Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.
Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.
Pluripotent stem cells are a powerful tool for generating and studying cells from a host of difficult to access tissues for basic research and pre-clinical studies, especially in humans1-5. Proper modulation of developmental signaling pathways can direct the differentiation of pluripotent stem cells to the desired phenotypic fate. Many protocols have been developed to generate cardiomyocytes (CMs) from pluripotent stem cells6-14. These protocols generally involve modulation of TFGβ superfamily (Activin, BMP, and TGFβ) and Wnt pathways through timed addition of exogenous growth factors and/or small molecules10,12-15. These protocols are generally effective at increasing the percentage of cells that become CMs, but lack specificity, generating a mixed population of cells representing atrial, ventricular, and nodal/conduction system lineages. In order to study specific cardiac subtypes a more directed differentiation approach is required.
Gremlin2 (Grem2, also called Protein Related to Dan and Cerberus or PRDC for short) is a secreted BMP antagonist that is necessary for proper cardiac differentiation and atrial chamber formation during cardiac development in zebrafish16. Treating differentiating embryonic stem cells with Grem2 at differentiation day 4, just after peak expression of mesodermal markers T-Brachyury and Cerberus like 1, increases the yield of CMs and generates a pool of cells predominantly of the atrial lineage14.
Recombinant Grem2 is used to treat the differentiating cells and can be made using standard protein production techniques17 or can be purchased commercially. It is highly soluble in aqueous solutions and may be added exogenously to cultures at the desired time point.
Differentiation can be tracked using RT-qPCR to quantify expression of markers representative of cardiovascular progenitors, cardiac progenitors, and committed CMs. Immunofluorescence can also be used to identify and visualize spatial distribution of cardiac cell types.
Applications that require pure populations are more readily carried out when using a reporter system to identify and isolate CMs. For this purpose, we have introduced the αMHC-DsRedNuc construct into the mouse CGR8 ES cell line14. CGR8 cells grow and remain pluripotent without feeder cells, facilitating expansion and differentiation assays18. The ES cell line contains a DSRed fluorescent protein coding sequence with a nuclear localization signal under the cardiac-specific alpha Myosin Heavy Chain (αMhc or Myh6) gene promoter. Using these cells, CMs can be easily identified and isolated for quantification, cell sorting, electrophysiology, drug screens, and studying mechanisms of atrial differentiation.
1. Preparation of Cell Culture Media, Solutions, and Reagents.
2. Preparation of Gelatin Coated 10 cm Tissue Culture Dishes
3. Preparation of Gelatin Coated 6-Well Tissue Culture Plates
4. Embryonic Stem Cell Culture
5. Preparation of Embryoid Bodies
NOTE: All steps except centrifugation are performed in a sterile tissue culture hood.
6. Plating and Treatment of EBs With Grem2
7. Cell Dissociation for RT-qPCR Analysis
8. Electrophysiological Analysis
Prior to differentiation, pluripotent stem cells should be compact and free of spontaneous differentiation. The cells shown in Figure 1A are ready to be singularized and used for hanging drops as described in 5.1 of the protocol section. The cells shown in Figure 1B are spontaneously differentiating and should not be used for preparation of hanging drops.
The panels included in Figure 2 show successfully formed EBs at differentiation day 2. Quality EBs generally form best within evenly spaced, well rounded hanging drops as shown in Figure 2. By differentiation day 2, EBs should be visible to the naked eye as spherical arrangements of differentiating stem cells at the tips of the hanging drops (Figure 2). These EBs are ready for wash-down as described in 5.3 of the protocol section.
During days 2-4, washed-down EBs in suspension should continue to grow in size. At day 4, well-formed EBs should be free-floating and homogeneous in size and shape (Figure 3). The EBs shown in Figure 3 are ready to be transferred to pre-coated plates and treated with Grem2 as described in 6.1-6.3 of the protocol section.
Once plated, EBs should flatten as cells migrate out from the EB onto the surface of the tissue culture plate. Shown in Figure 4 are examples of properly attached EBs at differentiation days 8 (left panel) and 10 (right panel). Cells should continue to be monitored daily to be sure they remain attached and to check for cardiac differentiation as described below.
Contracting cells, indicating the presence of CMs, may be observed as early as day 6 and as late as day 9. Typically, contracting cells will be present at day 8 of differentiation. The numbers of contracting cells in each well should continue to increase through differentiation day 14. In non-treated control wells small, slowly contracting patches are observed (Video S1) while in Grem2 treated wells large patches with a relatively fast contracting rate are common (Video S2). Occasionally, cardiac differentiation fails to occur. In such cases the timing of Grem2 treatment, the activity of the protein, and the state of the cells prior to differentiation should be re-evaluated to be sure that each of these conditions is optimized as described in this protocol (see discussion).
Figure 5 shows results typical of Grem2-treated cultures and untreated controls. RT-qPCR analysis of gene expression in cells collected and lysed as described in 7.1-7.2 of the protocol section typically reveal high levels of cardiac structural and regulatory genes in Grem2-treated cultures (Figure 5A). If the αMHC-DsRed-Nuc cell line is being used, the increased yield of CMs can be observed visually as higher numbers of DsRed-labeled nuclei in Grem2-treated wells (Figure 5B, bottom panel).
In addition to generating an increased number of CMs, Grem2 treatment also biases differentiation towards an atrial like phenotype. The atrial phenotype is usually confirmed in two ways. First, RT-qPCR analysis of cells collected as described in 7.1-7.2 of the protocol section should reveal that genes characteristic of atrial CMs are upregulated while ventricular genes are downregulated (Figure 6A). Second, patch clamp measurements of cellular action potential duration (as described in 8.1-8.2 of the protocol section) reveals that a short, atrial-like action potential predominates among Grem2 treated cells (Figure 6B).
Figure 1. Representative mouse embryonic stem cells (mESCs). Pluripotent mouse embryonic stem cells prior to EB formation with characteristic large nucleus, compact colony-forming morphology (A, black arrows), and white phase borders (A, orange arrows). Spontaneous differentiation of mouse ES cells is indicated by larger, single cells separated from colonies (B, black arrows) and loss of phase border around colonies (B, orange arrow). Images captured using an inverted phase contrast microscope. Scale bars represent 50 µm. Please click here to view a larger version of this figure.
Figure 2. Embryoid bodies (EBs) at day 2 of differentiation. Hanging drops suspended from a petri dish lid (left, scale bar is 2 cm) are evenly spaced and homogeneous in size. EBs in hanging drops are observed as compact spheres in the center of each drop (right, scale bar is 1 mm). Please click here to view a larger version of this figure.
Figure 3. EBs in suspension at day 4 of differentiation. Ideal EBs should be homogeneous in size and shape with minimal adherence to each other or the bottom of the plate. Images were captured using a dissecting scope under sterile conditions. Scale bar represents 100 µm. Please click here to view a larger version of this figure.
Figure 4. Typical EB morphology after plating. Plated EBs at days 8 (left) and 10 (right) of differentiation. As EBs attach to gelatinized plates, they flatten out and appear as dense centers surrounded by an increasingly confluent monolayer of cells. Small pockets of contracting cells are generally observed around differentiation days 6-8 near the center of the EB. These pockets continue to expand throughout the differentiation protocol to form larger sheets of contracting cells in the surrounding monolayers. Images captured using an inverted phase contrast microscope and. Scale bars represent 200 µm. Please click here to view a larger version of this figure.
Figure 5. Cardiac differentiation in Grem2-treated and non-treated control wells. qPCR analysis of cardiac markers indicates an increase in cardiac gene expression in EBs treated with Grem2 as early as day six and continuing through day 10 of differentiation (A). The engineered αMHC-DsRed-Nuc cell line shows large pools of DsRed-labeled CMs in cultures treated with Grem2 (B, bottom) vs. non-treated controls (B, top). Figures adapted with permission from14. Error bars represent SEM from at least 3 replicate experiments. Please click here to view a larger version of this figure.
Figure 6. Characterization of atrial-like cardiac phenotype. qPCR analysis indicates an increase in expression of atrial genes and downregulation of genes associated with ventricular CMs in Grem2-treated samples (A). Control cultures produce CMs with action potential duration characteristic of atrial and ventricular cells while treatment with Grem2 generates cell populations with the short action potential duration characteristic of atrial myocytes (B). Samples were compared using student's t test. *, p < 0.05; ***, p < 0.001 Figures adapted with permission from14. Error bars represent SEM from at least 3 replicate experiments. Please click here to view a larger version of this figure.
Video S1. Non-treated control wells (Right click to download). Small, slowly-contracting pockets or patches of cardiac myocytes around the periphery of the EB (white arrows). Video was taken at differentiation day 10.
Video S2. Grem2-treated wells (Right click to download). Typical results observed in Grem2-treated cells. Large patches of quickly contracting cells are observed throughout the plated EB. Video was taken at differentiation day 10.
Gene | Forward Primer (5' to 3') | Reverse Primer (5' to 3') |
Actin | CTACGAGGGCTATGCTCTCCC | CCGGACTCATCGTACTCCTGC |
Gapdh | CTCACTCAAGATTGTCAGCAATG | GAGGGAGATGCTCAGTGTTGG |
Gata4 | ACAAGGTCCAAGCCTACTCCA | CTGCGATGTCTGAGTGACAGG |
Gja1 | ACAAGGTCCAAGCCTACTCCA | CCGGGTTGTTGAGTGTTACAG |
Gja5 | ATAACAGTGGGCAGTTGAACAGCAG | TACCCAATAACGAATGTGGGAGATG |
Myh6 | TACACTCTTCTCTACCTATGCTTCT | CACTATCTTCTTGAACTCAATGC |
Myl2 | AGAGATCGATGAAATGATCAAAGAG | CAGAGCCAAGACTTCCTGTTTATT |
Myl7 | AAATCAGACCTGAAGGAGACCTATT | CAGAGAGACTTGTAGTCAATGTTGC |
Nkx2.5 | GTCTCAATGCCTATGGCTAC | CTACGTCAATAAAGTGGGATG |
Tnnt2 | CAGAGGAGGCCAACGTAGAAG | CTCCATCGGGGATCTTGGGT |
Table 1. List of qPCR primer sequences. Primer sequences are listed alphabetically by gene name. Sequences are provided 5' to 3' for all genes analyzed in Figures 5 and 6.
This protocol routinely produces cultures with a high percentage of CMs that are characteristic of the atrial lineage. As with any differentiation protocol, the quality of the mESCs prior to differentiation should be given particular attention. mESCs should be routinely monitored for proper morphology (Figure 1A). Any spontaneous differentiation that occurs prior to formation of EBs will severely limit the efficiency of cardiogenesis and should be removed before passaging (Figure 1B). EB size also affects cardiogenesis. Starting cell numbers between 200 and 1,000 per EB have been tested and 500 cells per EB routinely produces the highest numbers of CMs. Cells that are passaged the day prior to EB formation also tend to differentiate more efficiently.
The "hanging drop" method is used to generate EBs in this protocol25. Other methods for making EBs used for cardiac differentiation have been reported26-29. The "hanging drop" method is simple and inexpensive, readily adopted in any laboratory with common cell culture equipment and materials, and can be conducted by anyone with cell culture experience. It is also versatile, producing EBs that may be easily manipulated, transferred, plated, or collected for RNA analyses according to the needs of the investigators. It is also scalable, producing small or large numbers of EBs as needed.
The protocol dictates the plating of EBs onto gelatin coated plates at Day 4 of differentiation. This step converts differentiating EBs into the more typical monolayer format common to tissue culture. In some cases it may be more convenient and or necessary to leave the EBs in suspension rather than plating. If suspension EBs are preferred for downstream applications the cells may be left in suspension throughout the differentiation process instead of being plated at day 4. When treating with Grem2, the EBs are placed into 1.5 ml centrifuge tubes and allowed to settle by gravity. The media is then carefully removed with a P1000, leaving a small amount behind to prevent aspiration of the EBs, and 1.5 ml Grem2 media is added to the tube. This suspension is then transferred to a 6 cm petri dish and placed back at 37˚C. The media is changed using the micro centrifuge tube method indicated above every two days.
Differentiation day 4 was chosen for treatment of cells with Grem2 based on expression analysis of genes generally associated with major developmental events. Addition of Grem2 after peak expression of the gastrulation marker genes T Brachyury and Cerberus like 1 and at the onset of expression of cardiac progenitor cell markers such as Nkx2-5 is critical for both cardiogenesis and atrial specification. Because peak expression of these genes may vary slightly among cell lines it is recommended to monitor expression of these genes during differentiation to determine optimal timing for Grem2 addition. Of the lines tested for this protocol, most responded to treatment with Grem2 between days 4 and 5 of differentiation.
As with any recombinant protein, the activity of Grem2 varies from lot to lot. It is therefore recommended that Grem2 from the same lot is used for each set of experiments to maintain consistency. When a new lot is purchased, effectiveness may be assessed by titrating the dose in the range of 1-5 µg/ml. This protocol yields CMs from the atrial lineage of sufficient number for analysis and culture. Cells produced using this protocol may be analyzed via flow cytometry, electrophysiology, RT-qPCR, or re-cultured for use in live cell assays. To facilitate the identification and isolation of CMs after culture the αMHC-DsRed-Nuc reporter line was developed and is routinely used by our laboratory.
This protocol uses serum to maintain healthy cell cultures throughout the differentiation process. While this protocol routinely produces robust, atrial-like cardiac differentiation, use of serum introduces an undefined element to the differentiation process. This may limit usefulness of the protocol in cases where a more defined media is required. If a more defined media preparation is required, serum may be replaced by KnockOut Serum Replacement30. When switching to a defined culture media it is recommended that timing and concentration of Grem2 treatment be re-optimized as already described in this section.
RT-qPCR is used to quantify the expression of genes specific to atrial and ventricular myocytes. Grem2 treatment leads to an induction of atrial specific genes while down regulating or not affecting the expression of ventricular genes. This result is typical for Grem2-induced CMs. A suggested set of genes for assessing atrial identity is included in this protocol. An expanded set of genes and a discussion of their relevance can be found in works referenced by this protocol14,16. As the field continues to evolve and our understanding of cardiac differentiation improves it is recommended that this list of atrial and ventricular identity markers is assessed and revised as needed.
Generating homogeneous cultures of CMs will facilitate clinical research efforts focused on diseases known to affect specific cardiac subtypes and provide a model system for basic research focused on understanding the mechanisms of chamber specification in vertebrate hearts.
The authors have nothing to disclose.
This work was supported by NIH grants HL083958 and HL100398 (A.K.H.) and 2T32HL007411-33 "Program in Cardiovascular Mechanisms: Training in Investigation" (J.B.).
GMEM | Life Technologies | 11710 | |
FBS | Life Technologies | 10082 | |
Glutamax | Life Technologies | 35050 | |
LIF | EMD Millipore | ESG1107 | |
ß-Mercaptoethanol | Sigma | M3148 | |
IMDM | Sigma | I3390 | |
Non-Essential Amino Acids | Sigma | M7145 | |
10 cm Tissue Culture Plates | Sarstedt | 83.3902 | |
10 cm Bacterial Petri Dishes | VWR | 25384-342 | |
6 cm Bacterial Petri Dishes | VWR | 25384-092 | |
6-well tissue culture plates | Sarstedt | 83.3920 | |
Gremlin 2 recombinant protein | R&D Systems | 2069-PR-050 | |
Sterile filter units | Thermo Fisher | 09-741-02 | |
Gelatin (from porcine skin) | Sigma | G1890 | |
10X PBS, Sterile | Sigma | P5493 | |
BSA | Sigma | 5470 | |
0.05% Trypsin-EDTA solution | Life Technologies | 25300054 | |
DPBS, no Calcium, no Magnesium | Life Technologies | 14200 |