Patent Application
20230296590
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Mar. 6, 2023 having the file name “21-0110-US.xml” and is 13,847 bytes in size.
As robust protocols were developed in the last decade to differentiate human induced pluripotent stem cells (hiPSCs) into beating cardiomyocytes (hiPSC-CMs), expectations were set in to create human adult-like cardiac tissue constructs for applications in regenerative medicine, toxicity screens, basic science research, and precision medicine. However, differentiated beating cardiomyocytes exhibit immature phenotypes reminiscent of very early stages of heart development with limited applicability. To date, metabolic maturation, a hallmark of matured cardiac tissue and a fundamental functional necessity, is not yet convincingly demonstrated on engineered heart tissue constructs. Adult cardiomyocytes have a unique physiology, both at the cellular and tissue levels, rendering them highly specialized in generating sufficient and repeated forces for long durations.
In a first aspect, the disclosure provides a construct including a patterned scaffold at submicron resolution. The patterned scaffold includes a polymeric hydrogel substrate including a plurality of wrinkles. The wrinkles include linear or branched folds directionally aligned over a centi-meter length scale. The polymeric substrate has a viscoelasticity between about 15 kPa and about 100 MPa, or about 15 kPa to about 75 MPa. The construct further includes one or more cardiac matrix ligands conjugated to the patterned scaffold. The one or more cardiac matrix ligands comprises 1, 2, 3, 4 or more of Nephronectin, GRGDS (Gly-Arg-Gly-Asp-Ser), GFOGER, GFPGER and/or other peptides containing one or more RGD motifs.
In a second aspect, the disclosure provides a method for making the construct of the first aspect. The method includes creating a patterned substrate comprising a plurality of wrinkles, wherein the plurality of wrinkles comprise linear or branched folds directionally aligned over a centimeter length scale; transferring the patterned substrate to a mold and then transferring the patterned substrate from the mold onto a polymeric hydrogel. The transfer to the polymeric hydrogel creates a patterned scaffold at submicron resolution comprising a plurality of wrinkles.
In a third aspect, the disclosure provides a method for making the construct of the first aspect. The method includes dual exposure patterning (DEP).
In a fourth aspect, the disclosure provides methods for generating cardiomyocytes, including culturing cardiomyocyte precursors on the construct of the first aspect of the disclosure, wherein the culturing is carried out for a time and under suitable conditions to generate differentiated cardiomyocytes.
In a fifth aspect the disclosure provides methods for using the construct of the first aspect of the disclosure.
All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
As used herein, the term “about” means+/−5% of the recited value.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
In a first aspect, the disclosure provides a construct, comprising: (a) a patterned scaffold at submicron resolution, wherein the patterned scaffold comprises a polymeric hydrogel substrate comprising a plurality of wrinkles, wherein the wrinkles comprise linear or branched folds directionally aligned over a centi-meter length scale, wherein the polymeric substrate has a rigidity or viscoelasticity between about 15 kPa and about 100 MPa, or about 15 kPa to about 75 MPa; and (b) one or more cardiac matrix ligands conjugated to the patterned scaffold, wherein the one or more cardiac matrix ligands comprises 1, 2, 3, 4 or more of Nephronectin (see SEQ ID NO: 13), GRGDS (Gly-Arg-Gly-Asp-Ser) (SEQ ID NO: 10), GFOGER (SEQ ID NO: 11), GFPGER (SEQ ID NO: 12) and/or other peptides containing one or more RGD motifs.
As disclosed herein, the inventors provide constructs and methods to accelerate maturation of human pluripotent stem cell (induced, or embryonic) derived cardiomyocytes (hiPSC-CMs) by maintaining them on a substrate (referred to herein as Cardiac Mimetic Matrix (CMM)), which combines ligand presentation, anisotropic ultrastructure, and matrix elasticity that are surprisingly shown to synergistically mature hiPSC-CMs, resulting in a systemic adult-like manifestation of gene expression, metabolism, electrophysiological, redox and calcium handling, and force generation in an accelerated time frame. The resultant cardiac tissue is more matured than prolonged cultures, and more closely matches the transcriptomic state of late fetal and adult hearts, in respect to well established and expected structural, mechanical, and metabolic readouts. Specifically, the matured tissue developed using the methods and constructs of the present disclosure has more physiologically a) enhanced oxidative stress handling; b) enhanced calcium handling; c) expression of ion channels resulting in adult-like action potential profile, and responsiveness to to ion channel inhibitor drugs; d) increased expression of cardiac development related transcription factors and genes e) structural maturation with manifestation of fused elongated mitochondria alongside aligned with sarcomeres; f) mitochondrial maturation, and change in energetics with higher mitochondrial electron transport chain (ETC) respiration, increase Adp stimulated respiration, fatty acid oxidation, and metabolic substrate plasticity; and g) increased mechanical contractile, force generation, and increased electromechanical coupling.
In various embodiments, the construct comprises Nephronectin, RGD, and GFOGER (SEQ ID NO: 11) conjugated to the patterned scaffold. In one, non-limiting embodiment, the Nephronectin, RGD, and GFOGER (SEQ ID NO: 11) are present in about an equimolar ratio.
The construct can further comprise laminin, including but not limited to laminin 511/521 and/or laminin 211/221, conjugated to the patterned scaffold. As used herein, “aligned wrinkle ridges”, or “aligned wrinkles” refer to raised portion of the polymer scaffold, arranged in highly parallel arrangements with average distance between adjacent ridges between 400 nm to 3 um, which can branch off in space to create new wrinkles while maintaining high degree of parallelness, with angle of branching ranging from 0 to 30 degrees.
In various embodiments, the one or more cardiac matrix ligands comprises proteins comprising the following amino acid sequence:
1. Nephronectin (SEQ ID NO: 13)
2. RGD (rgd) repeated 1-100 times in sequence
3. GFOGER (Gly-Phe-Hyp-Gly-Glu-Arg) (SEQ ID NO: 11) repeated 1-20 times in sequence.
In various embodiments the polymeric hydrogel substrate comprises aligned wrinkle ridges. The aligned wrinkle ridges can be arranged in one or more (i.e.: 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) continuous and ordered patterns.
The ridges may be of any suitable height between 10 nm to 4 μm; the ridges may all be of approximately the same height over the entire scaffold, of approximately the same height in each discrete section of the scaffold, or may vary between different sections or within a given section. In one embodiment, the height of ridges ranges between about 10 nm and about 2 μm. In one specific embodiment, the ridges are all of approximately the same height over the entire scaffold, or of approximately the same height in each discrete section of the scaffold. Valleys are present between the ridges.
In various, non-limiting embodiments a height of ridges ranges between about 10 nm and about 4 μm, and optionally the ridges are all of approximately the same height over the entire scaffold, or of approximately the same height in each discrete section of the scaffold. In various, non-limiting embodiments the valley to valley distances and/or ridge peak to ridge peak distances of between about 400 nm and about 3 μm. For example, the valley to valley distances and/or ridge peak to ridge peak distances of between about 400 nm and about 500 nm, about 400 nm and about 600 nm, about 400 nm and about 700 nm, about 400 nm and about 800 nm, about 400 nm and about 900 nm, about 400 nm and about 1 μm, about 400 nm and about 1.5 μm, about 400 nm and about 2 μm, or about 400 nm and about 2.5 μm.
As used herein, “valley to valley distance” means the distance between adjacent valleys separated by ridges (i.e.: ridges next to each other).
As used herein, “ridge peak to ridge peak distance” means the distance between immediately adjacent ridges (i.e.: ridges next to each other).
The valley to valley and/or peak to peak distances may all be the same, or may vary from one valley to valley or peak to peak distance to another.
Any suitable polymer may be used that is not toxic to cardiomyocytes, and can be fabricated at high fidelity at submicron scale, and can be fabricated within the rigidity or viscoelasticity range of about 10 kPa-75 MPa as Young's Modulus. In various, non-limiting embodiments the patterned polymeric scaffold comprises a polyacrylamide (PA) or polyethylene glycol (PEG) hydrogel, or poly lactic-co-Glycolic Acid (PLGA), or their chemical branch derivatives. In one embodiment, the patterned polymeric scaffold comprises a polyacrylamide (PA) hydrogel having a rigidity of between about 16-24 kPa. In various, non-limiting embodiments the polymer, such as PA, binds to the one or more cardiac matrix ligands via covalent binding, or polymer, such as PEG, binds to one or more cardiac matrix by functional group conjugation with peptide (e.g. NHS).
In various, non-limiting embodiments the construct comprises embedded fluorescent beads to calculate cellular traction force generation by detecting displacement of beads upon contraction, spontaneous or upon electrical stimulation. Inclusion of embedded fluorescent beads in the construct can be used, for example, to directly calculate cellular traction force, spontaneous or upon electrical stimulation, by detecting displacement of the fluorescent beads. The fluorescent beads may be embedded in the construct using any suitable technique. In one embodiment, the fluorescent beads are embedded in the hydrogel by coating poly-L-lysine (PLL) on plasma treated coverslips followed by bead placement, followed by replica molding of nanowrinkle pattern on polyacrylamide solution over the beads.
The patterned polymeric scaffold can be prepared by shrinking of hydrogel, or stretching of an elastic membrane, preferably polydimethylsiloxane (PDMS), and oxygen plasma administration after stretching to create a mold, which can be then transferred directly to a hydrogel, or via an intermediate oxygen impermeable mold using capillary force lithography, or nanoimprinting. In one non-limiting embodiment, the PDMS membrane has a rigidity of about 580 kPa (created by mixing about 10:1 ratio of base to crosslinker). In another non-limiting embodiment, the membrane may be stretched with the force of about 10.44 N to yield about a 30% strain to create anisotropic nanowrinkles. Shrinking or expansion of expansive hydrogels (e.g. polyacrylate) with prepared nanowrinkles can be utilized to achieve the desired feature sizes ranging from 0.1 μm to 10 μm. The nanowrinkles can be isotropic or non-aligned nanowrinkles and can be created by non-directional stretching, either by stretching in orthogonal directions, or in a circular pattern. Shrinking or expansion of hydrogel can be used to achieve desired feature size, to be used directly, or before transfer to a mold.
The construct can further comprise cardiomyocytes or precursors thereof seeded on the construct. In one non-limiting embodiment, the cardiomyocytes or precursors thereof comprise induced pluripotent stem cell (iPSC, such as human iPSC) derived cardiomyocytes. In a further non-limiting embodiment, the cardiomyocytes or precursors thereof comprise cardiomyocytes. In another non-limiting embodiment, the cardiomyocytes or precursors thereof comprise human cardiomyocytes or precursors thereof. In yet another non-limiting embodiment, the cardiomyocytes or precursors thereof comprise electrochemically connected cardiomyocytes. The cardiomyocytes on the construct may establish and maintain cellular/electrical communications such as gap junctions, such that they can serve as a model of cardiac tissue. In one embodiment, the electrically connected cells are capable of spontaneous synchronized contractions across all or part of the construct, or are capable of being paced in a synchronized fashion by external pacing.
In various, non-limiting embodiments the construct can further comprise cardiac fibroblasts or precursors thereof, endothelial cells, vascular smooth muscle cells and/or macrophages and/or other immune cells seeded on the construct. These embodiments can result in a construct which is similar to the in vivo environment including improved maturation of differentiated human cardiomyocytes, promotion of cell spreading, higher strain energy and contractility, decreased myofibroblast phenotype, maturation in calcium transients and electrophysiological parameters, and increased remodeling.
In a second aspect, the disclosure provides methods for making the construct of any embodiment or combination of embodiments disclosed herein. In one embodiment, the methods includes creating a patterned substrate comprising a plurality of wrinkles, wherein the plurality of wrinkles comprise linear or branched folds directionally aligned over a centimeter length scale; transferring the patterned substrate to a mold and then transferring the patterned substrate from the mold onto a polymeric hydrogel. The transfer to the polymeric hydrogel creates a patterned scaffold at submicron resolution comprising a plurality of wrinkles.
In one embodiment, the method includes two transfer steps; the double transfer involves replica transfer of patterns to a primary mold made of polymer without oxygen permeability, the fabricated pattern is then transferred to the hydrogel. As oxygen interferes with hydrogel polymerization, double transfer allows pattern replication with high fidelity and yield. Anisotropic patterned scaffolds are similar to the aligned bundles of collagen fibers in the human heart. Creating a patterned substrate comprising a plurality of wrinkles can occur using any suitable method, including, but not limited to, nanowrinkling nanoindentation, nanoetching, electron beam lithography, or photolithography, hot embossing, dual exposure patterning, and these methods can also be combined with shrinking or expansion to control feature size. Exemplary techniques are described in the Examples. The wrinkles can be isotropic or non-aligned wrinkles or nanowrinkles and can be created by non-directional stretching, either by stretching in orthogonal directions, or in a circular pattern. Shrinking or expansion of hydrogel can be used to achieve desired feature size. The transferring can occur using any suitable method according to the methods of the invention, including but not limited to capillary force lithography or replica molding. Exemplary techniques are described in the Examples.
In a third aspect, the methods for making the construct of any embodiment or combination of embodiments disclosed herein include dual exposure patterning (DEP). DEP can include sequential photo-crosslinking of hydrogel precursors in which (a) primary photo-crosslinking involves a flood light exposure to partially crosslink the precursors; (b) secondary photo-crosslinking involves a masked light exposure to fully crosslink the residual precursors. DEP can be utilized to rapidly achieve centimeter to meter-scale micro/nanopatterned hydrogel. In one embodiment, DEP is used to create anisotropic patterns to culture myocytes in an aligned fashion with directional expression of My12 (
In various non-limiting embodiments, the patterned substrate comprises polydimethylsiloxane (PDMS) and/or the mold comprises polyurethane (PUA). In various other non-limiting embodiments, the polymeric hydrogel comprises polyacrylamide (PA) or polyethylene glycol (PEG) hydrogel, or poly lactic-co-Glycolic Acid (PLGA), polyurethane (PUA), polyacrylate (PA) or their chemical branch derivatives. In various non-limiting embodiments, the polymeric hydrogel substrate has a viscoelasticity between about 10 kPa and about 100 MPa, or about 15 kPa to about 75 MPa. For example, the polymeric hydrogel substrate has the viscoelasticity between about 15 kPa and about 95 MPa, about 15 kPa and about 90 MPa, about 15 kPa and about 85 MPa, about 15 kPa and about 80 MPa, about 15 kPa to about 70 MPa, about 15 kPa and about 65 MPa, about 15 kPa and about 60 MPa, about 15 kPa and about 55 MPa, about 15 kPa and about 50 MPa, about 15 kPa and about 45 MPa, about 15 kPa and about 40 MPa, about 15 kPa and about 35 MPa, about 15 kPa and about 30 MPa, about 15 kPa and about 25 MPa, about 15 kPa and about 20 MPa, about 15 kPa and about 15 MPa, about 15 kPa and about 10 MPa, or about 15 kPa and about 5 MPa. In various, non-limiting embodiments the patterned polymeric scaffold comprises a polyacrylamide (PA) or polyethylene glycol (PEG) hydrogel, or poly lactic-co-Glycolic Acid (PLGA), or their chemical branch derivatives. In one embodiment, the patterned polymeric scaffold comprises a polyacrylamide (PA) hydrogel having a rigidity of between about 16-24 kPa. For example, the patterned polymeric scaffold comprises the PA hydrogel having the rigidity of about 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, or 24 kPa.
As described in the first aspect, the construct can comprises fluorescent beads. According to this embodiment, the fluorescent beads can be embedded in the hydrogel. In one-limiting example of this embodiment, the fluorescent beads can be embedded in the hydrogel by coating poly-L-lysine (PLL) on plasma treated coverslips followed by bead placement, followed by replica molding of nanowrinkle pattern on polyacrylamide solution over the beads.
The methods further comprise conjugating one or more cardiac matrix ligands to the patterned scaffold. In various embodiments, the one or more cardiac matrix ligands comprise 1, 2, 3, 4 or more of Nephronectin (SEQ ID NO: 13), GRGDS (SEQ ID NO: 10), GFOGER (SEQ ID NO: 11), GFPGER (SEQ ID NO: 12), and/or other peptides containing one or more RGD motifs.
The methods can further comprise seeding cardiomyocytes or precursors thereof seeded onto the patterned scaffold. In various, non-limiting embodiments, the cardiomyocytes or progenitors thereof comprise induced pluripotent stem cell (iPSC) derived cardiomyocytes. In one non-limiting embodiment, the cardiomyocytes or progenitors thereof comprise human cardiomyocytes or precursors thereof.
The methods can further comprise seeding cardiac fibroblasts, endothelial cells, vascular smooth muscle cells and/or macrophages and/or other immune cells alone, or in combination, seeded onto the patterned scaffold.
In a fourth aspect, the disclosure provides methods for generating cardiomyocytes, comprising culturing cardiomyocyte precursors on the construct of any embodiment or combination of embodiments disclosed herein, wherein the culturing is carried out for a time and under suitable conditions to generate differentiated cardiomyocytes. In various, non-limiting embodiments, and according to the methods of the disclosure, the cardiomyocytes precursors comprise induced pluripotent stem cell (iPSC, such as human iPSC) from healthy or patients with genetic mutation of interest (e.g. any mutation causing hypertrophic cardiomyopathy, dilated cardiomyopathy, or other cardiovascular diseases). In one non-limiting embodiment, the cardiomyocytes precursors comprise human cardiomyocyte precursors. In various, non-limiting embodiments, and according to the methods of the disclosure, generating cardiomyocytes comprises generating electrically and/or chemically connected cardiomyocytes. According to the methods of the disclosure, the methods may generate cardiomyocytes on the construct that establish and maintain cellular/electrical communications such as gap junctions, such that they can serve as a model of cardiac tissue. In one embodiment, the electrically connected cells are capable of spontaneous synchronized contractions across all or part of the construct. The methods according to the disclosure may generate an electrochemically coupled cardiac layer, expressing key potassium, calcium, or sodium channels in adult ventricular cardiomyocytes, sensitive to caffeine, dependent on fatty acid oxidation, structurally express gap junctions, aligned sarcomeres, fused mitochondria, sensitive to ion channel inhibitors in regulating action potential duration profile, and generating high contractile mechanical force. In various, non-limiting embodiments, and according to the methods of the disclosure, the cardiomyocytes possess one or more of the naturally expressed gene and/or protein characteristics disclosed in the Examples, including but not limited to those listed in Tables 1-4. Specific non-limiting examples include CAMK2D, CASQ2, PLN, TRDN, MYOM2, TTN, MYBPC3, CAV3, PFKM, PDHB, NEFL2, NNT, NOS1, GSR TNNI3, MYOD1, MYPN, MYH2, XIRP2, RYR1, RYR2, ACTN1, DAG1, NBR1, TRIM63, ACTN2, CAV3, GSK3A, MYBPC1/3, GATA4, MEF2C, MYOCD, SRF, KCNJ2, MYL2, MFN1, MFN2, DNM1L, OPA1, LTCC, SERCA, SCNA5, KCNA4, GATA4/5, PPARG, TBX5, MEF2A, MYL7, CICR, and MYOC.
In a fifth aspect the disclosure includes methods for using the construct of any embodiment or combination of embodiments of the disclosure for any suitable purpose, including but not limited to those disclosed in Examples. Examples of suitable methods include, but are not limited to testing the effect of candidate drugs on the construct as a model of the heart (such as human heart, healthy or diseased), studying heart development, and finding therapies for heart diseases (such as human heart disease), or testing toxicity of drugs on human cardiac tissue construct. In one embodiment, the methods may comprise contacting a construct of the disclosure that comprises cardiomyocytes or precursors thereof seeded on the construct with one or more candidate compounds to assess an effect of the one or more candidate compounds on the cardiomyocytes or precursors thereof. In this embodiment, the methods may be used to, for example, identify candidate compounds that elicit a desired effect, or that elicit an undesirable effect, on the cardiomyocytes or precursors thereof. Such methods are useful for identifying candidate compounds to treat heart disorders, as well as identifying candidate compounds that may be toxic to the cardiomyocytes or precursors thereof. Non-limiting effects that can be assessed in these methods include, but are not limited to changes in metabolic readouts, electrophysiological readouts, action potential profile, traction, force generation, calcium and redox handling readouts, optical mapping, transcriptomic and proteomic readouts, and/or mitochondrial function.
In a further non-limiting embodiment, the methods comprises testing candidate drugs for pro-fibrotic and anti-fibrotic effect of candidate drugs on the construct as a model of fibrosis, finding therapies for acquired and/or genetic diseases, and/or using the construct as a model of scarring or fibrotic cardiac tissue at different stages of fibrosis.
The constructs used in the methods may be any embodiment or combination of embodiments of the constructs of the disclosure that are seeded with cardiomyocytes or precursors thereof. In some embodiments, the cardiomyocytes or precursors thereof are human cardiomyocytes or precursors thereof. In other embodiments, the cardiomyocytes or precursors thereof are electrically connected. In other embodiments, the constructs can further comprise cardiac fibroblasts or precursors thereof, endothelial cells, vascular smooth muscle cells and/or macrophages and/or other immune cells seeded on the construct. These embodiments can result in a construct which is similar to the in vivo environment, closely mimicking natural heart collagen matrix architecture, including improved maturation of differentiated human cardiomyocytes, promotion of cell spreading, higher strain energy and contractility, decreased myofibroblast phenotype, maturation in calcium transients and electrophysiological parameters, and increased remodeling. These methods combine the ligand chemistry, elasticity, and anisotropic ultrastructure of the stromal matrix within the heart in a scalable, inexpensive, reproducible, and convenient platform for testing the effects of one or more candidate compounds or drugs on an animal heart model—at the tissue, individual myocytes and on an organelle level.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.
Human iPSC culture and cardiac differentiation. Human iPSCs (WTC-11) were cultured using chemically defined medium and published protocols (Burridge et al., 2014; Tohyama et al., 2013). Undifferentiated iPSCs were seeded (125,000 cells per cm2) on Geltrex (ThermoScientific)-coated six-well plates and maintained in E8 medium (Life Technologies) for 4 days when they reached 80-85% confluence. Medium was then changed to cardiac differentiation medium (CDM), consisting of RPMI 1640 medium (11875, ThermoScientific), 500 μg/ml O. sativa—derived recombinant human albumin (A9731, Sigma-Aldrich), and 220 μg/ml L-ascorbic acid (A4544-Sigma-Aldrich), containing B27 supplement-insulin free (A1895601 ThermoScientific) to initiate differentiation. On d0-d2, medium was supplemented with 6 μM CHIR99021 (LC Laboratories) for induction of mesoderm. On d4-d5, medium was supplemented with 10 uM IWR (Selleck Chemicals) for cardiac differentiation. Contracting cells are typically noted, starting on d7-d8. To purify cardiac myocytes, a variant of RPMI 1640 medium without D-glucose (11879, Life Technologies) was supplemented with 4 mM sodium lactate (L4263, Sigma-Aldrich) for 2 days on day 10 of differentiation followed by culture in cardiac media, containing B-27™ Supplement with insulin (17504001, ThermoScientific) in CDM medium (Tohyama et al., 2013). Cells were dissociated on d13-d14 using TrypLE (ThermoScientific) and plated/cultured on either flat/anisotropic cell culture surfaces for 15-16 further days (d30 for flat and CMM) or 45 days (d60 for flat culture surface) in cardiac culture media. Cardiac culture media was used in all the conditions from day 13-d14 onwards, and it was composed of RPMI-1640, lipid-enhanced Cellastium S, 220 μg/ml L-ascorbic acid and B27 with insulin. Control flat tissue-culture surfaces were coated with fibronectin at a concentration of 4 μg/cm2. For hypertrophy induction, day 30 differentiated cells were treated with 10 nM endothelin 1 (E7764 Millipore Sigma) for 48 hrs in cardiac culture media.
Fabrication of Nanowrinkled Molds for Cardiac Mimetic Matrix.
Polydimethylsiloxane (PDMS) was prepared by mixing the pre-polymer and curing agent in a 10:1 ratio (Dow Corning Sylgard 184), cured at RT or 24 h on a horizontal surface followed by thermal curing of 4 h at 65° C. PDMS slabs (4 (L)×2 (W)×0.3 (H) cm) were then uniaxially stretched to yield 30% strain using a home-made mechanical stretcher and plasma treated with a plasma cleaner (Harrick Plasma PDC32G) with medium RF power for 5 minutes. PDMS nanowrinkles were obtained upon releasing the PDMS slabs from the mechanical stretcher. A library of PDMS nanowrinkles with various periodicity and depth can be achieved by modulating the strain, RF power, and plasma treatment time. The nanowrinkled slab was then transferred to a polyurethane (PU) mold by replica molding. This was achieved by drop dispensing 100 μl PU prepolymer (NOA 76) onto a clean glass slide, and covering the drop with PDMS slab and placing a 3-gram weight on the slab for 5 minutes. Cross-link NOA76 with a UV Cross linker (UV Stratalinker 188; 365 nm) for 20 minutes. Cool the sample at room temperature before peeling off the PDMS mold. SEM images of PDMS and PU molds were obtained using a Hitachi TM1000 tabletop SEM.
Fabrication of Cardiac Mimetic Matrix Substrates. PU molds, bonded to PET sheets for ease of handling, were used as a replica mold to transfer polyacrylamide (PA) topographic patterns. PEG with a similar elasticity was also tested, and no difference between either PEG or PA as measured by calcium response to caffeine, or RT-PCR of a panel of cardiac genes was found. PA precursor was prepared by mixing 10% acrylamide and 0.225% bis-acrylamide solution to yield an expected 23 kPa gel. After degassing for 30 min, 0.05% v/v tetramethylethylenediamine (TEMED) and 0.5% v/v 10% ammonium persulphate (APS) was mixed with precursor solution by gently pipetting. Precursor solution was dispersed onto PU mold, and covered with salinized coverslip for 20 min in a wet chamber. After cross-linking the hydrogel was immersed in 1× PBS for 1 h before peeling off from PU mold. All the samples were stored in 1× PBS solution at 4° C. AFM imaging of the surface topography of the hydrogel was performed using Asylum Research Cypher AFM with a PNP-TR probe in 1× PBS.
Functionalization of CMM and Raman Spectroscopy. Samples were functionalized with 1.3 mg/ml Sulfo-SANPAH under UV for 10 minutes, and incubated in GRGDS (SEQ ID NO: 10) (Peptides International PFA-4189-v), GFPGER (SEQ ID NO: 12) (Sigma-Aldrich 165044K), and nephronectin (SEQ ID NO: 13) (Novus 9560-NP-050) solutions at 4° C. overnight. The samples were washed with 1× PBS for 3 times and stored in PBS at 4° C. before Raman spectroscopy or cell culture. Raman spectroscopy of functionalized hydrogel was performed using WITec alpha300 R Raman microscopy with a 40× immersive objective in 1× PBS using a 785 nm laser. Briefly, five kind of samples were prepared: hydrogel functionalized with (i) no ligands, (ii) GRGDS (SEQ ID NO: 10), (iii) GFPGER (SEQ ID NO: 12), (iv) nephronectin (SEQ ID NO: 13), and (v) GRGDS/GFPGER/nephronectin (SEQ ID NOs: 10, 12, and 13). Individual spectrums of samples (i)-(iv) were obtained with integration time of 1 s and accumulation of 60 times. Distribution of each component in sample (v) was obtained by true component analysis of the large-area Raman scan (50 μm×50 μm, with resolution of 50 pixel×50 pixel) based on the individual spectrums of samples (i)-(iv).
Statistical analysis. Students t-test was performed unless otherwise mentioned with each result. Data is presented as ±SD or ±SEM and mentioned in each results section. Statistical significance is defined as the p<0.05 (*) p<0.01 (**) or p<0.001 (***).
For gene ontology analyses, statistical significance and z-scores for the enrichment of differentially expressed genes in Gene Ontology gene sets was computed using the following method. First, the individual gene level p-values were transformed to z scores using the inverse of the normal distribution, and the sign assigned by the direction of the fold change. Then, a p-value was evaluated for the gene set by the Student's t-test performed for the genes inside and outside the test. P-values were corrected for multiple testing using false discovery rate (Benjamin-Hochberg) method.
Mitochondrial number. Mitochondrial number was quantified by estimating the amount of mitochondrial DNA (mt-ND1 & mt-ND4) relative to nuclear DNA (B2M) using probe-based Taqman Real Time PCR as described earlier (primer/probes sequence and concentration)(Phillips et al., 2014).
RNA Sequencing and analysis. RNA was isolated using RNeasy Mini Kit™ (Qiagen). Bioanalyzer 2100 (Agilent) was used to check the RNA integrity and samples with RIN <8 were used for library preparation. Library preparation and RNA sequencing were performed by Yale Center for Genome Analysis (YCGA). Reads were aligned to the NCBI GRCh38 genome assembly using the HISAT2 pipeline with default parameters. Reads were counted using HTSeq. Fold changes and statistical significance (p-values) for differential expression were calculated using DESeq2. P-values for differential expression were calculated for the Wald test.
For each functional category the following gene sets were used from the Gene Ontology(Harris et al., 2004), KEGG(Wixon and Kell, 2000), Msigdb(Subramanian et al., 2005), WikiPathways(Slenter et al., 2018), and EBI(Huntley et al., 2015) were used to select the genes to include in the transcriptomic analysis.
Hierarchical clustering using UPGMA method with eucledian distance were performed on samples and z-scores of all differentially regulated genes in either conditions, sorted using Anova analysis with FDR correction of 0.05) and 9 main clusters were observed. Ontologies were evaluated in each cluster in GO, Wikipathways, Kegg and Reactome using gprofiler2(Kolberg et al., 2020).
Immunostaining. Cultured cells were washed twice with PBS before fixing them with 4% paraformaldehyde (pH 7.4) for 15 min at room temperature. After fixation, cells were washed with cold PBS before permeabilizing them with PBS buffer containing 0.2% Triton™ X-100 and 0.1% BSA for 15 minutes followed by one-hour incubation with blocking buffer (1% BSA in PBS). Cells were then incubated overnight with primary antibodies with subsequent 30 minutes incubation with secondary antibody. Following primary antibodies were used for labeling; Alexa fluor™ 594 phalloidin (ThermoFisher A12381), Connexin™ 43 (ThermoFisher 35-5000), sarcomeric alpha actinin (ThermoFisher MA1-22863), cardiac troponin T (ThermoFisher MA5-12960), cardiac troponin I (Abcam ab47003), MYL2 (Abcam ab79935), and Wheat Germ Agglutinin, Alexa Fluor™ 488 Conjugate (ThermoFisher W11261). Following Alexa Fluor Secondary Antibodies (ThermoFisher) were used: Alexa Fluor™ 568 (A-11004, A-11011), Alexa Fluor™ 488 (A32723, A32731). Cells were counterstained with DAPI (ThermoFisher D21490) before imaging. The data was analyzed using ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD, USA).
Confocal and Airyscan Imaging. Confocal Images were acquired using a laser scanning confocal microscope, Zeiss LSM 880. Images were taken using a 63× oil objective. Imaging was performed on LSM 880 laser scanning confocal microscope (ZEISS) equipped with 63X Plan Apochromatic 1.40 NA oil objective, an Airyscan super-resolution module, GaAsP detectors and Zen Black acquisition software (ZEISS). The pixel dwell time, laser intensity and detector gain were kept low to avoid saturation and photobleaching during the image acquisition. To increase signal-to-noise ratio and resolution, acquired images were processed by 3D Airyscan filter strength 7.0 with Zen Black software.
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Banding patterns prevalent as signatures of cardiac maturity were measured using ImageJ and analyzed using custom MATLAB scripts. Banding frequency was measured as the spatial metric between peak intensities of myosin light chain in the respective α-Actinin and cardiac troponin (cTnT) images.
Flow Cytometry. Cells were detached from the substrate with TrypLE Airyscan (Gibco), quenched with excess medium, and washed thrice with phosphate-buffered saline (PBS). Isolated cells were either labeled with the requisite dye (Mitotracker-Green at 100 nM for 15 minutes, and washed twice with PBS), or fixed in 4% paraformaldehyde in PBS and stained with primary and secondary antibodies with method described previously(Hubbi et al., 2013; Kshitiz et al., 2012). Primary antibodies used were: Myosin Light Chain 2 (Abcam ab79935), sarcomeric alpha actinin (ThermoFisher MA1-22863) and cardiac troponin T (ThermoFisher MA5-12960). Cells were analyzed in a BD FacsARIA II, and analyzed using Flowing software (Turku University). Gating was performed using the requisite negative controls in each channel with unlabeled cells.
Immunoblots. Three different biological replicates (batches) were generated using separate cultures/cardiac differentiations of iPSCs for immunoblots. Samples were harvested and lysed in buffer containing radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology 9806), protease inhibitors (Sigma-Aldrich P8340) and phosphatase inhibitors (Sigma-Aldrich 4906845001-PhosSTOP). Protein concentration was quantified using bicinchoninic acid assay kit (Thermo Fisher Scientific). Proteins were denatured 95° C. for 5 minutes in SDS and 20 μg of samples were loaded on 4-12% NuPAGE™ Bis-Tris Gel (Thermo Fisher Scientific NP0322BOX). They were transferred to polyvinylidene difluoride (PVDF) membranes, and subsequently blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4° C. with primary antibody—cardiac troponin I (Abcam ab47003); Troponin T (ThermoFisher MA5-12960), PDK1 (Cell Signaling Technology 5662), AceCS1 (Cell Signaling Technology 3658), FAS (Cell Signaling Technology 3180), PFPK (Cell Signaling Technology 12746). For Total OxPhos primary antibody (Abcam ab110413), samples were prepared in lysis buffer (as above) but loaded onto the gel without denaturation. Proteins samples were transferred onto PVDF membranes at 4° C. and equal protein loading was verified with Ponceau S staining solution (Cell Signaling Solution 59803). Following the primary antibody incubation, membranes were washed several times before incubation with GAPDH (Cell Signaling Technology 5174) for 1 h at room temperature. Subsequently, samples were incubated with horseradish peroxidase-linked anti-rabbit or mouse IgG secondary antibody (GE healthcare NA9340 or NA9310) for 1 h at room temperature. An enhanced chemiluminescence reagent (Thermo Fisher Scientific 34095) was used to visualize the bands. Semi-quantification of protein was conducted by comparison against the GAPDH bands using ImageJ software.
Mitochondrial respiration. Cellular energetics was monitored using the Seahorse Bioscience XF instrument in intact cells (non-permeabilized cells) and permeabilized cells(Afzal et al., 2017; Salabei et al., 2014). Intact cell respiration was monitored to evaluate the contribution of oxidative phosphorylation versus glycolysis, whereas permeabilized cell respiration was used to monitor electron transport chain (ETC) complex function/activity using ETC complex specific substrates and fatty acid oxidation.
Respiratory rates were measured as basal rates (in the absence of added compounds/metabolic inhibitors) and after injection of compounds through injection ports of Seahorse XF analyzer during the assay run. Specific components of ETC or glycolysis (in intact cells) were inhibited to investigate components of metabolism. Oligomycin (404) was used to inhibit mitochondrial FIFO-ATP synthase, rotenone (204) to inhibit Complex 1 of ETC, antimycin A (204) to inhibit complex 3 of ETC, FCCP (204) to uncouple mitochondria for quantification of maximum respiratory capacity and iodoacetate (100 μM) to inhibit glycolysis (glyceraldehyde-3-phosphate dehydrogenase). The compounds were prepared as stock solutions and dissolved in the assay media immediately before the experiment. For intact cell respiration, Seahorse XF Base Medium (Part #102353-100) with addition of D-glucose (11 mM) and glutamine (2 mM) were used.
For permeabilized cell respiration, iPSC-CMs were permeabilized using 2 nM of Seahorse XF Plasma Membrane Permeabilizer (Part #102504-100). In permeabilized cells only OCR is measured. Mitochondria respiration were assayed using Buffer (pH 7.2) containing 137 mM KCl, 2 mM KH2PO4, 0.5 mM EGTA, 2.4 mM MgCl2, 20 mM HEPES with 0.2% fatty acid-free BSA (Sigma). Respiration rates were normalized to the protein concentration using BCA assay. Complex I respiration were evaluated by 5 mM glutamate and 5 mM malate (G/M) to evaluate State 4 respiration; 4 mM ADP was then injected to evaluate State 3 respiration. Maximal respiratory capacity (uncoupled mitochondria) was measured by injecting 204 FCCP. Coupling of Electron Transport Chain respiration to ATP synthesis were evaluated using the ATP synthase inhibitor oligomycin (4 uM). Complex II respiration were measured using 5 mM succinate. Fatty acid oxidation was measured by injecting 200 uM of Palmitoyl-1-carnitine chloride/2.5 mM Malate and assessing the increase in OCR.
Particle Image Velocimetry (PIV) analysis. Time lapse movies of three given conditions were taken using a 4× magnification lens to capture the contraction dynamics over a 2.2×1.5 mm field of view (FOV). The images were taken using the EVOS™ Imaging system with a camera (X) equipped for image acquisition at video rate (60 Hz) over a period of 20 seconds. Particle image velocimetry metrics was employed to track the dynamical movements of cell monolayers during the period of contraction. From the time-lapse images, the local contrast was sufficient to track movement of the monolayers over the 140×140 um interrogation window. Further spatial and temporal filtering was employed to assess correct measurement of particle velocities(Thielicke, 2014). These metrics were used to assess the instantaneous velocities of the monolayer over the field of view and the beating frequency. Furthermore, metrics over localized areas were used to measure the temporal signatures in the contractile moments and the directional movement of the monolayer during contraction.
Transmission Electron Microscopy. Transmission Electron Microscopy was performed by Dr. Maya Yankova at the UConn Health Central Electron Microscopy Facility. To assess the subcellular organizational changes of cardiomyocytes cultures for the two varying topographies, samples were prepared for Transmission Electron Microscopy using standard protocols. Briefly, post culturing cells in flat and CMM, samples were fixed in 2%/2.5% paraformaldyhyde/glutaraldehyde solution overnight. Following fixation, samples were treated with 1% osmium tetroxide and embedded in Epon resin for sectioning. Thin, 70 nm sections were imaged using X imaging setup at various locations using Hitachi H-7650 EM. Organelle structures such as mitochondria size and fusion, sarcomeric structures and banding were visually assessed and imaged.
Traction Force Microscopy. Polyacrylamide substrates were prepared and functionalized to measure traction forces generated by cardiomyocytes from standard gel preparation protocols(Aratyn-Schaus et al., 2010; Fischer et al., 2012; Wang and Pelham, 1998). Briefly, coverslips for gel attachment were cleaned with ethanol and sonication, treated with air plasma, and silane-activated with 0.5% glutaraldehyde and 0.5% (3-Aminopropyl)triethoxysilane. Coverslips (for TFM) and nanopatterned poly(urethane acrylate) molds (for nanoTFM) for beads coating were treated with air plasma, coated with 0.01% poly-L-lysine (PLL), and then coated with carboxylate-modified fluorescent microspheres (0.2 um; Thermo Fisher). Gel precursor solution containing 7.5% acrylamide and 0.15% bis-acrylamide was degassed for 30 mins and mixed with 0.1% tetramethylethylenediamine and 0.1% ammonium persulfate before sandwiched between silane-activated coverslips and beads-coated coverslips or molds for 20 mins. Gels were functionalized using 1 mg/mL suflo SANPAH (ThermoFisher) for 10 min under UV lamp (UV StratAligner 1800) and incubated in 30 ug/ml collagen type I (Thermo Fisher) overnight at 4° C. Gels were sterilized under UV for at least 2 hours before cell seeding. Harvested cardiomyocytes from flat and nano substrates were seeded on functionalized gels and incubated for 36 hours prior to imaging. Differential Interference Contrast (DIC) and fluorescent beads were imaged using a Zeiss Observer Z 1. Monolayer contractions were imaged over a period of 20 seconds to accurately assess contraction dynamics of at least 4-6 cell cycles. Cells were paced at different rates by coverslips with cells in a chamber connected to grass stimulator with 4.0 ms pulse duration and 5V of current at which one to one pacing was observed at different pacing rates. Cells were then detached by trypsinization, and stress-free reference images were recorded. Traction stress calculations were performed by comparing images containing beads position displaced by cellular traction force and reference images using particle image velocimetry as described in detail (Sabass et al., 2008). Strain energy calculations were made as mentioned earlier to assess contractile energies of cardiomyocytes (Sabass et al., 2008).
Cyto-calcium, Action potential and redox handling. Cytoplasmic calcium and redox status of iPSC-CMs using spinning disk confocal microscope (Olympus/Andor Revolution XD) was monitored. 3rd gen. lentiviral probes using ViraPower™ Lentiviral mix was generated and transduced iPSC-CMs using MOI 5 (multiplicity of infection) that generated good signal to noise ratio. Cytoplasmic targeted roGFP-Grx1 probes(Gutscher et al., 2008), Calcium probe (genetically encoded Ca2+ indicators (GECIs)-GCamP6f(Hubbi et al., 2013), Arclight(Jin et al., 2012) and Varnam(Kannan et al., 2018) were obtained from Addgene and transferred to pENTR™/SD/D-TOPO® vector using PCR. The vector was sequence verified and then transferred to pLEX_307 vector (Addgene plasmid #41392) that contains the EFlα promoter using the LR reaction. The vector was sequence verified and transferred to pLEX_307 vector using the LR reaction. The virus was generated for each probe in HEK293-FT cells using an optimized mix of three packaging plasmids (pLP1, pLP2, and pLP/VSVG). The virus was concentrated using PEG-it™ Virus Precipitation Solution (SBI biosciences) that also removes HEK293-FT cell medium. MOI was calculated using qRT-PCR and MOI=5 was found to be optimal for these probes in iPSCs-CMs.
To monitor the florescence signal from cells transduced with probes, cells were plated at a density of 200,000 per cm2 on cultured surfaces for at least 10-15 days prior to imaging, to achieve cell-cell coupling. During the experiment, continuous perfusion of modified Tyrode solution was performed at 37C with pH 7.4 containing (in mM) 130 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2), 20 Na-HEPES, 11 glucose, 2 pyruvate, 0.1% fatty acid free-BSA.
Florescence from cells (transduced with probes) was collected separately at a frame rate of 5112×512 using an electron-multiplying charge-coupled device (EMCCD) camera using Andor Revolution X1 Spinning Disk confocal inverted microscope. Data was acquired with 2×2 pixel binning. ROS (roGFP2-Grx) probes were excited with 405 nm and 488 nm laser and emission was detected using bandpass filter of 500 nm-554 nm. The image 405 nm image was divided by 488 nm image (pixel by pixel), and the values are reported as the ratio of 405/488. Recovery of glutathione pool was monitored for 180-240 sec before the utilization of diamide (oxidized) and DTT (reduced) to obtain min and max signal. Recovery timepoint of 60 seconds were selected as the signal was stable 60 sec in most cells. Cells transduced with cytoplasmic GCamp6f were excited at 488 nm and emission was detected using bandpass filter of 500 nm-554 nm. Cells transduced with Arclight were excited at 488 nm and emission was collected using bandpass filter of 500 nm-554 nm. The data was analyzed using image J (NIH).
To investigate calcium content of the sarcoplasmic reticulum (SR), Fura2 (ThermoFisher 3 uM) loaded cells were perfused with caffeine. Cells were initially perfused with modified Tyrode solution at 37C (pH 7.4) containing (in mM) 130 NaCl, 5 KCl, 1 MgCl2, 1.2 CaCl2, 20 Na-HEPES, 10 glucose, 1 pyruvate, 0.1% fatty acid free-BSA. After pacing the cells at 0.5 Hz for 30-40 sec, cells were perfused with the modified Tyrode buffer containing caffeine (10 mM). Images were analyzed using image J (NIH). Line plots (trace plots) were generated for both action potential and calcium transients by calculating average intensity (and standard deviation) from 3 biological batches with >100 cells for each timepoint and plotting it against the time (using R).
Steiner tree based Protein-Protein Interaction Map. Networks intending to generate hypotheses for proteins mediating the transcriptomic response related to specific ontologies from the ECM coated nanofabricated substrate was derived. Our method prioritizes including the genes from a particular gene set that are differentially up-regulated in the nanofabricated structures and the ECM receptors (integrins) that are supposed to cause the response.
The networks are generated from the protein-protein interactions taken from BioGRID. In order to get the gene set related subnetwork of differentially expressed genes, the Prize Collecting Steiner Tree algorithm was modified. The prize for including a differentially regulated gene that is a member of the gene set under consideration is set as
p
g=α(1+fgIf
where fi is the log 2 fold change of the gene i, If
Then, the cost of each edge (i,j) is set as
where If>0 is again the indicator function. The numerator is the equivalent of symmetrical degree normalization of the adjacency matrix, while the denominator has the effect of decreasing the cost for edges that connect up-regulated edges. Overall, promiscuous and non up-regulated genes are penalized. The indicator function was used so that it isn't, per se penalizing down-regulation because down-regulation may either mean the shutting down of a signaling pathway or the participation of a particular gene in some complex dynamics.
The Prize Collecting Steiner Tree algorithm then attempts to join the genes with prizes using edges and any other out of set genes as needed to maximize the total gene prizes minus the total edge costs. A small set of genes needed to connect the gene set will be included by the algorithm, prioritized by their up-regulation and specificity (i.e., low degree in the PPI network). Omicslntegrator2 was used to arrive at a solution to the optimization problem (Kedaigle A and E., 2018).
For
Patch Clamp Electrophysiology. Cardiac cells differentiated on flat and CMM substrate (d30 and CMM cells) were harvested as single cell and plated on fibronectin coated cover slips followed by few days of culture in cardiac media to recover them before performing patch clamp and action potential recording. Action potential was recorded under whole-cell current clamp mode using an Axon Axopatch 200B amplifier and pclamp9 software (Molecular Devices, USA) at room temperature. Patch pipettes were pulled from borosilicate glass tubes to give a resistance about 10 MΩ when filled with pipette solution. Data were low-pass filtered at 1 kHz and digitized at a rate of 10 kHz. The bath solution contained 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH7.41 and the pipette solution was 145 mM KCl, 5 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 3 mM MgATP, 4 mM EGTA, and 10 mM HEPES, pH7.35. A 0.2 nA stimulation was applied for 2 ms to induce action potential firing. Nifedipine at 100 nM was used through a gravity-perfusion system until reach the maximum inhibition was achieved. MDP, MDR, APD20, APD50 and APD90 were analyzed using the pclamp10 software.
Cell culture and Treatment. Normal human ventricular cardiac fibroblasts were purchased from Lonza biosciences (CC-2904). Cells were cultured and expanded in cardiac fibroblast media (Millipore Sigma, 316-500). Experiments were performed at passage 4 of sub culture. Cells were replated on anisotropic and flat surfaces on an uncoated surface for 7 days before starting any treatment.
TOP (5 ug/ml) treatment was performed in serum free Dmem/f12 containing ITS (insulin transferrin selenium) solution (Thermo Fisher 41400045) and 0.1% BSA. Cells were washed twice with PBS before starting the Tgfβ treatment for 48 hours. Multiple biological batches were used for experiments but the passage number (p4) and confluency (70-80%) were kept the same in the experiments.
RNA isolation, RNA Sequencing and analysis. RNA was isolated using RNeasy™ Mini Kit (Qiagen). Bioanalyzer 2100 (Agilent) was used to check the RNA integrity and samples with RIN ˜8 were used for library preparation. Library preparation and RNA sequencing were performed by Novogene. The data was aligned against NCBI GRCh38 genome assembly using the HISAT2 pipeline with default parameters and reads were counted using HTSeq. Deseq2 was used to calculate Fold changes and statistical significance (p-values) for differential expression. P-values for differential expression were calculated for the Wald test. IPA (ingenuity pathway analysis-Qiagen) was used for canonical pathway and predicted activation of transcriptional factors.
Gene sets from the Gene Ontology(12), KEGG(13), Msigdb(14), WikiPathways(15), and EBI(16) were used in the relevant pathway/ontology related transcriptomic analysis.
qRT PCR. RNA isolated using RNeasy™ Mini Kit (Qiagen) was converted into cDNA using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher). Predesigned primers were purchased from integrated DNA technologies (IDT, PrimeTime qPCR Primer Assays) to run real-time PCR using intercalating dye with melting curve at the end of each assay. PowerUp SYBR Green Master Mix (ThermoFisher) was used in Biorad CFX384 thermal cycler to estimate the relative gene expression.
Statistical analysis. Students t-test was performed for most of the analysis unless otherwise mentioned. Data is presented as ±SD or ±SEM. Statistical significance is defined as the p<0.05 (*) p<0.01 (**) p<0.001 (***) or p<0.0001 (****).
For ontology related analyses, statistical significance and z-scores for the enriched differentially expressed genes in gene sets were calculated by transforming the individual gene level p-values to z scores using the inverse of the normal distribution, and then the sign was assigned by the direction of the fold change. Student's t-test was used to calculate p-value and correction for multiple testing was performed using false discovery rate (Benjamini-Hochberg) method.
Immunostaining and imaging. Cultured fibroblasts were washed multiple times with PBS before treating them with 4% paraformaldehyde (pH 7.4) for 15 min at room temperature for cell fixation. Cells were subsequently washed with cold PBS and permeabilized with permeabilization buffer containing PBS, 0.2% Triton X-100 and 0.1% BSA for 15 minutes. Permeabilized cells were then blocked with one-hour incubation with blocking buffer (1% BSA in PBS). Overnight incubated was performed with primary antibodies with subsequent 30 minutes incubation with secondary antibody. Following primary antibodies were used for labeling; Alexa fluor™ 594 phalloidin (ThermoFisher). Following Alexa Fluor Secondary Antibodies (ThermoFisher) were used: Alexa Fluor 568 (A-11004, A-11011), Alexa Fluor 488 (A32723, A32731). Image J (National Institutes of Health, Bethesda, MD, USA) was used to analyze the data.
Immunoblots. Cardiac fibroblasts were harvested and lysed in cell lysis buffer containing radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology 9806), protease inhibitors (Sigma-Aldrich P8340) and phosphatase inhibitors (Sigma-Aldrich 4906845001-PhosSTOP). Bicinchoninic acid assay kit (Thermo Fisher Scientific) was used for protein concentration. For collagen, non-denatured samples (10 ug) were loaded on the tris acetate gels (Thermo Fisher Scientific) while other immunoblots were performed with denatured samples (95° C. for 5 minutes in SDS) before loading samples on 4-12% NuPAGE Bis-Tris Gel (Thermo Fisher Scientific). Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes, and subsequently blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4° C. with primary antibody. GAPDH loading control was used (Cell Signaling Technology 5174). Horseradish peroxidase-linked anti-rabbit or mouse IgG secondary antibody (GE healthcare NA9340 or NA9310) for 1 h at room temperature was used to visualize the protein bands using enhanced chemiluminescence reagent (Thermo Fisher Scientific). Protein quantification was performed using ImageJ software.
Enzyme-linked immunosorbent assay (Elisa). Cell culture supernatant was collected at the end of treatment (48 hours following treatment in serum free media) from 6 wells (100 ul from each well). Colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kits were used from R&D Systems (Igf1 DY291, MMP1 DY901B, Postn DY3548B) and Abcam (Vegfa ab119566, Opn ab100618). Elisa was performed following manufacturer's instructions and BioTek Synergy microplate reader was used to measure the absorbance. Protein secretion was estimated after normalizing the data with cell number from each well. Cell numbers were calculated using Countess 3 FL Automated Cell Counter (ThermoFisher) after harvesting the cell from each well following 48 hours of treatment and media collection. Per well protein concentration was estimated after calculating the calibration curve for each Elisa kit
A substratum was created matching the chemistry, elasticity, and topographic ultrasctructure of the extracellular matrix that adult cardiomyocytes respond to. Integrin alpha and beta subunits combine to form more than 20 different heterodimers with varying ligand specificity, and transduce extracellular chemical and mechanical information to intracellular signaling modulating various cellular phenotypes, including cell fate and differentiation (Kshitiz et al., 2012; Mamidi et al., 2018). Adult human hearts transcriptomic data was used (Choy et al., 2015; He et al., 2016; Lopez-Acosta et al., 2018; Zhao et al., 2019), and identified genes encoding integrin receptor subunits upregulated in comparison to hiPSCs (
It was surmised that combining nano-architectured substrates, elasticity matching heart tissue, and adult-cardiomyocytes inspired ligand chemistry would, in combination, affect hiPSC-CM maturation. Based on transcriptomic analysis of integrins present in the adult human heart, RGD, GFOGER (SEQ ID NO: 11) (a commercial alternative to GFOGR (SEQ ID NO: 14)), and Nephronectin (SEQ ID NO: 13) was conjugated in equal amounts with hydrogel patterns into nano-architectured arrays produced by anisotropic nanowrinkle pattern transfer to create a cardiac mimetic matrix (CMM) substrate (
Considering the short period of culture on CMM, many gene sets were found to be different in CMM vs d30 (p<0.05), surprisingly most being related to cardiac function (
Tracing individual gene expression change along the stages of maturation (d30 to d60 to CMM to fetal to adult) confirmed the general trend of a transcriptomic cardiac maturation vector (
CMM is a composite platform, consisting of specific ligand chemistry, matrix mechanics and ultrastructure. To assess their relative contribution and identify potential signaling intermediaries involved, a novel networking analysis method was creasted using the Prize Collecting Steiner Tree formulation (see Example 1) (Akhmedov et al., 2017; Bienstock et al., 1993). The resultant subnetwork connecting CMM constituents (ligand chemistry, and mechanics) with the genes related to cardiac development through BIOGRID interactome (Stark et al., 2006). Genes in actomyosin organization ontology were used as a surrogate for those influenced by anisotropic matrix and physiological elasticity, while adult-heart specific integrins were used as the second input into the PPI network, prioritizing inclusion of genes differentially upregulated in CMM vs Control. A comprehensive network was generated connecting the 5 integrin genes in adult-heart, and top 5 genes (
It was then experimentally tested the prediction of synergistic effect of individual components of CMM for cardiac transcripts, and mitochondrial maturation. Cells were placed on flat (d30), matrix conjugated (d30+matrix), anisotropic nanowrinkled (ANW) surfaces, and ANW surfaces conjugated with matrix (CMM) (
It was first characterized whether CMM induced changes in gene expression resulted in accompanying maturation in the structural and mechanical characteristics. Focusing on ontologies related to cardiac structure, it was found that CMM caused increase in gene expression even more than d60 (
Cardiomyocytes are contractile cells, capable of force generation upon electrical stimulation. Increased structural maturation and high mitochondrial content in hiPSC-CMs on CMM suggested increased capability to produce contractile force. Particle image velocimetry on time-lapsed images of cells revealed contraction velocity vectors being randomly aligned on control substrate, while being more sustained and highly directional on CMM (
Cardiac tissue is highly metabolically active, dependent on mitochondrial oxidative respiration(Lopaschuk and Jaswal, 2010), with a high plasticity in substrate utilization, essential to rapidly generate ATP, which is limiting; ˜10 mM lasting for a few contractions (Ingwall, 2009; Stanley et al., 2005). Transcriptomic data showed that hiPSC-CMs on CMM follow a more adult cardiac metabolic program, with increased expression of key transcripts in electron transport chain (ETC), FAO and OxPhos (
Dependence on OxPhos and a high rate of ATP generation result in a high burden of reactive oxygen species, which adult-like cardiac cells can scavenge by maintaining 100 fold higher reduced glutathione (GSH) than the oxidized species (oxidized GSH, GSSG and mixed disulphide, GSSR) (Burgoyne et al., 2012; Santos et al., 2011) (Aquilano et al., 2014). Using a pulse of 1 mM-hydrogen peroxide (H2O2) in Tyrode buffer, the recovery of oxidized glutathione pool of hiPSC-CMs was evaluated using cytoplasmic Grx1-roGFP2 probe (Gutscher et al., 2008; Meyer and Dick, 2010). Grx1-roGFP2 signal intensity was normalized by respective addition of diamide, and Dithiothreitol (DTT) for max and min signal(Meyer and Dick, 2010). It was found that stable Grx1-roGFP2 signal (400 nm/485 nm ratio) at 60 seconds in all the conditions before calibration, and found that cells on CMM showed consistently lower Grx1-roGFP2 ratio (400 nm/485 nm) of 0.423±0.092, indicating rapid recovery of glutathione pool compared to cells on d30 cells (0.7163±0.08) (
To support increased ATP generation from OxPhos, cells require high mitochondrial number, which are typically fused and elongated in adult cardiomyocytes, as well as upregulation of ETC subunits. MFN1, MFN2, DNM1L, OPA1, and other genes related to mitochondrial fusion were up-regulated on CMM (
Cardiac tissue development leads to coordinated electrical excitation coupled to contraction known as excitation contraction coupling (ECC)(Bers, 2002). The interplay of ions for ECC requires specific expression of proteins including L-type Ca2+ channels (LTCC), ryanodine receptors, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and Na+/Ca2+ exchanger(Bers, 2002; Liu et al., 2016). Cardiac maturation on CMM led to a synchronous but slow beating rate of cells (
Efficient calcium cycling is crucial to convert electrical signal to mechanical force in the myocytes(Bers, 2002), and calcium transient profiles inform the extent of maturation (Liu et al., 2009). SERCA had higher abundance CMM vs control (
Single cell patch clamp was performed and it was found that CMM significantly increased action potential duration (APD) APD90 and APD50 vs d30 (
With establishment of transcriptional, metabolic, redox, and calcium handling characteristic of a matured cardiac tissue, the effect of a key pathological stimulus, endothelin-1 (ET-1) treatment on cardiac cells was tested (
Using IPA pathway analysis, it was found that ET-1 treatment in d30 exhibited differential regulation of gene expression related to adrenergic signaling, metabolism, hypertrophic signaling, calcium and NOS signaling (
The attenuated response of ET-1 on hiPSC-CMs matured on CMM for key characteristics of cardiac function: metabolism (energetics and redox), calcium handling and electrophysiology was confirmed. ET-1 treatment significantly increased both OxPhos and glycolysis on d30, but the levels of OxPhos was still lower than cells on CMM (
This application claim priority to U.S. Provisional Patent Application Ser. No. 63/321,393, filed Mar. 18, 2022, incorporated by reference here in its entirety.
This invention was made with government support under CA248161 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63321393 | Mar 2022 | US |
