Patent Application
20230000924
The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and lack of robust in vitro derivation methods. There is a need for methods to isolate cells with SAN characteristics, or to generate cells from stem cells that exhibit SAN characteristics so that effects of various therapeutics on SAN function can be investigated.
Previous efforts have focused on in vivo direct conversion or an NKX2.5 negative selection approach to obtain nodal like cells. However, due to lack of a purification method, the studies were limited in characterization and validation of nodal identity for the cells at cellular and molecular levels (Kapoor et al., Nat Biotechnol 31, 54-62 (2013); Protze et al., Nat Biotechnol 35, 56-68 (2017), and therefore use of such cells for drug discovery is limited.
The present disclosure provides compositions and methods for generating cells with sinoatrial node (SAN) characteristics from pluripotent cells, such as human pluripotent stem cells. The method is based on a strategy using a dual SHOX2:GFP; MYH6:mCherry knock-in reporter line to generate and purify human pluripotent stem cell-derived SAN cells (hPSC-SAN), displaying molecular and electrophysiological characteristics of bona-fide nodal cells.
The method was developed by using the steps comprising i) providing a population of pluripotent stem cells, such as human pluripotent cells, ii) introducing into the cells, a reporter for SHOX2 expression, iii) incubating the cells in a medium comprising retinoic acid, an EGFR/FGFR inhibitor, Wnt inhibitor, and optionally, a STAT3 inhibitor, iv) optionally, further incubating the cells in a medium further comprising HDAC inhibitor, and v) sorting for cells that are positive for the reporter for SHOX2. This process generates SAN-like cells. These cells (SAN-like cells) display SA node like biochemical and electrophysiological characteristics. In an embodiment, the method uses media further comprising Activin Receptor inhibitor. The cells may be used for implantation or for screening (such as in a high throughput screening) to identify agents that can affect the functioning of SAN cells.
In an embodiment, SAN-like cells may be generated by incubating PSCs (such as hPSCs) with a medium comprising a GSK inhibitor, and one or more members of the TGF-β superfamily of proteins (member of TGF-β SFP), for a sufficient period of time to generate pre-cardiac mesoderm cells. The pre-cardiac mesoderm cells are incubated in a medium comprising retinoic acid signaling activator, Wnt inhibitor, Activin receptor (ALK) inhibitor, member of TGF-β SFP and/or EGFR inhibitor, and optionally, STAT3 inhibitor, for a period of time sufficient to generate pacemaker progenitor cells. The pacemaker progenitor cells are then incubated in a medium comprising a GSK inhibitor, and EGFR inhibitor and optionally, a HDAC inhibitor for a sufficient period of time to generate SA-node like cells. In an embodiment, the pacemaker progenitor cells are first incubated for a period of time in a medium comprising GSK inhibitor and a EGFR inhibitor, and after about 3-7 days, the medium may additionally comprise a HDAC inhibitor. After this, the cells can then be continued in culture in a serum-free, growth factor fee medium and by day 30 (total time in culture) exhibit pacemaker markers and electrophysiological properties.
In an embodiment, the SAN-like cells generated by the present methods can be used to evaluate cell type specific toxicity upon treatment with therapeutics for various diseased conditions. For example, cancer treatments cause cardiovascular toxicities including arrhythmias and cardiomyopathy. Doxorubicin (DOXO) remains a key component of regimens to treat a wide spectrum of solid tumors and leukemia. DOXO-induced cardiomyopathy ranges from 10-20% and sub-clinical systolic dysfunction is estimated at 40% of treated patients. The SAN-like cells generated by the present methods can be used to evaluate therapeutic candidates for their cardiac toxicity effects, and also to evaluate candidate agents that could mitigate cardiac toxicity associated with therapeutics like anthracyclines, including doxorubicin.
Further, using this technique, we discovered 3 new genetic loci associated with increased sensitivity to DOXO-induced hPSC-SAN death. Genetic variants in these loci were associated with significantly higher early arrhythmia risk in patients receiving DOXO, confirmed by an unbiased PheWAS analysis. The in vitro DOXO assay enables an unbiased drug screening platform and identification of candidate therapeutic that can partially block DOXO-mediated cardiac toxicity.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Every numerical range given throughout this specification includes its upper and lower values, all values to the tenth of the lower value, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
As used in this disclosure including the claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein.
The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Treatment does not have to lead to complete cure, although it may. For example, treatment does not have to lead to complete mitigation of drug-induced cardiac toxicity. Treatment can mean alleviation of one or more of the symptoms or markers of the indication. The exact amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amount can be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation. Within the meaning of the disclosure, “treatment” also includes prophylaxis and treatment of relapse, as well as the alleviation of acute or chronic signs, symptoms and/or malfunctions associated with the indication. Treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, over a medium term, or can be a long-term treatment, such as, for example within the context of a maintenance therapy. Administrations may be intermittent, periodic, or continuous.
The present disclosure provides compositions and methods for generation of heart pacemaker cells, such as human heart pacemaker cells. The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and lack of robust in vitro derivation methods. This disclosure provides an efficient method to generate purified human pluripotent stem cell-derived SAN cells (hPSC-SAN). In an embodiment, the method comprises using a reporter for SHOX2 expression, (e.g., dual SHOX2:GFP; MYH6:mCherry knock-in reporter line). The method generates pluripotent stem cell-derived SAN cells displaying molecular and electrophysiological characteristics of SAN cells. These cells can be used for screening of agents that affect SAN cell functioning. For example, therapeutic agents may be screened for side effects on cardiac toxicity, or agents may be identified that can be used as protectants against therapeutic drugs that cause cardiac toxicity. As an example, agents may be identified that can protect against anthracycline-induced cardiac toxicity.
The present method provides an improvement over previous methods. Protze et al. (2017, Nat Biotechnol 35, 56-68) used an NKX2.5-GFP reporter as a negative marker to enrich SAN-like cells, which is far from ideal. Since NKX2.5 is a transcription factor, and not a surface marker, investigators would need to generate such a reporter for any hPSC line they use for differentiation. Also, since it uses negative selection to eliminate non-SAN-like cells (primarily cardiomyocytes) it has limited capacity to expand the desired cell type, rather than eliminate the wrong cell type, which seriously limits capacity to scale. Another method by Schweizer et al. (2017, Stem Cell Res Ther 8, 229) derived pacemaker cells by co-culturing hESC-derived cells with a visceral endoderm-like cell line END-2, which severely limits any application for transplantation or cell therapy purposes. The present method used a dual reporter in pluripotent cells (such as hESC line) to screen for conditions promoting pacemaker differentiation, and to enrich and expand the desired SAN-like population, and established a highly efficient, chemically defined condition to derive functional pacemaker cells. In an embodiment, the dual reporter system SHOX2:eGFP; MYH6:mCherry was used. While the reporter is used for positive output quantification, it is not needed once the protocol is established because it is not a positive selection feature.
The cells used may be human embryonic stem cells or human induced pluripotent stem cells, which are herein together referred to as human pluripotent stem cells (hPSCs). The human pluripotent stem cells may be derived from any suitable cell type, including lung, fibroblasts (e.g. skin fibroblasts), keratinocytes, blood progenitor cells, bone marrow cells, hepatocytes, gastric epithelial cells, pancreatic cells, neural stem cells, B lymphocytes, ES derived somatic cells, and embryonic fibroblasts.
The term “pluripotent stem cell” (also referred to as “PSC”) as used herein refers to a cell having an ability to differentiate into any type of cell of an adult (pluripotency) and also having self-renewal capacity which is an ability to maintain the pluripotency during cell division. “PSCs” include Embryonic Stem Cells (ESCs), which are derived from inner cell mass of blastocysts or morulae, including cells that have been serially passaged as cell lines. Embryonic stem cells, regardless of their source or the particular method used to produce them, can be identified based on their ability to differentiate into cells of all three germ layers, expression of at least Oct4 and alkaline phosphatase, and ability to produce teratomas when transplanted into immunodeficient animals. The term PSCs also includes induced PSCs (iPSCs), which are cells converted from somatic cells by a variety of methods, such as a transient overexpression of a set of transcription factors. A PSC may be a cell of any species with no limitation, and preferably a mammalian cell. It may be a rodent or primate cell. For example, it may be a monkey, mouse or a human pluripotent stem cell. The term “human pluripotent stem cells” or hPSCs includes human embryonic stem cells and human induced PSCs. Human embryonic stem cells may be obtained from established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines H1, H7, and H9 (WiCell).
iPSCs can be generated by reprogramming adult cells using lentivirus or plasmids. Any type of human somatic cells (e.g. skin fibroblasts or cells from a biological fluid) can be reprogrammed to iPSC using lentivirus (Maherali et al. Cell Stem Cell 3, 340-345 (2008)) or plasmids (Okita et al. Nat. Methods 8, 409-412 (2011)). For example, human dermal fibroblasts can be infected for a suitable period of time (such as 16 hr) with lentiviruses to express the following: hOct4, hSox4, hKlf4, hNanog, and c-Myc, at specific times. Infected cells can be plated on feeder cells in suitable culture media, and transgenes can be induced. Clones with hESC morphology generally appear after about 3 weeks.
The present disclosure describes a new and efficient strategy to derive and purify SAN-like cells from hPSCs. The ability to generate human SAN-like cells in vitro can facilitate study of genetic and iatrogenic arrhythmias and may allow scaled production and transplantation as biological pacemakers. Our reporter strategy enabled direct visualization and standardized in vitro assays for developmental studies, purification and drug discovery. Using the reporter strategy allowed the development of a method that generated a unique population of SAN-like cells.
The term “sinoatrial node-like cells” or “SAN-like cells” as used herein refers to cardiomyocytes or cardiac cells that express sinoatrial nodal (SAN) cell specific markers TBX18, TBX3, SHOX2 and ISL1 and which are capable of displaying pacemaker activity (e.g., display “funny current”). The SAN cells are the only cells in the heart that can initiate beating, and they alone display a “funny current”, which is a mixed sodium-potassium current that activates upon hyperpolarization at voltages in the diastolic range (normally from −60/−70 mV to −40 mV). The funny current flows through an HCN4-dependent channel. The SAN-like cells express much lower levels of cardiac working muscle markers such as myosins and troponins. Unlike cardiomyocytes, they do not express Cx40.
For culturing of cells, any medium that is routinely used for culturing animal cells can be used, except that no growth factors or serum should be present or are added in the media. Examples of suitable culture media include mTeSR1, Essential 8 medium, BME, F-12, BGJb, MCDB131, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, DMEM, Ham, RPMI 1640, and Fischer's media, but other similar media can also be used. Non-growth factor additives, such as antibiotics, B-27 supplement (with or without insulin), amino acids, salts, ascorbic acid and thioglycerol can be added to the media.
Incubation conditions for cell cultures are known in the art. For example, the conditions typically include culturing at a temperature of between about 32-40° C., for example, at least or about 32, 33, 34, 35, 36, 37, 38, 39 or 40° C. The CO2 concentration is generally about 1 to 10%, for example, about 2 to 7%, or about 5% or any range or value between 1 and 10%. The oxygen tension is adjusted to generally to provide normoxic conditions and is preferably about 20%.
The cells, starting with the pluripotent stem cells may be cultured on suitable substrates. For example suitable substrates include Matrigel, collagen IV, fibronectin, laminin, collagen, vitronectin, polylysine, iMatrix-511, iMatrix-521 and the like. These materials are commercially available and routinely used for cell culture.
In an embodiment, the present disclosure provides a method of producing a population of sino-atrial node like cells, comprising or consisting essentially of (a) incubating PSCs with medium comprising or consisting essentially of one or more GSK inhibitors, one or more members of the TGF-β superfamily of proteins, (b) incubating the cells from (a) with a medium comprising or consisting essentially of one or more retinoic acid signaling activators, one or more Wnt inhibitors, one of more ALK inhibitors, and one or more TGF-β superfamily of proteins and/or EGFR inhibitors, and optionally, one or more STAT3 inhibitors, for a period of time to generate SAN progenitor (also referred to as pacemaker progenitor cells that express SHOX2, ISL1 TBX3, TBX5, TBX18 and HCN4; and (c) incubating the SAN progenitor cells in a medium comprising or consisting essentially of one or more GSK inhibitors, one or more EGFR inhibitors, and optionally, one or more HDAC inhibitor for a period of time to generate a population of SAN-like cells that remain enriched for SHOX2, ISL1, TBX3, TBX5, TBX18, and HCN4, but will now also express channels for unique electrophysiological activity. If continued in culture, (such as by 30 days), the cells express CAV3.1, CAV1.3 and Cx30.2 and electrophysiological properties similar to SA node cells. The cells do not express Cx40. The cells may be continued in culture for 30-60 days and beyond that.
Compounds described herein for use for differentiation along the SA node lineage includes pharmaceutically acceptable salts thereof. Other compounds having these functions (i.e., functional analogs) are included within the scope of this disclosure. A “functional analog” as used herein means a compound that has a similar physical, chemical, biochemical, or pharmacological property as compared to another compound. Functional analogs may or may not have similar structures as compared to one another. For example, any functional analog of the particular inhibitors or activators used in the specific embodiments may be used.
Examples of GSK3 inhibitors include CHIR99021, SB2116763, CHIR-98014, and LY2090314.
Examples of Wnt inhibitor include XAV939, IWR-1, IWP-2, and Wnt-C59.
Examples of Activin receptor (ALK) inhibitor include SB431542, LDK378, TAE684, and ASP3026.
Examples of EGFR inhibitors include SU 5402 (Torcis), Tyrphostin AG-490.
Examples of STAT3 (signal transducer and activator of transcription 3) inhibitors include cucurbitacin, Stattic, and Cryptotanshinone.
Examples of HDAC inhibitors include chidamide, Vorinostat, Panobinostat, and Trichostatin A.
Examples of activators of Retinoic acid signaling include retinoic acid (RA), TTNPB, EC23, AM580, Ch55.
Examples of members of the TGF-β superfamily of proteins includes BMP-4 and Activin A.
These inhibitors or activators are generally used at concentrations from 0.01 to 10 μM.
In an embodiment, PSCs may be cultured on a substrate and then upon reaching about 70-80% confluence, incubated with a medium comprising or consisting essentially of a GSK inhibitor, and one or more members of the TGF-β superfamily of proteins for a sufficient period of time (such as 2-3 days) to generate pre-cardiac mesoderm cells. The pre-cardiac mesoderm cells express fetal liver kinase 1 (FLK1) and platelet derived growth factor receptor (PDGFR)-alpha. The pre-cardiac mesoderm cells are incubated in a medium comprising or consisting essentially of a retinoic acid signaling activator, a Wnt inhibitor, an ALK inhibitor, a member of the TGF-β superfamily of proteins and/or an EGFR inhibitor, and optionally, a STAT3 inhibitor, for a sufficient period of time (such as 3 days) to generate pacemaker progenitor cells. The pacemaker progenitor cells express TBX3, TBX5, TBX18, SHOX2, and ISL1. The pacemaker progenitor cells are then incubated in a medium comprising a GSK inhibitor, an EGFR inhibitor (such as 3-9 days), and optionally, a HDAC inhibitor for a sufficient period of time to generate SA-node like cells. The HDAC inhibitor may be added at the same time as the GSK inhibitor and the EGFR inhibitor or maybe added subsequent to an initial culture period with GSK inhibitor and the EGFR inhibitor (such as initial period of 3-6 days with GSK inhibitor and EGFR inhibitor, and another 1-3 days with GSK inhibitor, EGFR inhibitor and HDAC inhibitor). The SA-node like cells express SHOX2, TBX5, TBX3, ISL1 and TBX18, as well as CAV3.1, CAV1.3 and Cx30.2, and HCN4, and display the funny current based on electrophysiological analysis The SA-node like cells can be continued in culture in a serum-free, growth factor free medium for an extended period of time (such as at least 3 months).
In embodiments, the various factors referred to for the steps may be the only differentiation/growth factors present in the culture medium. For example, PSCs may be incubated in a medium in which the only differentiation factors are a GSK inhibitor, and one or more members of the TGF-β superfamily of proteins to generate pre-cardiac mesodermal cells, the pre-cardiac mesodermal cells may be incubated in a medium in which the only differentiation factors are retinoic acid signaling activator, Wnt inhibitor, ALK inhibitor, a member of the TGF-β superfamily of proteins and/or an EGFR inhibitor, and optionally, STAT3 to generate pacemaker progenitor cells, and the pacemaker progenitor cells may be incubated in a medium in which the only differentiation factors are GSK inhibitor, an EGFR inhibitor, and optionally, a HDAC inhibitor to generate SAN-like cells. Non-growth/differentiation factor additives, such as antibiotics, B-27 supplement (with or without insulin), amino acids, salts, ascorbic acid and thioglycerol, may be present.
In an embodiment, PSCs may be cultured on a substrate and then upon reaching about 70-80% confluence, incubated with a medium comprising or consisting essentially of a CHIR99021, BMP4 and/or Activin A for a sufficient period of time (such as 3 days) to generate pre-cardiac mesoderm cells. The pre-cardiac mesoderm cells are incubated in a medium comprising or consisting essentially of cucurbitacin, retinoic acid, XAV939, SB431542, MP4 and/or μM SU5402 for a sufficient period of time (such as 3-6 days) to generate pacemaker fate cells. The pacemaker fate cells are then incubated in a medium comprising or consisting essentially of a CHIR99021, Tyrphostin AG 490 and/or 1-10 μM chidamide for a sufficient period of time (such as 1-3 days) to generate SA-node like cells. The SAN-like cells exhibit SAN-like electrophysiological characteristics, such as ‘funny channel’ by 30 days (total time in culture, starting from PSCs). The SA-node like cells can be continued in culture for an extended period of time (such as at least 3 months). It was observed that while it was preferred for some cell lines to include both BMP-4 and Activin A in the medium for generating pre-cardiac mesoderm cells, in other cell lines only BMP-4 or Activin A was sufficient. Thus, these components may be optimized for a particular cell line.
In an embodiment, PSCs may be cultured on a substrate and then upon reaching about 80% confluence (in about 72 hours), incubated with a medium comprising or consisting essentially of a 0.5-3 μM CHIR99021, 5-100 ng/mL BMP4 and/or 5-50 ng/mL Activin A in RPMI (Cellgro) supplemented with B27 minus insulin, 2 mM GlutaMAX, 1× NEAA and/or 1× Pen/Strep for 3 days (RB27-INS) to generate pre-cardiac mesoderm cells. The pre-cardiac mesoderm cells are incubated in a medium comprising or consisting essentially of 0-5 M cucurbitacin, 0.1-10 μM retinoic acid, 1-10 μM XAV939, 1-10 μM SB431542, 5-100 ng/mL BMP4 and/or 0.1-10 μM SU5402 in RB27-INS for the next 3-6 days to generate pacemaker fate cells. The pacemaker fate cells are then incubated in a medium comprising or consisting essentially of a 0.5-3 μM CHIR99021, 1-10 μM Tyrphostin AG 490 and optionally, 1-10 μM chidamide in RB27-INS from day 7 to day 9 to generate SA-node like cells. The SA-node like cells can be continued in culture in RPMI+B27 medium. for an extended period of time (such as at least 3 months). Variations of the protocol are shown in
In an embodiment, of the many potential applications, we used the hPSC-SAN platform for in vitro prediction of pacemaker cell toxicity following anthracycline treatment, since supraventricular arrhythmias and/or atrial fibrillation can be early manifestations of the toxicity. One striking finding from the present results was a significant sensitivity to DOXO treatment in cell lines harboring genetic variation in the CBR3 locus. The platform also identified new loci that may potentially be associated with increased arrhythmia susceptibility in patients receiving anthracyclines. This platform can be exploited as a high throughput in vitro safety test for future potential drugs that might affect SAN-like cells. Finally, the ability of physcion to modulate doxorubicin-related toxicity on SAN-like cells represents proof-of-concept for the use of human SAN-like cells for the identification of compounds that can counteract arrhythmogenic drugs.
In an aspect, this disclosure provides a method of selectively producing and identifying SAN-like cells comprising providing a population of human pluripotent stem cells and introducing into the cells, a reporter for SHOX2 expression, and based on the expression of SHOX2, identifying and isolating hPSC-SAN cells. In an embodiment, the method comprises i)) providing a population of human pluripotent stem cells, ii) introducing into the cells, a reporter for SHOX2 expression, iii) providing the cells from ii) with a GSK inhibitor, and one or more members of TGF-β superfamily of proteins, iv) providing cells from iii) with retinoic acid, Wnt inhibitor, ALK inhibitor, a TGF-β superfamily of proteins and/or EGFR inhibitor, and optionally, a STAT3 inhibitor, v) providing the cells from iv) with GSK inhibitor and EGFR inhibitor, and optionally, additionally providing to the cells with an HDAC inhibitor, and vi) sorting for cells that are positive for the reporter for SHOX2. The cells may be used for implantation or for screening (such as in a high throughput screening) to identify agents that can affect the functioning of SAN cells.
Introduction of the reporter, such as SHOX2 permits the refinement of the technique and also allows for validation. However, the introduction of the reporter is not required for the generation of SAN-like cells from PSCs. Thus the steps of the method may be carried out without introducing the reporter for SHOX2 and without screening for the presence of SHOX2.
In an aspect, this disclosure provides a population of cells derived from human pluripotent stem cells which exhibit SAN like characteristics. If the SAN-like cells are derived from hPSCs, they may be referred to as hPSC-SAN cells or hPSC-SAN-like cells. In embodiments, the SAN-like cells, such as hPSC-SAN cells, may be at least 50% pure in the population. In embodiments, the population may be at least 60%, at least 70%, at least 80%, at least 90% or at least 95% or 99% pure for SAN-like cells, such as hPSC-SAN cells. In an embodiment, this disclosure provides a population of cells produced by the methods described herein.
In an aspect, this disclosure provides a method for identifying drugs that induce cardiac toxicity by exposing the candidate agent to a population of hPSC-SAN cells generated as described in the present disclosure and evaluating for effects on the functioning of the cells. The functioning may be evaluated in terms of chemical, biological, biochemical or electrophysiological characteristics.
In an aspect, this disclosure provides a method for identifying agents that can protect against drug induced cardiac toxicity by exposing the particular drug or therapeutic agent to a population of hPSC-SAN cells in the presence or absence of candidate protecting agents and evaluating for effects on the functioning of the SAN-like cells, and identifying agents that minimize or eliminate adverse effects of the therapeutic agent. The functioning may be evaluated in terms of chemical, biological, physical, biochemical or electrophysiological characteristics.
In an aspect, this disclosure provides a method of reducing cardiac toxicity side effects of a therapeutic agent comprising administering to an individual in need of treatment the therapeutic agent and a cardiac toxicity protective agent, which exhibits protection action on SAN-like cells. An example of a cardiac toxicity protective agent is physcion.
In an embodiment, this disclosure provides a method of treatment of cancer by chemotherapeutic agents comprising administering a chemotherapeutic agent in conjunction with a cardiac toxicity protective agent, wherein the cardiac toxicity protective agent reduces cardiac toxicity side effect of the chemotherapeutic agent, but does not adversely affect its cell growth inhibiting efficacy. In an embodiment, the chemotherapeutic agent may be an anthracycline (such as doxorubicin) and the cardiac toxicity protective agent may be physcion.
In an embodiment, this disclosure provides compositions and methods for the treatment of cardiac toxicity, the compositions comprising a therapeutically effective dose of physcion or derivatives thereof. The compositions may further comprise pharmaceutically acceptable carriers, additives, excipients, stabilizers, enhancers, adjuvants and the like. A therapeutically effective amount of physcion may be determined by a person skilled in the art given the benefit of the present disclosure and known principles of clinical therapeutics. In embodiments, compositions comprising physcion may be administered in conjunction with the therapeutic agent whose cardiac toxicity is desired to be muted. The two may be administered together or separately, by the same route or different routes, and over the same period of time or different periods of time.
The structure of physcion is as follows.
Derivatives of physcion include glycosylation at either of the alcohols, general functionalization on the alcohols with protecting groups (e.g., trimethyl silyl groups or acetyl groups), and dimerization of physcion (either through one of the carbonyls of the anthraquinone or through substitution on one of the flanking rings). Physcion derivatives may be produced by derivatizing physcion at one or more of its four main substituents (the methoxy group, two alcohol groups, and methyl group) providing a structure as follows.
where R1 and R4 are chosen from substituted and unsubstituted alkyl groups and substituted and unsubstituted alkoxy groups, and R2 and R3 are chosen from hydrogen, substituted and unsubstituted alkyl groups, substituted and unsubstituted alkylcarbonyl groups, substituted and unsubstituted alkylsulfonyl groups, and substituted and unsubstituted alkylphosphonyl groups.
Physcion and/or derivatives thereof may be provided as pharmaceutical compositions comprising pharmaceutically suitable carriers. Suitable carriers include excipients, or stabilizers which are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; and/or non-ionic surfactants such as Tween or polyethylene glycol (PEG). Examples of suitable pharmaceutical preparation components can be found in Remington: The Science and Practice of Pharmacy 20th edition (2000). The pharmaceutical compositions may comprise other therapeutic agents.
The subject to be treated may be an individual who has one or the more of the following SNPs: a) rs1056892 in CBR3, b) rs2229774 in RARG, c) rs885004 in SLC28A3, d) rs9559211 in LIG4, e) rs7314566 in ANO2, f) rs17267852 in NRXN1.
In an aspect, the present invention provides a method for treating a subject suffering from a defect related to SA-node malfunction. The method comprises culturing pluripotent stem cells, differentiating the pluripotent stem cells in vitro into SAN-like cells, and implanting the cells into the SA-node area within the heart of the individual.
Cells may be implanted into an appropriate site in a recipient. The amount of cells used in implantation depends on a number of various factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art. The number of cells for transplantation may be used from 1 million to 20 million and all integer values therebetween. In embodiments, the number of cells used may be 1, 2, 5, 7.5, 10, 12.5, 15, 17.5 and 20 million cells. In an example, the differentiated SAN-like cells may be implanted as dispersed cells or formed into clusters that may be infused into the appropriate cardiac site via a suitable method, such as catheterization. Alternatively, cells may be provided in biocompatible degradable polymeric supports, porous non-degradable devices or encapsulated to protect from host immune response. Support materials suitable for use for implantation of cells of the present disclosure include tissue templates, conduits, barriers, and reservoirs, such as those used for tissue repair. For example, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures may be used.
Some examples in the following paragraphs provide non-limiting embodiments of the present disclosure.
Example 1) A method to generate cells with sinoatrial node (SAN) characteristics from human pluripotent stem cells comprising: a) providing a population of human pluripotent stem cells; b) transfecting the cells with a reporter for SHOX2 expression;
c) providing retinoic acid, an EGF inhibitor, an FGF inhibitor, and a STAT3 inhibitor; d) providing an HDAC inhibitor; and e) sorting for cells positive for the reporter for SHOX2.
Example 2) The method of Example 1, wherein the initial population of cells are embryonic pluripotent cells.
Example 3) The method of Example 1 wherein the initial population of cells are induced pluripotent cells.
Example 4) The method of Example 1, wherein 5 μM EGF inhibitor is used, and said inhibitor is Tyrphostin AG490.
Example 5) The method of Example 1 wherein 0.1 μM STAT3 inhibitor is used, and said inhibitor is cucurbitacin.
Example 6) The method of Example 1 wherein 5 μM of FGFR inhibitor is used, and said inhibitor is SU5402.
Example 7) The method of Example 1 wherein 1 μM RA is used.
Example 8) The method of Example 1, where cells are incubated with X for about 7 days, after which the HDAC inhibitor is administered.
Example 9) The method of Example 1 wherein the HDAC inhibitor is chidamide.
Example 10) The method of Example 1, where the sorting in Example if occurs around 45 days after step 1d.
Example 11) A plasmid encoding SHOX2:GFP
Example 12) A method to test drugs for cardiac side effects by testing them on the hPSC-SAN cells.
Example 13) A method to screen for drug leads using the hPSC-SAN cells, including drug leads that prevent cardiac side effects of other drugs.
Example 14) A method to prevent anthracycline-induced cardiotoxity by co-administering physcion.
Example 15) Method of Example 14, wherein the subject has any of the following SNPs: a) rs1056892 in CBR3, b) rs2229774 in RARG, c) rs885004 in SLC28A3, d) rs9559211 in LIG4, e) rs7314566 in ANO2, and f) rs17267852 in NRXN1.
The following examples are provided for illustrative purposes and not intended to be limiting.
We developed a protocol that maximizes the generation of pluripotent stem cell-derived SAN pacemaker lineages (PSC-SAN). Based on previous reports (Hoogaars et al., Genes Dev 21, 1098-1112 (2007); Ionta et al., Stem Cell Reports 4, 129-142 (2015); McNally et al., Circ Res 104, 285-287 (2009), SAN progenitors are expected to be enriched for expression of key pacemaker transcription factors TBX3, TBX18, and SHOX2 (TTS). Recently, murine embryonic stem cell (ESC) lines that were engineered for conditional expression of GATA4, GATA5 or GATA6, to efficiently generate diverse cardiac cell populations including, nodal, atrial and ventricular cells4. Among these cell lines, GATA5 induction resulted in the highest expression levels of TTS (
These results were next translated into a human embryonic stem cell (hESC) differentiation protocol, including two additional pathway modulators. Retinoic acid (RA) was included to shift regional identity of cardiac progenitors toward a more caudal phenotype, since pacemaker cells are thought to arise from a more caudal population of cardiac progenitors. In addition, an FGF inhibitor was used to augment the expression of ISL1, a known regulator of the nodal program in early mouse development. Indeed, the full combination (condition 8) of 5 μM EGF inhibitor (Tyrphostin AG490), 0.1 μM STAT3 inhibitor (cucurbitacin), 5 μM FGFR inhibitor (SU5402), and 1 μM RA significantly increased human TTS expression levels (
To quantify and purify SAN-like cells using this directed differentiation protocol, a dual knock-in SHOX2:GFP; MYH6:mCherry reporter line was created using CRISPR/Cas-based gene-targeting techniques (
To better validate the nodal identity of the SHOX2:GFP+ cells, the GFP+ (regardless of mCherry expression) and GFP−mCherry+ populations were sorted at Day 25 or Day 40 of differentiation and analyzed by RNA-seq. The data was compared by hierarchical clustering with RNA-seq databases generated using HCN4:GFP+ cells from dissected murine SAN tissue and GFP− cells from adjacent right atrial tissue (GSE65658). Whether the clustering analysis used whole transcriptomic data (
We maintained differentiated cells for up to 60 days and evaluated gene expression patterns. At day 30, most SHOX2:GFP+ cells stained positive by immuno-fluorescence for ISL1, TBX18, TBX5 and HCN4 (
A major function of the SAN is to regulate heart rhythm under the control of autonomic neurons (AN). ESC-derived cardiomyocytes show poor connectivity with ESC-derived AN12. We probed the functionality of in vitro-derived hPSC-SAN cells by assessing connectivity with AN (
One key functional property of a nodal cell is its characteristic action potential with fast spontaneous firing rates and slow maximum upstroke velocities (<30 V/s). SHOX2:GFP+ cells generated from the differentiation protocol at day 30 were sorted and patch-clamping experiments showed 8 out of 10 demonstrated the characteristic electrophysiologic phenotype of nodal cells (
The hPSC-SAN cells were analyzed in a single-cell RNAseq experiment to explore heterogeneity of the cell population and identify lineage-restricted transcriptome features. For this purpose SHOX2:GFP+ cells in the hPSC-SAN population were sorted from the pacemaker differentiation culture while MYH6:mCherry+ cells were sorted following cardiomyocyte directed differentiation to generate a ventricular-like population (hESC-V). After pooled sequencing, tSNE plots (
High-SHOX2 cells were significantly enriched in transcripts encoding structural proteins TPM1, TNNT2, MYL7, MYH6, MYL6, ACTA2, DSTN, ACTC1, NEXN, NEBL, MYH11 and MYLK (
The high-SHOX2 population showed relatively homogenous expression of HCN4, ISL1, TBX5 and TBX3 genes compared to TBX18 (
With an effective protocol to derive human SAN-like cells, we next established a platform to model sensitivity of pacemaker cells to drug toxicity. Some of the most common forms of arrhythmias are iatrogenic and occur as a consequence of chemotherapy treatment, requiring a permanent pacemaker replacement in severe cases. We focused on doxorubicin (DOXO) because it has well known adverse effects on the heart, thereby limiting its application for management of breast cancer and other tumors. Importantly, individual patients display different levels of sensitivity to this drug, indicating a contribution of genetic variation to this toxicity. This can be tested by modeling drug toxicity in human pacemaker cells derived from various genetic backgrounds (
To identify genetic variation that associates with the sensitivity of pacemaker cells to DOXO-induced toxicity, we performed an unbiased screen using a library of hPSC-SAN cells differentiated from 48 distinct iPSC lines. Cells differentiated for 35 days were treated with 0.16 μM DOXO for 3 days. Due to the lack of a robust antibody to SHOX2, an antibody recognizing ISL1 that is co-expressed with SHOX2:GFP (
The remaining lines, including several high-sensitive lines, had no known risk alleles associated with DOXO-induced cardiotoxicity. Therefore, we sought to identify novel genetic variants associated with chemo-toxicity of SAN tissue. Genomic DNA from each of the iPSC lines was hybridized to Illumina arrays to genotype SNPs across the human genome. The SNP data was then analyzed for associations with cell toxicity (
These findings demonstrate that hPSC-SAN cells can be used to model DOXO-induced cytotoxicity and discover novel genetic variants that are associated with arrhythmia phenotypes. With this validated platform, we carried out a small molecule screen to identify compounds capable of rescuing the toxicity effects observed with DOXO-treatment of hPSC-SAN cells. 1472 compounds were tested consisting of FDA-approved drugs and natural products. Hit targets were selected based on a reduced percent ratio of GFP+ cleaved caspase-3+/GFP+ cells (
Methods:
Culture of hESC lines. hESC line H9 and the derivative (SHOX2:GFP; MYH6:mCherry) line, and 48 iPSC clones were grown on matrigel-coated plates and maintained in mTeSR medium (Stem Cell Technologies).
Pacemaker and cardiomyocyte induction. Differentiation was initiated 72 hours after plating when the culture was approximately 80% confluent. Step 1: Cells were differentiated with 1.5 μM CHIR99021 (CHIR, Stem-RD, Ch), 20 ng/mL BMP4 (B) and 20 ng/mL Activin A (AA) in RPMI (Cellgro) supplemented with B27 minus insulin, 2 mM GlutaMAX, 1× NEAA and 1× Pen/Strep for 3 days (RB27-INS). Step 2: For cardiomyocyte differentiation, cells were treated for an additional 3 days with 5 μM XAV939 (Tocris). For hPSC-SAN differentiation, Step 1 was followed by addition of 0.1 μM cucurbitacin (Cu, Sigma Aldrich), 1 μM retinoic acid (RA), 5 μM SU5402 (SU, Fi, Tocris) in RB27-INS from day 3-6. 5 μM XAV939 (Wi) was added from day 5-6. From day 6 onward, both cardiomyocyte and hPSC-SAN differentiation were carried out in RPMI supplemented with B27, 2 mM GlutaMAX, 1× NEAA and 1× Pen/Strep (RB27+INS). Step 3: hPSC-SAN differentiation included additional treatment with 5 μM Tyrphostin AG 490 (Sigma Aldrich) from day 6-9 in RB27+INS. The HDAC inhibitor, chidamide, was added to the differentiation cocktail from day 7-9 with a final concentration 5 μM. Variations are shown in
Gene expression analysis. Total RNA was isolated using the Qiagen RNeasy mini kit according to manufacturer instructions. cDNA synthesis was performed using TAKARA PrimeScript 1st strand cDNA synthesis kit. qRT-PCR reactions were generated using Roche SYBR green PCR mix (Roche). Each data point represents at minimum 3 independent biological replicates. Primary antibodies and working dilutions are listed in Supplementary Table 1.
FACS and Immuno-fluorescence. For FACS analysis, the cells were dissociated with Accutase (Innovative Cell Technologies) for 15 min at 37° C. and fixed and permeabilized with the FOXP3 Fixation/Permeabilization set (eBiosciences). Subsequently, they were washed, blocked and permeabilized using FOXP3 permeabilization buffer (eBiosciences) according to the manufacturer's protocol. The cells were stained with primary (overnight 4 degrees) and secondary (45 mins, room temperature) antibodies and analyzed using a flow cytometer. For immunofluorescence, cells were fixed with 4% PFA (eBiosciences) for 20 min at room temperature. Next, cells were blocked and permeabilized in 5% horse serum (Invitrogen), 0.3% Triton X in PBS for one hour at room temperature, followed by primary antibody incubation overnight. After several rounds of washing, secondary antibodies were added for 1 hour at room temperature. Nuclei were stained with DAPI. Primary antibodies and working dilutions are listed in Supplementary Table 1.
Generation of the SHOX2:NLS-eGFP;MYH6:mCherry dual hESC reporter line. The αMHC:mCherry reporter is described in a complementary manuscript. For targeting the SHOX2 locus, sgRNA sequences were designed using the website http://crispr.mit.edu/and targeted the sequence: 5′-GGCGTTGGCGTCACAGACCC-3′ (SEQ ID NO:37). sgRNA was cloned into the PX459 vector (Addgene, plasmid #42230). For constructing the SHOX2:NLS-eGFP donor plasmid, the NLS-eGFP (from Tol2kit plasmid), codon optimized P2A, and left and right homology arms (from H9 genomic DNA) were PCR amplified and assembled using the In-Fusion kit (Clontech) and then cloned into the zero-blunt plasmid (Thermo Fischer). H9 hESCs were disassociated into single cells with Accutase for 5-7 min. Cells were electroporated using human stem cell nucleofector kit2 (Lonza, #VPH-5022) according to the manufacturer guidelines. Briefly, 1 million cells were resuspended in 100 μL nucleofector mix to which was added 2 μg CRISPR targeting plasmid and 4 μg donor plasmid. Cells were re-plated on matrigel coated plates with ROCK inhibitor. Two days after electroporation cells were treated with puromycin (0.5 μg/mL) for two days. About two weeks after electroporation individual colonies were picked and expanded for PCR genotyping and sequencing for the reporter lines. The sequences for all primers used for generating the donor plasmids are listed in Supplementary Table 2.
RNA Sequencing and single cell Drop-seq Analysis. For RNA sequencing, total RNA was isolated using the Agilent nano kit according to manufacturer instructions. The quality of RNA samples was examined using an Agilent bioanalyzer. cDNA libraries were generated using TruSeq RNA Sample Preparation (Illumina). Each library was sequenced using a single-read 50 bp in HiSeq4000 (Illumina). Gene expression levels were analyzed with TopHat and Cufflinks by the Weill Cornell Genomic Core facility. Raw data were normalized to that from SHOX2:GFP negative cells to define fold-change. Gene lists were filtered based on expression level differences ≥4 or ≤−4. Murine sinoatrial and atrial tissues were obtained from a published database (GSE65658). False discovery rate (FDR) q values <0.25 or nominal (NOM) p values <0.05 were considered significant. The expression data were normalized for each gene by subtracting their mean across samples and then dividing by their standard deviation. The normalization was performed separately on three sets of samples (D25 samples, D40 samples and public samples) to remove batch effects introduced by experiments. The heatmap plot was generated using heatmap.2 in the R “gplots” package.
Single cell sequencing. Cells were differentiated into hPSC-SAN or cardiomyocyte lineages. hPSC-SAN cells were sorted based on eGFP, while hPSC-V cells were sorted based on mCherry. Single-cells were captured using a Drop-Seq system. The two aqueous suspensions—the single-cell suspension and the barcoded primer beads suspended in a lysis buffer, were loaded into 3 mL plastic syringes (BD) respectively. Droplet generation oil (BioRad) was loaded into a 10 mL plastic syringe (BD). The three syringes were connected to a 125 μm co-flow device by 0.38 mm inner-diameter polyethylene tubing (Scientific Commodities, Inc.), and injected using syringe pumps (KD Scientific), resulting in ˜125 μm emulsion drops with a volume of ˜1 nL each. The flow was monitored under an inverted microscope. Droplets were collected in 50 mL falcon tubes; the collection tube was changed out after every 1 mL of combined aqueous flow volume. During droplet generation, the beads were kept in suspension by continuous, gentle magnetic stirring (V&P Scientific). The uniformity in droplet size and the occupancy of beads were evaluated by observing aliquots of droplets under the microscope. The oil from the bottom of each aliquot of droplets was removed after which 30 mL 6×SSC (Life Technologies) at room temperature was added. Collected droplets were broken by adding perfluorooctanol in 30 mL 6×SSC to destabilize the oil-water interface and the microparticles were disrupted. RNA-hybridized beads were extracted. The extracted beads were then washed and resuspended in a reverse transcriptase mix, followed by a treatment with exonuclease I to remove unhybridized, non-extended primers. The beads were then washed, counted, and aliquoted into PCR tubes for PCR amplification. 15-18 cycles of PCR amplification were applied to reach cDNA concentration at 400-1000 μg/μL after pre-PCR test step. The final PCR products were purified and pooled, and the amplified cDNA quantified on an Agilent 2100 BioAnalyzer High Sensitivity Chip. The cDNA was fragmented and amplified for sequencing with the Nextera XT DNA sample prep kit (Illumina) using custom primers. The libraries were purified, quantified, and sequenced on the Illumina NextSeq 500 at >50,000 reads/cell.
After quality assessment of the sequencing with FastQC v0.11.3, all single-cell RNA-seq samples were processed according to the Drop-seq Computational Protocol with the Drop-seq Toolkit v1.2 created by the McCarroll Lab. Sequences were first tagged with their corresponding cell and molecular barcodes (unique molecular identifiers, UMI) using dropseq TagBamWithReadSequenceExtended. Cell and molecular barcodes were required to have a minimum base quality of 10 across their lengths, otherwise they were discarded. SMART adapter sequences were trimmed from the 5′ end of each read whenever there were at least five continuous bases exactly matching the SMART adapter (dropseq TrimStartingSequence). Putative poly(A) tails were trimmed from the 3′ end whenever there were stretches of at least 6 adenosines with zero mismatches (dropseq PolyATrimmer). The UMI-tagged and trimmed reads were then aligned with default parameters to the human reference genome (GRCh38) using STAR (v2.4.2a). The final file containing alignment information plus the cell and UMI tags was generated with Picard MergeBamAlignment and Picard MergeBamAlignment. Sequences that mapped to more than one locus were excluded from further analysis by filtering reads with a mapping quality lower than 10.
To determine the numbers of detected transcripts per gene and cell, the overlaps of the aligned reads with gene annotation (Ensembl release 76) were counted and summarized using dropseq TagReadWithGeneExon and dropseq DigitalExpression. Individual gene expression matrices from each sample were combined in R to create a single gene count matrix for all cells and genes.
The count matrix was read into R; basic quality metrics such as the distributions of the numbers of gene counts per cell and gene were determined using in-house scripts and the scater package. We excluded cells with fewer than 100 UMI or fewer than 200 genes per cell, and more than 15% of the UMI mapping to mitochondrial genes. This resulted in 1,571 GFP-negative and 2,057 GFP-positive cells (representing about equal numbers per technical replicate). After filtering the cells, we excluded genes that were expressed in fewer than 5 cells per condition. Additionally, genes had to be covered by at least 10 UMI across all cells of the same condition. 13,448 genes met these filtering criteria and were used for downstream analyses. The UMI counts were adjusted for the differences in the individual cells' coverage by dividing every gene count by the total number of UMI counts of the respective cell (library size) and multiplying with the median of all library sizes. These scaled UMI counts were then further de-noised using the MAGIC algorithm. All expression values shown in the manuscript are log 10-transformed values (with an offset of 0.01 to avoid taking the log of zeros) after applying MAGIC. t-SNE was performed with the Rtsne package. Hierarchical clustering was applied to all cells and a subset of genes and visualized with the pheatmap function. Genes and cells were clustered with the base R hclust function using Euclidean distances and the agglomeration function “complete”.
Calcium imaging. hESC-derived cells were plated on ibidi plates coated with gelatin. The cells were loaded with 2 μM Fluo-4 AM dissolved in 1:1 (v/v) of 20% Pluronic®-F127 and DMSO with stock concentration of 1 mM for 45 min at RT in Tyrode solution consisting of (mM): 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2), 10 glucose and 10 HEPES at pH 7.4. Calcium transients of hESC-derived beating cardiac clusters were recorded on a heated stage using a confocal scanning microscope (Zeiss LSM 710) at intervals of 200 ms (5 frames per second). They were then quantified as the background subtracted fluorescence intensity changes normalized to the background subtracted baseline fluorescence using MetaXpress software.
Cellular electrophysiology and characterization. Spontaneous action potentials were recorded over 10 seconds with the perforated patch clamp technique using an AM-Systems (WA, USA) model 2400 amplifier in current-clamp mode and the software platform Real-Time eXperiment Interface (RTXI; rtxi.org). Cells were superfused at 35° C. with a Tyrode's solution containing in mM: 137 NaCl, 5.4 KCl, 2 CaCl2), 1 MgSO4, 10 HEPES, and 10 glucose at pH 7.35 (NaOH). Whole cell access was achieved using 480 μg/mL Amphotericin-B (Sigma-Aldrich, MO, USA) in a pipette solution also containing in mM: 5 NaCl, 20 KCl, 120 K-aspartate, and 10 HEPES at pH 7.2 (KOH). A liquid junction potential of 14 mV was corrected. Pipettes were pulled from 1.5 mm capillary tubes (AM-Systems, WA, USA) to a resistance between 2.5-3.5 MΩ. Cells were characterized as nodal-like if they exhibited spontaneity and a slow upstroke (dV/dtmax<30 V/s).
SNP array and GWAS analysis. High-throughput genotyping of 906,600 SNPs was performed on Affymetrix Genome-Wide Human SNP Array 6.0 (Santa Clara, Calif., USA) at Core Facility of Albert Einstein College of Medicine following the manufacturer's instructions (Affymetrix, Inc., Santa Clara, Calif., USA). Genotyping was performed using the default parameters in the Birdseed v2 algorithm of Genotyping Console (GTC) 4.2 software (Affymetrix). As a quality control for the genotyping, Contrast QC values were calculated as implemented in the GTC 4.2, and samples used passed the recommended values. Genome annotations applied in data analysis referred to the human reference assembly GRCh37/hg19 as provided by the Affymetrix annotation file. In this study, SNPs with low minor allele frequencies (<0.05), low call rates (<95%), and inconsistent genotype frequencies with Hardy-Weinberg equilibrium (P<1.0×10−5) were excluded. In addition, allosome SNPs were not analyzed. After quality control, a total of 691,822 common SNPs were included in the association analysis. An iterative procedure was used to simultaneously estimate principal components (PCs) reflecting population structure. Linear regression was applied to examine associations of SNPs and phenotypes under an additive model adjusted for sex and two principal components. Analyses were performed using R (version 3.3.2 R Foundation).
Association with anthracycline-association arrhythmia phenotypes in humans. Variants identified in cell-based screens were tested for association in a human cohort of subjects (N=384) treated with anthracyline-based chemotherapy. The cohort was developed from BioVU, the Vanderbilt University biobank linking DNA samples (and any related genotype data) extracted from blood leftover after routine clinical care at Vanderbilt University Medical Center to a de-identified version of the electronic health record (EHR) intended to support research. Details of the cohort as well as the development and implementation of BioVU have been described (PMID: 28542097, 18500243). Subjects with atrial fibrillation (AF) or pacemaker implantation after chemotherapy initiation were identified using key word searches and automated queries of billing and procedure codes. Subjects were genotyped in the Vanderbilt Technologies for Advanced Genomics (VANTAGE) Core using the Illumina Omni 1-Quad platform using standard quality control filters at the sample and variant level. Genotypes were imputed to 1000 Genomes Phase 1 version 3 (Apr. 19, 2012) using reference panels from all 1000 Genomes populations. Variants were tested for association with AF and pacemaker implantation separately using multiple logistic regression, implemented in PLINK, adjusted for age, race, gender. As this was a hypothesis-generating “replication” from our cell-based “discovery” analyses, we chose a significant threshold of 0.025 (0.05/2 phenotypes).
PheWAS analysis. To assess whether genes from identified loci have broader phenotypic relevance (i.e., outside patients treated with chemotherapy) we conducted phenome-wide association studies (PheWAS) in subjects with available genotype data [25,539 on the Illumina HumanExome BeadChip v.1.0 (‘exome chip’) and 13,331 on the Illumina Multi-Ethnic Genotyping Array (MEGA) platform] generated as part of ongoing research efforts. All subjects were of European decent and subject overlap between the two platforms was modest (2,729). The PheWAS method utilized a validated medical phenome of hierarchically grouped International Classification of Disease (ICD-9) billing codes into ˜1,800 phenotypes (‘phecodes’), each with defined control groups. Logistic regression was used to serially test for association between genetic variants in candidate genes and each phecode phenotype. All analyses were adjusted for age and gender. As these were exploratory analyses, we chose a liberal threshold of 0.05 for evidence of nominal association with each phenotype.
Chemical screen. The high-throughput chemical screening was performed by Prestwick FDA approved and natural products libraries. The 30 day old hES-SAN cells derived from the dual-reporter line were replated into matrigel coated 384-well plates. The DOXO was added at final concentration as 0.16 μM. At the same time, individual compound from chemical library was added at final concentration as 10 μM and maintained for 3 days. The plates were fixed and stained with primary antibodies against GFP and Caspase-3. The compounds that decreased the cell death rate by more than two-fold of the standard deviation below the average of DMSO-treated samples were selected as primary hits. For validation, the cells treated with varying doses of the hit compound and a dose curve was generated. Finally, the sensitive iPS lines were treated with 10 μM physcion and DOXO to validate the efficacy on different cell lines and stained with ISL1 and Caspase-3 for scoring.
Data availability statements. The datasets generated during and analyzed during the current study are available in the GEO repository, which will be available to public upon publication. The reviewer link will be sent to editor once GEO number is assigned.
This example provides further studies on characterization of the SAN-like cells generated by the present methods and the use of these cells to identify cardiac toxicity reducing agents.
Biased pacemaker cell death has yet to be reported as a cardiotoxic side effect of Doxorubicin. To corroborate this in vitro discovery, we assayed Doxorubicin toxicity in vivo in mice. C57BL/6 mice were treated with 20 mg/kg Doxorubicin by intraperitoneal injection. 9 days post injection, the mice were euthanized and whole heart imaging of mouse heart revealed cell death localized to the HCN4+ SAN cells (
The restorative effect of Physcion identified in our drug screen was gauged in the bona fide SAN. doxorubicin toxicity in vivo is a multifactorial process that impedes the differentiation between correlative and causative factors. Thus, we used an ex vivo model to document the direct effect of Doxorubicin and Physcion on the murine SAN. Whole, intact atrial explants were incubated with 1 μM doxorubicin or 1 uM doxorubicin+20 μM Physcion for 3 hrs. Consistent with our in vivo doxorubicin treatment studies, control explants lacked cell death and had rhythmic contractions; while doxorubicin-treated explants exhibited localized SAN cell death and irregular contractility (
Although specific embodiments and examples are provided those skilled in the art will recognize that routine modifications to these embodiments can be made which are intended to be within the scope of the invention.
This application claims priority to U.S. Provisional patent application No. 62/942,505, filed on Dec. 2, 2019, the disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/062919 | 12/2/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62942505 | Dec 2019 | US |
