Patent Grant
11576949
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 24, 2017, is named 167914_010305USCON_Sequencelisting.txt and is 9,209 bytes in size.
Despite recent advances in treating ischemic injuries, stroke and myocardial infarction continue to kill or disable vast numbers of people each year. In the United States alone, 600,000 new myocardial infarctions and 320,000 recurrent attacks occur annually. About 38 percent of the people who experience a myocardial infarction in a given year will die, while many of those who survive will experience some loss in cardiac function.
Certain cell types, including muscle cells and neurons are particularly vulnerable to ischemic injury in connection with myocardial infarction and stroke. Technologies associated with the identification, isolation, and culture of stem/progenitor cells now provides many candidate cells for cell replacement applications in regenerative medicine. Notably, however, transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. As such, current cell replacement strategies for treating myocardial infarction involving the injection of stem/progenitor cells result in modest improvements in cardiac function, at best. Low levels of engraftment, survival, and cell replacement after injection of adult or embryonic stem cells into the injured left ventricle wall are important issues that reduce the potential effectiveness of cell replacement strategies after myocardial infarction. Moreover, intravenous infusion of cultured adult stem/progenitor cells can be accompanied by microembolism and cardiac arrhythmias. Accordingly, improved methods of treating tissue injury, particularly ischemic injuries associated with myocardial infarction, are urgently required.
As described below, the present invention features compositions and methods for treating or preventing cardiac tissue damage, including damage associated with an ischemic event.
In one aspect, the invention provides a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1.
In another aspect, the invention provides a composition containing a human C-terminal CTGF peptide that enhances cardiac progenitor cell survival and/or proliferation.
In still another aspect, the invention provides a method for increasing cardiac cell survival or proliferation, the method involving contacting a cardiac cell at risk of cell death with a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1.
In yet another aspect, the invention provides a method for stabilizing or reducing cardiac tissue damage in a subject, the method involving contacting a cardiac cell of the subject with a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1, thereby stabilizing or reducing cardiac tissue damage in the subject.
In still another aspect, the invention provides a method for enhancing engraftment of a cardiac cell or progenitor thereof (e.g., an adult cardiac myocyte, adult cardiac endothelial cell, adult cardiac smooth muscle cell, adult cardiac fibroblast, adult cardiac stem cell, adult cardiac progenitor cell, adult vascular stem cell, adult epicardial cell, adult sub-epicardial cell, adult bone marrow-derived stem or progenitor cell, cardiac derivative from embryonic stem (ES) cells, and/or induced pluripotent stem (iPS) cell), the method involving contacting the cell (i.e., to be primed) with a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1, thereby enhancing engraftment.
In another aspect, the invention provides a method for enhancing engraftment of a cardiac cell or progenitor thereof in a subject, the method involving: contacting the cardiac cell or progenitor in vitro with a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1, thereby generating a primed cell; and administering the primed cell to the subject, thereby enhancing engraftment of the cell in the subject.
In one aspect, the invention provides a cellular composition containing an isolated cardiac progenitor cell, cardiac stem cell, mesenchymal stem cell or progeny cell thereof contacted with a composition containing one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and one or more of insulin and IGF-1.
In another aspect, the invention provides an isolated polynucleotide encoding CTGF-D4.
In still another aspect, the invention provides an isolated polypeptide comprising CTGF-D4.
In yet another aspect, the invention provides an isolated cardiac progenitor cell, cardiac stem cell, mesenchymal stem cell or progeny cell thereof expressing recombinant CTGF-D4. In certain embodiments, the isolated cardiac progenitor cell, cardiac stem cell, mesenchymal stem cell or progeny cell also expresses insulin and/or IGF-1.
In various embodiments of any of the aspects delineated herein, the cardiac cell or progenitor is contacted in vitro or in vivo. In various embodiments of any of the aspects delineated herein, the cardiac cell or progenitor is contacted with the composition or primed cell for 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, 90, 120, 150, 180, 210, 240 min, or 3, 4, 5, or 6 hrs. In various embodiments of any of the aspects delineated herein, the cardiac cell or progenitor is autologous or heterologous. In various embodiments of any of the aspects delineated herein, following contact of the cardiac cell or progenitor in vitro, the cell is administered to a subject.
In various embodiments of any of the aspects delineated herein, the composition or primed cell is administered to a subject directly to a site of cardiac tissue damage or cardiac disease or is administered systemically. In particular embodiments, the composition or primed cell is administered by intra-arterial infusion.
In various embodiments of any of the aspects delineated herein, the method reduces cell death or increases cardiac function. In particular embodiments, the method increases cardiac cell number or reduces cardiac cell death. In various embodiments of any of the aspects delineated herein, the method increases cardiac cell number by at least about 5% compared to a corresponding untreated control cardiac tissue or heart.
In various embodiments of any of the aspects delineated herein, the subject has a disease or disorder selected from the group consisting of myocardial infarction, congestive heart failure, stroke, and ischemia. In various embodiments of any of the aspects delineated herein, the method prevents or ameliorates ischemic damage. In various embodiments of any of the aspects delineated herein, the method reduces apoptosis or increases cell proliferation. In various embodiments of any of the aspects delineated herein, the method prevents or ameliorates ischemic damage in a cardiac tissue post-myocardial infarction.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
By “increasing epicardial cell proliferation” is meant increasing cell division of an epicardial progenitor cell or a cell derived from an epicardial progenitor cell in vivo or in vitro. Increasing epicardial cell proliferation may also include promoting, supporting, or inducing the differentiation and/or migration of epicardial cells. For example, an increase in cell number may be at least about a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% increase in the number of epicardial cells relative to the number of cells present in a naturally-occurring, corresponding cardiac tissue or heart.
By “cardiac protective activity” is meant any biological activity that maintains or increases the survival or function of a cardiac cell or cardiac tissue in vitro or in vivo.
By “cardiac function” is meant the biological function of cardiac tissue or heart (e.g., contractile function). Methods for measuring the biological function of the heart are standard in the art (e.g., Textbook of Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co., 2000) and are also described herein. By “increasing in cardiac function” is meant an increase in a biological function of the heart by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to the biological function present in a naturally-occurring, corresponding cardiac tissue or heart.
By “cell survival” is meant cell viability.
By “reducing cell death” is meant reducing the propensity or probability that a cell will die. Cell death can be apoptotic, necrotic, or by any other means.
By “cellular factor” is meant any biological agent produced by a cell. While cellular factors isolated from culture media are typically secreted by cells in culture, the scope of the invention is intended to include any factor released from a cultured cell into growth media. In one embodiment, a cellular factor of the invention is secreted by a cell or is released into culture media when a cell breaks open and releases its contents into the growth media. Exemplary cellular factors include connective tissue growth factor (CTGF), human C-terminal CTGF peptide, insulin and IGF-1.
By “secreted cellular factor” is meant any biologically active agent that a cell secretes during in vitro culture.
By “connective tissue growth factor (CTGF) polypeptide” is meant a protein or fragment thereof having at least about 85% identity to NCBI Accession No. NP_001892, or that binds an antibody generated against the CTGF antigen. An exemplary full-length CTGF polypeptide is provided below.
By “CTGF nucleic acid molecule” is meant a polynucleotide encoding a CTGF polypeptide.
In one preferred embodiment, a CTGF polypeptide is CTGF module 4 (CTGF-D4), which is provided below.
In another preferred embodiment, a CTGF polypeptide is CTGF-D4 that includes a signal peptide (bold), which is provided below.
maaasmgpvr vafvvllalc srpavggpsc gpercpdepk kcirtpkisk pikfelsgct
An exemplary CTGF nucleic acid molecule encoding the above CTGF-D4 polypeptide and signal peptide sequence (bold) is provided below.
atggccgccg ccagtatggg ccccgtccgc gtcgccttcg tggtcctcct cgccctctgc
agccggccgg ccgtcggcca gaactgcagc gggccgtgcc ggtgcccgga cgagccaaaa
By “insulin polypeptide” is meant a protein or fragment thereof having at least about 85% identity to NCBI Accession No. NP_000198, or that binds an antibody generated against the insulin antigen. An exemplary full-length insulin polypeptide is provided below.
In one preferred embodiment, the insulin is a dimer of insulin A-chain and insulin B-chain of the full-length insulin polypeptide (A-chain: amino acids 90-110; B-chain: amino acids 25-54) linked by 2 disulfide bonds. Active or mature insulin is processed from the full-length insulin polypeptide, also termed preproinsulin. The insulin signal peptide (amino acids 1-24) is cleaved from preproinsulin to generate proinsulin. The proinsulin polypeptide folds in the endoplasmic reticulum, including the formation of intramolecular disulfide bonds. Cleavage of insulin C-chain (amino acids 57-87) from the folded proinsulin polypeptide produces a dimer of the A-chain and B-chain linked by 2 interchain disulfide bonds, which is the mature or active form. The A-chain of the active or mature dimer contains an intrachain disulfide bond. Active insulin is commercially available (Sigma-Aldrich I9278; CAS No. 11061-68-0; MDL No. MFCD00131380). Insulin A- and B-chain sequences are provided below.
By “insulin nucleic acid molecule” is meant a polynucleotide encoding an insulin polypeptide.
By “Insulin-like growth factor 1 (IGF-1) polypeptide” is meant a protein or fragment thereof having at least about 85% identity to NCBI Accession No. NP_000609, or that binds an antibody generated against the IGF-1 antigen. An exemplary full-length CTGF polypeptide is provided below.
By “IGF-1 nucleic acid molecule” is meant a polynucleotide encoding a IGF-1 polypeptide.
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.”
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
By “deficiency of a particular cell-type” is meant fewer of a specific set of cells than are normally present in a tissue or organ not having a deficiency. For example, a deficiency is a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% deficit in the number of cells of a particular cell-type (e.g., cardiomyocytes, epicardial progenitor cells, embryonic stem cells, endothelial cells, endothelial precursor cells, fibroblasts, neurons, adipocytes) relative to the number of cells present in a naturally-occurring, corresponding tissue or organ. Methods for assaying cell-number are standard in the art, and are described in (Bonifacino et al., Current Protocols in Cell Biology, Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif., 1999; Robinson et al., Current Protocols in Cytometry Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif., October 1997).
“Derived from” as used herein refers to the process of obtaining a cell from a subject, embryo, biological sample, or cell culture.
“Detect” refers to identifying the presence, absence or amount of the object to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include any disease or injury that results in a reduction in cell number or biological function, including ischemic injury, such as stroke, myocardial infarction, or any other ischemic event that causes tissue damage.
By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a ischemic injury varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “engraftment” is meant the integration of an exogenous cell into a tissue of a subject. In one embodiment, a primed cell (e.g., cardiac progenitor cell) is engrafted into the heart of a subject in need thereof (e.g., post-myocardiac infarction).
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
By “increase” is meant to alter positively by at least 5%. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. When a cellular factor is “isolated” from a cultured epicardial progenitor cell the cellular factor is typically separated from cells and cellular debris. It need not be purified to homogeneity. In fact, the composition comprising an isolated cellular factor typically comprises any number of cellular factors whose presence contributes to the biological activity (e.g., growth promoting, survival promoting, or proliferation promoting activity) of the composition. In one embodiment, a composition of the invention comprises or consists of conditioned media from which cells and cellular debris have been removed.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
By “reduce” is meant to alter negatively by at least 5%. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
By “reference” is meant a standard or control condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or there between.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “primed cell” is meant a cell that is contacted with any one or more of connective tissue growth factor (CTGF), human C-terminal CTGF peptide, insulin and/or IGF-1 prior to administration to a subject (e.g., for engraftment in the subject). In various embodiments, a primed cell is contacted with CdM. In various embodiments, a primed cell is contacted with any one or more of connective tissue growth factor (CTGF), human C-terminal CTGF peptide, insulin and/or IGF-1 for 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, 90, 120, 150, 180, 210, 240 min, or 3, 4, 5, or 6 hrs., prior to administration for engraftment in a subject
By “repair” is meant to ameliorate damage or disease in a tissue or organ.
By “tissue” is meant a collection of cells having a similar morphology and function.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
The invention features compositions comprising (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1; and methods of using such compositions to reduce cardiac tissue damage associated with an ischemic event or to enhance engraftment of a cell in a cardiac tissue.
The present invention is based, at least in part, on the discoveries that priming of cells for engraftment in p75MSC CdM or a defined combination of CTGF-D4, insulin, and/or IGF-1 provides a useful way to boost graft success for clinical application of CSCs or cultured stem/progenitor cells derived from other tissues or sources. Accordingly, the invention provides therapeutic and prophylactic compositions comprising agents secreted by mesenchymal stem cells (e.g., CTGF, insulin, and IGF-1) and methods of using such compositions to reduce cardiac cell death, preserve cardiac function after an ischemic event, and to generally prevent cardiac damage and promote cardiac healing or regeneration.
Mesenchymal Stem Cells
Paracrine activity from mesenchymal cells such as fibroblasts and other stromal cells promotes tissue repair after injury and also regulates, in part, stem cell niches. In the bone marrow, endothelial cells and stromal derivatives from non-hematopoietic progenitor cells (multipotent stromal cells, MSCs) support hematopoietic stem cells (HSCs) by providing critical structural and regulatory components of the hematopoietic niche. The niche components include cellular substrate, e.g. extracellular matrix, as well as multiple growth factors, cytokines, and hormones that influence HSC self-renewal, proliferation, survival, and function. Due to their supportive roles, feeder layers of stromal cells (e.g. MSCs or fibroblasts) are commonly used to support the culture of HSCs, other types of adult stem/progenitor cells, and ES cells.
Human bone marrow contains a subpopulation of MSC that can be isolated by magnetic-activated cell sorting against CD271 (p75 low-affinity nerve growth factor receptor, p75MSCs). Human p75MSCs secrete diverse growth factors and cytokines that promote cell survival, angiogenesis, and stem cell engraftment. In transplantation studies, co-infusion of human HSCs and p75MSCs into immunodeficient mice provided a 10-23 fold improvement in multi-lineage engraftment of bone marrow compared with co-infusion of HSCs and typical (non-selected) human MSCs. CD271+ cells characteristics of bone marrow p75MSCs are rapidly mobilized into the blood of patients with acute MI. Without being bound to a particular theory, it was hypothesized that circulating CD271+ cells participate in cardiac repair/remodeling after myocardial infarction, in part through paracrine activity. Experiments were performed to investigate the effects of stromal cell-derived ligands on cardiac stem/progenitor cells (CSCs/CPCs). It was found that conditioned medium (CdM) from human p75MSCs supported the proliferation and survival of adult rat CSCs/CPCs. Furthermore, priming of CSCs in p75MSC CdM for 30 min. prior to transplantation markedly improved CSC grafts after MI. By screening p75MSC CdM for molecules that protected CPCs under hypoxic conditions, two ligands with synergistic effects on CSC survival, CTGF and Insulin, were identified. Priming of CSCs with a defined combination of human CTGF C-terminal peptide (domain 4) and Insulin promoted graft success after MI. Short-term priming of human CSCs with p75MSC CdM or CTGF-D4 and Insulin/IGF-1 may improve graft success and cardiac regeneration in patients with myocardial infarction.
Insulin and Insulin Growth Factor 1 (IGF-1)
Priming in IGF-1 was shown to improve the survival of cardiac cell grafts with adult and embryonic stem cells. Cultured adult rat CPCs were protected by Insulin or IGF-1 during hypoxia. Insulin and Insulin-like growth factor 1 (IGF-1) bind to tyrosine kinase holoreceptors and promote cell survival and proliferation by signaling through the PI3K/Akt and Ras/MAP kinase pathways. Partial functional redundancy for Insulin and IGF-1 signaling is evidenced by signaling through receptor heterodimers IR/IGF-1R and bidirectional cross-talk between ligands/receptors. Although human p75MSC CdM did not contain detectable IGF-1, it had sufficient residual bovine Insulin to significantly improve CPC survival under hypoxic conditions. CdM contains Insulin because MSCs actively sample their environment and internalize fetal calf serum components such as albumin, IgG, and Insulin from their growth medium. Despite washing, they release some components back into the base medium used for CdM production.
Connective Tissue Growth Factor (CTGF)
CTGF is a secreted “matricellular” protein with multiple functions in mammalian development and tissue remodeling/repair after injury, including angiogenesis and fibrosis. During pancreatic development, CTGF promotes the proliferation of beta cell progenitors in islets. Cardiac expression of CTGF increases significantly after MI and it is expressed by interstitial fibroblasts and cardiac myocytes. By interacting with the extracellular matrix, integrins and several cell surface receptors (e.g. LRP-1, LRP-6, TrkA), CTGF mediates numerous cellular functions including: adhesion, proliferation, migration, differentiation, and survival. To regulate coincident processes after injury such as angiogenesis and fibrosis, CTGF physically associates with numerous other secreted proteins including VEGFA, Slit3, von Willebrand Factor, PDGF-B, BMP-4, IGF-1, IGF-2, TGF alpha and TGF beta.
CTGF controls fibrosis in multiple tissues after injury, in part, by interacting with TGF beta, IGF-1 or IGF-2 and promoting the differentiation of fibroblasts into myofibroblasts. The N-terminal (1st) and 2nd domains of CTGF interact with IGFs and TGF beta or BMP4, respectively. Notably, due to its numerous binding partners, the effects of CTGF are context-dependent. In the presence of cellular mitogens such as EGF, CTGF does not induce fibrosis, even when pro-fibrotic mediators like TGF beta or IGF-2 are present. C-terminal domain of CTGF (CTGF-D4) and Insulin were found to act in synergy to promote the proliferation and survival of cultured CPCs under hypoxic conditions. Furthermore, a defined combination of CTGF-D4 and Insulin promoted graft success with adult CSCs. Importantly, the CTGF-D4/Insulin priming method described herein is unlikely to promote myofibroblast differentiation or fibrosis from transplanted CSCs as CTGF-D4 is known to promote cell adhesion and proliferation, but lacks N-terminal functions in fibrosis.
Formulations
In one embodiment, a composition of the invention comprises or consists essentially of (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1. In another embodiment, a composition of the invention comprises a cell contacted with (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1.
The biologically active agents present in the conditioned media, the cells, or a combination thereof, can be conveniently provided to a subject as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Cells and agents of the invention may be provided as liquid or viscous formulations. For some applications, liquid formations are desirable because they are convenient to administer, especially by injection. Where prolonged contact with a tissue is desired, a viscous composition may be preferred. Such compositions are formulated within the appropriate viscosity range. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
Sterile injectable solutions are prepared by compositions comprising a secreted cellular factor isolated from cultures of stromal or epicardial progenitor cells in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the cells or agents present in their conditioned media.
The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent, such as methylcellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form). Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert.
Compositions comprising (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1 or cells contacted with such agents are administered in an amount required to achieve a therapeutic or prophylactic effect. Such an amount will vary depending on the conditions. Typically, biologically active cellular factors present will be purified and subsequently concentrated so that the protein content of the composition is increased by at least about 5-fold, 10-fold or 20-fold over the amount or protein originally present in the media. In other embodiments, the protein content is increased by at least about 25-fold, 30-fold, 40-fold or even by 50-fold. Preferably, the composition comprises an effective amount of (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1.
The precise determination of what would be considered an effective dose is based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
Optionally, the methods of the invention provide for the administration of a composition of the invention to a suitable animal model to identify the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit tissue repair, reduce cell death, or induce another desirable biological response. Such determinations do not require undue experimentation, but are routine and can be ascertained without undue experimentation.
Methods of Delivery
Compositions comprising (a) one or more of connective tissue growth factor (CTGF) and human C-terminal CTGF peptide; and (b) one or more of insulin and IGF-1 or cells contacted with such agents may be delivered to a subject in need thereof. Modes of administration include intramuscular, intra-cardiac, oral, rectal, topical, intraocular, buccal, intravaginal, intracisternal, intra-arterial, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, e.g., fibers such as collagen, osmotic pumps, or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal, intragonadal or infusion.
The compositions can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). Compositions of the invention can be introduced by injection, catheter, or the like. Compositions of the invention include pharmaceutical compositions comprising cellular factors of the invention and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous.
If desired, biologically active agents present in conditioned media are incorporated into a polymer scaffold to promote tissue repair, cell survival, proliferation in a tissue in need thereof. Polymer scaffolds can comprise, for example, a porous, non-woven array of fibers. The polymer scaffold can be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to a cell of the invention. Polymer scaffolds can comprise a fibrillar structure. The fibers can be round, scalloped, flattened, star-shaped, solitary or entwined with other fibers. Branching fibers can be used, increasing surface area proportionately to volume.
Unless otherwise specified, the term “polymer” includes polymers and monomers that can be polymerized or adhered to form an integral unit. The polymer can be non-biodegradable or biodegradable, typically via hydrolysis or enzymatic cleavage. The term “biodegradable” refers to materials that are bioresorbable and/or degrade and/or break down by mechanical degradation upon interaction with a physiological environment into components that can be metabolized or excreted, over a period of time from minutes to three years, preferably less than one year, while maintaining the requisite structural integrity. As used in reference to polymers, the term “degrade” refers to cleavage of the polymer chain, such that the molecular weight stays approximately constant at the oligomer level and particles of polymer remain following degradation.
Materials suitable for polymer scaffold fabrication include polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulphonated polyolefins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, and shape memory materials, such as poly(styrene-block-butadiene), polynorbornene, hydrogels, metallic alloys, and oligo(ε-caprolactone)diol as switching segment/oligo(p-dioxyanone)diol as physical crosslink. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989).
Methods for Evaluating Therapeutic Efficacy
In one approach, the efficacy of the treatment is evaluated by measuring, for example, the biological function of the treated organ (e.g., cardiac cell function). Such methods are standard in the art and are described, for example, in the Textbook of Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co., 2000). In particular, a method of the present invention, increases the biological function of a tissue or organ by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or even by as much as 300%, 400%, or 500%. Preferably, the tissue is cardiac tissue and, preferably, the organ is heart.
In another approach, the therapeutic efficacy of the methods of the invention is assayed by measuring an increase in cell number in the treated or transplanted tissue or organ as compared to a corresponding control tissue or organ (e.g., a tissue or organ that did not receive treatment). Preferably, cell number in a tissue or organ is increased by at least 5%, 10%, 20%, 40%, 60%, 80%, 100%, 150%, or 200% relative to a corresponding tissue or organ. Methods for assaying cell proliferation are known to the skilled artisan and are described, for example, in Bonifacino et al., (Current Protocols in Cell Biology Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif.). For example, assays for cell proliferation may involve the measurement of DNA synthesis during cell replication. In one embodiment, DNA synthesis is detected using labeled DNA precursors, such as [3H]-Thymidine or 5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals) and then the incorporation of these precursors into genomic DNA during the S phase of the cell cycle (replication) is detected (Ruefli-Brasse et al., Science 302(5650):1581-4, 2003; Gu et al., Science 302 (5644):445-9, 2003).
In another approach, efficacy is measured by detecting an increase in the number of viable cells present in a tissue or organ relative to the number present in an untreated control tissue or organ, or the number present prior to treatment. Assays for measuring cell viability are known in the art, and are described, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol. 62, 338-43, 1984); Lundin et al., (Meth. Enzymol. 133, 27-42, 1986); Petty et al. (Comparison of J. Biolum. Chemilum. 10, 29-34, 0.1995); and Cree et al. (AntiCancer Drugs 6: 398-404, 1995). Cell viability can be assayed using a variety of methods, including MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop, Bioorg. & Med. Chem. Lett. 1: 611, 1991; Cory et al., Cancer Comm. 3, 207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays for cell viability are also available commercially. These assays include but are not limited to CELLTITER-GLO® Luminescent Cell Viability Assay (Promega), which uses luciferase technology to detect ATP and quantify the health or number of cells in culture, and the CellTiter-Glo® Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH) cytotoxicity assay (Promega).
Alternatively, or in addition, therapeutic efficacy is assessed by measuring a reduction in apoptosis. Apoptotic cells are characterized by characteristic morphological changes, including chromatin condensation, cell shrinkage and membrane blebbing, which can be clearly observed using light microscopy. The biochemical features of apoptosis include DNA fragmentation, protein cleavage at specific locations, increased mitochondrial membrane permeability, and the appearance of phosphatidylserine on the cell membrane surface. Assays for apoptosis are known in the art. Exemplary assays include TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays, caspase activity (specifically caspase-3) assays, and assays for fas-ligand and annexin V. Commercially available products for detecting apoptosis include, for example, Apo-ONE® Homogeneous Caspase-3/7 Assay, FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, Calif.), the ApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, Calif.), and the Quick Apoptotic DNA Ladder Detection Kit (BIOVISION, Mountain View, Calif.).
Methods for Evaluating Cardiac Function
Compositions of the invention may be used to enhance cardiac function in a subject having reduced cardiac function. Methods for measuring the biological function of the heart (e.g., contractile function) are standard in the art and are described, for example, in the Textbook of Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co., 2000). In the invention, cardiac function is increased by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to the cardiac function present in a naturally-occurring, corresponding tissue or organ. Most advantageously, cardiac function is enhanced or damage is reversed, such that the function is substantially normal (e.g., 85%, 90%, 95%, or 100% of the cardiac function of a healthy control subject). Reduced cardiac function may result from conditions such as cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy (e.g., hypertrophic cardiomyopathy originating from a genetic or a secondary cause), post ischemic and post-infarction cardiac remodeling and cardiac failure.
Any number of standard methods are available for assaying cardiovascular function. Preferably, cardiovascular function in a subject (e.g., a human) is assessed using non-invasive means, such as measuring net cardiac ejection (ejection fraction, fractional shortening, and ventricular end-systolic volume) by an imaging method such echocardiography, nuclear or radiocontrast ventriculography, or magnetic resonance imaging, and systolic tissue velocity as measured by tissue Doppler imaging. Systolic contractility can also be measured non-invasively using blood pressure measurements combined with assessment of heart outflow (to assess power), or with volumes (to assess peak muscle stiffening). Measures of cardiovascular diastolic function include ventricular compliance, which is typically measured by the simultaneous measurement of pressure and volume, early diastolic left ventricular filling rate and relaxation rate (can be assessed from echoDoppler measurements). Other measures of cardiac function include myocardial contractility, resting stroke volume, resting heart rate, resting cardiac index (cardiac output per unit of time [L/minute], measured while seated and divided by body surface area [m2])) total aerobic capacity, cardiovascular performance during exercise, peak exercise capacity, peak oxygen (O2) consumption, or by any other method known in the art or described herein. Measures of vascular function include determination of total ventricular afterload, which depends on a number of factors, including peripheral vascular resistance, aortic impedance, arterial compliance, wave reflections, and aortic pulse wave velocity, Methods for assaying cardiovascular function include any one or more of the following: Doppler echocardiography, 2-dimensional echo-Doppler imaging, pulse-wave Doppler, continuous wave Doppler, oscillometric arm cuff, tissue Doppler imaging, cardiac catheterization, magnetic resonance imaging, positron emission tomography, chest X-ray, X ray contrast ventriculography, nuclear imaging ventriculography, computed tomography imaging, rapid spiral computerized tomographic imaging, 3-D echocardiography, invasive cardiac pressures, invasive cardiac flows, invasive cardiac cardiac pressure-volume loops (conductance catheter), non-invasive cardiac pressure-volume loops.
Kits
Compositions comprising a cell of the invention (e.g., a cardiac or mesenchymal stem/progenitor cell that expresses or is primed with CTGF, insulin, or IGF-1) or a composition comprising biologically active agents (e.g., CTGF, insulin, or IGF-1) is supplied along with additional reagents in a kit. The kits can include instructions for the treatment regime, reagents, equipment (test tubes, reaction vessels, needles, syringes, etc.) and standards for calibrating or conducting the treatment. The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert. Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if whether a consistent result is achieved.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Conditioned medium (CdM) was collected from human mesenchymal stem cells (MSCs), p75MSCs, and dermal fibroblasts. CdM from each of the cell types supported CPC proliferation (
STAT3 activation is important for the self-renewal of ES cells, adult HSCs, and adult neural stem cells. Accordingly, levels of phosphorylated-STAT3 (p-STAT3) in CPCs exposed to CdMs from MSCs, p75MSCs, or fibroblasts were assayed. Levels of p-STAT3 were significantly higher in CPCs at 1 and 2 days after CdM treatment compared with SFM treatment (
To determine whether p-STAT3 and/or p-Akt mediated the effects of CdM on CPCs, CPCs were treated with pharmacological inhibitors. AG490, a Jak2/STAT3 pathway inhibitor, reduced the number of CPCs treated with MSC CdM in a dose-dependent manner: control (CdM+DMSO), 100±1.5%; 1 μM, 96.3±0.9%; 5 μM, 89.5±0.9%; 10 μM, 43.4±2.4% (cell number ratio to control cell number [121,863 cells], mean±SEM, n=3 to 6). The inhibitory effect of AG490 (10 μM) was also observed for CPCs treated with CdM from p75MSCs or fibroblasts (
Insulin and Insulin-like growth factor 1 (IGF-1) bind to tyrosine kinase holoreceptors and promote cell survival and proliferation by signaling through the PI3K/Akt and Ras/MAP kinase pathways. Partial functional redundancy for Insulin and IGF-1 signaling is evidenced by signaling through receptor heterodimers IR/IGF-1R and bidirectional cross-talk between ligands/receptors. CSCs/CPCs did not proliferate in the absence of medium supplements containing Insulin (e.g. Insulin-Transferrin-Selenium [ITS] or fetal calf serum), even when in the presence of other mitogenic components from CSC/CPC growth medium such as LIF, EGF or bFGF (
Because cells transplanted to the heart after myocardial infarction (MI) may encounter hypoxic environments, it was examined whether CdM could protect CPCs during exposure to 1% oxygen for 48 hrs ex vivo. CdM from MSCs and p75MSCs significantly promoted the survival of CPCs compared with SFM (
Control CPCs cultured in CPC growth medium were negative for α-smooth muscle actin and von Willebrand Factor staining, whereas about 60% expressed α-sarcomeric actin (
Based on the results with CdM and cultured CPCs, it was examined whether infusion of CdM after MI would increase the number of endogenous CPCs to improve cardiac repair and function. MI was produced in C57bl6/J mice by permanent ligation of the left anterior descending coronary artery (LAD). The following day, mice were randomized to receive treatment with intra-arterial infusion (left ventricle lumen) of 30×p75MSC CdM or vehicle (SFM). Mice from each group were euthanized at 1 day or 1 week after treatment. Hearts obtained 1 day after treatment were sectioned and processed for TUNEL and immunohistochemistry to detect c-Kit. TUNEL assays showed that CdM infusion significantly reduced cardiac apoptosis/necrosis (SFM, 17.2±8.1%; CdM, 3.7±2.2%; P<0.05,
Based on the observations with p75MSC CdM and its ability to increase proliferation and survival of cultured CSCs/CPCs, it was determined whether prior exposure of CSCs to p75MSC CdM could foster the grafting of CSCs to the injured heart. MI was produced in Fischer rats by permanent ligation of the LAD. One day after MI, syngeneic GFP-positive rat CSCs were primed for 30 min. on ice in p75MSC CdM (30×CdM, n=6 rats) or vehicle (SFM, n=5 rats). The chest wall was re-opened and rats were randomized to treatment with co-injections of CSCs/CdM or CSCs/SFM (125,000 cells/5 μl injection, 2 sub-epicardial injections, 1 per border zone). At 1 week after MI, rats were euthanized and their hearts were processed as frozen serial-sections from apex to base. For each heart, GFP+ cells were quantified in the tissue section with the most GFP+ cells. For 2 rats in CSCs/SFM co-injection group, GFP+ cells were not detected. Furthermore, hearts from CSC/SFM rats with the highest level of engraftment contained less than 40 GFP+ cells/section (
To identify factors in p75MSC CdM that promoted the proliferation and survival of CPCs, Affymetrix gene expression profiles were examined from human p75MSCs that were freshly sorted from marrow aspirates and from p75MSCs cultured adherently for 2 passages (Bakondi et al., Mol Ther 2009; 17(11):1938-1947). Antibody blocking/neutralization studies for selected secreted factors were carried out with 10×p75MSC CdM and CPCs exposed to hypoxic conditions (1% oxygen, 48 hrs). Under hypoxia, neutralizing antisera specific to human CTGF prevented CdM from protecting CPCs (P<0.001,
CTGF (CCN2, IGFBP8) consists of 4 domains and the C-terminal (4th) domain alone was reported to increase cell adhesion and proliferation (Steffen et al., Growth Factors. 1998; 15(3):199-213; Gao et al. J Biol Chem. 2004; 279(10): 8848-8855). In experiments with recombinant peptides, combined treatment with human C-terminal CTGF peptide (CTGF-D4) and Insulin (i.e., alpha chain-beta chain dimer) had synergistic effects on CPC survival and proliferation under hypoxic conditions. For example, CTGF-D4 or Insulin alone (1 ng/ml in SFM, each) did not protect CPCs against 48 hr of hypoxia (
Having observed synergistic protective effects after CTGF-D4/Insulin treatment of cultured CPCs exposed to simulated ischemia (
While examining different signaling pathways that might be stimulated by p75-CdM and/or CTGF-D4, it was found that epicardial-derived cells (EPDC) expressed the Wnt co-receptor Lrp5/6 and that both p75-CdM and CTGF-D4 promoted signaling through the canonical Wnt pathway, as evidenced by phosphorylation of GSK3β and accumulation of nuclear β-catenin (
Furthermore, incubation of EPDCs in Dkk-1, a secreted inhibitor of Lrp6, significantly reduced the protection normally conferred by CTGF-D4 when EPDCs were incubated under hypoxic conditions (1% oxygen, 48 hr)(p<0.05,
It was also investigated whether signaling through CTGF-D4/Lrp6 played a role in graft success after MI. A two-color competitive grafting assay for EPDCs was developed to be able to evaluate inhibition effects (e.g. primed with CTGF-D4 and anti-Lrp6) on a separate aliquot of cells from the same isolate that was used for control (e.g. primed with CTGF-D4/Insulin and non-specific IgG). Importantly, labeling of EPDCs with cell-tracking dye did not affect their ability to graft and migrate into the heart after MI (see distance traveled from subepicardial injection site at 1 week in
These results show that the Wnt pathway is involved in the effects of p75-CdM and CTGF-D4 on epicardial-derived cells.
In ex vivo adhesion assays with EPDCs, CTGF-D4 (40 ng/ml) promoted adhesion of EPDCs to fibronectin (
To examine EPDC CdM-mediated vascular protection, cardiac endothelial cell protection assays were performed under conditions of simulated ischemia: low glucose medium and 1% oxygen for 24 or 48 hrs. Compared with incubation in MEM (vehicle control), unconcentrated CdM (1×CdM) generated from EPDC protected cardiac endothelial cells from hypoxic/ischemic injury for 24 hr in this assay. CdM concentrated 10-fold (10×CdM) provided a greater level of cell protection than did 1×CdM, protecting coronary artery endothelial cells, microvascular endothelial cells and aortic endothelial cells for 48 hr.
To determine whether EPDC CdM could provide vascular protection in vivo, adult male Fischer rats underwent 2 hr of ischemia followed by 24 hr of reperfusion. They were then treated with either MEM (vehicle control, MEM) or 30×EPDC CdM at the time of reperfusion. Two hours prior to harvesting the heart, each rat was injected with 0.5 ml of FITC-albumin (5 mg/ml) through the tail vein. Hearts were then harvested and homogenized. Twenty-four hours after ischemia/reperfusion we quantified the amount of FITC extravasation in each treated animal by normalizing it to extravasation in sham-operated rats that were also injected with FITC-albumin. Heart homogenates from EPDC CdM-treated rats contained 37.8% greater FITC fluorescence that those from rats that received vehicle. The amount of extravagated FITC-albumin in the vehicle-treated group was 527.4%±109.33 of sham (n=5), while in the CdM-treated group it was 359.7724%±78.82 of sham (n=5)(
To understand the effects of EPDC CdM on blood vessels, immunoblotting was performed on the soluble fraction from left ventricle homogenates. VE-Cadherin plays a key role in maintaining endothelial barrier integrity, and the level of phosphorylated VE-Cadherin (pVE-Cadherin) is a marker for increased vascular permeability. At 1 day after MI, reperfusion, and treatment, the level of pVE-Cadherin was significantly higher in the MEM group of animals than in the EPDC CdM group (p≤0.05, n=3;
Materials and Methods
Preparation and Isolation of Human Stromal Progenitor Cells and Fibroblasts.
Human MSCs, p75MSCs and dermal fibroblasts were prepared with protocols approved by an Institutional Review Board (Bakondi et al., Mol Ther 2009; 17(11):1938-1947). To obtain MSCs, bone marrow aspirates were taken from the iliac crest of healthy adult donors. Mononuclear cells were isolated with the use of density gradient centrifugation (Ficoll-Paque, Amersham Pharmacia Biotech) and resuspended in complete culture medium consisting of Alpha-MEM (GIBCO/BRL, Grand Island, N.Y.); 17% FBS (Atlanta Biologicals, Norcross, Ga.); 100 units/ml penicillin (GIBCO/BRL); 100 μg/ml streptomycin (GIBCO/BRL); and 2 mM L-glutamine (GIBCO/BRL). Cells were plated in 20 ml of medium in a 150 cm2 culture dish and incubated in a humidified incubator (Thermo Electron, Forma Series II, Waltham, Mass.) with 95% air and 5% CO2 at 37° C. After 24 hr, nonadherent cells were removed. Adherent cells were washed twice with PBS and incubated with fresh medium. The primary adherent cells were cultured and propagated.
To obtain p75MSCs, bone marrow stem/progenitor cells were isolated by MACS using antibodies against the p75LNGFR. Freshly isolated bone marrow mononuclear cells from the Ficoll gradient were resuspended in 0.4 ml of PBS containing 0.5% bovine serum albumin and 2 mM EDTA. After adding mouse anti-human p75LNGFR antibody conjugated to magnetic beads (CD271, Miltenyi Biotech, Auburn, Calif.), the sample was incubated for 30 min. at 4° C., and then applied to a magnetic column (LS Column; Miltenyi Biotech). The bound fraction was eluted with 5 ml of MACS buffer and the cells were concentrated by centrifugation at 1000×g for 8 min. After re-suspension, the entire isolate was cultured in complete culture medium. MSC-like cells appeared as small colonies after about 1 week, and the cells were expanded.
Isolation and Culture of Adult Rat Cardiac Stem/Progenitor Cells.
Adult CSCs were isolated from the ventricles of Fischer 344 rats and labeled with retroviral vector for GFP (Bakondi et al., Mol Ther 2009; 17(11):1938-1947). CSCs were cultured as floating spheres in DMEM/F12 supplemented with bFGF (10 ng/ml), EGF (20 ng/ml), LIF (10 ng/ml) and ITS. To grow adherent CPCs, CSCs were plated at 500 cells/cm2 and cultured in CSC medium supplemented with 2% FBS (CPC growth medium).
Isolation and Culture of Adult Rat Cardiac Fibroblasts.
Ventricular fibroblasts were prepared with protocols approved by an Institutional Animal Care and Use Committee. Ventricular fibroblasts were isolated from hearts of adult Sprague-Dawley rats. The hearts were minced and enzymatically-dissociated into single cell suspension. Non-myocytes were separated by discontinuous density gradient centrifugation and cultured in DMEM/F-12 supplemented with 10% FCS. Passage 2 cells were used for experiments.
Preparation of Serum Free Conditioned Medium (CdM).
Passage 4 to 8 human MSCs, p75MSCs or dermal fibroblasts were cultured to 80 to 90% confluence in 150 cm2 dishes with complete culture medium (Bakondi et al., Mol Ther 2009; 17(11):1938-1947). To generate CdM, cells were washed twice with PBS and incubated with 20 mls of fresh serum-free α-MEM in standard conditions without any supplements or growth factors for 48 hrs. Medium was then collected, filtered, and stored at −80° C. For some experiments, CdM was concentrated up to 10- or 30-fold with the use of a Labscale™ TFF diafiltration system (Pellicon XL 5 kDa cut-off filters, Millipore, Bedford, Mass.).
Short-Term Priming of CSCs.
CSCs were cultured as spheres in serum-free CSC growth medium. CSC spheres were trypsinized and centrifuged at 1000×g for 8 min. After re-suspension in 1×PBS, cells were passed through a 40 micron filter to isolate single CSCs (cell strainer, Fisher Scientific). Cells were counted on a hemocytometer, centrifuged again, and re-suspended in 30×p75MSC CdM or Alpha-MEM (CdM vehicle control), or CTGF-D4 (3 ng/ml)/Insulin (30 ng/ml)/1% BSA, or 1% BSA in Alpha-MEM (vehicle control for recombinant peptides). CSCs were incubated in the above conditions for 30 minutes on ice prior to sub-epicardial injection.
Myocardial Infarction Surgery and CSC Transplantation in Rats.
Fischer 488 rats (males, 7 weeks of age) were weighed, shaved, anesthetized under 4% isoflurane, and endotracheally-intubated. Rats were ventilated at a respiration rate of 65 beats per min under a peak inspiration pressure of 15 cm H2O (Kent Scientific). Body temperature was maintained at 37° C. with a heating pad (Gaymar). Through a dermal incision, a blunt dissection of the fascia was performed and the intercostal muscles were separated. The heart was exposed by retraction of the pericardium to expose the LAD. The LAD was occluded with a 6-0 nylon suture and occlusion was confirmed by blanching of the anterior free wall of the LV. The animals were allowed to recover off the ventilator.
After 24 hours, rats were re-intubated, ventilated, and the chest wall was re-opened. Hearts were exposed to reveal the border zones of the infarct. For each rat, we performed 2 sub-epicardial injections of CSCs (5 ul each, one per border zone) with a 30 gauge Hamilton syringe. The needle was introduced tangentially to the wall of the LV and with the bevel facing upward. The syringe was advanced only as far as the bevel edge to access the sub-epicardial surface of the heart and so as not target the underlying myocardium of the LV. After the injections, the chest wall was closed and rats recovered for 7 days prior to euthanization.
Cell Culture in CdM and Evaluation of Cell Number.
Adult rat CPCs and cardiac fibroblasts were plated at 500 cells/cm2 and cultured in their respective growth mediums. Three days after plating, the medium was removed, the wells were washed twice with PBS, and the cells were then exposed to CdM or to fresh serum-free medium (Alpha-MEM). For time course proliferation studies, CdM and serum-free medium were changed every 2 days. In signal transduction inhibitor studies, the following pharmacological inhibitors were used: AG490, inhibitor of Jak2/STAT3 pathway; Stattic, inhibitor of STAT3; LY294002, inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt pathway; and PD98059, inhibitor of extracellular signal-regulated kinase (ERK). All of the inhibitors were purchased from Calbiochem (Darmstadt, Germany) and were dissolved in dimethyl sulfoxide (DMSO). CPCs were cultured in CdM with the inhibitors or with the equivalent volume of DMSO as a control for 48 hrs. In cell protection studies, 3 days after plating, medium was replaced with either the CdM or serum-free medium and the cells were exposed to hypoxia in a specialized incubator (1% oxygen) for 48 hrs. The hypoxia incubator was a model that measured both CO2 and O2 (Thermo Electron, Forma Series II, model 3130). Oxygen was maintained at 1% by the injection of nitrogen gas and was monitored continuously.
Cell numbers were quantified by the fluorescent labeling of nucleic acids (CyQuant dye; Molecular Probes, Carlsbad, Calif.) and with a microplate fluorescence reader (FLX800; Bio-Tek Instruments Inc., Winooski, Vt.) set to 480 nm excitation and 520 nm emission. Each experiment was repeated a minimum of 3 times.
Immunocytochemistry.
CPCs were fixed with 4% paraformaldehyde in 1×PBS. Non-specific binding was limited by 1 hour incubation in PBS containing 5% goat serum and 0.4% triton X-100. Primary antibodies were incubated overnight at 4° C. After washing 3×5 min with PBS, secondary antibody that was diluted 1:1000 (Alexa 594, Molecular Probes) was applied for 1 hr at room temperature (RT). After 3×5 min washes, slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, Calif.). Epifluorescence images were taken using a Leica DM6000B microscope equipped with a CCD camera (Leica DFC350Fx) and FW4000 software. The primary antibodies for immunocytochemistry were as follows: phospho-STAT3 (Tyr705, 1:50, Cell signaling, Danvers, Mass.); α-sarcomeric actin (1:500, Sigma); α-smooth muscle actin (1:800, Sigma); and von Willebrand factor (1:100, Chemicon, Temecula, Calif.). For quantification of differentiation, cells positive for α-sarcomeric actin, α-smooth muscle actin and von Willebrand factor and total cells were counted at least in three fields per slide. The percentage of positive cells was calculated for each slide (n=3 in each group).
DNA Replication Assay.
Three days after the plating, CSCs were cultured in growth medium, CdM or serum-free medium for 24 hr, and BrdU (BD Biosciences) was added at a final concentration of 10 μM. Immunocytochemistry was carried out with BrdU antibody (Sigma) and BrdU-positive cell numbers were quantified as described herein above.
Immunoblotting.
Cells were lysed in a buffer that consisted of 0.1% sodium dodecyl sulphate (SDS) and complete protease inhibitor cocktail (Roche, Basel, Switzerland) in PBS. Protein concentration was determined by the DC protein assay (Biorad, Hercules, Calif.). Twenty of protein was separated by SDS-PAGE. After electrophoresis, the gels were electroblotted to polyvinylidene difluoride (PVDF) membranes. All electrophoresis and electroblotting used Novex reagents and systems (Invitrogen, Carlsbad, Calif.). The blots were blocked for 1 hr at RT in 5% nonfat dry milk in PBS with 0.1% Tween 20 (PBST), washed 3×5 min in PBST, and incubated in primary antibodies in PBST with 5% BSA overnight at 4° C. After 3×5 min washes in PBST, the blots were incubated in secondary antibody conjugated to horseradish peroxidase conjugate (1:2000, Sigma) in PBST for 1 hr at RT. Unbound secondary antibody was removed and positive bands were detected with a chemiluminescent reaction. The primary antibodies for immunoblotting were Ki67 (clone SP6, 1:200, Abcam, Cambridge, Mass.); p-STAT3 (Tyr705, 1:1000), total STAT3 (1:1000), p-Akt (Ser 473, 1:1000)(Cell signaling); and β-actin (1:5000, Sigma).
ELISAs for IGF-1, Insulin, and CTGF.
For assay of human IGF-1, 1×p75 CdM was assayed by ELIAS using commercial ELISA reagents according to manufacturer's protocol (R and D Systems). For assay of Insulin and CTGF, high protein-binding plates were incubated with 1 or 10×p75 CdM overnight at room temperature to capture antigens from CdM. Wells were then washed with mild detergent (0.05% Tween-20 in PBS) followed by blocking with 1% BSA in PBS for 1 hour. After blocking buffer was thoroughly washed off from the wells, samples were incubated with 100 μl of biotin-conjugated polyclonal antibody to CTGF at 5 μg/100 μl (Peprotech) for 2 hours at room temperature. Polyclonal mouse anti-Insulin antibody (Santa Cruz) was incubated for 2 hours at room temperature, followed by 3 washes with wash buffer. The wells were then incubated in anti-mouse biotin conjugated IgG (Sigma Aldrich) for 2 hours at room temperature. After washing in wash buffer 3 times, samples for CTGF and Insulin ELISA were incubated in Streptavidin conjugated HRP (1:2000) for 2 hrs at room temperature, followed by washing and addition of 100 μl of substrate ABTS (Thermo Scientific; #37615) for 20 minutes. Absorbance was measured (450 nm) on a Synergy HT plate reader.
Myocardial Infarction Surgery in Mice.
Male mice at 8-10 weeks of age underwent permanent ligation of the Left Anterior Descending Coronary Artery (LAD) to induce myocardial infarction (C57bl6 mice, Taconic, Hudson, N.Y.). Mice were not included in the study if they did not survive the initial MI surgery, did not achieve a successful MI (blanching observed at time of treatment), or died during treatment application. Following all procedures, mice were given analgesia (buprenorphine, 0.05-0.1 mg/kg i.p.) and monitored for signs of distress until termination of the study. All procedures were done in accordance with protocols approved by an Institutional Animal Care and Use Committee.
For permanent LAD ligation surgery, mice were anesthetized with 2-4% Isoflurane, shaved, weighed, intubated, and then maintained for the duration of the procedure on a sterile surgical field with the use of a mechanical ventilation system (MiniVent, Harvard Apparatus, Holliston, Mass.). Throughout the surgery and during the recovery period, body temperatures were maintained with a heated water pad system (Gaymar T-Pump TP-500, Gaymar Industries, Orchard Park, N.Y.). Viewing the chest through a dissecting microscope (Stemi 2000-C, Carl Zeiss MicroImaging, Thornwood, N.Y.) a dermal incision was made, the underlying fascia were removed, and the thoracic musculature was retracted to expose the left ribcage. Next the intercostal muscles were retracted and the outer (parietal or visceral) pericardium was removed to expose the LAD. The LAD was then ligated (2.0-3.0 mm from left atrial apex) with 8.0 nylon suture (Henry Schein, Melville, N.Y.) and blanching within the myocardium of the left ventricle was noted. The intercostals were rejoined with a 6.0 nylon suture (Henry Schein), the lungs were reinflated, and overlying dermis rejoined with a 6.0 nylon suture. All mice were recovered to an ambulatory state prior to any subsequent treatment procedure. Survival after the MI surgery was >90%. Sham-operated mice underwent all procedures except that the suture was placed under the LAD but was not ligated.
Infusion of p75MSC CdM after MI in Mice.
To evaluate p75 CdM treatment in an unbiased manner, all animals were randomized to treatment (after LAD ligation). Following 24 hour recovery of an animal after the first surgery, the mouse was then again anesthetized, intubated, and the chest opened. Once the intact suture and area of blanching were confirmed, 30×p75 CdM (200 μL) or vehicle (Alpha-MEM, 200 μL) warmed to 37° C. was delivered to the entire cardiovascular arterial tree by injecting the solution into the lumen of the left ventricle (LV). Injections were performed slowly (over a period of 1 minute) with a 30.5 gauge needle inserted below the great cardiac vein (LV apex) at an angle 45° to the myocardium. Following treatment with either CdM or vehicle, the needle was removed and the intercostals were rejoined using 6.0 chromic gut suture (Ethicon, Johnson and Johnson, Inc., Livingston, UK), lungs then reinflated, and overlying dermis rejoined with 6.0 nylon suture. All mice were then recovered to an ambulatory state and transferred to the vivarium for the remaining duration of the experiment.
Echocardiography.
Two dimensional, Doppler, and M-Mode echocardiography was performed with a Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, ON, Canada). Data were recorded from sham-operated mice, vehicle-treated mice with MI, and CdM-treated mice with MI while under isoflurane anesthesia. All left ventricular dimensions in systole and diastole were measured from M-mode images obtained at the mid-papillary muscle level. Echocardiographic data were coded for unbiased measurements and determination of ECHO wall motion scores. ECHO scores were determined from functional assessment of 13 segments with a model based on the American Society of Echocardiography 17 segment model. Systolic wall motion scores were assigned to 4 quadrants each of 3 short axis segments taken at apical, mid papillary, and basal levels, with an additional segment at the apex (13 total). Scores were assigned as: 1=normal, >25% motion; 2=hypokinetic, 10-25% motion; 3=akinetic, <10% motion. Pulmonary arterial Doppler flow velocities and volumes were quantified (Baumann et al. Echocardiography 2008; 25:739-748).
Following ECHO, the mice were killed humanely by exsanguination and the hearts were removed and rinsed in PBS. For CK assays, left ventricular tissue was dissected away from the atria and the aorta, further separated into anterior LV and posterior LV/septum, and immediately snap frozen by submersion of cryovials in liquid N2. The LV tissues were maintained at −80° C. until the day of the CK assay (see below).
TUNEL Assay.
TUNEL was performed as reported previously (French et al. FASEB J 2009; 23:1177-1185). Quantification of TUNEL-positive cells within zones of infarction was performed in an unbiased fashion by a viewer that was blinded to slide (sample) identity. Cells were counted with Image Pro Plus Software as reported previously (French et al. FASEB J 2009; 23:1177-1185).
Creatine Kinase Assay.
The remaining creatine kinase (CK) activity in left ventricular tissues was assessed to determine the extent of infarction in mouse hearts as reported previously (Kjekshus et al. Circ Res 1970; 27:403-414; Zaman et al. Exp Biol Med. 2011; 236:598-603). The loss of CK activity directly reflects the loss of viable myocardium after MI. The percentage of left ventricle with infarction was calculated based on observed total LV CK activity (IU/mg protein) in left ventricles of normal hearts without infarction. The percent of MI=100×[NL CK-LV CK]/Δ, where NL CK is the amount of CK in tissue from normal LV (IU/mg of soluble protein), LV CK is total remaining CK activity in the LV after MI (IU/mg soluble protein), and Δ is the difference between the amount of CK in normal zones of myocardium and in zones of myocardium with infarction.
Immunohistochemistry.
Rats were euthanized under isoflurane, their hearts harvested and washed in PBS to remove remnant blood. Hearts were fixed in 4% paraformaldehyde overnight and equilibrated in 15 and 30% sucrose consecutively for cryoprotection. After mounting in OCT (Tissue-Tek), serial sections were performed from apex to base at 20 microns (Leica CM1800 Cryostat) and sections were mounted on glass slides. Slides were dried at 37° C. and washed twice in 1×PBS. Primary antibodies were against Ki67 (clone SP6, 1:100; Abcam), CD31, (1:50, BD Biosciences), and smooth muscle alpha actin (1:500, Sigma). Primary antibodies were detected with secondary antibodies conjugated to Alexa 594 (1:2000). Slides were mounted in Vectashield with DAPI (Vector Labs). Sections were imaged by epifluorescence deconvolution microscopy (Leica DM6000B; Leica) with Leica FW4000 software.
statistical Analysis.
Comparisons of parameters among the three groups were made with one-way analysis of variance (ANOVA) followed by Scheffé's multiple comparison test. Comparisons of parameters between two groups were made by unpaired Student's t-test. P<0.05 was considered significant.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
This application may be related to U.S. patent application Ser. No. 13/220,555, which is a continuation-in-part application of International Patent Application No.: PCT/US2010/001540, filed May 26, 2010, which claims the benefit of U.S. Provisional Application Ser. No. 61/181,071, filed May 26, 2009, the disclosures of which are hereby incorporated herein in their entireties by reference.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application is a continuation of U.S. patent application Ser. No. 15/623,025, filed Jun. 14, 2017, which is now U.S. Pat. No. 10,675,329, issued on Jun. 9, 2020, which is a continuation of U.S. Ser. No. 14/771,747, filed on Aug. 31, 2015, which is now U.S. Pat. No. 9,707,271, issued on Jul. 18, 2017, which is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT international application Ser. No. PCT/US2014/022094, filed Mar. 7, 2014, designating the United States and published in English, which claims priority to and the benefit of U.S. Provisional Application No. 61/775,285, filed Mar. 8, 2013, which is incorporated herein by reference in its entirety.
This work was supported by the following grant from the National Institutes of Health, Grant No: HL085210. The government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6696238 | Murphy et al. | Feb 2004 | B2 |
| 9132155 | Spees | Sep 2015 | B2 |
| 9707271 | Spees | Jul 2017 | B2 |
| 10675329 | Spees | Jun 2020 | B2 |
| 20110275563 | Attramadal et al. | Nov 2011 | A1 |
| 20180140671 | Spees | May 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 2007066823 | Jun 2007 | WO |
| 2010138180 | Dec 2010 | WO |
| 2011057249 | May 2011 | WO |
| Entry |
|---|
| Ahmed et al., “Mechanisms of novel cardioprotective functions of CCN2/CTGF in myocardial ischemia-reperfusion injury,” American Journal of Physiology—Heart and Circulatory Physiology, Apr. 2011, vol. 200, No. 4, pp. H1291-H1302. |
| Bork, Peer, “Powers and Pitfalls in Sequence Analysis: The 70% Hurdle,” Genome Research, Apr. 2000, vol. 10, No. 4, pp. 398-400. |
| Brenner, Steven E., “Errors in genome annotation,” Trends in Genetics, Apr. 1999, vol. 15 No. 4, pp. 132-133. |
| Doerks et al., “Protein annotation: detective work for function prediction,” Trends in Genetics, Jun. 1998, vol. 14, No. 6, pp. 248-250. |
| Ellison et al., “Endogenous Cardiac Stem Cell Activation by Insulin-Like Growth Factor-1/Hepatocyte Growth Factor Intracoronary Injection Fosters Survival and Regeneration of the Infarcted Pig Heart,” Journal of the American College of Cardiology, Elsevier, New York, NY, US, May 24, 2011, vol. 58, No. 9, pp. 977-986. |
| Grotendorst et al., “Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation,” FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, May 7, 2005, vol. 19, No. 7, pp. 729-738. |
| Iso et al., “Priming with Ligands Secreted by Human Stromal Progenitor Cells Promotes Grafts of Cardiac Stem/Progenitor Cells After Myocardial Infarction,” Stem Cells, Mar. 2014, vol. 32, No. 3, pp. 674-683. |
| Kofidis et al., “Insulin-Like Growth Factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration,” Stem Cells, Dec. 2004, vol. 22, No. 7, pp. 1239-1245. |
| Liu et al., “Early stem cell engraftment predicts late cardiac functional recovery: preclinical insights from molecular imaging,” Circulation: Cardiovascular Imaging, Jul. 1, 2012, vol. 5, No. 4, pp. 481-490. |
| Liu et al., “Role of connective tissue growth factor (CTGF) module 4 in a regulating epithelial mesenchymal transition (EMT) in HK-2 cells,” Clinica Chimica Acta, Elsevier BV, Amsterdam, NL, Nov. 1, 2006, vol. 373, No. 1-2, pp. 144-150. |
| Martinov et al., “CCN2:CTGF as a Novel Stimulator of Proliferation and Survival of Cardiac Progenitor Cells/Stem Cells,” Circulation, Nov. 1, 2010, vol. 122, Supplement 21, pp. A19874. |
| Ngo et al., “The Protein Folding Problem and Tertiary Structure Prediction, Chapter 14: Computational Complexity Protein Structure Prediction, and the Levinthal Paradox,” 1994, pp. 433-440 and 492-495. |
| Skolnick et al., “From genes to protein structure and function: novel applications of computational approaches in the genomic era,” Trends in Biotechnology, Jan. 2000, vol. 18, No. 1, pp. 34-39. |
| Takeda et al., “Cellular interplay between cardiomyocytes and nonmyocytes in cardiac remodeling,” International Journal of Inflammation, 2011, vol. 2011, pp. 1-13. |
| Wells, James A., “Additivity of mutational effects in proteins,” Biochemistry, Sep. 1990, vol. 29, No. 37, pp. 8509-8517. |
| Communication pursuant to Article 94(3) EPC in corresponding European Patent Application No. 14760147.0, dated Feb. 20, 2018 (5 pages). |
| Communication pursuant to Article 94(3) EPC in corresponding European Patent Application No. 14760147.0, dated Apr. 26, 2019 (5 pages). |
| Extended European Search Report issued in European Patent Application No. 14760147.0, dated Jul. 29, 2016. |
| Notification of Transmittal of the International Search Report and the Written Opinion of the International Searching Authority of corresponding PCT/US2014/022094, dated Jun. 18, 2014 (11 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20200338163 A1 | Oct 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61775285 | Mar 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15623025 | Jun 2017 | US |
| Child | 16858154 | US | |
| Parent | 14771747 | US | |
| Child | 15623025 | US |
