Patent Application
20250163380
This disclosure generally relates to a method of generating one or more EMLOC gastruloids, one or more EMLOC gastruloid compositions, and/or a combined human gastruloid model of cardiogenesis and neurogenesis. In some embodiments, the disclosure provides compositions and methods employing stem cell-derived heart-like structures. In some embodiments, methods of generating heart-like tissues from human stem cells and the resulting tissues are provided. In some embodiments, uses of such tissues for research, compound screening and analysis, and therapeutics are provided.
The complex nature of in vivo cardiogenesis underlies the difficulties in establishing in vitro cardiac developmental models with human cells. The heart is the first organ to form in the mammalian embryo, caudal to the embryonic brain and within the developing trunk. It becomes contractile as a tube prior to complex morphogenesis into septated chambers and co-developmental population by neurons for innervation (Harvey, 2002; Hasan, 2013). In order to accommodate both contractility and structural rearrangement, the developing heart undergoes alternating phases of cardiac differentiation and morphogenesis (Ivanovitch et al., 2017). Calcium handling properties become refined during cardiac differentiation (Tyser et al., 2016). The cardiac crescent is the first bilateral structure to form and precedes epithelialization and formation of the transversal heart tube. At this stage, the heart tube remains open at the dorsal aspect, bound by dorsal mesocardium, and then seals during formation of the closed linear heart tube and outflow tracts. Intrinsic cell-driven forces within the tube and extrinsic physical constraints are known to mediate the establishment of left-right asymmetries required for heart function (Desgrange et al., 2018). Such complexity in cardiogenesis lays the framework for lifelong functioning of the adult heart, but also underlies the propensity for congenital heart disease in humans where developmental errors induce cardiac malformations (van der Linde et al., 2011; Desgrange et al., 2018). The ability to generate in vitro models of heart development that mimic essential aspects of multi-lineage input to cardiogenesis is needed to benefit biomedical treatments of heart disease and progress towards ex vivo organogenesis.
Organoid technology is revolutionizing the study of human development and disease, recapitulating key aspects of spatiotemporal tissue morphogenesis (Clevers, 2016; Olmsted and Paluh, 2021c). Most current organoid technologies are directed towards single tissue endpoints that lack the cellular contextual diversity present in normal organogenesis through inductive and mechanical interactions. As such, the ability to generate organotypic human cardiac organoids that form according to the in situ developmental signaling blueprint and integrate with the developing nervous system has not been achieved. The existing human cardiac organoid models derive primarily from pre-differentiated cardiomyocytes and their spheroid aggregates that form irrespective of developmental timelines (Nguyen et al., 2014; Giacomelli et al., 2017; Polonchuk et al., 2017; Andersen et al., 2018), or models that rely on integrated bioengineering efforts to constrain morphogenetic patterning (Ma et al., 2015; Lind et al., 2017; Macqueen et al., 2018; Hookway et al., 2019). These models lack identified critical inductive tissues indispensable to natural heart development such as the foregut, described as a central organizer of cardiogenesis in multiple species and acting through both inductive and structural interactions between endoderm and splanchnic mesoderm (Nascone and Mercola, 1995; Schultheiss et al., 1995; Varner and Taber, 2012; Anderson et al., 2016; Kidokoro et al., 2018; Han et al., 2020).
Two recent studies with human iPSCs succeeded in the co-production of cardiac and gastrointestinal tissue in single organoids without organized chambers (Silva et al., 2021; Drakhlis et al., 2021). As well, Hofbauer et al. (2021) succeeded in generating self-organized, isolated cardioids exhibiting chamber-like structures from human pluripotent stem cells that were used to model cardiac injury (Hofbauer et al., 2021). Although important advances to the cardiogenesis field, neural cells were not co-generated in these systems and were absent. One murine study generated chambered cardiac organoids from mESCs by embedding in exogenous extracellular matrix (ECM) with supplied FGF4 (Lee et al., 2020). More recently, Rossi et al. (2021) used mESC-derived gastruloids to recapitulate aspects of early cardiogenesis including first and second heart field contributions without extracellular matrix (ECM) embedding. Gastruloid research has been broadly applicable for conducting multi-lineage interaction studies in the trunk (van den Brink et al., 2014; van den Brink et al., 2020; Veenvliet et al., 2020; Olmsted and Paluh, 2021a). However, no study with human cells has succeeded in generating a de novo model to recapitulate cardiogenesis in an embryo-like, multi-lineage context and, in particular, with neuronal cooperative development that is a vital functional component. What is needed are human gastruloids that capture numerous key developmental aspects of human cardiogenesis and neurogenesis along with endoderm-derived primitive gut tube and other lineages.
A unique human trunk model system referred to as elongating multi-lineage organized (EMLO) gastruloids (Olmsted and Paluh, 2021a, 2021b) has been described (See e.g., Olmsted, Z. T., Paluh, J. L. Co-development of central and peripheral neurons with trunk mesendoderm in human elongating multi-lineage organized gastruloids. Nat Commun 12, 3020 (2021) https://doi.org/10.1038/s41467-021-23294-7) (herein incorporated entirely by reference). Neural crest lineage in EMLOs reveals insights into enteric development and the formation of the enteric nervous system. The enteric multi-lineage niche in EMLOs achieved only limited embryonic cardiogenesis that included generation of cardiomyocytes anterior to the gut tube. What is needed is extended or improved embryonic cardiogenesis.
In embodiments, the present disclosure includes a method of generating elongating multi-lineage organized cardiac (EMLOC) gastruloids, including: contacting a colony of human induced pluripotent stem cells with hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2) under culture conditions for a first duration to form a first plurality of gastruloids; and contacting the first plurality of gastruloids with vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and ascorbic acid under culture conditions for a second duration to form EMLOC gastruloids. In embodiments, the growth factor (HGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF) are provided in an effective amount to coax the colony of human induced pluripotent stem cells to form a first plurality of gastruloids. In embodiments, the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and ascorbic acid are provided in an effective amount to coax the first plurality of gastruloids to form EMLOC gastruloids. In embodiments, the human induced pluripotent stem cells are contacted under culture conditions with 2 ng/ml hepatocyte growth factor (HGF), 2 ng/ml insulin-like growth factor (IGF), 10 ng/ml fibroblast growth factor (FGF) for a first duration, and 5 ng/ml vascular endothelial growth factor (VEGF), 30 ng/ml fibroblast growth factor (FGF), and 0.5 mM ascorbic acid for a second duration. In embodiments, the contacting occurs in an environment characterized as cardiac permissive. In embodiments, the colony of human induced pluripotent stem cells is characterized as trunk-biased stem cells. In embodiments, the first duration is 24 hours, and the second duration is 2-7 days. In embodiments, the colony of human induced pluripotent stem cells are trunk-biased stem cells.
In some embodiments, the present disclosure relates to one or more EMLOC gastruloids formed by one or more methods of the present disclosure. In embodiments, the present disclosure includes one or more EMLOC gastruloids, including a three-dimensional structure including or consisting of contractile innervated tissue. In some embodiments, an EMLOC gastruloid of the present disclosure is characterized by having human heart characteristics. In some embodiments, an EMLOC gastruloid of the present disclosure mimics human heart at natural human development timepoints. In embodiments, an EMLOC gastruloid of the present disclosure expresses human heart genes at development timepoints substantially similar to natural human heart at substantially similar timepoints.
In some embodiments, the present disclosure relates to one or more EMLOC gastruloids formed by one or more methods of the present disclosure. In embodiments, the present disclosure includes one or more EMLOC gastruloids, including a three-dimensional structure including or consisting of contractile innervated tissue. In some embodiments, an EMLOC gastruloid of the present disclosure is characterized by having human heart characteristics. In some embodiments, an EMLOC gastruloid of the present disclosure mimics human heart at natural human development timepoints. In embodiments, an EMLOC gastruloid of the present disclosure expresses human heart genes at natural human development timepoints.
In embodiments, the present disclosure includes a method of generating a contractile innervated human heart tissue or organ, including: coaxing or inducing an exogenous population of stem cells to divide and differentiate to an innervated cardiac fate by contacting the exogenous population of stem cells with hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2) under culture conditions for a first duration to form a first plurality of gastruloids; and contacting the first plurality of gastruloids with vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and ascorbic acid under culture conditions for a second duration.
In embodiments, the present disclosure includes a method of generating an organized co-developed neuro-cardiac gastruloid, including: contacting an exogenous population of stem cells with an effective amount of hepatocyte growth factor (HGF), insulin-like growth factor (IGF-1), fibroblast growth factor (FGF-2) for a first duration, and subsequently contacting the exogenous population of stem cells with an effective amount of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), and ascorbic acid for a second duration. In embodiments, the present disclosure includes methods for utilizing dissociated cells of the elongating multi-lineage organized cardiac (EMLOC) gastruloids and reseeding the dissociated cells onto mammalian scaffold material to initiate structures similar in EMLOCs for cardiogenesis or innervation. In embodiments, the present disclosure includes methods for utilizing the elongating multi-lineage organized cardiac (EMLOC) gastruloids with mammalian cells and initiating structures similar in EMLOCs for cardiogenesis or innervation. In embodiments, the exogenous population of stem cells are characterized as adherent and/or induced by contacting the cells with a biologically active stem cell differentiation and reprogramming reagent and a fibroblast growth factor 2 (FGF-2).
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments.
The present disclosure is directed towards compositions, kits, and methods of extending/refining elongating multi-lineage organized (EMLO) gastruloids to cardiac (EMLOC) gastruloids, and the generation of interconnected neuro-cardiac lineages in a single gastruloid model.
Stem cell technology enables unprecedented studies of human multi-lineage development through the ability to recapitulate embryonic and post-embryonic dynamic regulatory processes in a multicellular context. As such, it is expected to accelerate ex vivo strategies in organ development. Reproducing human cardiogenesis remains challenging, requiring spatiotemporal paracrine inductive and mechanical cues by multi-lineage tissues. Here, elongating multi-lineage organized (EMLO) gastruloids has been extended to cardiac (EMLOC) and the present disclosure describes the generation of interconnected neuro-cardiac lineages in a single gastruloid model. In embodiments, the contractile EMLOCs of the present disclosure recapitulate numerous interlinked developmental features of heart tube formation and specialization, cardiomyocyte differentiation and remodeling phases, epicardium, ventricular wall morphogenesis, and formation of a putative outflow tract. Along with cardiogenesis in EMLOCs that originates anterior to the gut tube primordium, neurons were observed that progressively populate the cardiogenic region in a pattern that mirrors spatial distribution of neurons in heart innervation. In embodiments, the EMLOC model represents an important multi-lineage advancement for the study of human cardiogenesis with co-developed neuronal integration.
The present EMLOC gastruloid technology arises through directed morphogenic cellular changes. Three-dimensional cell aggregates progress from spherical to an ovoid shape that develop into an elongated structural form. During morphogenesis changes to the cells are co-directed to neural and cardiac cell lineages that are spatially compartmentalized. This polar state is recognized by cell lineage specific biomarkers and distinct cardiac-morphogenic events. Integration of the cell lineages occurs when neuronal progenitors migrate into the cardiac region to organize neurogenesis of the heart tissue. This elongation of cell structure is not seen in other cardiac organ models.
In embodiments, EMLOs are coaxed or caused developmentally, by providing the necessary cues, towards more extended cardiac differentiation with reproducible morphogenesis. In embodiments, EMLO formation techniques are altered to include angiocrine and pro-cardiogenic factors such as factors previously detailed (Rossi et al., 2021). The present disclosure now provides a gastruloid strategy for neuro-cardiac co-developed tissues that recapitulate aspects of early human heart morphogenesis with neuronal integration. In embodiments, multiple events are tracked in cardiomyocyte differentiation from splanchnic mesoderm and spontaneous contractility, and chamber precursor formation was observed along with early constrictions and septations, epicardium, and putative structures resembling the outflow tracts. Importantly, EMLOC embodiments, of the present disclosure not only retain the interacting neural compartment but achieve neurogenesis to generate an organized co-developed neuro-cardiac gastruloid. It has now been demonstrated that EMLOCs are suitable for use as an advanced model for human cardiogenesis and the integration with endoderm and neurons towards the goal of organ innervation.
As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a compound” include the use of one or more compound(s). “A step” of a method means at least one step, and it could be one, two, three, four, five or even more method steps.
As used herein the terms “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval [Cl 95%] for the mean) or within ±10% of the indicated value, whichever is greater.
As used herein the terms “under culture conditions” or “under culturing conditions,” as known in art, for example, includes plastic dished in CO2 chambers to culture cells.
As used herein the term “contractile innervated tissue,” refers to the multichambered heart that has neurons integrated from the neural region of the EMLOC over the heart chambers and the ability of the whole heart organ to contract in beats.
As used herein, the term “forming a mixture” refers to the process of bringing into contact at least two distinct species such that they mix together and interact. “Forming a reaction mixture” and “contacting” refer to the process of bringing into contact at least two distinct species such that they mix together and can react, either modifying one of the initial reactants or forming a third, distinct, species, a product. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. “Conversion” and “converting” refer to a process including one or more steps wherein a species is transformed into a distinct product.
As used herein the term “effective amount” as used herein means that amount of an agent that elicits the biological or medicinal response in a cell, tissue, organ, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician. In some embodiments, the effective amount is a “therapeutically effective amount” for the alleviation of the symptoms of the disease or condition being treated. In some embodiments, the effective amount is a “prophylactically effective amount” for prophylaxis of the symptom s of the disease or condition being prevented.
The term “elongating multi-lineage organized cardiac gastruloids,” or “EMLOC gastruloids,” refers to a multi-step process of morphological and cell lineage differentiation steps that mimics normal human heart development. The morphological changes of the cardiac gastruloids encompass various forms as they develop. The cardiac gastruloid forms include, but are not limited to, round to ovoid to tube-like and/or hour-glass shapes. These forms/shapes coincide with heart development.
The term “trunk biased” refers to the exclusion of anterior neural tissue/structures, and the term “cardiac permissive” refers to the atypical aspect of the presently disclosed protocol that allows for the co-generation of both cardiac and neural cell types without restriction to one or the other. Cardiac permissive directed generation within a domain of progenitor cells necessary for cardiac morphogenesis.
The term Trunk-biased stem cells refers to the discovery of distinct lineages in mammals, including humans for stem cells of the body trunk that can be mimicked in vitro, and excluding anterior structures such as brain and brainstem.
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.
Before embodiments are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
In embodiments, the present disclosure relates to EMLOC kits, EMLOC compositions, or one or more methods of making EMLOCs.
In embodiments, the present disclosure includes a method of generating elongating multi-lineage organized cardiac (EMLOC) gastruloids, including: contacting a colony of human induced pluripotent stem cells with hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2) under culture conditions for a first duration to form a first plurality of gastruloids; and contacting the first plurality of gastruloids with vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and ascorbic acid under culture conditions for a second duration to form EMLOC gastruloids. In embodiments, the growth factor (HGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF) are provided in an effective amount to coax/stimulate/induce the colony of human induced pluripotent stem cells to form a first plurality of gastruloids. In embodiments, the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and ascorbic acid are provided in an effective amount to coax the first plurality of gastruloids to form EMLOC gastruloids. In embodiments, the human induced pluripotent stem cells are contacted under culture conditions with 2 ng/ml hepatocyte growth factor (HGF), 2 ng/ml insulin-like growth factor (IGF), 10 ng/ml fibroblast growth factor (FGF) for a first duration, and 5 ng/ml vascular endothelial growth factor (VEGF), 30 ng/ml fibroblast growth factor (FGF), and 0.5 mM ascorbic acid for a second duration. In embodiments, the contacting occurs in an environment characterized as cardiac permissive. In embodiments, the colony of human induced pluripotent stem cells is characterized as trunk-biased stem cells. In embodiments, the first duration is 24 hours, and the second duration is 2-7 days. In embodiments, the colony of human induced pluripotent stem cells are trunk-biased stem cells.
In some embodiments, the present disclosure relates to one or more EMLOC gastruloids formed by one or more methods of the present disclosure. In embodiments, the present disclosure includes one or more EMLOC gastruloids, including a three-dimensional structure including or consisting of contractile innervated tissue.
In embodiments, the present disclosure includes a method of generating a contractile innervated human heart tissue or organ, including: coaxing or promoting an exogenous population of stem cells to divide and differentiate to an innervated cardiac fate by contacting the exogenous population of stem cells with hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2) under culture conditions for a first duration to form a first plurality of gastruloids; and contacting the first plurality of gastruloids with vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and ascorbic acid under culture conditions for a second duration.
In embodiments, the present disclosure includes a method of generating an organized co-developed neuro-cardiac gastruloid, including: contacting an exogenous population of stem cells with an effective amount of hepatocyte growth factor (HGF), insulin-like growth factor (IGF-1), fibroblast growth factor (FGF-2) for a first duration, and subsequently contacting the exogenous population of stem cells with an effective amount of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), and ascorbic acid for a second duration. In embodiments, the exogenous population of stem cells are characterized as adherent and/or induced by contacting the cells with a biologically active stem cell differentiation and reprogramming reagent and a fibroblast growth factor 2 (FGF-2).
In embodiments, the present disclosure includes hiPSC technology for formation of a contractile innervated human heart from a three-dimensional microenvironment. In embodiments, the present disclosure establishes the 3D microenvironment and developmental regulators and signaling molecules necessary to recapitulate human embryonic temporal events in heart formation. In embodiments, the technology enables three-dimensional innervation studies to non-neural tissue and organs in an embryo-like model. The technology has broad applications to provide fundamental insights into congenital heart disease, cardiac pathophysiology, cardiac hypoxia, and pharmacotherapy relevant to neuromodulation of cardiomyocytes, neurocardiogenic syncope, and other neural-based arrhythmia pathologies for embryonic to adult biomedical analysis. This technology further has biomedical relevance for neuro-gastro-cardiac development.
In some embodiments the present disclosure relates to gene expression from a single cell RNA sequence and data related thereto.
In embodiments, EMLOCs of the present disclosure mirror in vivo development of distinct cardiac, neural and epithelial lineages including primitive foregut, in signaling networks, adhesion proteins and transcription factors.
In embodiments, EMLOCs of the present disclosure generate trunk and neuroectoderm/spinal cord progenitors, trunk neurons, peripheral glia/Schwann cells.
In embodiments, EMLOCs of the present disclosure recapitulate numerous key features of human cardiogenesis including cardiac crescent transformation into the contractile heart tube, cardiomyocyte differentiation versus remodeling phases, and formation of chamber- and outflow tract-like structures. Cardiogenesis occurs anterior to primitive gut tube-like endodermal cells that in vivo are thought to be required.
In embodiments, EMLOCs of the present disclosure generate intermediate mesoderm, metanephric mesenchyme, genitourinary/renal epithelium.
In embodiments, EMLOCs of the present disclosure retain splanchnic mesoderm biomarkers: GATA4, GATA6, FOXF1, PDGFRA, TWIST1, and PRRX2.
In embodiments, EMLOCs of the present disclosure generate diverse cell types arising from splanchnic mesoderm, including cardiomyocytes that form myocardium, cells of the proepicardium and epicardium, and cardiac fibroblasts.
In embodiments, EMLOCs of the present disclosure generate cardiomyocytes, epicardial cells and cardiac fibroblasts and vascular endothelium.
In embodiments, EMLOCs of the present disclosure generate primarily ventricular cardiomyocytes at day 16 timepoint.
In embodiments, EMLOCs of the present disclosure express first heart field (FHF) and second heart field (SHF) genes, including: TBX5 and HAND2 (both) and NKX2-5, HAND1 (FHF) and MEF2C, ISL1, TBX18 (SHF).
In embodiments, EMLOCs of the present disclosure express genes for regulating ventricle growth and morphogenesis.
In embodiments, EMLOCs of the present disclosure express fetal heart development genes, including: KRT8/KRT18, APOE, PLAC9 and S100A10.
In embodiments, EMLOCs of the present disclosure express epicardial genes including: WT1, TCF21, TPJ1, LHX2, LHX9, TBX18, PLAC9.
In embodiments, EMLOCs of the present disclosure express upregulated cardiac fibroblast genes, including IGFBP5, a biomarker associated with cardiac fibroblast activation and BTS2, and/or a biomarker of mature cardiac fibroblasts.
In embodiments, EMLOCs of the present disclosure express cardiomyocyte and fibroblast-derived ECM genes, including biomarkers of cardiac jelly ECM and its spatiotemporal degradation by day 16 (VCAN, ADAMTS1, ANGPT1).
In embodiments, EMLOCs provide for the presence of cardiogenic mesoderm arising cells, including: contractile cells (TNNT1-slow type troponin, TNNT2-cardiac troponin, MYL7), outflow tract cells (PDE5A, ISL1, FN1, MEGF6, MSX2, SEMA3C, EMILN1, CNN1, TAGLN) and atrioventricular conduction and organization (GJA1, CACNA1H, TBX3, NRP2, CXCL12, DSP).
In embodiments, by comparing calcium transients per minute in EMLOC system versus the mESC cardiogenesis study in gastruloids, a similar species-specific ratio to that between the resting adult heart rate in human versus mouse (˜8× higher in the murine model) that have similar processes of cardiogenesis was observed or provided.
In embodiments, EMLOCs of the present disclosure express anterior foregut progenitor cells (HHEX, SHH, FOXA2) and anterior foregut identity (FOXA2, NKX2-1, SHH, EPCAM).
In embodiments, EMLOCs of the present disclosure express cardiac neural crest cell biomarkers (ETS1, EDNRA, TGIF1, HOXA3).
In embodiments, EMLOCs of the present disclosure express genes involved in left-right asymmetry specification during in vivo cardiogenesis by day 16 (IRX3, HAND1, PITX2, RTTN).
In embodiments, EMLOCs of the present disclosure express genes that are biomarkers for smooth muscle (CNN1/TAGLN), outflow tract development (ISL1/PDE5A/CDH11) and well-differentiated vascular endothelium (KDR/FLT1/ESAM/CDH5), and Nodal and valvar biomarkers (POSTN/TBX3/NPR3/NFATC4).
In embodiments, EMLOCs of the present disclosure display an emergence of neurons at day 7 and neuronal expansion by day 16 (>40 fold) that includes neural progenitors (ZIC1, RFX4, HES5, FABP7, EDNRB, NTRK2, OLIG3, MSX1) and specialized neuronal subtypes (INSM1, ELAVL3, DLG4, CAMK2A, SLC18A3, SLC17A6, CHRNA3, NTRK3).
In embodiments, EMLOCs of the present disclosure express, by day 16 neural crest-derived Schwann cells (SOX10, PLP1, MPZ, S100B, TFAP2B, NGFR).
In embodiments, EMLOCs of the present disclosure contains by day 16 combined INSM1/ISL1 expression that indicates that sympathetic neurogenesis occurs in EMLOCs, which is particularly relevant to developing cardiac innervation. Restricted expression of HOXC6 and HOXC9 to these clusters supports spinal cord and trunk identity.
In embodiments, EMLOCs of the present disclosure contain biomarkers for specialized neuronal subtypes of autonomic neurons that is ASCL1 (93/151 neurons, ˜62%) and PHOX2B (14/151 neurons, ˜9%) that predominated versus sensory neurons POU4F1/BRN3A (38/151 neurons, ˜25%) or motor neurons MNX1/HB9 (6/151 neurons, ˜4%). This finding is distinct from EMLOs (Olmsted and Paluh, 2021a), in which motor neurons were primarily generated.
In embodiments, EMLOCs of the present disclosure express biomarkers of cardiac innervation: neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), semaphorin 3A (SEMA3A), peripherin (PRPH), endothelin receptor type A (EDNRA), and ISL1. Genes involved in autonomic neurogenesis and cardiogenesis such as ISL1 also play a role in development and innervation of cardiac pacemaker cells that dictate automaticity and participate in the conduction system apparatus.
In embodiments, EMLOCs of the present disclosure express neuronal fiber patterning on cardiomyocytes and reflect normal developmental patterning and ability to respond to intrinsic spatial cues.
In embodiments, EMLOCs of the present disclosure show terminating neuronal fibers on cardiomyocytes identified by phosphor-tau (Ser214) immunostain.
In embodiments, EMLOCs of the present disclosure are formed in a cardiac permissive microenvironment created by two-day treatment of trunk-biased stem cells aggregated by orbital shaking in suspension with factors added in starting concentrations of: 2 ng/ml HGF, 2 ng/ml IGF-1, 10 ng/ml FGF-2 and switching to 5 ng/ml VEGF, 30 ng/ml FGF-2, 0.5 mM ascorbic acid from day 2 to day 7.
In embodiments, elongating multi-lineage organized (EMLO) gastruloids can act as trunk-biased, cardiac-permissive starting material.
In embodiments, methods of the present disclosure provide a rapid timeline of 3D cardiogenesis from beating cardiomyocytes at day 7 to innervated cardiac region by day 25.
In embodiments, methods of the present disclosure provide neuronal co-emergence by day 7 that are repelled from the cardiogenic region.
In embodiments, methods of the present disclosure provide for timing of neuronal interaction with reduced cardiac jelly extracellular matrix between day 7 and day 16 to populate the cardiogenic region.
In embodiments, methods of the present disclosure provide EMLOCs with innervation mirroring human embryonic heart patterning by day 25.
In embodiments, methods of the present disclosure provide EMLOCs with cardiac crescent formation from day 4 to day 6.
In embodiments, methods of the present disclosure provide EMLOCs with progression of GATA4+/GATA6+ expressing cardiogenic region into a cTnT+ co-expressing region indicative of cardiomyocyte formation.
In embodiments, methods of the present disclosure provide EMLOCs with embryonic-similar (human weeks 3-5) heart tube formation and cell type specialization.
In embodiments, methods of the present disclosure provide EMLOCs with cardiomyocyte transitions between flat or rounded cardiomyocyte cell shapes consistent with alternating phases of cardiomyocyte morphogenesis and differentiation in ventricular wall morphogenesis.
In embodiments, methods of the present disclosure provide EMLOCs with formation of communicating cardiac chambers with embryonic-similar organization (myocardium, cardiac jelly, endocardium).
In embodiments, methods of the present disclosure provide EMLOCs with formation of CD144 endocardium interior chamber lining and valve-like outflow tract.
In embodiments, methods of the present disclosure provide EMLOCs with cardiomyocyte contractility with innervation in 3D heart model.
In embodiments, methods of the present disclosure provide EMLOCs with contractility relative to human beats/s (median ˜10 calcium transients per minute, range 5-20 calcium transients per minute).
In embodiments, methods of the present disclosure provide EMLOCs with formation of a channel positional to the cardiac chamber outflow tract.
In embodiments, methods of the present disclosure provide EMLOCs with an ability to switch from growth factor-applied to growth factor-free medium after contractility begins at day 7.
In embodiments, methods of the present disclosure provide EMLOCs with neuron-cardiomyocyte synapse formation in a 3D heart mode.
In embodiments, methods of the present disclosure provide EMLOCs with cardiogenesis anterior to formation of gut tube primordium in a 3D heart model.
In some embodiments, the present disclosure includes a method of preparing heart-like tissue, including: contacting a colony of human induced pluripotent stem cells with hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2) under culture conditions for a first duration; and subsequently contacting the colony of human induced pluripotent stem cells with vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and ascorbic acid under culture conditions for a second duration to form heart-like tissue. In some embodiments, the heart-like tissue is characterized as including a plurality of EMLOC gastruloids. In embodiments, the colony of human induced pluripotent stem cells are contacted with an effective amount of hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and combinations thereof. In embodiments, the colony of human induced pluripotent stem cells are contacted with an effective amount of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), ascorbic acid, and combinations thereof. In embodiments, a heart-like tissue is formed by the methods of the present disclosure. In some embodiments, a “heart-like” tissue refers to tissue differentiated in vitro (e.g., from a stem cell) that has one or more properties of a heart, such as a human heart. In embodiments, the heart-like tissue is characterized as synthetic or non-naturally occurring. In embodiments, the heart-like tissue is characterized as innervated and contractile.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present disclosure.
It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.
Experimental Models And Subject Details: Human induced pluripotent stem cells. hiPSC lines were derived from Coriell de-identified human fibroblast samples from consenting donors, including the Hispanic-Latino H3.1.1 line used in this study (Chang et al., 2015; Tomov et al 2016). Line H3.1.1 was reprogrammed with Yamanaka factors by the laboratories of Dr. Paluh and Dr. Jose Cibelli from these donor fibroblasts and comprehensively characterized for pluripotency (immunofluorescence, RT-PCR), G-band karyotype, teratoma formation, multi-lineage differentiation, bulk RNA-Seq, ChIP-Seq, and used in multiple studies from this lab. Recent G-band karyotype validation and pathogen analysis was performed by Cell Line Genetics, Inc. (NY, NY). H3.1.1 hiPSC colonies were cultured in mTeSR Plus supplemented with 1× penicillin-streptomycin (P-S) on hESC-qualified Matrigel (1:100 dilution; Corning) in a humidified incubator at 37° C., 5% CO2. Cultures were passaged 1:6 in 6-well plates every 4-7 days using Gentle Cell Dissociation Reagent (GCDR, STEMCELL Technologies). Cells were cryopreserved in mFreSR.
Method Detail EMLOC Formation: EMLOs were formed similarly to as previously described (Olmsted and Paluh, 2021a, 2021b) with several important differences detailed as follows. H3.1.1 adherent hiPSC colonies were maintained in mTeSR Plus pluripotency medium as described above. At ˜60% confluency, pluripotency medium was changed to induction medium (N2B27 basal medium supplemented with 3 μM CHIR 99021, 40 ng/ml basic fibroblast growth factor FGF2). N2B27 basal medium: 1:1 DMEM/F-12:Neurobasal Plus medium, 2% (v/v) B27 Plus supplement, 1% (v/v) N2 supplement, 1× GlutaMAX, 1×MEM Non-Essential Amino Acids, 1× P-S. Adherent hiPSC colonies were induced for two days the one exchange of fresh medium at 24 h. On the day of aggregation, cells were dissociated with 1:1 Accutase:HBSS (Ca—Mg free) at 37° C. for 5 min followed by manual trituration with a P-1000 pipette. Six-well plates were pre-treated with Anti-Adherence Rinsing Solution (STEMCELL Technologies) for 5 min incubation at room temperature followed by two rinses with equal volumes of HBSS. Cells were resuspended in N2B27 supplemented with 10 ng/ml FGF2, 2 ng/ml IGF-1, 2 ng/ml HGF (R&D Systems) and 50 μM Y-27632 (Tocris Bioscience). For aggregation, the single cell suspensions were added at a density of 2×106 cells/ml (2 ml per well, 4×106 total cells). Gastruloids were aggregated overnight using an orbital shaker at 75 rpm clockwise in a humidified incubator with 5% CO2. The next day, one-half volume of medium was replaced with fresh medium N2B27 supplemented with 4 ng/ml IGF-1, 4 ng/ml HGF, 20 ng/ml FGF2 to maintain the same concentration of growth factors in the culture medium after one-half volume addition. At 48 h, the entire volume of medium was replaced with N2B27 basal medium supplemented with 5 ng/ml VEGF, 30 ng/ml FGF2, and 0.5 mM ascorbic acid (Rossi et al., 2021). EMLOCs were induced in this medium to day 5. At day 7, the EMLOCs were maintained in non-supplemented N2B27. For orbital shaking culture, cells were aggregated and induced at 80 rpm. Speed was reduced to 75 rpm on day 7.
EMLOC single-cell dissociation by cold activated protease for scRNAseq. Type here (UB): Day 7 and Day 16 EMLOCs were dissociated on their respective time points in differentiation according to a previously protocol described (Olmsted and Paluh, 2021a). In brief, ˜25 EMLOCs from each time point were pooled in a 2 ml centrifuge tube and exposed to 1 ml dissociation solution composed of 10 mg/ml Bacillus licheniformis protease and 125 U/ml DNase in ice-cold 1×PBS supplemented with 5 mM calcium chloride. EMLOCs were incubated on ice in dissociation solution and triturated with a P-1000 pipette every 30-60 s for 8 min. Dissociation to single cells was verified by optical inspection and the reaction was terminated by addition of 1 ml ice-cold 1×PBS with 10% fetal bovine serum (FBS). Cells were pelleted by centrifugation at 1,200×g for 5 min, resuspended in fresh 1× PBS/10% FBS, counted, and centrifuged once more. Supernatant was aspirated completely and cells were resuspended in CryoStor CS10 cryopreservation medium to a final concentration of 1×106 cells per ml, filtered through a 40 μm cell strainer, and transferred to a 1.8 ml Nunc cryo-storage tube. Cells were frozen at −80° C. overnight and transferred to a liquid nitrogen dewar. When samples from both time points were dissociated and stored, samples were shipped overnight on dry ice to University of Buffalo Genomics and Bioinformatics Core at the New York State Center of Excellence in Bioinformatics and Life Sciences.
Single-cell sequencing with CellPlex, cluster annotation and analysis: When samples were received, they were immediately stored at −80° C. On the day of cell capture for sequencing, day 7 and day 16 EMLOC samples were thawed in a 37° C. water bath. Individual time point samples were transferred to separate 15 ml tubes. RPMI1640+10% FBS pre-warmed media was added dropwise to a final volume of 10 ml per tube. Cells were centrifuged at 300×g for 5 min. This washing procedure was performed a total of three times. After the final wash, medium was completely removed and cell samples were separately resuspended in 100 μl of Cell Multiplexing Oligo (10× Genomics). The two populations were suspended with two different oligos as directed by the manufacturer's instructions. After a brief incubation, cells were washed 3× with ice cold 1×PBS (pH 7.4)+1% bovine serum albumin (BSA). Cells were then resuspended in 250 ul 1×PBS/1% BSA and counted on a Logos Biosystems LUNA II in bright field mode with 0.4% trypan blue. The two cell populations with different barcodes were then pooled to 10,000 cells (5,000 from each time point) and recounted. The combined single cell suspension was provided as input for the 10× Genomics Single Cell v3.1 protocol with Feature Barcode technology. After libraries were prepared, they were loaded onto an Illumina NextSeq in high-output mode with a general target of 50,000 reads per cell to provide for sufficient depth and transcriptomic saturation. Post sequencing, data was demultiplexed and provided as input into the 10× Genomics Cell Ranger multipipeline (ver 4), which quantifies the transcriptomic profile of each cell against a reference genome. Sequence saturation, detected barcodes per cell, percent of transcripts in cell, and general alignment statistics were evaluated for quality. Cell Ranger matrix files were then used as input into the R Bioconductor package Seurat (ver 4). Cells with outlier-status, abnormal gene detection rates, and high mitochondrial transcript load that is an indicator of cellular stress were filtered from the analysis. After filtering, the data was underwent Seurat normalization and integrated using the SCTransform and integration protocol, followed by UMAP (Uniform Manifold Approximation and Projection) or the alternative PHATE dimensionality reduction for visualization (Moon et al., 2019). Using the called clusters, cluster-to-cluster differential expression testing using the Wilcoxon Rank Sum Test was used to identify cluster-defining biomarkers, as well as further exploratory analysis with known biomarker genes (gene lists provided in Table S1). Data was then exported from Seurat for further analysis in the Loupe browser.
Calcium Imaging Of Contractility: The EMLOCs were incubated with Fluo-4 AM dye as described above in 1 ml of medium for 30 min. Cells were rinsed once in HBSS and imaged in BrainPhys medium without Phenol red. Timelapse series were acquired at 50 ms exposure using a 488 nm LED at 200 ms intervals for 1.5 min duration. Analysis of calcium spike transients was performed using ImageJ. A wide field fluorescence microscopy was performed using a Zeiss Axio Observer.Z1 inverted fluorescence microscope (20×/0.8 air objective for live cell calcium imaging). Images were acquired using an Hamamatsu ORCA ER CCD camera and Zeiss AxiovisionRel software (ver. 4.8.2).
Phase Contrast And Whole Mount Immunofluorescence: Phase contrast microscopy was performed at room temperature directly in the biosafety hood. Images were acquired using a Zeiss Invertoskop 40C (5×/0.12 CP-Apochromat, 10×/0.25 Ph1 A-Plan and 20×/0.30 Ph1 LD A-Plan, 40×/0.50 Ph2 LD A-Plan) mounted with an Olympus DP22 color camera and cellSens acquisition software. Whole-mount immunofluorescence preparation was performed as previously described (Veenvliet et al., 2020; Olmsted and Paluh, 2021). EMLOCs were pooled on the day of fixation, rinsed once with 1× phosphate-buffered saline (PBS), and fixed in 10% neutral buffered formalin solution at 4° C. for 2 h. Samples were washed three times in 1×PBS for 5 min at room temperature. Samples were then permeabilized by three successive incubations in 0.2% Triton X-100 in 1×PBS (PBST) for 20 min at 4° C., and blocked overnight in 1% BSA in PBST. For primary antibody incubation, samples were distributed evenly to 12-well plates in 1 ml blocking solution per well. Primary antibodies were added to requisite dilutions in 1% BSA (1×PBS): anti-SOX2 (goat, 5 μg/ml); anti-GATA4 (5 μg/ml); anti-GATA6 (5 μg/ml); anti-CDH1/E-Cadherin (5 μg/ml); anti-FOXA2 (5 μg/ml); anti-β-III-tubulin (rabbit, 1:2,000); phospho-Tau Ser214 (rabbit, 1 μg/ml); CDH2/N-Cadherin (1:200); anti-Collagen Type 1 (1:500, 1 mg/ml stock); anti-Laminin (1:500, 1 mg/ml); anti-Desmin (5 μg/ml); anti-Cardiac Troponin-T (25 μg/ml). Plates were left rocking at 4° C. for 24-48 h, rinsed three times in blocking solution, then three times in PBST for 5 min each at room temperature (2 ml centrifuge tubes). Secondary antibodies were incubated 1:1,000 with 2 drops of NucBlue fixed cell stain (Invitrogen) directly in the 2 ml tubes overnight at 4° C. Goat anti-mouse Cy5 secondary antibody was added the next day following washes steps to dilute donkey anti-goat AlexaFluor 594 secondary antibody for samples stained with three antibodies (mouse, rabbit, goat). Samples were again incubated overnight rocking at 4° C. Stained and rinsed EMLO samples were post-fixed in 10% neutral buffered formalin for 20 min at 4° C., and equilibrated in 0.1 M phosphate buffer (PB: 0.025 M NaH2PO4, 0.075 M Na2HPO4, pH 7.4) containing 0.2% Triton-X 100 by three successive incubations of 5 min at room temperature. To clear samples, 0.1 M PB was aspirated and replaced with 100 μl of 88% Histodenz solution (w/v) dissolved in 0.2 M PB and filter sterilized. Samples were left in the dark at 4° C. for 24 h, mounted on glass slides and sealed in clear nail polish for imaging. Samples were imaged on a Leica confocal TCS SP5 II system in conjunction with Leica Application Suite Advanced Fluorescence software. The SP5 II system was equipped with 10×/0.30 HCX PL FLUOTAR air, 20×/0.70 HC PL APO CS air or immersion, and 40×/1.25 HCX PL APO immersion objective lenses. Complete or partial Z-stacks were acquired at ˜2-2.5 μm separation distance. If necessary, images were corrected linearly for brightness in ImageJ. Maximally projected Z-stacks were performed directly in the Leica software and exported, or were made using Z-project in ImageJ.
Quantification Of Immunofluorescence Signal: Quantification immunofluorescence signals along the anterior-posterior axis was performed as described (Rossi et al., 2021). cTnT signal was quantified from maximal projection images (z-axis) and the FOXA2 was quantified from single Z-slices in order to capture the gut tube. The anterior-posterior axis length was measured from pole-to-pole for each gastruloid. Fluorescence intensity was determined using the plot profile tool in Fiji ImageJ, and was normalized along with gastruloid length to enable comparative analysis. FOXA2 and cTnT mean curves were plotted in GraphPad Prism 9 and juxtaposed. Curves were smoothed using a LOWESS function in GraphPad. Only the single channels in question were quantified.
Statistical analysis and reproducibility: Microsoft Excel (v16.16.27) and GraphPad Prism 9 (v9.0.2) were used for statistical analysis and data plotting. Data are reported as (mean+/−s.e.m.), analyzed using paired or unpaired two-tailed t-test as indicated. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, n.s. not significant (α=0.05 threshold for significance). Power analysis was not performed. Detailed information for each experiment is provided in Results and Figure Legends. Key resources including primary antibodies, chemicals and other reagents, software and equipment, and commercial kits are provided.
Angiocrine And Pro-Cardiogenic Factors Redirect Multi-Lineage EMLO Gastruloids For Human Developmental Cardiogenesis (EMLOCs): EMLO gastruloids were previously generated with co-developing central and peripheral neurons and trunk mesendoderm including components of the enteric nervous system (Olmsted and Paluh, 2021a, 2021b). To test the ability of EMLOs to model human developmental cardiogenesis (EMLOCs), exposure to growth factors was modified during early formation and polarization stages in a revised protocol (
EMLOC Gastruloids Generate Diverse Embryonic Cell Types Of The Human Trunk Revealed By scRNAseq Analysis: Single cell sequencing of H3.1.1 derived EMLOCs was performed at two time points that are day 7 and day 16 after initial aggregation in shaking culture. The integrated dataset of both time points was analyzed (2,859 cells) (
Multiple Derivatives Of Splanchnic Mesoderm In EMLOCs Identified By scRNAseq: Cardiogenic mesoderm gives rise not only to working cardiomyocytes but also contributes to epicardium, endocardium, connective tissue, outflow tract, valves and the conduction apparatus. Cluster annotation and analysis of scRNAseq data in day 7 and day 16 EMLOCs identified diverse cell types involved in cardiogenesis arising from splanchnic mesoderm (
Human EMLOC Multi-Lineage Gastruloids Form Chamber-Like Structures With Spontaneous Contractility And Calcium Signaling: Intracellular changes in Ca2+ couple cardiomyocyte depolarization with contraction. To demonstrate that EMLOCs express calcium-regulated contractile proteins and achieve calcium-mediated contractility, we performed 3D fixed and live cell imaging analysis (
Recapitulating Early Morphogenesis Events In Human Developmental Cardiac EMLOC Gastruloids: The first cardiogenic structure to form in the anterior aspect of the mammalian embryo is called the cardiac crescent, which fuses to form the transversal heart tube that seals dorsally to generate a closed tube with outflow tracts (
EMLOC Gastruloid Cardiac Morphogenesis Occurs Anterior To Primitive Gut Tube Endoderm: The developing anterior foregut derived from endoderm has been shown to be essential for cardiogenesis in multiple organisms through crosstalk with splanchnic mesoderm and by providing mechanical cues (
Whether EMLOCs recapitulate distinct phases of cardiomyocyte differentiation and morphogenesis that are ongoing developmentally (
EMLOCs exhibit specialization over heart tube length and multi-layering of chamber walls during morphogenesis: As the cardiac crescent is remodeled into the contractile primitive heart tube in vivo, specialization over the length of the tube establishes the future blueprints for the adult heart in terms of septated chambers and outflow tracts that transmit and receive blood (
In developing heart chambers in situ, the chamber walls are multi-layered, with myocardium composed of working contractile and conducting cardiomyocytes comprising the outermost layer, and endocardium lining comprising the innermost layer (
EMLOCs capture neurogenesis within a neuro-cardiac model of human trunk development: The EMLO approach (Olmsted and Paluh, 2021a, 2021b) was developed to study early neurogenesis events in trunk development. To investigate early neural lineage biomarkers in EMLOCs immunofluorescence and scRNAseq was performed (
In the integrated scRNAseq dataset, clusters 1, 8 and 10 predominantly represented the neural lineage. Neural progenitors (cluster 1; ZIC1, RFX4, HES5, FABP7, EDNRB, NTRK2, OLIG3, MSX1) became specialized neuronal subtypes (cluster 8; INSM1, ELAVL3, DLG4, CAMK2A, SLC18A3, SLC17A6, CHRNA3, NTRK3), and a population of neural crest-derived Schwann cells was present (cluster 10; SOX10, PLP1, MPZ, S100B, TFAP2B, NGFR) (
EMLOCs express biomarkers of cardiac innervation: In parallel with neurogenesis, axonal projections navigating the extracellular space to their target sites are expected to require spatial signals to generate the selective patterning on organs for innervation. Molecular and morphogenic features of the developing heart must therefore play an active role in establishing autonomic innervation, where the proper cellular milieu and receptive fields for innervation will dictate selective neuronal interactions. As such, several genes were identified with known roles in this process that were expressed in the cardiogenic region of the UMAP plot including and that code for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), semaphorin 3A (SEMA3A), peripherin (PRPH), endothelin receptor type A (EDNRA), and ISL-1 (
By immunofluorescence, neurons were not identified within the cardiac region at the earlier day 7 and day 8 time points. We therefore analyzed the degree to which cardiogenic and neurogenic regions of the EMLOCs co-develop and integrate. In EMLOCs at day 16 or more in formation, neurons were observed both in the posterior compartment and intercalated with cardiomyocytes anteriorly, resembling in vivo ganglionated plexuses that characterize heart innervation (Ashton et al., 2018) (
This protocol (See
Coat Cultureware With hESC-qualified Matrigel Timing: 24 h.: The description is for a 35 mm culture dish: Handle and store hESC-qualified Corning Matrigel according to manufacturer's instructions with attention to preparing and storing aliquots. Thaw 5 mL parafilm-wrapped Matrigel on ice placed within a 4° C. fridge overnight. The next day, 100 μL aliquots of thawed, undiluted Matrigel are prepared on ice and restored at −20° C. for later use. To prepare freshly coated cultureware, remove new 35 mm culture dish and UV sterilize with the top off for 30 min in a laminar flow tissue culture hood. After 30 min UV sterilization, pre-chill dish at −20° C., ˜20 min prior to use. Thaw a single aliquot (100 μL) of Matrigel on ice ˜45 min prior to use. Dilute Matrigel stock 1:100 in ice cold DMEM/F-12 (e.g., 20 μL/2 mL). Add 1 mL of diluted Matrigel per dish. To polymerize surface coating, incubate dish at 37° C. in a humidified incubator with 5% CO2 for at least 1 h prior to use. The steps should be carried out on ice to prevent premature gelling and/or non-uniform coating of matrix.
Human Induced Pluripotent Stem Cell (hiPSC) Culture: Timing: ˜3-7 days; To thaw hiPSCs cryopreserved in mFreSR cryopreservation medium, transfer 1 vial containing 1 mL of cell suspension from liquid nitrogen storage to 37° C. water bath. While cell suspension is thawing, remove DMEM/F-12 from the freshly coated Matrigel plate, rinse 1× with 1 mL DMEM/F-12, and replace with 2.5 mL mTeSR Plus hiPSC pluripotency medium containing 1× penicillin-streptomycin (mTeSR Plus is supplemented 1× with penicillin-streptomycin unless otherwise specified in this protocol). Before applying mTeSR Plus, bring working volume to room temperature for ˜15 min outside of water bath. In laminar flow tissue culture hood, carefully administer 1 mL of cells in thawed mFreSR dropwise to fresh mTeSR Plus (3.5 mL total), attempting to distribute cells over the surface area of the dish. Incubate seeded cells at 37° C. in a humidified incubator with 5% CO2 overnight. The next morning, visually inspect for stem cell colony adherence (
Passaging Human Induced Pluripotent Stem Cells: Timing: ˜24 h; Description is for passage from a 35 mm culture dish to 6-well plate. Remove media from the well to be passaged and immediately add 1 mL of Gentle Cell Dissociation Reagent (GCDR) to the empty well. Incubate at room temperature for ˜3 min. The GCDR incubation time requires cell line-specific optimization according to manufacturer's instructions. The incubation time here allows cells to be released in small ‘colony patches’ and not as single cells. Gently aspirate the GCDR without dislodging the cells. Immediately add 3 mL mTeSR Plus to the well that is being passaged. To dislodge colonies, use a 5 mL serological pipette oriented orthogonally to the plane of the plate. Perform a side-to-side scraping motion over the entire area of the well. Rotate the plate 90 degrees and repeat the side-to-side scraping motion to ensure that the bulk of cells are dislodged from the substrate. Mix 2× using a P-1000 blue tip. Gently transfer 0.5 mL of dislodged cells to each well of a Matrigel-treated 6-well plate already containing 1 mL of mTeSR Plus. Cells should be added dropwise quickly and serially to obtain an even distribution in each well (1.5 ml total). Incubate the plate overnight, undisturbed at 37° C. to allow colonies to settle and adhere. The next day, visually inspect cultures for adherence of small colonies. If positive, remove the 1.5 mL mTeSR Plus. Rinse 2× in 1 mL DMEM/F-12 to remove non-adherent cell debris. Add 2 mL fresh mTeSR Plus to each well. Return the plate to the incubator. Add fresh media changes every 2-5 days, until the colonies are expanded, and cultures are ˜50-60% confluent (
Preparation Of N2B27 Basal Media: Timing: ˜30 min; Prepare 1:1 DMEM/F-12:Neurobasal Plus medium appropriately supplemented to the following final concentrations: 2% (v/v) B-27 Plus, 1% (v/v) N-2, 1× GlutaMAX, 1×MEM Non-Essential Amino Acids, 1× penicillin-streptomycin. For 500 mL N2B27 add 235 mL DMEM/F-12, 235 mL Neurobasal Plus, 10 mL B-27 Plus, 5 mL N-2, 5 mL of 100× GlutaMAX, 5 mL of 100×MEM Non-Essential Amino Acids, 5 mL of 100× penicillin-streptomycin. Use a 0.2 μm pore filter to sterilize this solution and store at 4° C. Prewarm working volumes to room temperature as needed.
Step-By-Step Method Details: STEP 1: 2D induction of hiPSC colonies for EMLOC formation: The short induction time at this step yields mesendodermal-like cellular starting material that importantly is also primed for neural differentiation. Protocols for trunk biased uniformly committed neuromesodermal progenitors (NMPs) typically rely on more sustained exposure to FGF and CHIR signaling (e.g., 4-5 d induction period) (Olmsted et al., 2020). The induction factors for 2D adherent colonies and for subsequent aggregate formation and polarization were originally identified by the Gouti laboratory to generate neuromuscular trunk organoids using human stem cells (Faustino Martins et al., 2020). These factors were then optimized for elongating multi-lineage organized (EMLO) gastruloids by our laboratory with key protocol changes previously detailed (Olmsted and Paluh, 2021). The original EMLO gastruloids were not optimized for cardiogenesis.
Timing: ˜2 days: hiPSC colonies at ˜50-60% confluency in single wells of a 6-well plate are ready for 2D induction (
STEP 2: Transition to shaking culture and EMLOC polarization: Approximately 48 h after induction as adherent 2D colonies, cultures are primed to generate single cell suspensions to form 3D aggregates by orbital shaking. This transition helps to drive formation of uniformly and appropriately sized aggregates and introduces additional mechanical cues during the early polarization stage. Initial starting aggregates with small cell number (˜50-100 □m diameter aggregates; 50-100 cells each) are critical to establish the necessary axis length for local signaling and polarization (van den Brink et al., 2014).
Timing: 2 days: 48 h after the initial induction as 2D colonies in Induction Medium, cells can be used to generate 3D aggregates. Prepare a fresh 6-well plate by treatment with 1 mL Anti-Adherence Rinsing Solution per well for 10 min. Remove the Anti-Adherence Rinsing Solution from the wells and rinse 2× with 1 mL HBSS (CM-free). On the second rinse, do not remove the HBSS to prevent drying. Remove the Induction Medium from the wells containing primed colonies and rinse 2× with 1 mL HBSS (CM-free). For enzymatic dissociation to single cells, dilute Accutase 1:1 with HBSS (CM-free) and add 1 mL per well. Incubate at 37° C. for 5-10 min (cell line-dependent incubation time). After incubation, gently remove dissociation solution and add 1 mL of N2B27 (no supplements) to the empty well. Dislodge the cells using a side-to-side scraping motion over the entire surface area of the well with a serological pipette oriented orthogonally to the surface. Triturate the suspension manually with a P-1000 pipette ˜6× to generate a single cell suspension. Combine 2-3 wells of Accutase-treated hiPSC cells. To enhance collection of all cells, add the entire volume to a 15 mL conical tube and centrifuge at 350×g for 5 min to pellet the single cells. Aspirate the supernatant from the cell pellet and resuspend in EMLOC Polarization Medium (N2B27 basal medium supplemented with 10 ng/mL FGF2, 2 ng/mL IGF1, 2 ng/mL HGF, 50 μM ROCK inhibitor Y-27632) at the appropriate cell density. Remove remaining HBSS from treated wells and transfer cell suspension to the pretreated low adherence well plate. Note: ROCK inhibitor Y-27632 promotes differentiation of hiPSCs into neural crest-like progenitors (Kim et al., 2015). IGF-1 has effects in CNS neural induction by increasing anterior neural transcription factors (Dyer et al., 2016), and HGF stimulates motogenic and morphogenic activities in development via interaction with the C met tyrosine kinase receptor (Desole et al., 2021). The combined suspensions used to generate aggregates for EMLOCs should have ˜2×106 cells total in 2 mL medium (1×106 cells/mL). Too many cells will interfere with correct ratio of factors. Too few cells will prevent any aggregate formation. Total cell number per well may range from 2×106 to 4×106 cells depending on cell line in 2 mL total volume. The high ROCK inhibitor concentration is useful to ensure single cell survival, promote aggregation, and to induce the neural crest cell lineage. Place the plate on an orbital shaker at 80 rpm clockwise in a humidified incubator with 5% CO2. Visually inspect the orbital shaking cultures at 24 h post-aggregation. At this stage, round aggregates of similar size distribution (˜50-100 μm) should be visible (
STEP 3: EMLOC cardiac induction: EMLOC cardiogenesis is stimulated with defined angiocrine and cardiogenic factors in combination (FGF2, VEGF 165, ascorbic acid) to recapitulate morphological hallmarks such as thin walled, dilated chamber-like structures with spontaneous contractility. These factors were previously shown to stimulate cardiogenesis in mouse gastruloids (Rossi et al., 2021) and are applied here, adapting our original EMLO protocol (Olmsted and Paluh, 2021) to induce human cardiogenesis within the multi-lineage gastruloid framework. Timing: 5 days, 48 h post-aggregation, initiate cardiac induction by pooling aggregates in a 15 mL conical tube and allow them to settle by gravity for 10 min as described above. Completely aspirate the Polarization Medium and rinse with 10 mL HBSS (CM-free). Let the aggregates re-settle and aspirate the HBSS. Resuspend in Cardiac Induction Medium (N2B27 supplemented with 5 ng/mL VEGF, 30 ng/mL FGF2, 0.5 mM ascorbic acid). Note: VEGF regulates the development of the vascular endothelium and endocardium through the activation of Akt signaling in endothelial cells (Madonna and de Caterina, 2009), and ascorbic acid promotes cardiac differentiation by enhancing the proliferation of cardiac progenitor cells via the MEK-ERK1/2 pathway (Cao et al., 2011). Return cells to the orbital shaker at 80 rpm clockwise in a humidified incubator with 5% CO2. The Cardiac Induction Medium can be replaced with fresh media at 4-5 d post-aggregation. It is useful to replace procardiogenic media only once to allow intra-aggregate cell-cell and paracrine signaling. Visually monitor cultures for early polarization and cardiac crescent formation (
STEP 4: EMLOC Multi-Lineage Differentiation, Chamber Morphogenesis And Innervation: After 7 d post-aggregation, Cardiac Induction Medium is replaced with non-supplemented N2B27. This is intended to permit neurogenesis and cardiogenesis without further lineage restriction, favoring aggregate-derived signaling factors and self-organization. Timing: 18+ days: The maximal duration for continued maintenance and development of EMLOC analysis has not yet been determined beyond 25 d from induction. At 7 d post-aggregation, collect aggregates in a 15 mL tube and let settle at 37° C. for 10 min. Remove medium and rinse with HBSS (CM-free). Let re-settle and remove the HBSS. Exchange the medium to non-supplemented N2B27 and re-distribute EMLOCs evenly to new cultureware freshly treated with Anti-Adherence Rinsing Solution. Place on orbital shaker at 75 rpm clockwise in a humidified incubator with 5% CO2. Replenish N2B27 basal media every 3-5 d as needed to maintain the maturing EMLOCs for the remainder of the protocol.
Expected Outcomes: The EMLOC formation and multi-lineage differentiation protocol occurs in four general stages (
Stage 2 is characterized by exposure to FGF2, IGF1 and HGF in N2B27 basal medium for 48 h during aggregation of 2D primed colonies to 3D spherical aggregates. This necessitates dissociation of primed colonies to a single cell suspension using diluted Accutase. Prior to aggregation on orbital shaking culture in low-adhesion 6-well plates, a satisfactory single cell suspension should be verified by visual inspection using a tissue culture microscope. Single cells are applied at 2-4×106 cells/well depending on cell line in 2 mL of Aggregation Medium. After 24 h on the orbital shaker (80 rpm), spherical aggregates with size distribution of ˜50-100 μm should be present (
At Stage 3, early EMLOC aggregates undergo cardiac induction by exposure to the angiocrine and cardiogenic factors FGF2, VEGF and ascorbic acid in N2B27 basal medium. By 48 h after exposure to the Cardiac Induction Medium (day 4 post-aggregation), polarized EMLOCs are oblong with cardiac crescent structures evident on visual inspection (
The contractile, polarized EMLOCs undergo robust neurogenesis during Stage 4 and were maintained in the original publication to day 25. By this time point, the cTnT+ cardiac region should be populated by a subset of the neurons generated (
This protocol describes the generation of EMLOCs with the Hispanic-Latino H3.1.1 hiPSC line. In Olmsted and Paluh (2021), we previously demonstrated the use of this line and others to reproducibly generate enteric gut formation with neural integration in an elongating multi-lineage organized (EMLO) gastruloid model (Olmsted and Paluh, 2021). In Tomov et al. (2016) we comprehensively compared multiple ethnically-diverse hiPSC lines generated in collaboration with our laboratory (Chang et al., 2015), including cardiac differentiation and narrow effective windows in CHIR 99021 concentration gradient that are cell-line specific (Tomov et al., 2016). These publications may be helpful when replicating the protocol with a new hiPSC line, ideally at low passage number.
This study applies a unique reagent that is a Hispanic-Latino low passage hiPSC line previously generated and initially characterized with other ethnically-diverse hiPSC lines (Chang et al., 2015; Tomov et al., 2016). The ethnically diverse hiPSC lines are being made available via WiCell (Madison, Wisconsin).
The consistent lifelong critical functioning of the adult human heart is established during embryonic development in a process known as cardiogenesis. EMLOCs provide the first detailed insights into integrated neurogenesis and cardiogenesis in a human gastruloid developmental model. The complex process of cardiogenesis requires short-range interactions with surrounding tissues and occurs in conjunction with long-range input by neurons through progressive innervation (Harvey, 2002; Hasan, 2013). As the first organ to function in the embryo, the developing heart begins to supply blood to the growing fetal brain as a closed tube, even before undergoing dramatic structural reorganization and maturation into septated chambers with outflow tracts. Such complexity and dependence on multiple non-cardiac tissue inputs has made it difficult to recapitulate human heart development using traditional in vitro models, requiring instead refined gastruloid technologies.
The intracardiac nervous system is sometimes colloquially referred to as the “brain within the heart” (Campos et al., 2018). Using sophisticated methodologies such as optogenetic stimulation, the role of peripheral cardiac neural circuitry in pacemaking and conduction is beginning to be understood (Rajendran et al., 2019; Fedele and Brand, 2020). Innervation of the heart in vivo is predominately autonomic, where sympathetic neurons can directly innervate working cardiomyocytes in the ventricular wall, and are networked as so-called ganglionated plexuses (Zaglia et al., 2017). The neurons that begin to develop in EMLOCs at the time when spontaneous contractility is first observed (˜day 7) are unlikely to substantially contribute to contractile function, since at this stage they are relatively few in number and do not project into the cardiogenic region. This is consistent with in vivo development where contractility of the heart tube occurs prior to innervation that is established later (George et al., 2020). However, organized neuronal networks resembling ganglionated plexuses were observed as EMLOCs progressively matured. At the day 7 time point, neurons were localized distantly from the cardiogenic region, before expanding significantly in number to migrate, embrace and populate the myocardial layer over time. One potential explanation is a microenvironment switch from axon-repulsive to axon-permissive as ECM in the cardiac jelly is degraded. The ECM-rich cardiac jelly in vivo contains chondroitin sulfate proteoglycans and other components known to exert repulsive or pausing effects on axons during navigation and regeneration (Tom et al., 2004). Degradation of the cardiac jelly during development is physiologic and required for normal cardiac chamber morphogenesis (Kim et al., 2018). Differential regulation of SEMA3A expression may also play a role. Within this framework, our data support the adherence of EMLOC events to physiologic spatiotemporal developmental processes for establishing contractile chambers with supplied neurons (Hasan, 2013; George et al., 2020). Neuromuscular interactions between cardiac innervating neurons and cardiomyocytes at the “neuro-cardiac junction” remains poorly understood (Zaglia et al., 2017) including biomarkers. Synapses with cardiomyocytes are postulated to be mediated through an alternate structure other than the nAChR machinery in skeletal neuromuscular junctions (Sargent and Garrett, 1995). Traditional 2D hiPSC differentiation protocols that generate human neurons and cardiomyocytes separately and then co-culture these cells to obtain structural and functional detail are typically used to study innervation, as has been done for the skeletal muscle neuromuscularjunction (Darabid et al., 2014; Steinbeck et al., 2016). EMLOCs are expected to provide a developmental and spatiotemporal perspective of the neuro-cardiac junction.
Efforts to study heart development and function using human cells also focus on separate cardiac mechanisms and include combined tissue engineering platforms and solutions (Ma et al., 2015; Macqueen et al., 2018). Such top-down human intervention of biofabricated tissues and organs has not yet achieved developmentally patterned neuronal innervation (Das et al., 2020), but may benefit from this EMLOC study. Gastruloid models that more closely mimic embryogenesis are an exciting alternative to achieve and study organogenesis (van den Brink et al., 2014; Beccari et al., 2018; Moris et al., 2020). A recent study with mESCs made significant advances and achieved early key features of cardiogenesis (Rossi et al., 2021). Our developmental model of human cardiogenesis in gastruloids further advances cardiac models by including neuronal co-development and association with the myocardium. The scRNAseq analysis that indicates that we have established multiple prerequisites for innervation. In our previous study of EMLO gastruloids that generated CNS and PNS integration with mixed lineage trunk identity (Olmsted and Paluh, 2020a), we achieved self-organized spinal neurons, neural crest, and a primitive gut tube surrounded by splanchnic mesenchyme, thereby providing much of the ideal cardiogenic microenvironment. By modifying the EMLO protocol (Olmsted and Paluh, 2020b) to include pro-cardiogenic and angiogenic factors, VEGF and ascorbic acid, that were applied in the mESC in vitro cardiogenesis study (Rossi et al., 2021), we achieved coupled cardiogenesis and neurogenesis. Through comprehensive biomarker analysis and live cell calcium imaging, it is demonstrated here that EMLOCs recapitulate numerous key features of human cardiogenesis including cardiac crescent transformation into the contractile heart tube, cardiomyocyte differentiation versus remodeling phases, and formation of chamber and outflow tract-like structures. Cardiogenesis occurs anterior to primitive gut tube-like endodermal cells that in vivo are thought to be required (Nascone and Mercola, 1995; Schultheiss et al., 1995; Varner and Taber, 2012; Anderson et al., 2016; Kidokoro et al., 2018; Han et al., 2020). By comparing calcium transients per minute in the EMLOC system versus the mESC cardiogenesis study in gastruloids, a similar species-specific ratio to that between the resting adult heart rate in human versus mouse (˜8× higher in the murine model) that have similar processes of cardiogenesis (Krishnan et al., 2014) was observed.
EMLOCs will open new opportunities to study fundamental questions on neuromodulation of contracting cardiomyocytes with relevance to neurocardiogenic syncope and other neural-based arrhythmia pathologies (Ashton et al., 2018). As well, such a neuro-cardiac model system is expected to provide fundamental insights into the pathophysiology of congenital heart disease and potential treatments in addition to viral infection studies and in vitro pharmacotherapy testing and discovery. As a drastically needed component of in vitro stem cell systems, innervation in non-neural tissue, organ, and embryo models (Das et al., 2020; Sahu and Sharan, 2020) is beginning to be achieved in EMLO and EMLOC gastruloids to advance innervation research. Embodiments, of the present disclosure including models will have broad biomedical relevance for neuro-cardiac development and human organ innervation initiatives.
In this work a gastruloid model enabling co-development and self-integration of human neuronal and cardiac tissue precursors in multicellular and multi-lineage context was provided. These results extend previous work with neurogenesis and gut development in EMLO gastruloids to promote concomitant cardiogenesis that recapitulates multiple key features of in vivo heart development. A limitation of this study is the use of one hiPSC line and ability to evaluate only two developmental time points by scRNAseq, of the numerous stages analyzed and described. Nine lines previously evaluated for EMLO formation revealed reproducibility of structural organization and cell types but with differences in efficiency between lines that can be optimized (Olmsted and Paluh, 2021a; 2021b). The EMLOC system represents the first developmentally based human neuro-cardiac model that can be applied to advance knowledge. EMLOCs neurons are co-produced endogenously within the same gastruloid and in the context of the developing heart. This is as opposed to separate generation of neural or cardiac cells followed by combination by fusion or co-cultures (sometimes referred to as assembloids). The EMLOC generates a more natural path to neuron integration with the heart, both spatially and temporally, that mimics ex vivo what is seen in human embryos.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate embodiments of the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate and the inventors intend for embodiments of the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, the scope of the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the scope of the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The present application claims the benefit of prior-filed U.S. Provisional Application Ser. No. 63/311,498 that was filed on Feb. 18, 2022 and U.S. Provisional Application Ser. No. 63/419,507 that was filed on Oct. 26, 2022, the disclosure of both these applications is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US23/13415 | 2/20/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63311498 | Feb 2022 | US | |
| 63419507 | Oct 2022 | US |
