Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.
The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.
Congenital heart defects (CHDs) are the most common type of congenital defect in humans and affect approximately 1% of all live births1,2,3. Under most circumstances, the reasons for CHDs remain unknown. The ability to create human heart models in the lab that closely resemble the developing human heart constitutes a significant step forward to directly study the underlying causes of CHDs in humans rather than in surrogate animal models.
The epitome of laboratory-grown tissue models are organoids, 3D cell constructs that resemble an organ of interest in cell composition and physiological function. Organoids are often derived from stem cells or progenitor cells and have been successfully used to model many organs such as the brain4,5, kidney6,7, intestine8,9, lung10,11, liver12,13, and pancreas14,15, just to name a few. Recent studies have emerged demonstrating the feasibility of creating self-assembling heart organoids to study heart development in vitro. These models include using mouse embryonic stem cells (mESCs) to model early heart development16,17 up to atrioventricular specification18 and human pluripotent stem cells (hPSCs) to generate multi-germ layer cardiac-endoderm organoids19 and chambered cardioids20 with highly complex cellular composition.
This paper presents a novel 3-step WNT modulation protocol to generate highly complex hHOs in an efficient and cost-effective manner. Organoids are generated in 96-well plates, resulting in a scalable, high-throughput system that can be easily automated. This method relies on creating hPSC aggregates and triggering developmental steps of cardiogenesis, including mesoderm and cardiac mesoderm formation, first and second heart field specification, proepicardial organ formation, and atrioventricular specification. After 15 days of differentiation, hHOs contain all major cell lineages found in the heart, well-defined internal chambers, atrial and ventricular chambers, and a vascular network throughout the organoid. This highly sophisticated and reproducible heart organoid system is amenable to investigating structural, functional, molecular, and transcriptomic analyses in the study of heart development, and diseases, and pharmacological screening.
1. hPSC culture and maintenance
NOTE: The human induced PSCs (hiPSCs) or human embryonic stem cells (hESCs) need to be cultured for at least 2 consecutive passages after thawing before being used to generate EBs for differentiation or further cryopreservation. hPSCs are cultured in PSC medium (see the Table of Materials) on basement-membrane-extracellular matrix (BM-ECM)-coated 6-well culture plates. When performing medium changes on hPSCs in 6-well plates, add the medium directly to the inner side of the well rather than directly on top of the cells to prevent unwanted cell detachment or stress. Users should be wary of pre-warming PSC media that should not be warmed at 37 °C; all PSC media used in this protocol were thermostable.
2. Generation of 3D self-assembling human heart organoids
3. Organoid analysis
To achieve self-organizing hHO in vitro, we modified and combined differentiation protocols previously described for 2D monolayer differentiation of cardiomyocytes21 and epicardial cells22 using Wnt pathway modulators and for 3D precardiac organoids16 using the growth factors BMP4 and Activin A. Using the 96-well plate EB and hHO differentiation protocol described here and shown in Figure 1, the concentrations and exposure durations of the Wnt pathway activator CHIR99021 were optimized to yield highly reproducible and complex hHOs derived from human PSCs. hPSCs or hESCs cultured in PSC medium to 60-80% confluency in colony-like formation with minimal to no visible differentiation are ideal for EB generation (Figure 1B).
EBs were allowed to incubate for 48 h with a medium change after 24 h before starting differentiation at day 0. On day 0, the EBs should appear as a dark spherical aggregate at the center of each well under a light microscope (Figure 1B). The differentiation protocol starts on day 0 with the Wnt pathway activation and growth factor addition for exactly 24 h. This mesoderm induction followed by a cardiac mesoderm induction on day 2 using the Wnt pathway inhibitor Wnt-C59 will result in a significant enlargement of the organoid from ~200 µm in diameter to 500-800 µm in diameter at day 4 and to as much as 1 mm (organoids may experience a slight reduction in size by day 15 (Figure 1C)). The hHO will begin beating as early as day 6 (Video 1), with 100% of the organoids showing visible beating by day 10 (Video 2) (unless undergoing drug treatment or if inadequate hPSCs were used to generate the EBs). This has been observed in 5 distinct hPSC cell lines23.
To evaluate the capacity of the hHOs to represent various steps of the physiological development of the heart, we collected organoids at various time points throughout the differentiation protocol and looked for the presence and transcriptomic expression of heart field markers. Immunofluorescent staining for the first heart field (FHF) marker, HAND1, and the second heart field (SHF) marker, HAND2, revealed their nuclear presence in these cardiac progenitor cells arising at around day 3 and day 5, respectively (Figure 3A).
The expression of both markers happens at regions of the organoids that diminish in size after day 7 for the FHF and after day 9 for the SHF. Interestingly, high-magnification images of day 7 organoids revealed that most HAND1-expressing cells were cardiomyocyte in origin (as shown by the cardiomyocyte-specific marker TNNT2). In contrast, many of the HAND2-expressing cells did not express the cardiomyocyte marker (Figure 3B). This observation is in agreement with the precardiac organoids derived from mouse ESCs demonstrating the development of non-myocyte cells from SHF progenitor cells16. It is important to note that the RNA-Sequencing data show that the RNA transcripts for both HAND1 and HAND2 were expressed from day 3 onwards, with the FHF marker being more highly expressed between days 3 and 11 and the SHF marker being more highly expressed after day 13 (Figure 3C).
Immunofluorescence staining revealed the presence of markers of various cell-type lineages that make up the human heart. Myocardial tissue (identifiable using the cardiomyocyte-specific marker TNNT2) adjacent to epicardial tissue (marked by the nuclear transcription factor WT1 and the epithelial membrane marker TJP1) (Figure 4A). Endocardial cells expressing NFATC1 were detected lining the walls of internal chamber-like structures within the organoids (Figure 4B). Endothelial cells in a vessel-like network can be seen as early as day 13 of differentiation (Figure 4C). Lastly, we report the presence of cardiac fibroblasts intermixed throughout the organoid (Figure 4D). These cell-type markers were also observed in the RNA-Seq gene expression profiles (Figure 4E). The composition of cell types in the organoids, as measured by area of the organoid they occupy, were found to be ~58% cardiomyocytes, with the rest comprising of non-myocyte cardiac cells, including epicardial cells (~15%), endocardial cells (~13%), cardiac fibroblasts (~12%), and endothelial cells (~1%) (Figure 4F).
The electrophysiological function of the organoids was measured by live calcium imaging of individual cells in whole organoids. Fluo-4 fluorescence intensity varies over time due to calcium entry and exit from the cell, revealing regular action potentials (Figure 5A). Heatmaps showing calcium intensities over a high-magnification region of the organoid show the increased intensity because of calcium transients in individual cells (Figure 5B and Video 3).
Figure 1: Embryoid body generation and heart organoid differentiation steps. (A) (1–2) Dissociated cells are seeded into wells of a 96-well ultra-low attachment plate via a multichannel pipette. (3) The 96-well plate is then centrifuged, which allows the cells to aggregate in the center. (4) Over time, following the addition of growth factors and pathway modulators, the embryoid body begins to differentiate into several cardiac lineages and form spatially and physiologically relevant distinct cell populations surrounding internal microchambers. (B) Representative images of the progression of embryoid body generation, beginning with 2-dimensional iPSC culture (left) and ending with a Day 0 embryoid body (right); scale bar = 500 µm. (C) Summary of human heart organoid differentiation protocol, including chemical pathway modulators and inhibitors with respective time points, durations, and developing organoid images under light microscopy from day 1 to day 15; scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 2: Whole organoid mounting on slides for imaging. Steps for preparing slides and mounting organoids for imaging. (A) Placement of microbeads onto the periphery of a glass slide. (B) Covering the microbeads with mounting medium. (C) Transferring organoids onto the slide between the beads and removing excess liquid surrounding the organoids. (D) Covering the organoids with clearing/mounting medium. (E) Placing the coverslip on top of the slide with organoids and beads. Please click here to view a larger version of this figure.
Figure 3: First heart field and second heart field specification in hHOs recapitulates physiological human heart development. (A) Confocal immunofluorescence images of day 3 to day 11 hHOs showing the formation of FHF (HAND1, top) and SHF (HAND2, bottom), and cardiomyocytes (TNNT2) and nuclear dye DAPI; scale bars = 500 µm. (B) High-magnification images of day 7 organoids showing co-localization of HAND1 and HAND2 with the cardiomyocyte marker TNNT2; scale bars = 50 µm. (C) RNA-Seq gene expression profiles of FHF marker HAND1 (red) and SHF marker HAND2 (blue) from day 0 to day 19. Abbreviations: hHOs = human heart organoids; FHF = first heart field; SHF = second heart field; HAND = heart and neural crest derivatives expressed; TNNT2 = cardiac troponin T2; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 4: hHOs developmultiple cardiac lineages. (A–D) Confocal immunofluorescence images of day 15 hHOs showing the formation of cardiomyocytes (TNNT2) and staining with nuclear dye DAPI in non-myocyte cardiac cells. (A) Whole organoid and high magnification of epicardial marker WT1 (green) and epithelial membrane marker TJP1 (white) showing epicardial cells of epithelial origin on top and adjacent to myocardial tissue; scale bar = 500 µm, inset = 50 µm. (B) Endocardial marker NFATC1 (green) expression on the lining of chambers; scale bar = 500 µm. (C) Endothelial vessel network in hHOs shown by PECAM1 (green). Scale bar = 500 µm. (D) Cardiac fibroblasts markers THY1 and VIM shown in green and white, respectively, distributed throughout the organoid. Scale bar = 500 µm. (E) RNA-Seq gene expression profiles of the major cell types present in the hHOs from days 0 to 19 of differentiation. Abbreviations: hHOs = human heart organoids; TNNT2 = cardiac troponin T2; DAPI = 4',6-diamidino-2-phenylindole; WT1 = Wilm's tumor-1 transcription factor; NFATC1 = cytoplasmic nuclear factor of activated T cell; PECAM1 = platelet endothelial cell adhesion molecule-1; VIM = vimentin. (F) Pie chart of average tissue type composition in hHOs, calculated as the percentage area with the respective cell marker over an entire organoid by nuclear staining across three z-planes throughout the organoid using ImageJ. Please click here to view a larger version of this figure.
Figure 5: Fluo-4 live calcium transient recordings in live human heart organoids. (A) Representative calcium transient recordings of individual cardiomyocytes within whole organoids. (B) Heatmap showing low and peak calcium levels between action potentials as determined by Fluo-4 intensity; scale bars = 10 µm. Please click here to view a larger version of this figure.
Video 1: Live imaging of representative organoid derived from hPSCs at day 6 of differentiation under light microscopy at room temperature. Abbreviation: hPSC = human pluripotent stem cell. Please click here to download this Video.
Video 2: Live imaging of representative organoid derived from hPSCs at day 15 of differentiation under light microscopy at room temperature. Abbreviation: hPSC = human pluripotent stem cell. Please click here to download this Video.
Video 3: Live recording of day 10 organoid showing heatmap of calcium transients under a fluorescence microscope. Please click here to download this Video.
Recent advances in human stem cell-derived cardiomyocytes and other cells of cardiac origin have been used to model human heart development22,24,25 and disease26,27,28 and as tools to screen therapeutics29,30 and toxic agents31,32. Here, we report an easy-to-implement, highly reproducible protocol to generate and differentiate EBs into highly complex hHOs. This protocol has been successful in multiple cell lines, including hPSCs and hESCs23, showing consistent beating frequencies and cell type organization. This protocol draws aspects from previously described protocols for cardiomyocyte differentiation24, epicardial cell differentiation22, and precardiac organoids derived from mouse ESCs16 and optimizes the stepwise modulation of canonical WNT signaling using chemical inhibitors and growth factors in a fully defined medium. Several optimization methodologies were employed in the generation of this protocol.
First, the chemical inhibitor concentrations and exposure durations, as well as the addition of growth factors, have been optimized for the 3D environment and are discussed in previous work23. These were optimized to elucidate structures with physiological complexity and representation of the in vivo human heart, with physiological composition and ratios of cardiomyocytes to non-myocyte cardiac cell types (epicardial cells, cardiac fibroblasts). Second, the two-thirds medium change strategy allows minimal agitation of the EBs/organoids, as they sit in suspension near the bottom of the well, while also facilitating a gradient exposure to chemical inhibitors and growth factors when the medium is refreshed. The combination of cardiac mesoderm differentiation through Wnt pathway activation, followed by inhibition24, and the subsequent induction of proepicardial specification via a second Wnt pathway activation22, allows for a single protocol to yield highly complex hHOs. The organoids grow up to 1 mm after 15 days of differentiation and can be easily transferred for live or fixed analyses and assays. Third, given the large size of the organoids, the use of microbeads or other similar structures to maintain space between the slide and coverslip was found to better preserve the 3D structure of the organoids and improve the imaging process.
This developing human heart model allows access to otherwise inaccessible stages of heart development, such as early first and second heart field specification-observed between days 3 and 9 of differentiation-and organization into cardiac progenitor cells that give rise to heart tissues, including the myocardium, endocardium, epicardium, endothelial vasculature, and supporting cardiac fibroblasts, which were observed on day 15 of differentiation. The tissue types present in the heart organoids derived from this protocol are highly representative of the human fetal heart in both composition33 and transcriptomic profile23,34. They can therefore facilitate tissue-tissue and cell-cell higher-order interactions resembling that of the in vivo heart. This protocol was highly efficient and reproducible across experiments and cell lines, yielding organoids that comprise mostly of cardiomyocytes and include non-myocyte cardiac cells, such as epicardial cells, endocardial cells, cardiac fibroblasts, and endothelial cells, representing the physiological composition23,33,35.
Analyses of the ultrastructure of the forming cardiomyocytes via transmission electron microscopy and the development of chambers and a vascular network via optical coherence tomography and confocal imaging are discussed in detail in previous work23. A great advantage of this heart organoid protocol over other existing protocols recently published17,18,19,20,36,37 is the robust formation of an endothelial network throughout the organoid, allowing the ability to investigate vascular development and disease in the early human heart, without the need for further external inductions to the protocol. Lastly, functional analysis of the heart organoids is achievable through various approaches, including the use of a calcium-sensitive dye to track the calcium transients in the cardiomyocytes across the organoid. Using high-resolution microscopy, we recorded the fluorescence intensity of calcium entering and exiting cells and observed highly representative action potentials. Other possible functional analyses methods include the use of a transgenic line with a calcium-sensitive indicator or direct recording using a microelectrode array23.
The heart organoids described here are recapitulative of the developing human fetal heart, yet are limited in demonstrating more mature, adult-like features. Future protocols may build on the protocol described here to induce maturation in these organoids and yield constructs that better model the adult heart. Moreover, this protocol is designed to create miniature models of the human heart and is limited to research studies of heart development and disease or for pharmaceuticals screening and may not be suitable as a means of clinical intervention such as replacement of heart tissue via transplantation. Overall, we describe here an easy-to-follow and cost-effective protocol to generate highly reproducible and sophisticated human heart organoids that can facilitate research studies in human heart development, disease etiology, and pharmacological screening.
The authors have nothing to disclose.
This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award numbers K01HL135464 and R01HL151505 and by the American Heart Association under award number 19IPLOI34660342. We wish to thank the MSU Advanced Microscopy Core and Dr. William Jackson at the MSU Department of Pharmacology and Toxicology for access to confocal microscopes, the IQ Microscopy Core, and the MSU Genomics Core for sequencing services. We also wish to thank all members of the Aguirre Lab for their valuable comments and advice.
Antibodies | |||
Alexa Fluor 488 Donkey anti- mouse | Invitrogen | A-21202 | 1:200 |
Alexa Fluor 488 Donkey anti- rabbit | Invitrogen | A-21206 | 1:200 |
Alexa Fluor 594 Donkey anti- mouse | Invitrogen | A-21203 | 1:200 |
Alexa Fluor 594 Donkey anti- rabbit | Invitrogen | A-21207 | 1:200 |
Alexa Fluor 647 Donkey anti- goat | Invitrogen | A32849 | 1:200 |
HAND1 | Abcam | ab196622 | Rabbit; 1:200 |
HAND2 | Abcam | ab200040 | Rabbit; 1:200 |
NFAT2 | Abcam | ab25916 | Rabbit; 1:100 |
PECAM1 | DSHB | P2B1 | Rabbit; 1:50 |
TNNT2 | Abcam | ab8295 | Mouse; 1:200 |
THY1 | Abcam | ab133350 | Rabbit; 1:200 |
TJP1 | Invitrogen | PA5-19090 | Goat; 1:250 |
VIM | Abcam | ab11256 | Goat; 1:250 |
WT1 | Abcam | ab89901 | Rabbit; 1:200 |
Media and Reagents | |||
Accutase | Innovative Cell Technologies | NC9464543 | cell dissociation reagent |
Activin A | R&D Systems | 338AC010 | |
B-27 Supplement (Minus Insulin) | Gibco | A1895601 | insulin-free cell culture supplement |
B-27 Supplement | Gibco | 17504-044 | cell culture supplement |
BMP-4 | Gibco | PHC9534 | |
Bovine Serum Albumin | Bioworld | 50253966 | |
CHIR-99021 | Selleck | 442310 | |
D-(-)-Fructose | Millipore Sigma | F0127 | |
DAPI | Thermo Scientific | 62248 | 1:1000 |
Dimethyl Sulfoxide | Millipore Sigma | D2650 | |
DMEM/F12 | Gibco | 10566016 | |
Essential 8 Flex Medium Kit | Gibco | A2858501 | pluripotent stem cell (PSC) medium containing 1% penicillin-streptomycin |
Fluo4-AM | Invitrogen | F14201 | |
Glycerol | Millipore Sigma | G5516 | |
Glycine | Millipore Sigma | 410225 | |
Matrigel GFR | Corning | CB40230 | Basement membrane extracellular matrix (BM-ECM) |
Normal Donkey Serum | Millipore Sigma | S30-100mL | |
Paraformaldehyde | MP Biomedicals | IC15014601 | Powder dissolved in PBS Buffer – use at 4% |
Penicillin-Streptomycin | Gibco | 15140122 | |
Phosphate Buffer Solution | Gibco | 10010049 | |
Phosphate Buffer Solution (10x) | Gibco | 70011044 | |
Polybead Microspheres | Polysciences, Inc. | 73155 | 90 µm |
ReLeSR | Stem Cell Technologies | NC0729236 | dissociation reagent for hPSCs |
RPMI 1640 | Gibco | 11875093 | |
Thiazovivin | Millipore Sigma | SML1045 | |
Triton X-100 | Millipore Sigma | T8787 | |
Trypan Blue Solution | Gibco | 1525006 | |
VECTASHIELD Vibrance Antifade Mounting Medium | Vector Laboratories | H170010 | |
WNT-C59 | Selleck | NC0710557 | |
Andere | |||
1.5 mL Microcentrifuge Tubes | Fisher Scientific | 02682002 | |
15 mL Falcon Tubes | Fisher Scientific | 1495970C | |
2 mL Cryogenic Vials | Corning | 13-700-500 | |
50 mL Reagent Reservoirs | Fisherbrand | 13681502 | |
6-Well Flat Bottom Cell Culture Plates | Corning | 0720083 | |
8 Well chambered cover Glass with #1.5 high performance cover glass | Cellvis | C8-1.5H-N | |
96-well Clear Ultra Low Attachment Microplates | Costar | 07201680 | |
ImageJ | NIH | Image processing software | |
Kimwipes | Kimberly-Clark Professional | 06-666 | laboratory wipes |
Micro Cover Glass | VWR | 48393-241 | 24 x 50 mm No. 1.5 |
Microscope Slides | Fisherbrand | 1255015 | |
Moxi Cell Counter | Orflo Technologies | MXZ001 | |
Moxi Z Cell Count Cassette – Type M | Orflo Technologies | MXC001 | |
Multichannel Pipettes | Fisherbrand | FBE1200300 | 30-300 µL |
Olympus cellVivo | Olympus | For Caclium Imaging, analysis with Imagej | |
Sorvall Legend X1 Centrifuge | ThermoFisher Scientific | 75004261 | |
Thermoshaker | ThermoFisher Scientific | 13-687-711PM | |
Top Coat Nail Varish | Seche Vite | Can purchase from any supermarket |