Patent Application
20220162559
This invention generally relates to three-dimensional myocardial infarct organoids and methods of making and using the same.
While human organoid systems have provided a powerful platform in modeling diseases caused by genetic disorders1-4, non-genetic factors (such as lifestyle and environment) are the largest attributors to devastating diseases like cardiovascular disease (CVD), which is the leading cause of death worldwide.5 Specifically, myocardial infarction (MI) (i.e., heart attack) makes up about 8.5% of CVD and is a common cause of heart failure with a 40% five-year mortality after the first MI.5 Heart failure drugs have performed poorly in clinical trials during the last decade, which has been partially attributed to the distinct differences between human patient hearts and animal heart failure models.7-9 In addition, cardiotoxicity is a major concern for pre- and post-approval in the development for all systemically-delivered drugs.32 Specifically, the ability to detect drug-induced exacerbation of cardiotoxicity is an unmet need for all drug development to address safety concerns for patients with pre-existing cardiovascular conditions, as CVD is a common comorbidity of major diseases.32-35 Thus, there is a need to develop relevant human heart failure models for drug development.6
The present invention overcomes the shortcomings in the field by providing methods of making three-dimensional (3D) myocardial infarct organoids, which can be employed in drug screening and in personalized medicine related to cardiac disease.
One aspect of the invention relates to a three-dimensional (3D) myocardial infarct organoid, comprising cardiomyocytes and non-myocytes, wherein the 3D myocardial infarct organoid comprises:
(a) an apoptotic interior region due to lack of oxygen that is surrounded by a viable periphery comprising, consisting essentially of, or consisting of a region of about 20-75 μm from the organoid edge.
(b) a ratio of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive area to a 4′,6-diamidino-2-phenylindole (DAPI)-positive area ratio in the apoptotic interior region of in a organoid cross-section ranging from about 0.03 to 1.0;
(c) a contraction amplitude from about 0% to 5%;
(d) a beat rate of about 0 to 90 beats per minute;
(e) a calcium transient amplitude measured as a change in fluorescence divided by the starting fluorescence of about 0% to 40%;
(f) upregulated or downregulated fibrosis-related genes, wherein the downregulated genes comprise COL22A1, COL11A2, FGF12, SPINT1, COL9A2, MMP13, HPN, CTSS, FGF7, A2M, COL9A1, FBN2, FGF9, LAMA3, FLT1, SMOC2, COL2A1, FGF2, LAMB3, LAMA5, PDGFB, SMAD3, ICAM5, LAMB2, FGF18, MMP9, CXCL12, COL19A1, FGF13, COL4A5, COL26A1, F11R, COL14A1, COL9A3, FGF1, ICAM1, HBEGF, MDK, ITGA6, TGFB3, LAMA2, RHOQ, RND2, TGFB2, LAMC2, CCDC88A, ITGAE, JUP, ITGAM, COL4A2, CDH2, ITGB2, TGFBR3, BTG1, COL4A1, COL4A3, PDGFRA, FGFR4, SDC4, MMPI, FGF6, ITGA8, and/or COL4A4,
and upregulated genes comprise FGF8, ITGB1BP1, ITGA7, TGIF2, MMP12, PIK3CA, RHOH, COL18A1, ITGAL, LAMC1, RPS6KB1, TGFBR2, RHOD, PIK3CD, MMP3, RAC1, MMP8, ITGB4, ARPCSL, ITGA3, COL17A1, ADAM9, CD2AP, KDR, LAMB1, COL12A1, ITGAX, ABI1, MMP15, FGF14, TGFB1I1, SDC3, ITGAV, FGFR1, TNC, FGF11, FGFR3, RHOJ, LAMA4, FBLN1, CTSL, DDR2, PDGFRB, MMP24, CD151, ACAN, RHOU, ARF4, COL3A1, FGFR2, COL7A1, ADAM15, CD47, COL10A1, VTN, RHOG, CAPN2, BGN, CXCR4, HTRA1, ICAM2, JAM3, ANG, TGIF1, ITGB7, CD63, RHOA, RHOC, ITGA2, DPP4, COL6A3, COL15A1, SDC2, SPOCK2, DCN, BCAN, COL13A1, ITGB1, MATN3, CLDN1, TIMP2, ARPC5, FN1, CST3, TPM3, MATN1, CD44, HAPLN1, SERPINH1, TIMP3, ITGB3, PLOD3, L1CAM, COL11A1, SPARC, COL6A2, FGF10, P4HA1, IBSP, GREM1, COL6A1, HAS1, CTGF, BMP1, RHOB, VCAN, TGFBR1, MMP10, COL5A2, MFAP2, FGF5, DPT, COL8A1, ITGB5, BDKRB1, COL1A2, TGFB1, MMP11, SERPINE1, LOXL2, FSCN1, SPP1, ITGA4, POSTN, COL5A1, RELN, MMP16, CCDC80, LAMA1, COL1A1, FBN1, ITGA5, LOX, MMP17, LOXL1, LCP1, SDC1, MMP2, MMP14, FAP, TNXB, TGFBI, HAS2, MFAP5, and/or CTSK;
(g) upregulated or downregulated calcium signaling-related genes comprising genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway, wherein the downregulated genes comprise CACNA1G, EDNRB, CHRM1, ADRB1, PLCG2, ERBB4, RYR3, ERBB3, ATP2A2, ADRB2, P2RX7, PLCB1, ATP2A1, CAMK2A, RYR2, HRH2, PHKA1, PHKG1, ATP2B2, PDE1C, HTR4, CACNA1C, CAMK2B, SLC8A1, SLC25A5, CACNA1S, P2RX1, TBXA2R, CAMK2D, PRKACA, PHKA2, GRIN2C, PPIF, ADCY9, PTK2B, VDAC3, EGFR, VDAC2, PHKB, NOS2, PLCD1, GRIN2A, CALML4, P2RX6, TNNC2, VDAC1, PHKG2, CHRNA7, PRKCB, GRPR, SLC25A4, NOS1, CCKBR, ADORA2A, ADCY3, NTSR1, GRIN1, ADRA1A, PDGFRA, PPP3CB, NOS3, HTR2C, MYLK, TNNC1, and/or PLCG,
and the upregulated genes comprise GNA15, CACNA1H, GNAS, HTR5A, PTGFR, PTGER1, TACR1, RYR1, PRKACB, CCKAR, CD38, PTAFR, CALM2, PDE1A, PPP3R1, LHCGR, ADCY2, TACR2, PLCB3, GNA11, BDKRB2, PRKCG, STIM1, ADCY4, ATP2A3, GNA14, AVPR1A, CACNA1B, ITPR2, PPP3CC, HTR7, HTR2B, PPP3CA, PDGFRB, SPHK2, PRKCA, GRIN2D, PDE1B, GNAQ, CALM1, ITPKB, HRH1, CAMK4, P2RX4, PTGER3, ITPR1, ADCY7, ADORA2B, F2R, CACNA1E, BDKRB1, SPHK1, CACNA1A, ADRA1B, ADRB3, ITPR3, and/or ADCY8; and/or
(h) an elastic modulus of about 3 kPa to about 5 kPa.
A second aspect provides a method of making a 3D myocardial infarct organoid, the method comprising: culturing cardiomyocytes with non-myocytes for about 1 day to 20 days to form a self-assembled 3D cardiac organoid under normoxic conditions; and exposing the 3D cardiac organoid to hypoxic conditions for about 1 day to 20 days, thereby generating the 3D myocardial infarct organoid.
A third aspect of the invention provides a method of making a 3D myocardial ischemia-reperfused organoid, the method comprising: culturing cardiomyocytes with non-myocytes for about 1 day to 20 days to form a 3D cardiac organoid under normoxic conditions; exposing the 3D cardiac organoid under hypoxic conditions for about 1 day to 20 days to form a 3D myocardial infarct organoid, and exposing the 3D myocardial infarct organoid to normoxic conditions (and/or fresh culture media) for about 5 seconds to 20 days, thereby generating the 3D myocardial ischemia-reperfused organoid.
Further provided are methods for screening a compound for improving or diminishing cardiac function.
These and other aspects of the invention are set forth in more detail in the description of the invention below.
The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
Unless otherwise defined, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
The term “about,” as used herein, when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, “about X” where X is the measurable value, is meant to include X as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein.
As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
As used herein, the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
As used herein, the term “cardiomyocytes” refers to cardiac muscle cells that make up the cardiac muscle (heart muscle). Each myocardial cell contains myofibrils, which are specialized organelles consisting of long chains of sarcomeres, the fundamental contractile units of muscle cells. Cardiomyocytes show striations similar to those on skeletal muscle cells. Unlike multinucleated skeletal cells, the majority of cardiomyocytes contain only one nucleus, although they may have as many as four. Cardiomyocytes have a high mitochondrial density, which allows them to produce adenosine triphosphate (ATP) quickly, making them highly resistant to fatigue.
As used herein, the term “non-myocytes” refer to cells that are generally responsible for transmitting biochemical, mechanical and electrical cues, which makes them essential components in the cardiac microenvironment. Examples include, but are not limited to, fibroblasts (FBs), stem cells (e.g., human adipose derived stem cells (hADSCs)), endothelial cells (ECs) (e.g., human umbilical vein endothelial cells (HUVECs)), smooth muscle cells, neurons and immune cells, or any combination thereof.
As used herein, the term “contraction amplitude” refers to the ability of the cardiomyocytes present in a myocardial organoid and/or myocardial infarct organoid and/or myocardial ischemic-reperfused organoid to contract. Generally, the contraction amplitude of a heart measures the ability of a cardiac muscle to contract, which is essential for pushing blood through the heart and/or body of a mammal and is therefore a relevant measurement for the cardiac organoid. Specifically, for spherical microtissues, like cardiac organoids, contraction amplitude is measured from the percent change in fractional projected area change from peak contraction to relaxation calculated from videos of contraction.
As used herein, the term “beat rate” refers to the number of contractions per minute (bpm) of the cardiomyocytes in an organoid.
As used herein, the term “calcium transient amplitude” refers to the changes in calcium fluorescence signal as measured by fluorescent calcium probes, including but not limited to GCaMP6, indicating the relative calcium concentration in the organoid as the cardiomyocytes in the organoid contract and/or relax. In general, Ca2+ is released from the sarcoplasmic reticulum (SR) resulting in the efflux of Ca2+ from the SR into the cytoplasm resulting in contraction of the cardiomyocytes in the organoid. Relaxation is initiated by a reduction of [Ca2+] produced either by pumping back into the SR by the SR Ca2+-ATPase (SERCA) or out of the cell, largely by the sarcolemmal NatCa2+ exchange.
As used herein, the term “elastic modulus” describes the degree of stiffness and/or elasticity of a tissue. For myocardial tissue an increase in elastic modulus, i.e., stiffness, prevents contraction of the cardiomyocytes in the organoid and thus results in a decrease in cardiac function.
As used herein, the term “DAPI” stands for 4′,6-diamidino-2-phenylindole, which is a fluorescent stain that binds strongly to adenine-thymine rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower. Thus a “DAPI-positive area” would be the total area that stains positive for DAPI per organoid after fixation and permeabilization on 7 μm thickness frozen cross sections of cardiac organoids.
As used herein, the term “TUNEL” stands for terminal deoxynucleotidyl transferase dUTP nick end labeling, which is a method for detecting DNA fragmentation by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis. Thus, the TUNEL method may be used to detect apoptotic DNA fragmentation, therefore, may be used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells. The assay relies on the use of terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another marker, to 3′-hydroxyl termini of DNA double strand breaks. It may also be used to label cells in which the DNA is damaged by other means than in the course of apoptosis. Thus a TUNEL-positive area” would be an area that stains positive for TUNEL per organoid after fixation and permeabilization on 7 μm thickness frozen cross sections of cardiac organoids.
In the disclosure, the inventors combined non-genetic causal factors of MI with their previously established cardiac organoids to create the first human organoid model of cardiac infarction.10,11 In particular, the inventors leveraged the diffusion limitation in 3D microtissues to recreate the nutrient (e.g., oxygen) diffusion gradient across infarcted hearts (i.e., infarct-border-remote zones) in human cardiac organoids to induce cardiac organotypic response to infarction. This enabled the recapitulation of major MI hallmarks in human cardiac organoids at the transcriptomic, structural and functional level.
During a heart attack, a blocked artery limits the delivery of blood to downstream myocardium causing massive cell death, leading to reduced ability to pump blood to the body that triggers compensatory efforts by the nervous system to restore cardiac output (i.e., adrenergic stimulation via norepinephrine).12 Given the inability of the damaged heart to fully compensate or regenerate, this positive feedback causes chronic heart dysfunction and ultimately heart failure.12 With the understanding of major upstream causal factors in heart failure, the inventors leveraged inherent oxygen diffusion limitations in 3D microtissues and chronic adrenergic stimulation to induce organotypic response of myocardium to infarction with human cardiac organoids (
As human cardiac tissues post-MI are difficult to obtain28, human cardiac infarct organoids offer a model of the acute post-infarct heart tissue, a stage that is critical for the understanding the short-term post-MI injured state of both ischemia and ischemia/reperfusion (I/R) caused cardiac injury. While organoids have traditionally been prepared with embryonic bodies, the current disclosure demonstrates that the self-assembly of tissue-specific cell types provides a powerful alternative to prepare organoids with tissue-mimetic transcriptome, structure and function.29
Thus, one aspect of the invention relates to a three-dimensional (3D) myocardial infarct organoid, comprising cardiomyocytes and non-myocytes, wherein the 3D myocardial infarct organoid comprises, consists essentially of, or consists of:
(a) an apoptotic interior region due to lack of oxygen surrounded by a viable periphery that comprises, consists essentially of, or consists of a region of about 20-75 μm from the organoid edge
(b) a ratio of a TUNEL-positive area to a DAPI-positive area ratio in the apoptotic interior region of in a organoid cross-section ranging from about 0.03 to 1.0;
(c) a contraction amplitude from about 0% to 5%;
(d) a beat rate of about 0 to 90 beats per minute;
(e) a calcium transient amplitude measured as a change in fluorescence divided by the starting fluorescence of about 0% to 40%;
(f) upregulated or downregulated fibrosis-related genes', wherein the downregulated genes include, but are not limited to, COL22A1, COL11A2, FGF12, SPINT1, COL9A2, MMP13, HPN, CTSS, FGF7, A2M, COL9A1, FBN2, FGF9, LAMA3, FLT1, SMOC2, COL2A1, FGF2, LAMB3, LAMAS, PDGFB, SMAD3, ICAM5, LAMB2, FGF18, MMP9, CXCL12, COL19A1, FGF13, COL4A5, COL26A1, F11R, COL14A1, COL9A3, FGF1, ICAM1, HBEGF, MDK, ITGA6, TGFB3, LAMA2, RHOQ, RND2, TGFB2, LAMC2, CCDC88A, ITGAE, JUP, ITGAM, COL4A2, CDH2, ITGB2, TGFBR3, BTG1, COL4A1, COL4A3, PDGFRA, FGFR4, SDC4, MMPI, FGF6, ITGA8, and/or COL4A4,
and upregulated genes include, but are not limited to, FGF8, ITGB1BP1, ITGA7, TGIF2, MMP12, PIK3CA, RHOH, COL18A1, ITGAL, LAMC1, RPS6KB1, TGFBR2, RHOD, PIK3CD, MMP3, RAC1, MMP8, ITGB4, ARPCSL, ITGA3, COL17A1, ADAM9, CD2AP, KDR, LAMB1, COL12A1, ITGAX, ABU, MMP15, FGF14, TGFB1I1, SDC3, ITGAV, FGFR1, TNC, FGF11, FGFR3, RHOJ, LAMA4, FBLN1, CTSL, DDR2, PDGFRB, MMP24, CD151, ACAN, RHOU, ARF4, COL3A1, FGFR2, COL7A1, ADAM15, CD47, COL10A1, VTN, RHOG, CAPN2, BGN, CXCR4, HTRA1, ICAM2, JAM3, ANG, TGIF1, ITGB7, CD63, RHOA, RHOC, ITGA2, DPP4, COL6A3, COL15A1, SDC2, SPOCK2, DCN, BCAN, COL13A1, ITGB1, MATN3, CLDN1, TIMP2, ARPCS, FN1, CST3, TPM3, MATN1, CD44, HAPLN1, SERPINH1, TIMP3, ITGB3, PLOD3, L1CAM, COL11A1, SPARC, COL6A2, FGF10, P4HA1, IBSP, GREM1, COL6A1, HAS1, CTGF, BMP1, RHOB, VCAN, TGFBR1, MMP10, COL5A2, MFAP2, FGFS, DPT, COL8A1, ITGB5, BDKRB1, COL1A2, TGFB1, MMP11, SERPINE1, LOXL2, FSCN1, SPP1, ITGA4, POSTN, COL5A1, RELN, MMP16, CCDC80, LAMA1, COL1A1, FBN1, ITGA5, LOX, MMP17, LOXL1, LCP1, SDC1, MMP2, MMP14, FAP, TNXB, TGFBI, HAS2, MFAP5, and/or CTSK;
(g) upregulated or downregulated calcium signaling-related genes comprising genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway, wherein the downregulated genes include, but are not limited to, CACNA1G, EDNRB, CHRM1, ADRB1, PLCG2, ERBB4, RYR3, ERBB3, ATP2A2, ADRB2, P2RX7, PLCB1, ATP2A1, CAMK2A, RYR2, HRH2, PHKA1, PHKG1, ATP2B2, PDE1C, HTR4, CACNA1C, CAMK2B, SLC8A1, SLC25A5, CACNA1S, P2RX1, TBXA2R, CAMK2D, PRKACA, PHKA2, GRIN2C, PPIF, ADCY9, PTK2B, VDAC3, EGFR, VDAC2, PHKB, NOS2, PLCD1, GRIN2A, CALML4, P2RX6, TNNC2, VDAC1, PHKG2, CHRNA7, PRKCB, GRPR, SLC25A4, NOS1, CCKBR, ADORA2A, ADCY3, NTSR1, GRIN1, ADRA1A, PDGFRA, PPP3CB, NOS3, HTR2C, MYLK, TNNC1, and/or PLCG,
and the upregulated genes include, but are not limited to, GNA15, CACNA1H, GNAS, HTR5A, PTGFR, PTGER1, TACR1, RYR1, PRKACB, CCKAR, CD38, PTAFR, CALM2, PDE1A, PPP3R1, LHCGR, ADCY2, TACR2, PLCB3, GNA11, BDKRB2, PRKCG, STIM1, ADCY4, ATP2A3, GNA14, AVPR1A, CACNA1B, ITPR2, PPP3CC, HTR7, HTR2B, PPP3CA, PDGFRB, SPHK2, PRKCA, GRIN2D, PDE1B, GNAQ, CALM1, ITPKB, HRH1, CAMK4, P2RX4, PTGER3, ITPR1, ADCY7, ADORA2B, F2R, CACNA1E, BDKRB1, SPHK1, CACNA1A, ADRA1B, ADRB3, ITPR3, and/or ADCY8; and/or
(h) an elastic modulus of about 3 kPa to about 5 kPa.
In some embodiments, the cardiomyocytes may comprise induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiac progenitor cells, primary cardiomyocytes, or any combination thereof. In some embodiments, the cardiomyocytes and non-myocytes may be present in a ratio of about 95:5 to about 5:95 of cardiomyocytes to non-myocytes. In some embodiments, the cardiomyocytes and non-myocytes may be present in a ratio of about 60:40 to about 40:60 of cardiomyocytes to non-myocytes.
In some embodiments, the non-myocytes may comprise a combination of fibroblasts (FBs), endothelial cells (ECs) and mesenchymal stem cells (MSCs). In some embodiments, the non-myocytes may comprise FBs in amount of about 50% to 60% based on the total number of non-myocytes, ECs in an amount of about 25% to about 35% based on the total number of non-myocytes, and MSCs in an amount of about 10% to about 20% based on the total number of non-myocytes. In some embodiments, the cardiomyocytes and/or non-myocytes are derived from a human.
Another aspect of the invention relates to a method of making a 3D myocardial infarct organoid, the method comprising:
culturing cardiomyocytes with non-myocytes for about 1 day to 20 days to form a self-assembled 3D cardiac organoid under normoxic conditions; and
exposing the 3D cardiac organoid to hypoxic conditions for about 1 day to 20 days, thereby generating the 3D myocardial infarct organoid.
Another aspect of the invention relates to a method of making a 3D myocardial ischemia-reperfused organoid, the method comprising:
culturing cardiomyocytes with non-myocytes for about 1 day to 20 days to form a 3D cardiac organoid under normoxic conditions;
exposing the 3D cardiac organoid under hypoxic conditions for about 1 day to 20 days to form a 3D myocardial infarct organoid; and
exposing the 3D myocardial infarct organoid to normoxic conditions and/or exposing the 3D myocardial infarct organoid to fresh culture media for about 5 seconds to 20 days,
thereby generating the 3D myocardial ischemia-reperfused organoid.
In some embodiments, the cardiomyocytes may be cultured with the non-myocytes at a ratio of about 95:5 to about 5:95 of cardiomyocytes to non-myocytes. In some embodiments, the cardiomyocytes may be cultured with the non-myocytes at a ratio is about 60:40 to about 40:60 of cardiomyocytes to non-myocytes.
In some embodiments, the non-myocytes may comprise fibroblasts (FBs), endothelial cells (ECs), mesenchymal stem cells (MSCs), or any combination thereof. In some embodiments, the non-myocytes may comprise FBs in amount of about 50% to 60% based on the total number of non-myocytes, ECs in an amount of about 25% to about 35% based on the total number of non-myocytes, and MSCs in an amount of about 10% to about 20% based on the total number of non-myocytes. In some embodiments, the ECs may comprise human umbilical vein endothelial cells (HUVECs) and/or MSCs may comprise human adipose derived stem cells (hADSCs).
In some embodiments, the cardiomyocytes and the non-myocytes may be cultured at a total concentration of about 1×105 cells/mL to about 1×107 cells/mL. In some embodiments, the cardiomyocytes and/or non-myocytes are from a human.
In some embodiments, the cardiomyocytes and non-myocytes may be cultured in the presence of norepinephrine, angiotensin II, TNF-alpha, interfering RNAs, microRNAs, matrix metalloproteases, or any combination thereof. In some embodiments, the cardiomyocytes and non-myocytes may be cultured in the presence of norepinephrine at a concentration of about 0.01 μM to about 10 μM.
In some embodiments, the hypoxic conditions may comprise a partial pressure of oxygen in the gas phase that is less than about 15% of the total barometric pressure. In some embodiments, the normoxic conditions may comprise a partial pressure of oxygen in the gas phase of about 16% to about 20.9% of the total barometric pressure.
In some embodiments, the 3D myocardial infarct organoid and/or the 3D myocardial ischemia-reperfused organoid may comprise an average diameter of about 100 μm to about 1000 μm.
Another aspect of the invention relates to a method for screening a compound for improving cardiac function, the method comprising:
contacting the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention with the compound;
measuring in the 3D myocardial infarct organoid or 3D myocardial ischemia-reperfused organoid the size of an interior apoptotic region, a ratio of a TUNEL-positive area to a DAPI-positive area in the apoptotic region, a contraction amplitude, a beat rate, a calcium transient amplitude, and/or an elastic modulus; and
determining that the compound improves cardiac function when
(a) the interior apoptotic region is reduced by at least about 30% when compared a control;
(b) the ratio of TUNEL-positive area to DAPI-positive area is reduced by at least about 30% when compared to a control;
(c) the contraction amplitude is increased by at least about 30% when compared to a control;
(d) the calcium transient amplitude is increased by about 30% when compared to a control; and/or
(e) the elastic modulus is decreased by about 30% when compared to a control; wherein the control is the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention that has not been contacted with the compound.
Another aspect of the invention relates to a method for screening a compound for diminishing cardiac function, the method comprising:
contacting the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention with the compound;
measuring in the 3D myocardial infarct organoid or 3D myocardial ischemia-reperfused organoid the size of an interior apoptotic region, a ratio of a TUNEL-positive area to a DAPI-positive area in the apoptotic region, a contraction amplitude, a beat rate, a calcium transient amplitude, and/or an elastic modulus; and
determining that the compound diminishes cardiac function when
(a) the interior apoptotic region is increased by at least about 30% when compared to a control;
(b) the ratio of TUNEL-positive area to DAPI-positive area is increased by at least 30% when compared to a control;
(c) the contraction amplitude is decreased by at least about 30% when compared to a control;
(d) the calcium transient amplitude is decreased by about 30% when compared to a control; and/or
(e) the elastic modulus is increased by about 30% when compared to a control; wherein the control is the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention that that has not been contacted with the compound.
In some embodiments, the compound may be a therapeutic compound for treating, for example, cardiovascular disease, diabetes, liver disease, kidney disease, and/or cancer
One aspect of the invention relates to a three-dimensional (3D) myocardial infarct organoid comprising cardiomyocytes and non-myocytes, wherein the 3D myocardial infarct organoid can be characterized by one or more of the following characteristics: (a) size of the apoptotic region, (b) ratio of TUNEL-positive area to DAPI-positive area in the apoptotic region, (c) contraction amplitude, (d) beat rate, (e) calcium transient amplitude, (e) upregulated and/or downregulated fibrosis-related genes, (f) upregulated and/or downregulated KEGG calcium signaling pathway genes, and/or (h) elastic modulus.
In some embodiments, the cardiomyocytes include, but are not limited to, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiac progenitor cells, primary cardiomyocytes, or any combination thereof. In some embodiments, non-myocytes include, but are not be limited to, fibroblasts (FBs), endothelial cells (ECs), and mesenchymal cells (MSCs). Exemplary endothelial cells include, but are not limited to, human umbilical vein endothelial cells (HUVECs). Exemplary mesenchymal cells (MSCs) include, but are not limited to, human adipose derived stem cells (hADSCs).
In some embodiments, the cardiomyocytes and/or non-myocytes are from a mammal. A mammal may include but is not limited to a human, a nonhuman primate, a domesticated mammal (e.g., a dog, a cat, a rabbit, a guinea pig, a rat), or a livestock and/or agricultural mammal (e.g., a horse, a bovine, a pig, a goat). In some embodiments, the mammal is a human.
In some embodiments, the cardiomyocytes and non-myocytes are present in a ratio of about 95:5 to about 5:95, about 90:10 to about 10:90, about 85:15 to about 15:85, about 70:30 to about 30:70, or about 60:40 to about 40:60 of cardiomyocytes to non-myocytes (e.g., about 98:2, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, or about 2:98 of cardiomyocytes to non-myocyte cells.
The amount of fibroblasts (FBs), endothelial cells (ECs), and mesenchymal cells (MSCs) that make up the total amount of non-myocytes present in the 3D myocardial infarct organoid can vary. For example, in some embodiments, the non-myocytes may comprise FBs in amount of about 1% to about 100%, about 20% to about 80%, about 40% to about 70%, or about 50% to about 60% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes may comprise ECs in an amount of about 1% to about 100%, about 10% to about 80%, about 20% to about 50%, or about 25% to about 35% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes may comprise MSCs in an amount of about 1% to about 100%, about 5% to about 50%, or about 10% to about 20% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes may comprise fibroblasts (FBs), ECs, and MSCs in a ratio of about 4:2:1 of FBs:ECs:MSCs.
In some embodiments, the 3D myocardial infarct organoid comprises an apoptotic region that is due to lack of oxygen and is surrounded by a viable periphery comprising a region of about 20 μm to about 75 μm from the organoid edge, wherein the organoid edge is defined by the outermost DAPI stained nuclei.
In some embodiments, an organoid cross-section taken from the apoptotic region of the 3D myocardial infarct organoid comprises a ratio of a TUNEL-positive area to a DAPI-positive area may range from about 0.03 to about 1, about 0.1 to about 0.9, about 0.25 to about 0.75, or about 0.4 to about 0.6, wherein the ratio of the TUNEL-positive area to the DAPI-positive area of a region in a 3D cardiac organoid having no apoptotic region (e.g., control) is typically in a range from about 0 to about less than 0.03 (or less than about 0.01, 0.02, or about 0.025).
In some embodiments, the 3D myocardial infarct organoid may comprise a contraction amplitude from about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, or from about 0% to about 4% (e.g., about 1%, about 2%, about 3% about 4%, or about 5%), wherein the contraction amplitude of a 3D cardiac organoid having no apoptotic region (e.g., control) is typically in a range of about 0.5% to about 10%, about 6% to about 10%, or about 8% to about 10%.
In some embodiments, the 3D myocardial infarct organoid may comprise a beat rate of about 0 to about 90 beats per minute, about 0 to about 50, about 0 to about 40, about 0 to about 30, about 0 to about 20, or about 0 to about 10 (e.g., about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 beats per minute), wherein the beat rate of a 3D cardiac organoid having no apoptotic region (e.g., control) comprises a beat rate in a range from about 15 to about 75 beats per minute, about 55 to about 75 beats per minute, or about 60 to about 75 beats per minute.
In some embodiments, the 3D myocardial infarct organoid may comprise a calcium transient amplitude measured as a change in fluorescence divided by the starting fluorescence of about 0% to about 40%, about 0% to about 30%, about 0% to about 20%, or from about 0% to about 10% (e.g., about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, or about 40%), wherein the calcium transient amplitude of a 3D cardiac organoid having no apoptotic region (e.g., control) ranges from about 10% to about 100%, about 45% to about 100%, 55% to about 100%, about 65% to about 100%, about 75% to about 100%, or from about 85% to about 100%.
In some embodiments, the 3D myocardial infarct organoid may comprise upregulated or downregulated fibrosis-related genes, wherein the downregulated genes comprise, consists essentially of, or consists of COL22A1, COL11A2, FGF12, SPINT1, COL9A2, MMP13, HPN, CTSS, FGF7, A2M, COL9A1, FBN2, FGF9, LAMA3, FLT1, SMOC2, COL2A1, FGF2, LAMB3, LAMAS, PDGFB, SMAD3, ICAM5, LAMB2, FGF18, MMP9, CXCL12, COL19A1, FGF13, COL4A5, COL26A1, F11R, COL14A1, COL9A3, FGF1, ICAM1, HBEGF, MDK, ITGA6, TGFB3, LAMA2, RHOQ, RND2, TGFB2, LAMC2, CCDC88A, ITGAE, JUP, ITGAM, COL4A2, CDH2, ITGB2, TGFBR3, BTG1, COL4A1, COL4A3, PDGFRA, FGFR4, SDC4, MMPI, FGF6, ITGA8, and/or COL4A4,
and upregulated genes comprise, consists essentially of, or consists of FGF8, ITGB1BP1, ITGA7, TGIF2, MMP12, PIK3CA, RHOH, COL18A1, ITGAL, LAMC1, RPS6KB1, TGFBR2, RHOD, PIK3CD, MMP3, RAC1, MMP8, ITGB4, ARPC5L, ITGA3, COL17A1, ADAM9, CD2AP, KDR, LAMB1, COL12A1, ITGAX, ABIl, MMP15, FGF14, TGFB1I1, SDC3, ITGAV, FGFR1, TNC, FGF11, FGFR3, RHOJ, LAMA4, FBLN1, CTSL, DDR2, PDGFRB, MMP24, CD151, ACAN, RHOU, ARF4, COL3A1, FGFR2, COL7A1, ADAM15, CD47, COL10A1, VTN, RHOG, CAPN2, BGN, CXCR4, HTRA1, ICAM2, JAM3, ANG, TGIF1, ITGB7, CD63, RHOA, RHOC, ITGA2, DPP4, COL6A3, COL15A1, SDC2, SPOCK2, DCN, BCAN, COL13A1, ITGB1, MATN3, CLDN1, TIMP2, ARPC5, FN1, CST3, TPM3, MATN1, CD44, HAPLN1, SERPINH1, TIMP3, ITGB3, PLOD3, L1CAM, COL11A1, SPARC, COL6A2, FGF10, P4HA1, IBSP, GREM1, COL6A1, HAS1, CTGF, BMP1, RHOB, VCAN, TGFBR1, MMP10, COL5A2, MFAP2, FGF5, DPT, COL8A1, ITGB5, BDKRB1, COL1A2, TGFB1, MMP11, SERPINE1, LOXL2, FSCN1, SPP1, ITGA4, POSTN, COL5A1, RELN, MMP16, CCDC80, LAMA1, COL1A1, FBN1, ITGA5, LOX, MMP17, LOXL1, LCP1, SDC1, MMP2, MMP14, FAP, TNXB, TGFBI, HAS2, MFAP5, and/or CTSK.
In some embodiments, the 3D myocardial infarct organoid may comprise upregulated or downregulated calcium signaling-related genes comprising genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway, wherein the downregulated genes comprise, consists essentially of, or consists of CACNA1G, EDNRB, CHRM1, ADRB1, PLCG2, ERBB4, RYR3, ERBB3, ATP2A2, ADRB2, P2RX7, PLCB1, ATP2A1, CAMK2A, RYR2, HRH2, PHKA1, PHKG1, ATP2B2, PDE1C, HTR4, CACNA1C, CAMK2B, SLC8A1, SLC25A5, CACNA1S, P2RX1, TBXA2R, CAMK2D, PRKACA, PHKA2, GRIN2C, PPIF, ADCY9, PTK2B, VDAC3, EGFR, VDAC2, PHKB, NOS2, PLCD1, GRIN2A, CALML4, P2RX6, TNNC2, VDAC1, PHKG2, CHRNA7, PRKCB, GRPR, SLC25A4, NOS1, CCKBR, ADORA2A, ADCY3, NTSR1, GRIN1, ADRA1A, PDGFRA, PPP3CB, NOS3, HTR2C, MYLK, TNNC1, and/or PLCG,
and the upregulated genes comprise, consists essentially of, or consists of GNA15, CACNA1H, GNAS, HTR5A, PTGFR, PTGER1, TACR1, RYR1, PRKACB, CCKAR, CD38, PTAFR, CALM2, PDE1A, PPP3R1, LHCGR, ADCY2, TACR2, PLCB3, GNA11, BDKRB2, PRKCG, STIM1, ADCY4, ATP2A3, GNA14, AVPR1A, CACNA1B, ITPR2, PPP3CC, HTR7, HTR2B, PPP3CA, PDGFRB, SPHK2, PRKCA, GRIN2D, PDE1B, GNAQ, CALM1, ITPKB, HRH1, CAMK4, P2RX4, PTGER3, ITPR1, ADCY7, ADORA2B, F2R, CACNA1E, BDKRB1, SPHK1, CACNA1A, ADRA1B, ADRB3, ITPR3, and/or ADCY8.
In some embodiments, the 3D myocardial infarct organoid comprises an elastic modulus of about 3 kPa to about 5 kPa, about 3.6 kPa to about 5 kPa, about 4 kPa to about 5 kPa, or about 4.5 kPa to about 5 kPa (e.g., about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5 kPa), wherein the elastic modulus of a 3D cardiac organoid having no apoptotic region (e.g., control) ranges from about 2 kPa to less than 3.5 kPa, about 2 kPa to about 3 kPa, or from about 2 kPa to about 2.5 kPa (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, or about 3.4 kPa).
In some embodiments, a 3D myocardial infarct organoid of the invention may beat asynchronously. In some embodiments, a 3D myocardial infarct organoid of the invention may beat synchronously. In some embodiments, when synchrony of beat is measured in a population of 3D myocardial infarct organoids of the invention, all of the organoids in the population may beat synchronously. In some embodiments, when synchrony of beat is measured in a population of 3D myocardial infarct organoids of the invention, all of the organoids in the population may beat asynchronously. In some embodiments, when synchrony of beat is measured in a population of 3D myocardial infarct organoids of the invention, some of the organoids in the population may beat synchronously and others in the population may beat asynchronously. Thus, in some embodiments, a population of 3D myocardial infarct organoids may comprise a subpopulation of organoids that beat asynchronously. In some embodiments, a population of 3D myocardial infarct organoids of the invention may have a beat asynchrony of about 30% to 100% (e.g., about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the organoids in a population of 3D myocardial infarct organoids of the invention may beat asynchronously).
The 3D myocardial infarct organoid of the invention may comprise any one or more of the above described features in any combination thereof.
II. Methods of Making a Three Dimensional (3D) Myocardial Infarct Organoid and/or a 3D Myocardial Ischemia-Reperfused Organoid
One aspect of the invention relates to a method of making a 3D myocardial infarct organoid, the method comprising culturing cardiomyocytes with non-myocytes for about 1 to about 20 days to form a self-assembled 3D cardiac organoid under normoxic conditions and exposing the 3D cardiac organoid to hypoxic conditions for about 1 to about 20 days, thereby generating the 3D myocardial infarct organoid.
In some embodiments, the cardiomyocytes include, but are not limited to, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiac progenitor cells, primary cardiomyocytes, or any combination thereof. In some embodiments, non-myocytes include, but are not be limited to, fibroblasts (FBs), endothelial cells (ECs), and mesenchymal cells (MSCs). Exemplary endothelial cells include but are not limited to human umbilical vein endothelial cells (HUVECs). Exemplary mesenchymal cells (MSCs) include but are not limited to human adipose derived stem cells (hADSCs).
In some embodiments, the cardiomyocytes and/or non-myocytes are from a mammal. A mammal may be a human, a nonhuman primate, a domesticated mammal (e.g., a dog, a cat, a rabbit, a guinea pig, a rat), or a livestock and/or agricultural mammal (e.g., a horse, a bovine, a pig, a goat). In some embodiments, the mammal is a human. In some embodiments, the cardiomyocytes and/or myocytes are from a human. In some embodiments, the cardiomyocytes and myocytes may be from a subject (e.g., human) undergoing therapy or in need of therapy for a cardiac disease. In such cases, the organoids developed from these cells may be used for development of a personalized therapeutic protocol for the subject.
In some embodiments, the cardiomyocytes may be cultured with the non-myocytes at a ratio of about 95:5 to about 5:95, about 90:10 to about 10:90, about 85:15 to about 15:85, about 70:30 to about 30:70, or about 60:40 to about 40:60 of cardiomyocytes to non-myocytes (e.g., about 98:2, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, or about 2:98 of cardiomyocytes to non-myocytes.
In some embodiments, the amount of fibroblasts (FBs), endothelial cells (ECs), and mesenchymal cells (MSCs) comprising the total amount of non-myocytes that are being cultured with the cardiomyocytes can vary. For example, in some embodiments, the non-myocytes cultured with the cardiomyocytes may comprise FBs in amount of about 1% to about 100%, about 20% to about 80%, about 40% to about 70%, or about 50% to about 60% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes cultured with the cardiomyocytes may comprise ECs in an amount of about 1% to about 100%, about 10% to about 80%, about 20% to about 50%, or about 25% to about 35% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes cultured with the cardiomyocytes comprise MSCs in an amount of about 1% to about 100%, about 5% to about 50%, or about 10% to about 20% based on the total number of non-myocytes (e.g., about 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any value or range therein). In some embodiments, the non-myocytes cultured with the cardiomyocytes comprise FBs, ECs, and MSCs in a ratio of about 4:2:1 of FBs:ECs:MSCs.
In some embodiments, the cardiomyocytes (e.g., iPSC-CMs) may be cultured with the non-myocytes (e.g., FBs, endothelial cells, and/or mesenchymal stem cells) at a total concentration of about 1×105 cells/mL to about 1×107 cells/mL (e.g., about 1×105, 2×105, 3×105, 4×105, 5×105, 5×105 6×105, 7×105, 8×105,9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 5×106 6×106, 7×106, 8×106, 9×106, 1×107 cells/mL, or any value or range therein).
In some embodiments, the cardiomyocytes may be cultured with the non-myocytes in a cell suspension in microwells composed of non-fouling materials. The cell suspension may comprise one or more culture media suitable for culturing cardiomyocytes and/or non-myocytes. Culture media for culturing cardiomyocytes and/or non-myocytes are well known in the art. The type of culture media in a cell suspension can vary. For example, a cell suspension may comprise a larger amount of cardiomyocyte cell culture media when the amount of cardiomyocytes being cultured is greater than the amount of non-myocytes. In another example, the cell suspension may comprise a larger amount of non-myocyte cell culture media when the amount of non-myocytes being cultured is greater than the amount of cardiomyocytes being cultured in the cell suspension. Thus, the amounts of all the specific media may be ratiometric reflecting the cell ratio of the organoid.
The micro-wells employed in the inventive method can be any micro-wells comprising non-fouling materials known in the art that are suitable for microtissue fabrication. In some embodiments, the non-fouling materials comprise agarose or non-adhesive self-assembly plates, such as the InSphero Gravity TRAP ultra-low attachement plate. The non-fouling materials may comprise any suitable material, such as, for example, agarose gel, polyethylene glycol, alginate, hyaluronic acid, polyacryylic acid, polyacrylic amide, polyvinyl alcohol, polyhydroxyethyl methacrylate, methacrylated dextrans, poly(N-isopropylacrylamide), and any combination thereof. In some embodiments, the substrate may be any suitable unfouling hydrogel.
In some embodiments, the cardiomyocytes are cultured with the non-myocytes for about 1 to about 20 days, about 5 to about 15 days, or about 8 to about 12 days (e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, and any range or value herein).
In some embodiments, the cardiomyocytes are cultured with the non-myocyte cells thereby forming a self-assembled 3D cardiac organoid under normoxic conditions, wherein normoxic conditions comprise a partial pressure of oxygen in the gas phase of about 16% to about 20.9% of the total barometric pressure (or at least about 16%, about 17%, about 18%, about 19%, or at least about 20% of the total barometric pressure).
In some embodiments, the 3D cardiac organoid is exposed to hypoxic conditions, wherein hypoxic conditions comprise a partial pressure of oxygen in the gas phase of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or at least lower than about 1% of the total barometric pressure. In some embodiments, the hypoxic condition can include 0% oxygen of the total barometric pressure.
In some embodiments, the 3D myocardial organoid is exposed to the hypoxic conditions for 1 to about 20 days, about 5 to about 15 days, or about 8 to about 12 days (e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days, and any range or value therein).
In some embodiments, the cardiomyocytes are cultured with the non-myocytes in the presence of an additional agent selected from norepinephrine, angiotensin II, TNF-alpha, interfering RNAs, microRNAs, matrix metalloproteases, and any combination thereof. The amount of the additional agent can vary. For example, in some embodiments, the amount of the additional agent may range from about 0.01 μM to about 10 μM, about 1 μM to about 8 μM, or from about 3 μM to about 5 μM (e.g., about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or from about 10 μM, or any range or value therein).
Another aspect of the invention relates to a method of making a 3D myocardial ischemia-reperfused organoid, wherein the 3D myocardial infarct organoid of the invention is exposed to normoxic conditions. For example, in some embodiments, a method of making a 3D myocardial ischemia-reperfused organoid may comprise the steps of culturing cardiomyocytes with non-myocytes for about 1 to about 20 days to form a 3D cardiac organoid under normoxic conditions, exposing the 3D cardiac organoid under hypoxic conditions for about 1 day to 20 days to form the 3D myocardial infacrt organoid of the invention, which is exposed to normoxic conditions again and/or exposed to/contacted with fresh culture media for about 5 seconds to about 20 days. In some embodiments, the normoxic conditions employed in the exposure of the 3D myocardial cardiac organoid of the invention comprises a partial pressure of oxygen in the gas phase of about 16% to about 20.9% of the total barometric pressure (or at least about 16%, about 17%, about 18%, about 19%, about 20% of the total barometric pressure, or any range or value therein).
In some embodiments, the 3D myocardial infarct organoid may be exposed to the normoxic conditions for about 5 seconds to about 20 days, about lday to about 15 days, or about 8 days to about 12 days (e.g., about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or any range or value therein).
In some embodiments, the 3D myocardial infarct organoid may be exposed to the normoxic conditions for about 5 seconds to about 1 day, about 1 minute to about 1 day, about 2 minutes to about 1 day, about 5 minutes to about 1 day, about 10 minutes to about 1 day, about 20 minutes to about 1 day, about 30 minutes to about 1 day, about 40 minutes to about 1 day, about 50 minutes to about 1 day, about 1 hour to about 1 day, about 10 minutes to about 1 hour, about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 1 hour to about 12 hours, about 6 hours to about 10 hours (e.g., about 5 sec, 1 min., 5 min., 10 min., 20 min., 30 min., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or any range or value therein).
In some embodiments, a 3D myocardial infarct organoid may be contacted with fresh culture media, thereby exposing the 3D myocardial infarct organoid to the oxygen present in the fresh culture media and generating a 3D myocardial ischemia-reperfused organoid. In some embodiments, fresh culture media may be added to the culture medium of a 3D myocardial infarct organoid in addition to exposing the 3D myocardial infarct organoid to normoxic conditions (e.g., a partial pressure of oxygen in the gas phase of about 16% to about 20.9% of the total barometric pressure) to generate a 3D myocardial ischemia-reperfused organoid. The amount of fresh culture media added may vary.
A 3D myocardial infarct organoid and/or a 3D myocardial ischemia-reperfused organoid can be in any suitable shape. For example, in some embodiments, the 3D myocardial infarct organoid and/or the 3D myocardial ischemia-reperfused organoid can be in the shape of a spheroid. In some embodiments, the spheroid comprises an average diameter of about 100 to about 1000 μpm, about 200 to about 800 μm, or about 200 to about 400 μm (or of about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, or any value or range therein).
III. Method of Using a Three Dimensional (3D) Myocardial Infarct Organoid and/or a 3D Myocardial Ischemia-Reperfused Organoid
An aspect of the invention relates to employing a 3D myocardial infarct organoid of the invention and/or a 3D myocardial ischemia-reperfused organoid of the invention in a method of screening a compound for its ability to improve or diminish cardiac function. The ability of the compound to improve or diminish cardiac function is determined by contacting the 3D myocardial infarct organoid of the invention and/or the 3D myocardial ischemia-reperfused organoid of the invention with a compound followed by measuring one or more characteristics of the organoid that reflect modulation of cardiac function (e.g., size of the interior apoptotic region of the 3D myocardial infarct organoid and/or 3D myocardial ischemia-reperfused organoid, ratio of the TUNEL-positive area to the DAPI-positive area in the apoptotic region, contraction amplitude, beat rate, calcium transient amplitude, and/or elastic modulus). The measurements of these characteristics can then be compared with corresponding reference values for a 3D myocardial infarct organoid of the invention and/or a 3D myocardial ischemia-reperfused organoid of the invention that has not been contacted with the compound, thereby determining the effect(s) of the compound on one or more of the measured characteristics that reflect cardiac function. The compound can be any compound of interest, such as, for example, a therapeutic compound. Exemplary therapeutic compounds include, but are not limited to, a therapeutic compound for treating cardiovascular disease, diabetes, kidney disease, liver disease, and/or cancer. In some embodiments, the compound is a small-molecule, nucleic-acid based drug and/or protein-based drug.
In some embodiments, a method of screening a compound for improving cardiac function may comprise contacting the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention with the compound and measuring in the 3D myocardial infarct organoid or 3D myocardial ischemia-reperfused organoid one or more of : (a) size of the interior apoptotic region, (b) ratio of a TUNEL-positive area to a DAPI-positive area in the apoptotic region, (c) contraction amplitude, (d) beat rate, (e) calcium transient amplitude, and/or (0 elastic modulus.
In some embodiments, a compound may be determined to improve cardiac function when the size of the interior apoptotic region is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or a 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. The size of the apoptotic region can vary but typically ranges from about 20 lam to about 75 lam in a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to improve cardiac function when the ratio of TUNEL-positive area to DAPI-positive area is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. This ratio can vary but typically ranges from about 0.03 to about 1 in a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to improve cardiac function when the contraction amplitude is increased by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not contacted with the compound. This contraction amplitude can vary but typically ranges from about 0% to about 5% in a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not contacted with the compound.
In some embodiments, a compound may be determined to improve cardiac function when the calcium transient amplitude is increased by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. This calcium transient amplitude can vary but typically ranges from about 0% to about 40% in a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to improve cardiac function when the elastic modulus is decreased by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. The elastic modules of a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid can vary but typically ranges from about 3 kPa to about 5 kPa.
Another aspect of the invention relates to a method for screening a compound for diminishing cardiac function. For example, such compounds include therapeutic compounds used for treating diseases other than cardiovascular diseases. Screening of any cardiovascular effects of such compounds in a 3D myocardial infarct organoids and/or ischemic-reperfused myocardial organoid of the inventions provides useful information as to the potential cardiotoxicity associated with these compounds when administered to a mammals (e.g., a human) that is already cardio-compromised (i.e., wherein the heart is not functioning at full capacity). In some embodiments, the method may comprise contacting the 3D myocardial infarct organoid of the invention or the 3D myocardial ischemia-reperfused organoid of the invention with a test compound and measuring one or more of: (a) size of the interior apoptotic region, (b) ratio of a TUNEL-positive area to a DAPI-positive area in the apoptotic region, (c) contraction amplitude, (d) beat rate, (e) calcium transient amplitude, and/or (f) elastic modulus.
In some embodiments, a compound may be contact with a population of 3D myocardial infarct organoids or a population of 3D ischemia-reperfused organoids. In some embodiments, a population of 3D myocardial infarct organoids or a population of 3D ischemia-reperfused organoids may comprise about 2 to about 100, about 2 to about 80, about 2 to about 70, about 2 to about 50, about 2 to about 40, about 2 to about 35, about 2 to about 25 or about 2 to about 10 3D myocardial infarct organoids or 3D ischemia-reperfused organoids. In some embodiments, the number (percentage of the total population) of asynchronously beating 3D myocardial infarct organoids or 3D ischemia-reperfused organoids in the population may be determined after contacting the population with a test compound. In some embodiments, a compound may be determined to improve cardiac function when the percentage of asynchronously-beating organoids in the population (e.g., an asynchronously-beating subpopulation) is decreased by more than about 30%, about 40%, about 50% about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. In a control population of 3D myocardial infarct organoids and/or a control population of 3D myocardial ischemia-reperfused organoids that have not been contacted with the compound, the percentage of organoids that make up the asynchronously-beating subpopulation may vary but typically ranges from about 30% to about 100%.
In some embodiments, a compound may be determined to diminish cardiac function when the size of the interior apoptotic region is increased by at least about 30% about 40% about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that is not contacted with the compound. This size of the apoptotic region can vary but typically ranges from about 20 μm to about 75 μm in a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to diminish cardiac function when the ratio of TUNEL-positive area to DAPI-positive area is increased by at least 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. This ratio can vary but typically ranges from about 0.03 to about 1 in a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to diminish cardiac function when the contraction amplitude is decreased by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. This contraction amplitude can vary but typically ranges from about 0% to about 5% in a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to diminish cardiac function when the calcium transient amplitude is decreased by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. This calcium transient amplitude can vary but typically range from about 0% to about 40% in a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound.
In some embodiments, a compound may be determined to diminish cardiac function when the elastic modulus is increased by about 30%, about 40%, about 50%, about 60%, about 70% about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not contacted with the compound. This elastic modulus of a control 3D myocardial infarct organoid and/or a control 3D myocardial ischemia-reperfused organoid can vary but typically ranges from about 3 kPa to about 5 kPa.
In some embodiments, a compound may be contact with a population of 3D myocardial infarct organoids or a population of 3D ischemia-reperfused organoids. In some embodiments, a population of 3D myocardial infarct organoids or a population of 3D ischemia-reperfused organoids may comprise about 2 to about 100, about 2 to about 80, about 2 to about 70, about 2 to about 50, about 2 to about 40, about 2 to about 35, about 2 to about 25 or about 2 to about 10 3D myocardial infarct organoids or 3D ischemia-reperfused organoids. In some embodiments, the number (percentage of the total population) of asynchronously beating 3D myocardial infarct organoids or 3D ischemia-reperfused organoids in the population may be determined after contacting the population with a test compound. In some embodiments, a compound may be determined to diminish cardiac function when the percentage of asynchronously-beating organoids in the population (e.g., an asynchronously-beating subpopulation) is decreased by more than about 30%, about 40%, about 50% about 60%, about 70%, about 80%, about 90%, or about 100% when compared to a control 3D myocardial infarct organoid and/or control 3D myocardial ischemia-reperfused organoid that has not been contacted with the compound. In a control population of 3D myocardial infarct organoids and/or a control population of 3D myocardial ischemia-reperfused organoids that have not been contacted with the compound, the percentage of organoids that make up the asynchronously-beating subpopulation may vary but typically ranges from about 30% to about 100%.
The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.
A computational finite element model of nutrient diffusion and transport was developed to predict oxygen concentration profiles within cardiac spheroids (cardiomyocyte-only) based on Fick's Second Law of Diffusion. Given the developmental similarities between hiPSC-CMs and neonatal rat cardiomyocytes1, neonatal cardiomyocyte metabolic data was used according to the detailed oxygen consumption work of Brown and others.2 Specifically, the oxygen diffusivity value (DOx=3.0×10−6 cm2/s) and cardiomyocyte specific consumption rate constants for oxygen (Vmax=5.44×10−8 nmol/cell/s and Km=3.79 nmol/ml) were derived from Brown and others.2 Next, the concentration-dependent nutrient consumption rate of the cardiomyocytes was modeled by the Michaelis-Menten equation R=ρcVmax[C]/Km+[C], where ρc represents spheroid cell density, Vmax maximum rate at high substrate concentration, and Km Michaelis-Menten constant. Physiological (20%) and hypoxic (10%) culture conditions were accounted for in the boundary conditions of the model. In a spherical coordinate system, the internal oxygen concentration profile is governed by the equation D/r2∂/∂r(r2∂C/∂r)−R=0, where C represents nutrient concentration, r radial distance from spheroid center, D nutrient diffusivity, and R nutrient consumption rate. Upon compiling all of the relevant equations, the finite element model was numerically solved by the software COMSOL Multiphysics, from which the internal oxygen concentration profiles were determined in simulated cardiomyocyte spheroids. In evaluating the finite element model, the semi-circular concentration profiles obtained by solving the finite element model on COMSOL were reformatted into line graphs that showed the change in oxygen levels based on radial position from the spheroid center. These plots were then assessed for trends that indicated the effects of external oxygen concentration on the internal oxygen distributions within the cardiac spheroid.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (iCell Cardiomyocytes, Cellular Dynamics International-CDI, Madison, Wis., USA) were cultured according to the manufacturer's protocol. iCell Cardiomyocytes iPSC donor 01434 (CDI) were used for all experiments and iCell Cardiomyocytes iPSC donor 11713 were used where notated. Briefly, hiPSC-derived cardiomyocytes were plated on 0.1% gelatin coated 6-well plates in iCell Cardiomyocyte Plating Medium (CDI) at a density of about 3×105 to 4.0×105 cells/well and incubated at 37° C. in about 5% CO2 for about 4 days. Two days after plating, the plating medium was removed and replaced with 4 mL of iCell Cardiomyocytes Maintenance Medium (CDI). After 4 days of monolayer pre-culture, cells were detached using trypLE Express (Gibco Life Technologies, Grand Island, N.Y.) and prepared for spheroid/organoid fabrication. Human cardiac ventricular fibroblasts (FBs) (Lot#: 401462, Lonza, Basel, Switzerland) were cultured in FGM-2 media (Lonza) were used at passage 3-5 for spheroid/organoid fabrication. Human umbilical vein endothelial cells (HUVECs) (Lot#: 471466, Lonza) were cultured in EGM-3 media (Lonza) and were used at passage 2-4 for organoid fabrication. Human adipose-derived stem cells (hADSCs) (Lot#: 410257, Lonza) were cultured in low glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, 1% glutamine and 1% antimycin (Gibco Life Technologies, Grand Island, N.Y.). hADSCs were used at passage 5-7 for organoid fabrication.
Non-adhesive agarose hydrogel molds were used as microtissue fabrication molds made from commercial master micro-molds from Microtissues, Inc (Providence, R.I.). Working cell suspensions of each cell type were used at about 4.0×106 cells/ml to make organoid cell ratio mixtures of about 50% hiPSC-CMs and about 50% non-myocyte (4:2:1 ratio of FBs, HUVECs, hADSCs, respectively) and mixed with 1 volume media for a final concentration of about 2.0×106 cells/ml. hiPSC-CM only spheroids were fabricated using 100% hiPSC-CMs at a final concentration of about 2.0×106 cells/ml. Approximately 75 μl of the cell suspension was pipetted into each agarose mold. After the cells settled into the recesses of the mold (15 min), additional media was added to submerge the molds in a 12-well plate and exchanged about every 2 days for the length of the experiment (about 10 days). Day 0 (D0) of the experiment was marked after about 4 days of spheroid assembly. Culture media for cardiac organoids was comprised of a ratiometric combination of cell-specific media reflecting the cell ratio of the organoid. In organoid media, CM-specific media (i.e., CDI hiPSC-CM media supplied without glucose) was substituted with glucose-containing F12/DMEM media with 10% FBS, 1% glutamine, and 1% non-essential amino acids (Gibco).
For the cardiac infarction protocol, microtissues (about150 μm radius on D0) were placed in a hypoxia chamber within the incubator at about 10% O2 with 1 μM of norepinephrine (NE, A7257, Sigma) after 4 days of pre-culture. Media was changed with fresh NE every 2 days for the length of the experiment (10 days). For extended validation studies, 20 ng/ml of human recombinant transforming growth factor beta 1 (TGF-β1, ab50036, Abcam, Cambridge, UK), 10 nM of JQ1 (SML1524, Sigma), and 2 ng/ml of human recombinant vascular endothelial growth factor (VEGF, CC-4114A, Lonza) was added to the media every 2 days for infarct organoids for the length of the experiment (10 days).
Videos of spontaneously beating spheroids from each group were recorded immediately out of the incubator (to reduce temperature induced changes in beating) for each condition using a Carl Zeiss Axiovert A1 Inverted Microscope and Zen 2011 software (Zeiss, Gottingen, Germany). Threshold edge-detecting in ImageJ software (NIH—US National Institutes of Health) was used on high contrast spheroid picture series and graphed to realize beating profiles of fractional area change, from which contraction amplitude was calculated. Contraction amplitudes were calculated as the percent change in fractional area change amplitude between contraction and relaxation. Beat rate was calculated as the number of beats per second.
Total RNA was isolated one day after last media change (D11) according to the kit and protocol of an Omega bio-tek E.Z.N.A. Total RNA kit I (Omega bio-tek, Norcross, Ga.) with the addition of the Homogenizer Columns (Omega bio-tek) during the homogenization step for organoids. For each group, 30-35 organoids were used for RNA isolation. To prepare RNA-Seq libraries, the TruSeq RNA Sample Prep Kit (Illumina, San Diego, Calif., USA) was utilized; 100-200 ng of total input RNA was used in accordance with the manufacturer's protocol. High throughput sequencing (HTS) was performed using an Illumina HiSeq2000 with each mRNA library sequenced to a minimum depth of about50 million reads. A single end 50 cycle sequencing strategy was employed. Data were subjected to Illumina quality control (QC) procedures (>80% of the data yielded a Phred score of 30). RNA-Seq data has been submitted to the NCBI Gene Expression Omnibus, accession number GSE113871
Secondary analyses was carried out on an OnRamp Bioinformatics Genomics Research Platform as previously described (OnRamp Bioinformatics, San Diego, Calif., USA).4 OnRamp's Advanced Genomics Analysis Engine utilizes an automated RNA-Seq workflow to process data, including (1) FastQC to perform data validation and quality control; (2) CutAdapt5 to trim and filter adapter sequences, primers, poly-A tails and other unwanted sequences; (3) TopHat26 to align mRNA-Seq reads to h19 human genome using the ultra-high-throughput short read aligner Bowtie27; (4) HTSeq8 to establish counts which represent the number of reads for each transcript; and (5) DESeq29 to perform DE analysis, which enabled the inference of differential signals with robust statistical power. Transcript count data from DESeq2 analysis of the samples were sorted according to their adjusted p-value (or q-value), which is the smallest false discovery rate (FDR) at which a transcript is called significant. FDR is the expected fraction of false positive tests among significant tests and was calculated using the Benjamin-Hochberg multiple testing adjustment procedure and set to q≤0.1. Advaita Bio's iPathwayGuide was used to perform further characterization, including differential expressed (DE) gene summary, gene ontology, and pathway analysis.19
Previously published transcriptomic datasets were obtained through the Gene Expression Omnibus (GEO). Microarray data from a large human heart failure study11 (GSE5406, “nonfailing” and “ischemic” samples), a time-course mouse myocardial infarction study12 (GSE775, “lv-control”, “MI_ilv-below MI ligation site”, and “MI_nilv-above MI ligation site” samples), and a time-course porcine myocardial infarction study13 (GSE34569, “sham-operated”, “infarct core”, and “remote” samples) were analyzed using the interactive GEO web tool (limma-based), GEO2R, to obtain summary files of genes ordered by significance.14-16 For Venn diagram comparison across datasets, differentially expressed (DE) genes (p<0.05) were directly compared for common genes using Venny and graphed using VennDis.17,18 DE genes for Venn diagrams were obtained from comparisons of control vs infarct (organoids), nonfailing vs ischemic failing (human), control vs MI infarct zone at 1 wk (mouse), and sham vs MI infarct at 1 wk (porcine). RNA-seq datasets were obtained from GEO from a public human heart failure studyl9 (GSE46224, “ischemic cardiomyopathy (ICM)” and “nonfailing” samples) and a mouse 2 wk myocardial infarction study20 (GSE52313, “sham” and “MI” samples). Before merging gene lists for principal component analysis (PCA), organoid, human, and mouse RNA-seq data were normalized to the size of the library through the R package DESeq2 estimateSizeFactors function. For human RNA-seq data, starting counts were calculated based on the supplied RPKM and read counts/mapping details for nonfailing and ICM samples from the associated publication.19 After merging normalized organoid, human, and mouse RNA-seq data, the resulting 4,765 shared genes were loge-transformed followed by quantile normalized using the normalize. quantiles function in the preprocessCore package. PCA was then performed using the prcomp function in R and plotted as scatter plots (using jitter) with basic plotting tools in R. Principal component gene loadings were used as rankings for genes in gene set enrichment analysis (GSEA) with the gene ontology c5.all.v6.1.symbols.gmt file, run with default settings. GSEA terms were considered significant with a normalized p-value (NOM p-value) less than 0.05 and a false discovery rate (FDR) less than 0.25. Supplemental PCA using normalized mouse heart sham samples from another study (GSE96561) was performed after merging data into heart failure data, resulting in 4,244 shared genes.21
Given the lack of cardiac fibrosis pathway term and subjective nature of fibrosis in the heart, the fibrosis-related gene set was constructed based on the “extracellular matrix organization” GO term in addition to a “greedy”-based selection that incorporated common factors in fibrosis and (myo)fibroblast-related genes for a total of 349 genes. The calcium signaling-related gene set was defined as the genes contained in the “calcium signaling pathway” KEGG term 4020 for a total of 182 genes. Cardiac organoid RNA-seq and mouse 1 wk MI microarray (GSE775) were first intersected to isolate for common genes across platforms and then merged again with the filter gene sets, resulting in 208 fibrosis-related genes in organoids and mouse and 121 calcium-related genes in organoids and mouse. Heatmaps of fibrosis-related gene sets in organoids and mouse data were constructed separately using the pheatmap package in R with hierarchical clustering of samples (columns) with category-ordered genes (rows). Heatmaps of calcium handling-related gene sets in organoids and mouse data were constructed in like manner but with row order based on the organoid log-fold change.
Freshly collected organoids were flash frozen in Tissue-Tek OCT compound (Sakura, Torrance, Calif.). Embedded spheroids were cryosectioned into 7 μm thickness layers onto glass slides for immunofluorescence staining. The sections were fixed with pre-cooled acetone (−20° C.) for 10 min. After washing (2 times at 5 min) in PBS with 0.1% Triton X-100 (PBST) (Sigma), blocking buffer was made with 10% serum corresponding to host species of secondary antibody in PBST and added to sections for 1 hr at room temperature. Sections were incubated with primary antibody diluted in PBST (1:200) overnight at 4° C. or 2 hrs at room temperature: mouse anti-alpha smooth muscle actin (A5228, Sigma), mouse anti-alpha sarcomeric actinin (ab9465, Abcam), rabbit anti-collagen type I (ab34710, Abcam), rabbit anti-vimentin (ab92547, Abcam), rabbit anti-von Willebrand factor (ab6994, Abcam). After washing in PBST (2 times at 5 min), sections were incubated with complement secondary antibodies or conjugated primary antibodies diluted in PBST for 1 hr at room temperature: Alexa Fluor 488 phalloidin (A12379, Thermo), goat anti-mouse Alexa Fluor 546 (A1103, Thermo), goat anti-rabbit Alexa Fluor 647 (111-605-144, Jackson ImmunoResearch, West Grove, Pa.). After washing in PBST (2 times at 5 min), nuclei were counterstained with DAPI (Molecular Probes/Invitrogen, Eugene, Oreg.) diluted in PBST for 15 min at room temperature. Following the final wash procedure (PBST, 2 times at 5 min), glass cover slips were added using Fluoro-Gel (Electron Microscopy Sciences, Hatfield, Pa.) and stored in 4° C. until imaging. TCS SP5 AOBS laser scanning confocal microscope (Leica Microsystems, Inc., Exton, Pa.) was for imaging. Vimentin radial density was calculated using the Radial Profile ImageJ plugin and normalized to radius equal to 1. Each analysis consisted of high resolution images at 400× total magnification of cross sections of different organoids.
The Roche In Situ Cell Death Detection Kit (Sigma) was used to visualize apoptotic cells in frozen sections of cardiac organoids based on the Roche protocol. Briefly, cardiac organoid frozen sections were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Following washing in PBS for 30 minutes, samples were incubated in a permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate in PBS) for 2 minutes on ice. Then 50 μl of the TUNEL reaction mixture were added to samples and incubated at 37° C. for 1 hr. After washing in PBS (2 times at 5 min), nuclei were counterstained with DAPI (Molecular Probes/Invitrogen) diluted in PBS for 15 min at ambient temperature. Following the final wash procedure (PBS, 2 times at 5 min), glass cover slips were added to the slides using Fluoro-Gel (Electron Microscopy Sciences). TCS SP5 AOBS laser scanning confocal microscope (Leica Microsystems) was used for imaging.
NADH autoflourescence imaging of live cardiac organoids was performed in media at 37° C. within 1 hour of removal from culture conditions using an Olympus FV1200 laser scanning two-photon fluorescence microscope, which is equipped with a tunable ultrafast laser (Maitai, Newport) and two GaAsP PMTs. The excitation wavelength was tuned to 730 nm for autofluorescence imaging and a filter separated fluorescence with a passing band of violet (420-460 nm), which selected for NAD(P)H fluorescence.22 NADH index was calculated as the mean grey value of the sample (30-40 μm below surface) NADH autofluorescence minus the background mean grey value minus the mean grey value of the dead sample NADH autofluorescence.
A micropipette aspiration was performed in media similarly to previous studies using a custom-built fluid reservoir to generate a fixed pressure of 40 cm H2O (about 3.9 kPa) in pulled micropipette to apply the suction force on test organoids.23,24 Validation and stability of pressure changes were confirmed using an in-line 5 kPa 2-port pressure transducer with about 1 Pa sensitivity (Honeywell, Morristown, N.J.). Micropipettes were pulled to a final inner diameter of approximately 40-60 μm. Prior to and during testing, organoids were soaked in a 30 mM solution of 2,3-butanedione monoxime (BDM) (Sigma) in media for 5-10 min to eliminate contractions to reduce the effect of contractile status on tissue stiffness. Pressure was applied to the organoid surface and pictures were recorded until reaching equilibrium deformation (about 5 min) (
Research surrounding the human myocardium after MI has been limited to chronic end-stage ischemic cardiomyopathy (given the availability of donor tissue) and in vitro human models of related cellular mechanisms (e.g., adrenergic stimulation, cell death from reperfusion) (Tiburcy et al., 2017 Circulation 135: 1832-1847; Ulmer et al., 2018 Stem Cell Reports 10: 834-847; Prat-Vidal et al., 2013 PloS One 8: e54785; Tarnayski et al., 2004 Physiol. Genomics 16: 349-360; Chen et al., 2018 Tissue engineering: Part A 25(9-10):71-724). Whereas previous cardiac 2D/3D systems have contributed to the understanding of effects of individual MI-related factors (e.g., hypoxia, adrenergic stimulation, substrate mechanics) in homogenous (i.e., no gradient) environments, this study leveraged nutrient transport principles (e.g., oxygen diffusion) in 3D microtissues alone with chronic adrenergic stimulation to create a gradient of “apoptotic center-dysfunctional interior-functional edge” in human cardiac organoids, which recapitulate the “infarct-border-remote zones” of infarct hearts. This allowed the organoids to mimic organotypic myocardial response to infarction.
To gain an insight into oxygen distribution in cardiac organoids, a mathematical diffusion model was constructed using a 300 μm cardiac microtissue.14 In contrast to normoxia (20% oxygen), the microtissue in hypoxia (10% oxygen) experiences a gradient of viable to non-viable oxygen levels from edge to center (
To examine the global downstream effects of the cardiac infarction protocol (i.e., 10% O2 and 1 μM NE treatment) on gene expression, the transcriptomes from the control and infarct organoids were analyzed using RNA sequencing (RNAseq). When compared to ischemic cardiac injury transcriptomic data from public human (i.e., human ischemic dilated cardiomyopathy, ICM) and animal (i.e., 1 wk MI) studies, infarct organoid differentially expressed (DE) genes overlapped with >1,000 genes of human, mouse, and porcine DE genes, similar to the overlap between animal MI models and human ICM samples (
Whole transcriptome comparison between the organoid, human, and mouse data was performed using principal component analysis (PCA). After PCA of the 4,765 shared genes from the organoid data and two public RNA-seq datasets of human ICM and mouse MI (2 wks), the top PCs showed visible separations between samples (
With a translational relevance, we next investigated characteristics of ischemic cardiac injury observed in the organoid infarction platform. Fibrosis is one of the central hallmarks of ischemic heart failure in humans and animal MI models.17 Pathway analysis of DE genes in infarct organoids showed top hits that included “carbon metabolism” (p=5.24×106), “citrate cycle (TCA)” (p=9.25×10−6), and “glycolysis/gluconeogenesis” (p−1.67×10−3) (
The transcriptomic changes indicated a biomimetic fibrosis-like downstream response within the infarct organoids. This data was further characterized by structural analysis of fibroblast cellular organization. Infarct organoids showed a significant shift in vimentin+ organization (i.e., fibroblasts) toward the edge of the organoid compared to control organoids, seen by confocal imaging and radial density plots of vimentin+ area in organoid frozen sections (
Incorporation of TGF-β1 into the infarction protocol as a pro-fibrotic stimulus further increased collagen organization and the presence of αSMA+ myofibroblast-like cells with an associated significant increase in organoid stiffness. Immunoflouresent staining of myofibroblast-like cells in organoid sections using a pro-fibrotic stimulus of TGF-bl (20 ng/mL) during infarction protocol showed a visible shift in aSMA/F-actin (phalloidin) colocalization with fibrillary structure. (
In addition to fibrosis, calcium handling changes associated with MI serve as a functional hallmark of ischemic cardiac injury.25 Pathological calcium handling is linked to contractile dysfunction and contributes to the occurrence of arrhythmias after MI.26 At the transcriptomic level, “ion transport” was a top ten gene ontology term between control and infarct organoids, and top pathway hits included “calcium signaling pathway” and “arrhythmogenic right ventricular cardiomyopathy”, indicative of changes in the calcium handling (Tables 3-5). The “calcium signaling pathway” KEGG term 4020 was used to assemble a calcium handling-related gene set (Table 8). After filtering for the calcium handling-related gene set, infarct organoids showed an overall consistency with calcium handling gene expression of 1 wk post-MI mouse heart samples, including significant decreases in well-studied calcium-handling components ATP2A2 (sarco-endoplasmic reticulum Ca2+-ATPase), RYR2 (ryanodine receptor), CACNA1C (L-type calcium channel), SLC8A1 (sodium-calcium exchanger) and increase in ITPR3 (inositol 1,4,5-triphosphate receptor type 3) (
To functionally interpret the transcriptomic shifts in cardiomyocyte calcium handling and arrhythmogenesis, a two-photon, laser-scanning, light sheet (2PLS) microscope was used that allowed for deeper tissue penetration with high-speed imaging (50 frames/sec) and orthogonal selected plane (about 4 μm thickness) illumination.27 Given its strength for in situ imaging, the 2PLS microscope allowed for the visualization of calcium handling in the interior regions of 3D cardiac microtissues to study calcium handling and arrhythmogenicity across the organoids. Organoids were fabricated with GCaMP6-labeled hiPSC-CMs and calcium transient amplitudes (ΔF/F0) were measured from “cell-sized” regions of interest (ROIs) (representing individual cardiomyocytes) inside organoids. Imaging of control organoids displayed synchronized beating with an interconnected cardiomyocyte network. Specifically, imaging and calcium transient profiles of control and infarct organoid from selected imaging planes at >50 μM below organoid surface were carried out which showed unsynchronization of edge and interior cardiomyocytes regions of interest in infarct organoids. In contrast, imaging of infarct organoids revealed notable unsynchronized beating profiles (i.e., arrhythmias) between separated cardiomyocyte populations at the interior and the edge of the infarct organoids. Interior cardiomyocytes in the infarct organoids showed significantly lower max calcium transient amplitude in contrast to the control and infarct edge cardiomyocytes. In particular, imaging and calcium transient profiles of synchronized edge and interior cardiomyocyte ROIs from human induced pluripotent stem cell-derived-cardiomyocyte (hiPSC-CM) spheroids were cultured in the cardiac organoid infarction protocol. The difference in calcium synchronization between control and infarct organoids was supported by segregated αSA+ cardiomyocyte populations in infarct organoids in contrast to interconnected αSA+ cardiomyocytes in the control organoids was demonstrated using immunofluorescent staining of organoid sections showing interconnected alpha sarcomeric actinin (αSA)-positive cardiomyocytes in control organoids and separation of edge and interior cardiomyocytes by vimentin-positive cells in infarct organoids. We reasoned that fibrosis (i.e., vimentin+ cell-associated changes) separated interior cardiomyocytes in infarct organoids into an unsynchronized, smaller beating population that may experience hypoxia-induced aberrations in calcium handling, consistent with the in vivo contributors to ventricular arrhythmia post-MI.26 This was supported by hiPSC-CM-only spheroids (i.e., without fibroblasts) cultured in the infarction protocol that showed no indication of unsynchronized beating nor differences in edge-interior calcium transient amplitudes (
Previous research has shown that hiPSC-CMs from breast cancer patients with chemotherapy-induced cardiotoxicity were more sensitive to doxorubicin (DOX), a known cardiotoxic anticancer medication, than breast cancer patients without chemotherapy-induced cardiotoxicity, suggesting genetic basis for DOX-based cardiotoxicity (Benjamin et al., 2017 Circulation 135: 1832-1847). As a control, we performed 2D studies using hiPSC-CMs with DOX to evaluate the combined effects of an in vitro infarction protocol and DOX on hiPSC-CMs. Hypoxic culture (1%) with 1 μM NE in organoid media for 2 days prior to the 2 days DOX treatment causes an exacerbation of DOX-induced reduction in viability and reduction in contractile structures/organization. Nearly 100% of cells death was found for hiPSC-CMs after 4 days of the same hypoxic culture using hiPSC-CM media with galactose but no glucose, which has been attributed to the lack of glucose in the hiPSC-CM media. In addition, extended culture of 2D hiPSC-CMs in organoid media to mimic prolonged organoid infarction protocol led to minimal and irregular hiPSC-CM contractile behavior accompanied with the reduction of αSA+ cells. In contrast to the inherent difficulties of using 2D hiPSC-CM culture systems to model post-MI responses, human infarct organoids provide a robust, biomimetic 2D tissue-level context to evaluate the effects of DOX on the post-MI cardiac tissues.
Application of DOX showed functional toxicity with a reduced IC50 of contraction amplitude in infarct organoids (˜0.15 μM) compared to control organoids (˜0.37 μM) (
By leveraging nutrient diffusion limitations in 3D microtissues, we developed the first human cardiac organoid disease model that recapitulates the major hallmarks of myocardial infarction at a transcriptomic, structural and functional level. While human organoids have been widely used to study diseases caused by genetic mutation, this is the first demonstration of the use of tissue engineering principles (i.e., nutrient transport) to design an in vitro organotypic disease model with non-genetic upstream pathological stimuli. It is our belief that the focus on upstream pathological stimuli allowed for the recapitulation of major hallmarks of human myocardial infarction.
Through the integration of robust infarct organoid fabrication and imaging-based function analysis, we established a platform amenable for heart failure drug screening. The transcriptome- to function-level changes provided a multi-dimensional validation that illustrates the extent to which infarct organoids recreate responses of human cardiac tissue after infarction. As human cardiac tissues post-MI are difficult to obtain28, human cardiac infarct organoids offer personalized models for precision cardiovascular medicine and future investigation into genetic contributions of patients' tissue-level response to myocardial infarction. While organoids have traditionally been prepared with embryoid bodies, this study demonstrated that the self-assembly of tissue-specific cell types provides a powerful alternative to prepare organoids with tissue-mimetic transcriptome, structure and function.29
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
This patent application is a 35 U.S.C. § 371 national phase application of PCT Application PCT/US2019/040981 filed Jul. 9, 2019, which claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 62/696,660, filed on Jul. 11, 2018, the entire contents of each of which are incorporated by reference herein.
This invention generally relates to a three-dimensional myocardial infarct organoids and methods of making and using the same.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/40981 | 7/9/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62696660 | Jul 2018 | US |
