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.
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.
Large scale production of human pluripotent stem cells (hPSCs)-derived cardiac progenitors and cardiomyocytes is a pre-requisite for stem cell-based therapies1, disease modeling2,3 and the rapid characterization of novel pathways regulating cardiac differentiation4,5,6 and physiology7,8. Although a number of studies9,10,11,12,13,14,15 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.
1. Id1 Virus Preparation and Infection
2. hPSCsId1 Maintenance
3. Preparation of hPSCsId1 for Differentiation
4. Differentiation of hPSCsId1 into First Heart Field-like Cardiac Progenitors (FHF-L CPs)
5. Cryopreservation of FHF-L CPs
6. FHF-L CP Differentiation Into Ventricular-like Cardiomyocytes
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.
Generation of hPSCsId1lines
hPSCs are infected with a lentivirus mediating Id1 overexpression (Figure 1A). Once hPSCId1 are generated, transgene expression is quantified by qRT-PCR (Figure 1B). Only hPSCId1 lines expressing Id1 mRNA at levels greater than 0.005 fold of that of GAPDH should be used for differentiation.
hPSCsId1 Maintenance and Preparation for Differentiation
Optimal culture conditions are necessary and critical for successful first heart field-like cardiac progenitor differentiation (Figure 2A). hPSCId1 cultures should be monitored daily for stem morphology maintenance and continuous proliferation. Differentiation should be initiated when tightly packed stem cell colonies reach >90% confluency (Figure 2B). If differentiation is initiated prior to optimal confluency, cell death is enhanced and differentiation efficiency may decline (Figure 2C). Similarly, overgrown cultures will perform poorly (Figure 2D).
Generation of First Heart Field-Like Cardiac Progenitors (FHF-L CPs) From hPSCsId1
On day 1 (24 h post differentiation initiation), cells should be monitored to observe loss of stem morphology (cells appear flatter and darker than stem cells). Note that cell death is common during the first day of differentiation, but cell confluence should be maintained at >75% (Figure 2E). If after 24 h of differentiation, 50% or more of the covered surface area is lost, cells should be discarded.
On day 3, differentiating cells should remain clustered and have filled gaps created during the initial 24 h time period (Figure 2F). Flat cells growing independently are indicative of suboptimal differentiation conditions.
On day 4, optimal differentiation should show continued expansion and coverage of the plate.
On day 5, cells are harvested and cryopreserved. Optimal differentiations should result in tightly connected cell clusters with homogeneous morphology appearance (Figure 2G). Note that low density clusters of flat cells mark a suboptimal differentiation outcome. See Table 2 for the average yield of day 5 cardiac progenitors per well in various culture formats.
Generation of Ventricular-like Cardiomyocytes From FHF-L CPs
Day 5 FHF-L CPs are thawed at plate format-dependent densities described in Table 2, in order to maximize their cardiac differentiation potential (Figure 3A and Table 2). The viability after thawing needs to be monitored. Generally, viability ranges from 70–80%. If the viability is below 60%, the yield of cardiomyocytes would be affected.
On day 6 (24 h after resuming differentiation), FHF-L CPs should cover >90% of the available surface area and appear as a homogenous single layer of cells (Figure 3B). Low confluence on day 6 will reduce FHF-L CPs differentiation into cardiomyocytes.
Resulting cardiomyocytes start to beat by day 11–13 of differentiation (Figure 3C and Movie 1). During the day 14–16 replating process, see Table 2 for the average yield of cardiomyocytes per well in various culture formats. Differentiation efficiency is typically assessed by immunofluorescence for cardiac (ACTC1), fibroblast (TAGLN) and vascular endothelial markers (CDH5) and nuclear (DAPI) markers. Lineage quantification is performed using automated imaging and fluorescence quantification, as in Cunningham et al. (2017)5 (Figure 3D-E). Subtype characterization of resulting cardiomyocytes is performed by qRT-PCR (Figure 3F) and action potential transient analysis using kinetic imaging and voltage sensing probes (Figure 3G and Movie 2). Fluorescence quantification for the voltage probe and resulting action potential trace analysis (Figure 3H) is performed as described in McKeithan et al. (2017)7.
Figure 1: Generation of hPSCId1 lines. (A) Schematic representation of the hPSCsId1 generation protocol. (B) Examples of qRT-PCR results showing representative Id1 mRNA levels generated by lentiviral-mediated expression from distinct lines of hPSCId1 including one hESC line and two lines of hPSC lines. Dashed line represents Id1 expression threshold, below which hPSCId1 lines need to undergo an additional cycle of Id1-lentivirus infection. "p" indicates the passage number. The number in the parenthesis indicates the number of rounds of Id1-lentiviral infection. Error bars represent standard deviation of experiments performed in triplicate. Please click here to view a larger version of this figure.
Figure 2: Generation of First Heart Field-Like Cardiac Progenitors (FHF-L CPs) Differentiation From hPSCId1. (A) Schematic representation of the FHF-L CP generation protocol. Representative bright field pictures of hPSCId1 (day 0) at optimal (B), sub-confluent (yellow arrowheads indicate the void regions between cells) (C) and over-confluent (D) densities. Representative bright-field pictures of differentiating hPSCId1 at day 1, 3 and 5 respectively (E), (F), (G).Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Generation of Ventricular-like Cardiomyocyte From FHF-L CPs. (A)Schematic representation of the ventricular-like cardiomyocyte generation protocol. (B) Bright field image of day 6 differentiation cells (one day post-thawing). (C) Bright field image of a day 12 beating culture. (D) Representative immunofluorescence image of day 15 cultures (384-well plate format). ACTC1 marks cardiomyocytes, TAGLN: fibroblasts/smooth muscle, CDH5: vascular endothelial cells, and DAPI: nuclei (shown in the inset). (E) Lineage quantification of day 15 cultures. (F) qRT-PCR data time course expression from day 5 to day 25 of ventricular-specific markers (IRX4 and MYL2) and pan-cardiac marker (MYL7). (G) Representative action potential trace from ventricular-like cardiomyocytes at day 25 of differentiation. (H) Action potential duration measurements (APD50 and APD90) of resulting ventricular-like cardiomyocytes (n = 12). Error bars represent standard deviation of experiments performed in quadruplicate (unless otherwise noted). Scale bars = 100 µm. RFU, relative fluorescence unit. Please click here to view a larger version of this figure.
Medium | Base medium | Additive |
Stem Cell Media | mTesR1 | n/a |
Induction Media | RPMI 1640 Medium | B-27 Serum-Free Supplement without insulin, 1x |
Induction Media | RPMI 1640 Medium | Puromycin, 6 µg/ml |
Induction Media | RPMI 1640 Medium | Penicillin-Streptomycin, 1x |
Cardiogenic Media | RPMI 1640 Medium | B-27 Serum-Free Supplement, 1x |
Cardiogenic Media | RPMI 1640 Medium | Penicillin-Streptomycin, 1x |
Maintenance Media | RPMI 1640 Medium | B-27 Serum-Free Supplement, 1x |
Maintenance Media | RPMI 1640 Medium | KnockOut Serum Replacement, 2% |
Maintenance Media | RPMI 1640 Medium | Penicillin-Streptomycin, 1x |
Table 1: Media components (see Table of Materials for base media and additives).
384 well | 96 well | 12 well | 6 well | 100 mm | 150 mm | |
Coating volume (per well) | 10 µL | 50 µL | 500 µL | 1 mL | 5 mL | 10 mL |
Medium volume (per well) |
100 µL | 300 µL | 2 mL | 5 mL | 12 mL | 30 mL |
Differentiation day 0 activin A concentration (ng/mL) | 100 | 100 | 100 | 300 | ||
Cardiac progenitor day 5 yield (cells/well) |
1.0 – 1.2 x 105 | 2.5 – 3.0 x 105 | 1.0-1.5 x 107 | 2.0 – 3.0 x 107 | ||
Cardiac progenitor day 5 replating or thawing density (cells/well) | 2.0 x 104 | 7.5 x 105 | 4.5 x 106 | 8.0 x 106 | 2.2 x 107 | |
Ventricular-like cardiomyocyte day 14–16 yield (cells/well) |
2.0 – 5.0 x 104 | 0.8 – 1.2 x 106 | 3.5 – 5.0 x 106 | 0.8 – 1.2 x 107 | 1.8 – 2.5 x 107 | |
Ventricular-like cardiomyocyte replating density (cells/well) |
2.5 x 104 | 1.0 x 106 | 4.5 x 106 | 1.0 x 107 | 2.2 x 107 |
Table 2: Scalable differentiation parameters (coating volume, medium volume, Activin A concentration, yields, replating densities).
Gene Name | Gene Bank Accession | Sequence |
GAPDH | NM_001256799 | F: GGAGCGAGATCCCTCCAAAAT R: GGCTGTTGTCATACTTCTCATGG |
mouse Id1 | NM_010495 | F: CCTAGCTGTTCGCTGAAGGC R: CTCCGACAGACCAAGTACCAC |
IRX4 | NM_016358 | F: GGCTCCCCAGTTCTTGATGG R: TAGACCGGGCAGTAGACCG |
MYL2 | NM_000432 | F: TTGGGCGAGTGAACGTGAAAA R: CCGAACGTAATCAGCCTTCAG |
MYL7 | NM_021223 | F: GCCCAACGTGGTTCTTCCAA R: CTCCTCCTCTGGGACACTC |
Table 3: qRT-PCR primer sequences.
Movie 1: Bright field recording (100 frames/s) of day 15 cardiomyocytes where spontaneous contractions can be observed. Please click here to view this video. (Right-click to download.)
Movie 2: Kinetic imaging (100 frames/s) of voltage-sensitive fluorescent probe in day 25 id1-induced ventricular-like cardiomyocytes. Please click here to view this video. (Right-click to download.)
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 hPSCsId1, 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 hPSCsId1 as single cells and allowing them to form tightly clustered monolayers that grow to ~90% confluence during the course of 2 to 3 days. Conversely, if hPSCId1 cultures show poor cell and colony morphology, low colony compaction and the presence of large areas of differentiation within the culture, successful FHF-L CP differentiation are unlikely.
Media volume and Activin A concentrations are critical parameters of FHF-L CP differentiation and plate format-dependent (see Table 2). Also note that optimal Activin A concentrations may vary depending on used hPSC line and may require concentration optimization.
Id1 mRNA levels in hPSCs are crucial to efficiently promote FHF-L CP. Suboptimal Id1 levels will fail to produce robust FHF-L CP differentiation and massive cell death by day 1 of differentiation will be observed. The relative expression ratio of Id1-to-GAPDH must exceed 0.005 to promote efficient differentiation. Continued selection using higher doses of puromycin (6–10 µg/mL) is recommended in order to retain high levels of lentiviral-mediated Id1 expression. An evaluation of Id1 mRNA levels every 10 passages is recommended.
Plating densities of day 5 FHF-L CPsis also a critical parameter for cardiac differentiation efficiency. If day 5 FHF-L CPs are plated at low densities, the relative cardiac yield will decrease. Optimal plating densities are listed in Table 2 and are plate format-dependent.
The main limitation for this protocol is the use of lentiviral-mediated Id1 overexpression to promoteFHF-L CP differentiation which may restrict the therapeutic use of resulting progenitors and ventricular-like cardiomyocytes. Also, since lentiviruses may integrate at sites that could potentially affect hPSC maintenance and/or developmental potential, stemness of hPSCsId1 colonies should be monitored by evaluating stem gene expression and observing morphological signs of differentiation. Note that overexpressing Id1 using non-integrative vectors would overcome this limitation and is currently being investigated in our laboratory.
One convenient feature of this method is the simplicity of the differentiation protocol as it only requires one cytokine, Activin A, to generate FHF-L CPs from hPSCsId1. In comparison, other protocols 9,10,11,12,13,14,15 require complex sequences of combinations of cytokines and/or small molecules in order to promote and maintain cardiac differentiation. In addition, this protocol uniquely describes a convenient cryopreservation step which enables to uncouple FHF-L CP generation from subsequent cardiomyocyte production. This feature enables to biobank large batches of FHF-L CPs and to quickly generate cardiomyocytes from frozen progenitors, as cardiac differentiation resumes from day 5 upon thawing. This strategy allows to gain 1 to 2 weeks of time as compared to starting cardiac differentiation from hPSCs. In addition, and most importantly, this protocol is the first protocol to date to generate cardiac progenitors of defined heart field origin, while other protocols9,10,11,12,13,14,15 remain undefined in that regard.
In summary, this protocol outlines a robust and scalable method to produce large amounts (>108–109) of developmentally-relevant FHF-L CPs and ventricular-like cardiomyocytes. In turn, resulting cells can be used to identify novel regulatory pathways controlling cardiac differentiation and physiology, and for regenerative and disease modeling purposes.
The authors have nothing to disclose.
We thank members of the Colas lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NIEHS R44ES023521-02 and CIRM DISC2-10110 grants to Dr. Colas.
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) |