Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Summary

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Abstract

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70–90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Introduction

Large scale production of human pluripotent stem cells (hPSCs)-derived cardiac progenitors and cardiomyocytes is a pre-requisite for stem cell-based therapies¹, disease modeling^2,3 and the rapid characterization of novel pathways regulating cardiac differentiation^4,5,6 and physiology^7,8. Although a number of studies^{9,10,11,12,13,14,15} have previously described highly efficient cardiac differentiation protocols from hPSCs, none has addressed the heart field origin of resulting cardiomyocytes, in spite of the identification of significant molecular differences between left (first heart field) and right (second heart field) ventricular cardiomyocytes¹⁶ and the existence of heart field-specific congenital heart diseases; i.e., hypoplastic left heart syndrome¹⁷ or arrhythmogenic right ventricular dysplasia¹⁸. Thus, the generation of cardiac progenitors and cardiomyocytes of defined heart field origin from hPSCs is becoming a necessity in order to increase their relevance as therapeutic and disease modeling tools.

This protocol relies on the constitutive overexpression of Id1, a recently identified⁵ first heart field-specifying cue that in combination with Activin A, is both necessary and sufficient to initiate cardiogenesis in hPSCs. Notably, Cunningham et al. (2017)⁵ show that Id1-induced progenitors specifically express first heart field (HCN4, TBX5) but not second heart field markers (SIX2, ISL1) as they undergo cardiac differentiation. In addition, the authors also show that transgenic mouse embryos lacking the entire Id family of genes (Id1-4), develop without forming first heart field cardiac progenitors, while more medial and posterior cardiac progenitors (second heart field) can still form, thereby suggesting that Id proteins are essential to initiate first heart field cardiogenesis in vivo. Conveniently, Id1-induced progenitors can be cryopreserved and spontaneously differentiate into cardiomyocytes displaying ventricular-like characteristics, including ventricular-specific markers (IRX4, MYL2) expression and ventricular-like action potentials.

Here we describe a simple and scalable method to generate first heart field-like (FHF-L) cardiac progenitors and ventricular-like cardiomyocytes from Id1 overexpressing hPSCs. An important feature of this protocol is the possibility to uncouple cardiac progenitor generation from subsequent cardiomyocyte production using a convenient cryopreservation step. In summary, this protocol details the necessary steps to (1) generate Id1-overexpressing hPSCs, (2) generate FHF-L cardiac progenitors from hPSCs, (3) cryopreserve resulting progenitors, and (4) resume FHF-L cardiac progenitor differentiation and generate highly enriched (>70–90%) beating ventricular-like cardiomyocytes.

Protocol

1. Id1 Virus Preparation and Infection

1. Generate Id1 overexpressing lentivirus by co-transfecting pCMVDR8.74, pMD2.G, and pCDH-EF1-Id1-PGK-PuroR (Addgene: plasmid #107735) into HEK293T cells. Collect viral particles, filtrate, purify from the supernatant, and store at -80 °C as in Kitamura et al. (2003)¹⁹. Alternatively, produce Id1 lentivirus commercially by sending pCDH-EF1-Id1-PGK-PuroR to a lentivirus-producing vendor.
   Caution: Please follow lentiviral safety regulations and standard operation protocols.
   Note: IMPORTANT: Optimal virus titer should be at least 10⁸ to 10⁹ Transducing Units/mL (TU/mL).
2. Prior to infection, coat one well of a 96 well plate with 50 µl of coating reagent, typically a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (see Table of Materials).
3. Dissociate one well of pluripotent stem cells grown in a 6 well plate by adding 1 mL of PBS without Ca²⁺ and Mg²⁺ and with 0.5 mM EDTA.
   NOTE: Dissociation time varies from 3–10 min. When more than 80% of the cells are detached from the bottom of the plate, proceed to next step.
4. Collect cell suspension with sterile pipet into 15 mL plastic centrifuge tube.
5. Neutralize dissociation reagent with 5 mL of PBS-containing Ca²⁺ and Mg²⁺.
6. Pellet cells by centrifugation (200 x g for 3 min).
7. Remove supernatant and gently flick the tube in order to dislodge the pellet.
8. Re-suspend cells in 1 mL of stem cell media.
9. Count cells using an automated cell counter following vendor instructions.
10. Plate 20,000 viable cells per coated well (96-well plate) in 50 µL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor (see Tables 1 and 2).
11. After 24 h, monitor for cell attachment and replace media with 100 µl stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor and 6 ng/mL hexadimethrine bromide (see Tables 1 and 2), one h prior to lentiviral infection.
    NOTE: Cells should be firmly attached to the bottom of the plate.
12. Thaw Id1-lentivirus on ice.
13. Add 3 µL of purified lentivirus per well (virus titer should be between 10⁸ to 10⁹ TU/mL), and then transfer the plate back to cell culture incubator (37 °C, 5% CO₂, 20% O₂).
14. 24 h post-viral infection, add 100 µL of fresh stem cell media to infected cells.
15. 48 h post-lentiviral infection, replace virus containing media with 200 µL of fresh stem cell media supplemented with 1 µg/mL puromycin.
16. Refresh media daily with 200 µL of stem cell media supplemented with 1 µg/mL puromycin.
17. When cultures reach 80% confluency, dissociate cells using 200 µL of PBS without Ca²⁺ and Mg²⁺ + 0.5 mM EDTA (for one well of a 96-well plate).
18. Collect cell suspension into a centrifuge tube.
19. Neutralize dissociation reagent with 5 mL of PBS-containing Ca²⁺ and Mg²⁺.
20. Pellet cells by centrifugation (200 x g for 3 min).
21. Remove supernatant and gently flick the tube in order to dislodge the pellet.
22. Re-suspend cells in 1 mL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor.
23. Transfer all cells (1 mL) to a coated well of a 12-well plate.
24. After 24 h, monitor for cell attachment to the well and replace media with 1 mL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor and 6 ng/mL hexadimethrine bromide, one h prior to transduction.
25. Thaw Id1-lentivirus on ice.
26. Add 5 µL of purified virus per well (12-well plate).
27. 24 h post-lentiviral infection, replace virus containing media with 1 mL of fresh stem cell media supplemented with 3 µg/mL puromycin.
28. 48 h post lentiviral infection, replace virus containing media with 1 mL of fresh stem cell media supplemented with 6 µg/mL puromycin.
29. When cultures reach 80% confluency, dissociate cells using 500 µL of PBS without Ca²⁺ and Mg²⁺ and with 0.5 mM EDTA (for one well of a 12-well plate).
30. Transfer half (250 µL) of the cell suspension to one well of a coating reagent coated 6-well plate containing 2 mL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor.
31. Use the other half (250 µL) of the sample for RNA extraction and qRT-PCR in order to determine lentiviral-mediated Id1 expression levels, as in Colas et al. (2012)⁴.
    NOTE: IMPORTANT: If Id1 mRNA expression levels are lower than 0.005 fold of GAPDH expression levels (see Table 3 for qRT-PCR primers), repeat infection process as described above until Id1 expression levels exceed the threshold.

2. hPSCs^Id1 Maintenance

1. Culture hPSCs^Id1 in coated 6 well-plates and grow in 3 mL per well of stem cell media supplemented with 6 µg/mL puromycin.
2. When hPSCs^Id1 reach 80% confluency, passage cells as aggregates (1:6 split ratio) using enzyme-free dissociation reagent following vendor guidelines and grow in 3 mL per well of stem cell media supplemented with 6 µg/mL puromycin.

3. Preparation of hPSCs^Id1 for Differentiation

1. Coat a 12-well plate with 500 µL per well of coating reagent.
2. When hPSCs^Id1 reach 90% confluency dissociate cells by adding 1 mL of PBS without Ca²⁺ and Mg²⁺ and with 0.5 mM EDTA per well for 5–10 min. Check dissociation process every minute, and once 80% of the cells are dissociated from the plate, proceed to the next step.
3. Collect cell suspension into a centrifuge tube.
4. Neutralize dissociation reagent with 5 mL of PBS-containing Ca²⁺ and Mg²⁺.
5. Pellet cells by centrifugation (200 x g for 3 min).
6. Remove supernatant and gently flick the tube in order to dislodge the pellet.
7. Re-suspend cells in 2 mL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor.
8. Count cells using an automated cell counter.
9. Plate 300,000 cells per well in 1 mL of stem cell media supplemented with 2 µM RHO/ROCK pathway inhibitor and 6 µg/mL puromycin, and transfer plate into tissue culture incubator.
10. Refresh media daily with 2 mL of stem cell media supplemented with 6 µg/mL puromycin until cultures reach 90% confluency. At this point, initiate cardiac differentiation (next step).

4. Differentiation of hPSCs^Id1 into First Heart Field-like Cardiac Progenitors (FHF-L CPs)

1. For 12-well plate format, initiate differentiation (day 0) by replacing stem cell media with 1.5 mL of induction media (Table 1) supplemented with Activin A (100 ng/mL) (see Table 2 for recommended volumes and Activin A concentrations for different plate formats).
2. On day 1 (24 h after differentiation is initiated), replace media with 2 mL of induction media without Activin A (per well of a 12-well plate).
3. On day 3, replace media with 2 mL of induction media without Activin A (per well of a 12-well plate).
4. On day 5, collect FHF-L CPs for cryopreservation (next step).
   NOTE: IMPORTANT: Cryopreservation is preferred at this point, as it enables to uncouple cardiac progenitor generation from subsequent cardiomyocyte production and biobank large batches of cells. Note that alternatively, day 5 FHF-L CPs can be passed to a freshly coated plate to continue differentiation, please refer to Steps 5.1–5.5 to passFHF-L CPs and then proceed to Step 6.5 for differentiation.

5. Cryopreservation of FHF-L CPs

1. Aspirate all media from wells containing day 5 FHF-L CPs and quickly add 1 mL of warm (37 °C) 1x enzyme-containing dissociation reagent (see Table of Materials). Place plate in the tissue culture incubator.
2. After 1 min, shake plate side to side in the incubator.
3. After 2 min (total time), remove plate from the incubator and add 1 mL of 10% FBS media.
4. Gently pipet cells up and down with a 5 mL pipet to facilitate cell detachment from the plate.
5. Collect cell suspension to a centrifuge tube and pellet cells by centrifugation at 200 x g for 3 min
6. Re-suspend cell pellet in 2 mL of cryopreservation reagent.
7. Count cells using an automated cell counter.
8. Add cryopreservation reagent (see Table of Materials) at a sufficient volume to cryopreserve cells at desired concentration (typically 5–10 x 10⁶ cells/mL).
9. Transfer cells to cryopreservation vials and place vials in cooling device (1 °C/min) and place cooling device in -80 °C freezer overnight.
10. After 24 h, transfer frozen vials to liquid nitrogen for long term storage.
    NOTE: The protocol can be paused here.

6. FHF-L CP Differentiation Into Ventricular-like Cardiomyocytes

1. Transfer day 5 FHF-L CPs frozen vials from liquid nitrogen storage into dry ice container.
2. Place vials in 37 °C water bath for 2 to 3 min, until cell suspension is thawed completely.
   NOTE: IMPORTANT: The thawing process is time-sensitive. Exposing cells to the cryopreservation media for an excessive amount of time (i.e., 10 min) will decrease cell viability.
3. Transfer cells to 15 or 50 mL centrifuge tube, depending on the number of thawed vials.
4. Dispense dropwise (5 drops every 30 seconds) pre-warmed (37 °C) cardiogenic media (Table 1) to cell solution. Continue this process until a 1:3 cryopreservation media:cardiogenic media ratio is reached. At this point, increase the cardiogenic media addition rate until a 1:10 cryopreservation media:cardiogenic media ratio is reached.
   NOTE: IMPORTANT: Rapid osmolarity changes will increase cell death during the thawing process; therefore, dropwise addition of cardiogenic media as described above is highly recommended.
5. Centrifuge cell suspension and pellet cells by centrifugation (200 x g for 3 min).
6. Remove supernatant and gently flick centrifuge tube to dislodge cell pellet.
7. Re-suspend cells with cardiogenic media supplemented with 2 µM RHO/ROCK pathway inhibitor.
   Note: Cell viability can vary from thaw to thaw, and, in general, > 65% viability predicts good plating efficiency.
8. Dilute concentrated cell suspension with cardiogenic media supplemented with 2 µM RHO/ROCK pathway inhibitor to obtain the desired cell concentration for plating (see Table 2 for different plate formats).
   NOTE: IMPORTANT: Note that cardiac differentiation efficiency may decline if cells are seeded too sparsely.
9. Seed cells on plate.
10. Keep plate in the tissue culture hood for 20 min before transferring it to tissue culture incubator.
11. On day 6 of differentiation, monitor cell attachment.
    NOTE: Cardiac progenitors are cryopreserved on day 5 of the differentiation. 24 h after thawing the cells would be on day 6 of the differentiation.
    NOTE: The presence of floating (dead) cells is common.
12. On day 7, aspirate 50% of the media and replace with an equal amount of fresh cardiogenic media.
    NOTE: The addition of RHO/ROCK pathway inhibitor to the cardiogenic media is not necessary after this point.
13. Replace 50% of the media with cardiogenic media every other day.
14. Note spontaneous beating that appears between day 11 and day 13.
15. On day 15, quantify cardiac differentiation efficiency by immunofluorescence using DAPI (nucleus stain) and ACTC1 (pan-cardiac marker).

7. Passing and Maintenance of Ventricular-like Cardiomyocytes

NOTE: By day 14–16, a monolayer of spontaneously contracting ventricular-like cardiomyocytes should be obtained. At this point, it is suggested to dissociate and re-plate cardiomyocytes in order to homogenize the culture and prevent cells from detaching from the plate.

1. Aspirate all media from wells and quickly add 1 mL of pre-warmed (37 °C) 1x enzyme-containing dissociation reagent.
   NOTE: 1x enzyme-containing dissociation reagent volumes vary depending on plate format, but should completely cover the bottom of the well surface.
2. Transfer plate into a tissue culture incubator.
3. Gently shake plate side to side every 2 min inside the incubator to accelerate the detachment process.
4. After 5–15 min, when 80% to 100% of the cells are detached from the bottom of the plate, remove the plate from the incubator and inhibit 1x enzyme-containing dissociation reagent activity by adding an equal volume of 10% FBS media.
   NOTE: Incubation time with 1x enzyme-containing dissociation for this process will vary from batch-to-batch.
5. Collect cell suspension to a centrifuge tube.
6. Mix cell suspension with pipet and count cells using an automated cell counter.
7. Dilute cell suspension to desired cell concentration (see Table 2 for different plate formats) with maintenance media supplemented with 2 µM RHO/ROCK pathway inhibitor.
8. After seeding cardiomyocytes onto the plate, distribute the cells evenly and allow the cells to attach for 10–20 min before transferring plate to tissue culture incubator.
9. 24 h after re-plating, monitor cell attachment and recovery.
   NOTE: The presence of floating (dead) cells is common. 48–72 h after re-plating, cardiomyocyte should resume spontaneous contraction.
10. To maintain cardiomyocyte culture, replace 50% of the media with the maintenance media every other day until use of ventricular-like cardiomyocytes for subsequent experiments.

Results

Generation of hPSCs^Id1lines
hPSCs are infected with a lentivirus mediating Id1 overexpression (Figure 1A). Once hPSC^Id1 are generated, transgene expression is quantified by qRT-PCR (Figure 1B). Only hPSC^Id1 lines expressing Id1 mRNA at levels greater than 0.005 fold of that of GAPDH should be...

Discussion

For successful differentiations, make sure to closely follow instructions listed above. In addition, here we highlight key parameters that strongly influence differentiation outcomes. Before starting a differentiation, the following three morphological parameters should be observed: a stem morphology of hPSCs^Id1, a high cellular compaction and a high confluence (>90%) of the culture at day 0. In that regard, optimal differentiation conditions are best created by plating dissociated hPSCs^Id1 as s...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACTC1 antibody	Sigma	A7811
Activin A	Stem Cell Technologies	Hu Recom Activin A
Antibiotic Antimycotic (Anti-Anti)	Thermo Fisher Scientific	15240062
B27 supplement	Thermo Fisher Scientific	17504044
B27 supplement w/o - insulin	Thermo Fisher Scientific	A1895601
B27 supplement w/o - vitamin A	Thermo Fisher Scientific	12587001
CDH5 antibody	R&D Systems	AF938
CryoStor CS10	Stem Cell Technologies	7930	Cryopreservation reagent
DMEM high Glucose	Mediatech	10-013-CV
DPBS w/ Ca & Mg	Corning	21-030-CV
EDTA	Thermo Fisher Scientific	15575-038
FBS	VWR	89510-186
FluoVolt membrane potential kit	Thermo Fisher Scientific	F10488	For optical action potential acquisition, please refer to McKeithan et al. 2017
KnockOut Serum Replacement	Gibco	10828010
Matrigel, Growth Factor Reduced	Corning	356231	Coating reagent
mTeSR1 media kit	Stem Cell Technologies	5850
PBS w/o Ca & Mg	Corning	21-040-CV
Penicillin-Streptomycin	Gibco
Puromycin	Acros	227422500
ReLeSR	Stem Cell Technologies	5872	Enzyme-free dissociation reagent
RPMI 1640	Thermo Fisher Scientific	11875-093
TAGLN antibody	Abcam	ab14106
Thiazovivin	Stem Cell Technologies	72254	RHO/ROCK pathway inhibitor
TrypLE Express	Thermo Fisher Scientific	12605 -010	1X enzyme-containing dissociation reagent
Tyrodes solution mix packets	Sigma	T2145-10X1L	(For optical action potential acquisition, please refer to McKeithan et al. 2017)