Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Congenital heart defects (CHDs) are the most common type of congenital defect in humans and affect approximately 1% of all live births^1,2,3. 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 brain^4,5, kidney^6,7, intestine^8,9, lung^10,11, liver^12,13, and pancreas^14,15, 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 development^16,17 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.

Protokół

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.

1. To coat the well-plates with the BM-ECM, thaw one aliquot of the BM-ECM (stored at -20 °C according to the manufacturer's instructions) on ice and mix 0.5 mg of the BM-ECM with 12 mL of cold Dulbecco's modified Eagle's medium (DMEM)/F12 medium (stored at 4 °C). Distribute 2 mL of the DMEM/F12-BM-ECM mixture onto each well of a 6-well plate and incubate at 37 °C for at least 2 h.
2. To thaw the cells, first, thaw the hPSC cryovial in a 37 °C bead or water bath for 1-2 min until only a small amount of ice is visible. Transfer the thawed cells to a centrifuge tube and slowly add 8-9 mL of the PSC medium supplemented with 2 µM of the ROCK inhibitor, thiazovivin (Thiaz), and centrifuge at 300 × g for 5 min. Remove the supernatant and resuspend the cell pellet in the PSC medium supplemented with 2 µM of Thiaz. Distribute the cells in the culture medium into 1-2 wells depending on the cryovial cell concentration and culture at 37 °C, 5% CO₂ for 24 h before changing the PSC medium.
3. Change the medium on the cells at 48 h intervals. Perform washes and medium changes using DMEM/F12 (1 mL/well) and the PSC medium (2 mL/well), respectively.
   NOTE: Washes help remove cell waste and debris while fresh media changes provide cells with a renewed source of nutrients.
4. Passage the cells upon subconfluency (60-80% confluent) by aspirating the medium, then washing each well with 1 mL of 1x Dulbecco's phosphate-buffered solution (no calcium, no magnesium; DPBS). Aspirate the DPBS and add 1 mL of the dissociation reagent for hPSCs (see the Table of Materials), followed by the aspiration of all but a thin film of the reagent after 10 s.
5. Incubate for 2-5 min with the thin film of the dissociation reagent for hPSCs until gaps form between cells.
   NOTE: The time to stop the dissociation is cell-line-dependent.
6. Add 1 mL of the PSC medium supplemented with 2 µM of Thiaz (PSC medium+Thiaz) to the well and gently tap the plate to induce cell detachment. Pipette the detached cells in the medium 1-2 times to break up any large colonies, and resuspend the cells in PSC medium+Thiaz in a 1:6 well ratio (cells from 1 well resuspended in 12 mL of culture medium). Replate the cells on BM-ECM-coated wells.

2. Generation of 3D self-assembling human heart organoids

1. Embryoid body (EB) formation:
   NOTE: It is imperative to limit observable differentiated cells prior to embryoid body formation. Two to three wells of a 6-well plate at a 60-80% confluency will yield enough cells for a single 96-well plate of organoids. All media should be aliquoted and warmed in a 37 °C bead or water bath before any medium changes to minimize temperature shock to the EBs or organoids (this does not include cell dissociation reagents). See Figure 1A,B.
   1. Day -2
      1. To create EBs, on day -2, wash sub-confluent hPSCs (60-80% confluent) with DPBS for at least 10 s to wash any cell debris and aspirate the DPBS.
      2. To detach the cells and release them into a single-cell state, add 1 mL of room-temperature cell dissociation reagent (see the Table of Materials) to each well for 3-6 min. Gently tap the plate ~5 times every minute to induce detachment while checking under the microscope. Add 1 mL of PSC medium+Thiaz to stop the reaction.
      3. To collect the cells and break up any remaining aggregates, pipette the media up and down in the well 2-3 times to generate a single-cell suspension. Transfer the single-cell suspension to a centrifuge tube and spin for 5 min at 300 × g.
      4. To obtain the desired cell concentration, discard the supernatant and resuspend the cells in 1 mL of PSC medium+Thiaz. Count the cells using a cell counter or hemocytometer and dilute the cells in PSC medium+Thiaz to a concentration of 100,000 cells/mL.
      5. To distribute the cells for EB formation, use a multichannel pipette to add 100 µL (10,000 cells) to each well of a round-bottom ultra-low attachment 96-well plate. Centrifuge the plate at 100 × g for 3 min and incubate for 24 h at 37 °C, 5% CO₂.
   2. Day -1
      1. Carefully remove 50 µL of medium from each well and add 200 µL of fresh PSC medium warmed to 37 °C to achieve a final volume of 250 µL per well. Incubate the cells for 24 h at 37 °C, 5% CO₂.
         NOTE: Remove and add medium carefully on the side of the well to avoid disturbing the EBs at the bottom of the well. Due to the delicate nature of the EBs and the suspension culture, it is necessary to leave a small volume of liquid in each well when changing the medium to avoid disturbing the EBs.
2. Human Heart Organoid (hHO) Differentiation:
   NOTE: All media should be warmed in a 37 °C bead or water bath prior to any media changes. Remove and add medium carefully on the side of the well to avoid disturbing the developing organoids at the bottom of the well. Washes are not needed between media changes to minimize agitation and allow the gradual removal of inhibitors and growth factors. RPMI with 2% B-27 supplement (Table of Materials) was used throughout the differentiation protocol. B-27 supplement contains insulin unless specified (insulin-free in days 0-5). See Figure 1C.
   1. Day 0
      1. To initiate differentiation towards a mesoderm lineage, remove 166 µL of medium from each well (~2/3^rd of total well volume) and add 166 µL of RPMI 1640 containing insulin-free B-27 supplement, 6 µM CHIR99021, 1.875 ng/mL bone morphogenetic protein 4 (BMP4), and 1.5 ng/mL Activin A for a final well concentration of 4 µM CHIR99021, 1.25 ng/mL BMP4, and 1 ng/mL Activin A. Incubate for 24 h at 37 °C, 5% CO₂.
   2. Day 1
      1. Remove 166 µL of medium from each well and add 166 µL of fresh RPMI 1640 with insulin-free B-27 supplement. Incubate for 24 h at 37 °C, 5% CO₂.
   3. Day 2
      1. To induce cardiac mesoderm specification, remove 166 µL of medium from each well and add 166 µL of RPMI 1640 containing insulin-free B-27 supplement and 3 µM Wnt-C59 for a final well concentration of 2 µM Wnt-C59. Incubate for 48 h at 37 °C, 5% CO₂.
   4. Day 4
      1. Remove 166 µL of medium from each well and add 166 µL of fresh RPMI 1640 with insulin-free B-27 supplement. Incubate for 48 h at 37 °C, 5% CO₂.
   5. Day 6
      1. Remove 166 µL of medium from each well and add 166 µL of RPMI 1640 with B-27 supplement. Incubate for 24 h at 37 °C, 5% CO₂.
   6. Day 7
      1. To induce proepicardial differentiation, remove 166 µL of medium from each well and add 166 µL of fresh RPMI 1640 containing B-27 supplement and 3 µM CHIR99021 for a final well concentration of 2 µM CHIR99021. Incubate for 1 h at 37 °C, 5% CO₂.
      2. Remove 166 µL of medium from each well and add 166 µL of fresh RPMI 1640 containing B-27 supplement. Incubate for 48 h at 37 °C, 5% CO₂.
         NOTE: Extra caution is advised at this second medium change on day 7 as the organoids are more prone to movement because of the media changes.
      3. From day 7 onwards until collection or transfer for analyses or experimentation, perform medium changes every 48 h by removing 166 µL of medium from each well and add 166 µL of fresh RPMI 1640 containing B-27 supplement.
         ​NOTE: Organoids are ready for analyses and experimentation at day 15 unless earlier developmental stages are of interest. They can be cultured past day 15 for long-term culture or maturation experiments.

3. Organoid analysis

1. Transferring whole organoids (live or fixed)
   NOTE: For live organoid transfer, ensure that pipette tips used are sterile.
   1. Cut the tip off a P200 pipette tip 5-10 mm from the tip opening, resulting in a wide opening of ~2-3 mm diameter.
   2. Insert the tip straight into the round-bottom well containing the organoid so that the pipette is completely vertical (perpendicular to the plate). Ensure that the pipette plunger is already pressed all the way before inserting the tip into the medium.
   3. Slowly release the pipette plunger, taking up enough medium (100-200 µL) to collect the organoid.
   4. Transfer the organoid in medium to the target destination (e.g., for fixing, live imaging, electrophysiology recording, new plate culture).
2. Fixing organoids
   NOTE: Fixing and staining organoids can be done either in the 96-well culture plate or microcentrifuge tubes. Paraformaldehyde (PFA) should be handled only in a fume hood.
   1. For fixation in microcentrifuge tubes, transfer live organoids to separate tubes with 1-8 organoids per tube.
      NOTE: Do not exceed 8 organoids per tube.
   2. Carefully remove and discard as much medium from the tube as possible without touching the organoids.
   3. Add 4% PFA to each tube or well (300-400 µL per microcentrifuge tube and 100-200 µL per well of a 96-well plate). Incubate at room temperature for 30-45 min.
      NOTE: Incubation times over 1 h may require antigen retrieval steps and are not recommended.
   4. Safely discard the PFA without disturbing the organoids. Perform 3 washes with DPBS supplemented with 1.5 g/L glycine (DPBS/Gly), using the same volume used for the 4% PFA, waiting 5 min between washes. Remove DPBS/Gly and proceed to immunostaining or other analyses or add DPBS and store at 4 °C for future use for up to 2 weeks.
      NOTE: Storing fixed organoids for longer than 2 weeks may result in tissue degradation and contamination and is not recommended.
3. Whole-mount immunofluorescent staining
   1. Add 100 µL of blocking/permeabilization solution (10% normal donkey serum + 0.5% bovine serum albumin (BSA) + 0.5% Triton X-100 in 1x DPBS) to each well or tube containing the fixed organoids. Incubate at room temperature overnight on a shaker.
      NOTE: Do not exceed 8 organoids per tube.
   2. Carefully remove and discard as much of the blocking solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.
   3. Prepare the primary antibody solution (1% normal donkey serum + 0.5% BSA + 0.5% Triton X-100 in 1x DPBS) with the desired primary antibodies at the recommended concentrations. Incubate at 4 °C for 24 h on a shaker.
   4. Carefully remove and discard as much of the antibody solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.
   5. Prepare secondary antibody solution (1% normal donkey serum + 0.5% BSA + 0.5% Triton X-100 in 1x DPBS) with the desired secondary antibodies at the recommended concentrations. If the antibodies are fluorescently labeled, incubate at 4 °C in the dark (e.g., covered in aluminum foil) for 24 h on a shaker.
   6. Carefully remove and discard as much of the antibody solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.
   7. Prepare slides with beads (90-300 µm in diameter) mounted in a mounting medium (see the Table of Materials) near the edges of the slide where the coverslip with the organoids will be placed.
      NOTE: It is recommended to allow the mounting medium around the beads to dry before proceeding; this will prevent the beads from moving around. See Figure 2.
   8. Transfer the stained organoids using a cut pipette tip onto the slide, between the beads, ensuring spacing to avoid contact between the organoids once on the slide. Use the corner of a rolled-up laboratory wipe to carefully remove excess liquid around the organoid.
   9. Cover the organoids with mounting-clearing medium (fructose-glycerol clearing solution is 60% (vol/vol) glycerol and 2.5 M fructose)³⁷ using 120-150 µL of the mounting-clearing medium per slide.
      NOTE: It is recommended to use a cut pipette tip when working with the mounting-clearing medium as it is very viscous.
   10. Hover the coverslip over the slide with the organoids covered with mounting-clearing solution and slowly press the coverslip over the slide, ensuring the organoids are between the mounted beads.
   11. Seal the perimeter of the coverslip on the slide using top coat nail varnish. Allow the slide to dry in the dark at room temperature for 1 h. Store at 4 °C in the dark for long-term storage.
4. Calcium transient imaging in live heart organoids
   NOTE: According to the manufacturer's instructions, Fluo4-AM was reconstituted in dimethyl sulfoxide (DMSO) to a final stock solution concentration of 0.5 mM. Fluo4-AM was added directly to the organoid well in the 96-well plate.
   1. Perform 2 washes on the organoids using RPMI 1640 medium.
      1. Remove 166 µL of the spent medium from the well.
      2. Add 166 µL of warmed RPMI 1640 medium, remove 166 µL of medium, and add 166 µL of fresh RPMI 1640 medium.
         NOTE: The washes are done to remove waste material and cell debris. Two-thirds of the medium is removed from the wells during the washes to avoid disturbing the organoids at the bottom of the well before the functional assay.
   2. Add Fluo4-AM medium to the organoids.
      1. Add Fluo4-AM reconstituted in DMSO to RPMI 1640 containing B-27 supplement to prepare a 1.5 µM solution.
      2. Remove 166 µL of medium from the well.
      3. Add 166 µL of 1.5 µM Fluo4-AM in RPMI 1640 containing B-27 supplement for a final well concentration of 1 µM. Incubate at 37 °C, 5% CO₂ for 30 min.
   3. Perform 2 washes as in step 3.4.1.
   4. Add 166 µL of RPMI 1640 containing B-27 supplement to the well.
   5. Using a cut tip of a P200 pipette tip, transfer the organoid to a glass-bottom Petri dish (e.g., 8-well chambered cover glass with #1.5 high-performance coverglass) with 100-200 µL of medium.
      NOTE: See section 3.1 on transferring whole organoids.
   6. Image the organoids live under a microscope with a temperature- and CO₂-controlled chamber at 37 °C, 5% CO₂.
   7. Record several 10-20 s videos at various locations across the organoid, showing the increase and decrease in fluorescence intensity levels as the calcium enters and exits the cells.
      NOTE: For high-resolution recordings, it is recommended to record at a speed of 10 fps or faster; 50 fps is recommended.
   8. Analyze the videos using image analysis software (e.g., ImageJ) by selecting regions of interest and measuring intensity levels over time.
   9. Normalize the intensity recordings using ΔF/F₀ vs. time in milliseconds and plot.

Wyniki

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 a...

Dyskusje

Recent advances in human stem cell-derived cardiomyocytes and other cells of cardiac origin have been used to model human heart development^22,24,25 and disease^26,27,28 and as tools to screen therapeutics^29,30 and toxic agents^31,

Materiały

Name	Company	Catalog Number	Comments
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
Other
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
Thermal Mixer	ThermoFisher Scientific	13-687-717
Top Coat Nail Varish	Seche Vite		Can purchase from any supermarket