) in standard tissue culture. At both 2 and 6 days of culture, all cells stain positive for muscle. After 6 days of culture, hiPSC-CMs expanded. When seeded on fibroblast patches, at day 2 (C and E), hiPSC-CMs remain small in size, but by day 6 (D and F), hiPSC-CMs have developed into an intact layer with clearly present striations, suggesting that the fibroblast patches provide structural support to allow maturation of hiPSC-CMs in vitro.

[0132] Furthermore, we demonstrate that implantation of hiPSC-CM patches, either by endogenous means, cell transplantation, or a combination of the two, increases anterior wall thickness and increases viable myocardium (Figure 7).

[0133] We assessed mRNA expression via real-time PCR in CHF treated with human iPSC-derived cardiomyocyte patches. The data show that hiPSC-derived cardiomyocyte cardiac patches increase mRNA expression levels of angiopoietin 1 (ANG-1), connexin 43 (Cx43), and vascular endothelial growth factor (VEGF) in LV cardiac tissue (Table 2). 3DFC alone does not significantly increase VEGF and ANG-1 expression, but delivery of hiPSC-CM-3DFC results in a dose-dependent increase in expression. VEGF and ANG-1 expression may be mechanistic contributors for microvascular formation that can provide endogenous trophic support for tissue regeneration. Furthermore, Cx43 expression may be a confirmation of LV cardiomyocyte repopulation and functional recovery.

[0134] [Table 2] References 1. Thai, H. M., Juneman, E., Lancaster, J., Hagerty, T., Do, R., Castellano, L., Kellar, R., Williams, S., Sethi, G., Schmelz, M., Gaballa, M., & Goldman, S. (2009). Implantation of a three-dimensional fibroblast matrix improves left ventricular function and blood flow after acute myocardial infarction. Cell Transplant., 18(3), 283-295. PMC2739416:PM19558777. doi:10.3727 / 096368909788535004 2. Lancaster, J., Juneman, E., Hagerty, T., Do, R., Hicks, M., Meltzer, K., Standley, P., Gaballa, M., Kellar, R., Goldman, S., & Thai, H. (2010). Viable fibroblast matrix patch induces angiogenesis and increases myocardial blood flow in heart failure after myocardial infarction. Tissue Eng.Part A., 16(10), 3065-3073. PM20486785. doi:10.1089 / ten.TEA.2009.0589 3. Lancaster, J. J., Arnce, S. A., Johnson, N. M., Juneman, E. B., Thai, H. M., Kellar, R. S., Vitorin, J. E., Burt, J. M., Bahl, J. J., & Goldman, S. (2010). In vivo evaluation of a biologically active cardiomyocyte seeded scaffold to treat chronic heart failure. [abstract]. Paper presented at the Heart Failure Society of America: 14th Annual Scientific Meeting, San Diego,CA. , 16(8) S45. doi:10.1016 / j.cardfail.2010.06.155 4. Lancaster, J. J., Arnce, S. A., Johnson, N. M., Juneman, E. B., Thai, H. M., Kellar, R. S., Vitorin, J. E., Burt, J. M., Gaballa, M. A., Bahl, J. J., & Goldman, S. Tissue engineered scaffold seeded with cardiomyocytes improves cardiac function in rats with chronic ischemic heart failure disease. (Journal of Heart and Lung Transplantation).

Claims

1. A contractile construct comprising contractile cells derived from human pluripotent stem cells (PSCs), obtained by seeding immature contractile cells derived from human induced pluripotent stem cells (hiPSCs) on the surface of a three-dimensional scaffold comprising fibroblasts (3DFCS), the immature contractile cells are immature cardiomyocytes; The resulting construct is capable of spontaneous synchronous contraction across the surface of the 3D FCS; The contracting cells form striations on the construct. Construction.

2. The construct of claim 1, wherein the pluripotent stem cell (PSC) derived contractile cells have been engineered to reduce or eliminate expression of CD40 and / or HLA.

3. the contracting cells include immature cardiomyocytes and / or mature cardiomyocytes; (a) the immature cardiomyocytes and / or mature cardiomyocytes are 1.3×10 ５ cells / cm ２ ~2.9 x 10 ６ cells / cm ２ and the contractile cells are present on the surface of the 3DFCS at a ratio of 1:7 to 3:1 with the fibroblasts on the 3DFCS; or (b) the immature cardiomyocytes and / or mature cardiomyocytes are 2.9×10 ５ cells / cm ２ ~2.3 x 10 ６ cells / cm ２ are seeded onto the surface of said construct at a total density of A construct according to any one of claims 1 to 2.

4. The construct according to any one of claims 1 to 3, further comprising vascular progenitor cells.

5. A construct according to any one of claims 1 to 4 for use in the treatment of a disorder characterised by a dysfunction of contractile cells.

6. the contracting cells comprise immature cardiomyocytes, mature cardiomyocytes, or a combination thereof; The treatment comprises contacting the heart of a subject suffering from such a disorder with an effective amount of the construct to treat the disorder. The construct of claim 5.

7. The construct is attached to the epicardium of the subject; and / or The cardiomyocytes on the construct are electrically coupled to the patient's native cardiomyocytes. The construct of claim 6.

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