Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.
Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient's blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.
The discovery of human induced pluripotent stem cells has revolutionized modern cardiovascular medicine1,2. Human iPSCs are capable of self-renewing and generating all cell types in the heart, including cardiomyocytes, endothelial cells, smooth muscle cells and cardiac fibroblasts. Patient iPSC-derived cardiomyocytes (iPSC-CMs) can serve as indefinite resources for modeling genetically inheritable cardiovascular diseases (CVDs) and testing cardiac safety for new drugs3. In particular, patient iPSC-CMs are well poised to investigate genetic and molecular etiologies of CVDs that are derived from defects in cardiomyocytes, such as long QT syndrome4 and dilated cardiomyopathy (DCM)5. Combined with CRISPR/Cas9-mediated genome editing, patient iPSC-CMs have opened an unprecedented avenue to understand the complex genetic basis of CVDs including congenital heart defects (CHDs)6,7,8. Human iPSC-CMs have also exhibited potentials to serve as autologous cell sources for replenishing the damaged myocardium during a heart attack9. In recent years, it has become paramount to generate high-quality human iPSC-CMs with defined subtypes (atrial, ventricular and nodal) for cardiac regeneration and drug testing10.
Cardiac differentiation from human iPSCs has been greatly advanced in the past decade. Differentiation methods have gone from embryoid body (EB)-based spontaneous differentiation to chemically defined and directed cardiac differentiation11. Key signaling molecules essential for embryonic heart development, such as Wnt, BMP, Nodal, and FGF are manipulated to enhance cardiomyocyte differentiation from human iPSCs10,12. Significant advances include sequential modulation of Wnt signaling (activation followed by inhibition) for robust generation of cardiomyocytes from human iPSCs13,14. Chemically defined cardiac differentiation recipes have been explored to facilitate large-scale production of beating cardiomyocytes15,16, which have the potential to be upgraded to industrial and clinical level production. Moreover, robust expansion of early human iPSC-CMs is achieved by exposure to constitutive Wnt activation using a small chemical (CHIR99021)17. Most recently, subtype-specific cardiomyocytes are generated through manipulation of retinoic acid (RA) and Wnt signaling pathways at specific differentiation windows during cardiomyocyte lineage commitment from human iPSCs18,19,20,21,22.
In this protocol, we detail a working procedure for robust generation and proliferation of human CMs originating from patient peripheral blood mononuclear cells. We present protocols for 1) reprogramming human PBMCs to iPSCs, 2) robust generation of beating cardiomyocytes from human iPSCs, 3) rapid expansion of early iPSC-CMs, 4) molecular characterization of human iPSC-CMs, and 5) electrophysiological measurement of human iPSC-CMs at the single-cell level by patch clamp. This protocol covers the detailed experimental procedures on converting patient blood cells into beating cardiomyocytes.
The experimental protocols and informed consent for human subjects were approved by the Institutional Review Board (IRB) at Nationwide Children's Hospital.
1. Preparation of cell culture media, solutions, and reagents
2. iPSC reprogramming of PBMCs
3. Human iPSC maintenance and passaging
4. Chemically defined cardiomyocyte differentiation
5. Passage human iPSC-CMs
6. Expansion of human iPSC-CMs
7. Immunofluorescence
8. Flow cytometry sample preparation
9. Real time qPCR
10. Whole-cell patch clamp recording
Human iPSC reprogramming from PBMCs
After pre-culture with Complete Blood Media for 7 days, PBMCs become large with visible nuclei and cytoplasm (Figure 1B), indicating that they are ready for virus transfection. After transfection with the Sendai virus reprogramming factors, PBMCs will undergo an epigenetic reprogramming process for another week. Typically, we get 30-50 iPSC colonies from the transfection of 1 x 105 PBMCs and the reprogramming efficiency is 0.03%-0.05%. Completely reprogrammed cells will attach and start forming colonies when they are introduced to the complete E8 media (Figure 1C). These early iPSC colonies are expanded for another 7 days and then mechanically cut and picked up individually. Each iPSC colony is transferred to one well of a 6-well plate to establish individual iPSC lines. After 4-5 passages, iPSC colonies will become pure with very few differentiated cells around (Figure 1D). At this stage, most of the cells in iPSC colonies are OCT4 and NANOG positive (Figure 1E), demonstrating their pluripotency. Stable iPSC lines are established by the fifth passage.
Cardiac differentiation
The cardiac differentiation protocol is depicted in Figure 1F. Cardiac differentiation is initiated when iPSCs are maintained for at least 10 passages. The degree of iPSC confluency is critical when CHIR99021 is applied. The cell density is more than 90% confluent but not over confluent. If iPSC colonies become too crowded, they will start spontaneous differentiation which will negatively affect the directed cardiomyocyte differentiation efficiency. Beating cardiomyocytes are usually observed after day 12 of differentiation (Video 1). The date in which the onset of beating occurs varies and is dependent on the iPSC lines in use. After glucose starvation and replating, iPSC-CMs show spontaneous beating (Video 2) and aligned sarcomere structure with intercalated cardiac troponin T (TNNT2) and α-actinin (Figure 1G-H). In addition, the purity of iPSC-CMs is high, with more than 93% of cells being TNNT2+ as shown by FACS analysis (Figure 1I).
Although iPSC-CMs are relatively immature compared to adult cardiomyocytes, they show ventricular- and atrial-like action potentials measured by whole-cell patch clamp (Figure 2A,B). In a typical cardiac differentiation, day 30 iPSC-CMs are a mixture of ventricular-, atrial-, and nodal-like subtypes, with ventricular CMs accounting for the majority (60%, Figure 2C) using the abovementioned differentiation protocol (Figure 1F). Different differentiation protocols yield varying percentages of cardiomyocyte subtypes due to distinct signaling pathways activation during cell lineage determination10. Ventricular CMs are labeled with MYL2 (MLC2v, Figure 2D) whereas atrial iPSC-CMs are marked by NR2F2 (COUP-TFII, Figure 2E). These markers are highly expressed in more mature iPSC-CMs (>D30) rather than those in early stage.
Expansion of iPSC-CMs by Wnt activation
In mammals, adult cardiomyocytes do not actively divide for self-renewal. This phenomenon also takes place for human iPSC-CMs. Once mature beyond D30, cell division of iPSC-CMs is a rare event, thus limiting their ability for clinical- and industrial-level mass production. To mimic the developmental environment during embryonic cardiomyocyte proliferation, we activate the Wnt pathway by CHIR99021 to stimulate the multiplication of early iPSC-CMs. D12-14 iPSC-CMs (after purification by glucose deprivation) are seeded at a low density in the presence of 2 μM CHIR99021. Wnt activation stimulates cell division of iPSC-CMs and promotes the expression of cell cycle regulators such as Cyclin D1 (Figure 3A) which can push the cell cycle to advance through the G1 phase. Interestingly, CHIR99021 enables robust proliferation of early iPSC-CMs for 2 passages compared to the controls (Figure 3B). However, the proliferation ability of iPSC-CMs diminishes with extensive passage (Figure 3B), which is consistent with the limited and well-controlled cardiac proliferation during embryonic heart development. In addition, it does not appear that CHIR99021 is able to stimulate the expansion of more mature iPSC-CMs when they reach over 30 days of differentiation and develop stable sarcomere structures.
Figure 1: Human iPSC reprogramming and cardiomyocyte differentiation. (A) A schematic diagram showing the PBMC layer after separation of patient blood samples. (B) Enlarged PBMCs are ready for transfection. (C) Early human iPSC colonies. (D) An established iPSC line at passage 5. (E) Human iPSCs are positive for the pluripotency markers OCT4 (green) and NANOG (red). Nuclei are counterstained by DAPI (blue). (F) Overview of a cardiomyocyte differentiation protocol. (G-H) Sarcomere structure of iPSC-CMs is revealed by immunofluorescence staining using antibodies against TNNT2 (green) and α-actinin (red). Nuclei are counterstained by DAPI (blue). (I) FACS analysis of iPSC-CMs using an antibody against TNNT2. Scale bars: 200 μm (B-D), 50 μm (E and G) and 20 μm (H). Please click here to view a larger version of this figure.
Figure 2: Cardiomyocyte subtypes in human iPSC-CMs. (A-B) Representative action potential durations for ventricular-like (A) and atrial-like (B) iPSC-CMs. (C) Representative percentages of ventricular-, atrial- and nodal-like subtypes in human iPSC-CMs. (D-E) D30 iPSC-CMs are stained with antibodies against ventricular cardiomyocyte marker MYL2 (D) and atrial marker NR2F2 (E). Cells are simultaneously stained with a TNNT2 antibody. Nuclei are counterstained by DAPI (blue). Scale bars: 50 μm (D-E). Please click here to view a larger version of this figure.
Figure 3: Expansion of human iPSC-CMs by Wnt activation. (A) Percentage of Cyclin D1 positive iPSC-CMs is increased in the presence of CHIR99021. Cells are double stained with antibodies against TNNT2 (red) and Cyclin D1 (green). Nuclei are counterstained by DAPI (blue). (B) Cell number fold changes during the expansion of human iPSC-CMs with or without CHIR99021 in the first 3 passages. Y-axis shows the cell number fold changes. CHIR99021 stimulates the robust proliferation of early iPSC-CMs. Scale bars: 50 μm (A). Please click here to view a larger version of this figure.
Video 1. Beating human iPSC-CMs at day 18 of differentiation. Please click here to download this video.
Video 2. Beating human iPSC-CMs at day 25 after metabolic purification. Please click here to download this video.
During iPSC reprogramming, it is critical to culture PBMCs for 1 week until they are enlarged with clear nuclei and cytoplasm. Because PBMCs do not proliferate, an appropriate cell number for viral transduction is important for successful iPSC reprogramming. Cell number of PBMCs, multiplicity of infection (MOI) and titer of virus should be considered and adjusted to reach the optimal transduction outcomes. For cardiac differentiation, initial seeding density is critical for iPSCs to reach over 90% confluent on the day when CHIR99021 is administered. On one hand, if iPSCs are less confluent at the time of cardiac differentiation, CHIR99021 will be toxic and lead to substantial cell death. On the other hand, if iPSCs are over confluent, they will undergo spontaneous differentiation which will compromise the efficiency of directed cardiac differentiation. For the expansion of early iPSC-CMs, the timing and cardiomyocyte quality should be taken into account. Early iPSC-CMs can robustly multiply only when the purity of cardiomyocytes is high enough. Existing non-cardiomyocytes in the culture may also proliferate in response to CHIR99021 treatment, which will negatively affect the proliferation of early iPSC-CMs. In addition, it is crucial to stimulate cardiomyocyte expansion by day 20 of differentiation. Once iPSC-CMs pass over day 30, it will be difficult for them to resume robust dividing.
Human iPSCs were initially derived from dermal and lung fibroblasts via retrovirus-mediated transfection1,2. There are two major issues with these reprogramming methods that prevent the progress in clinical translation of patient iPSCs: 1) the retrovirus integrates into the host genome thus introducing potential genetic mutations; 2) patient-derived fibroblasts require skin biopsies which many patients may decline. In this protocol, we describe a protocol that utilizes commercial non-integration Sendai virus23 and PBMCs to robustly derive patient iPSCs. These iPSCs are free of exogenous reprogramming vectors and can be maintained with self-renewal and pluripotency indefinitely. In addition, patient blood samples are easily collected in clinical laboratories. Our protocol is versatile and can be used for mass production of patient- and disease-specific iPSCs for large-scale repository and clinical translations24.
Robust cardiomyocyte differentiation is achieved by sequential modulation of specific signaling pathways during cardiac differentiation from human iPSCs. Key pathways involved in cardiac specification and proliferation include Wnt, BMP, Activin, NOTCH, VEGF and retinoic acid (RA) 10,12. Here we present an efficient cardiac differentiation protocol by sequential modulation of Wnt signaling by small chemicals: first activation by CHIR99021 and then inhibition by IWR-113,14. Small chemicals are stable and give consistent differentiation outcomes compared to those using growth factors. Most iPSC-CMs generated by this protocol are ventricular-like cardiomyocytes, mixed with atrial- and nodal-like cells. Precision generation of subtype-specific cardiomyocytes is achieved through fine-tuning later differentiation steps10,12. For example, addition of RA immediately after IWR-1 treatment yields a high percentage of atrial-like cardiomyocytes whereas RA inhibition promotes generation of ventricular-like iPSC-CMs18,22. Wnt signaling activation at a later stage of differentiation promotes the induction of cardiac progenitor cells to nodal-like cardiomyocytes19,21, which is promising for the generation of patient-specific biological pacemaker cells.
Human iPSC-CMs are immature and have limited proliferation ability25. During embryonic cardiac development, the maturation proceeds while the proliferation diminishes. There is a narrow window when iPSC-CMs can be stimulated for robust proliferation, which is reflective of embryonic cardiomyocyte expansion. Here we use a Wnt activator CHIR99021 to promote the proliferation of early iPSC-CMs for a limited period, which is consistent with a recent report17. It is speculated that the Wnt signaling pathway affects cardiomyocyte proliferation possibly through the crosstalk with multiple upstream pathways such as NOTCH and Hippo26,27. NOTCH signaling promotes cardiomyocyte proliferation whereas the Hippo pathway restricts cardiac growth and heart size28,29,30. It is still unknown how the interaction between NOTCH and Hippo determines downstream Wnt activity and fine-tunes an appropriate degree of cardiac proliferation. Our protocol has provided an interesting model for cardiomyocyte proliferation to study disease mechanisms of congenital heart defects that are caused by the hypoplasia of ventricular cardiomyocytes, such as hypoplastic left heart syndrome (HLHS) and pulmonary atresia with intact ventricular septum (PA-IVS).
The authors have nothing to disclose.
This study was supported by the American Heart Association (AHA) Career Development Award 18CDA34110293 (M-T.Z.), Additional Ventures AVIF and SVRF awards (M-T.Z.), National Institutes of Health (NIH/NHLBI) grants 1R01HL124245, 1R01HL132520 and R01HL096962 (I.D.). Dr. Ming-Tao Zhao was also supported by startup funds from the Abigail Wexner Research Institute at Nationwide Children's Hospital.
ABI 7300 Fast Real-Time PCR System | Thermo Fisher Scientific | ||
Axon Axopatch 200B Microelectrode Amplifier | Molecular Devices | Microelectrode Amplifier | |
B27 supplement | Thermo Fisher Scientific | 17504044 | |
B27 supplement minus insulin | Thermo Fisher Scientific | A1895601 | |
BD Cytofix/Cytoperm Fixation/Permeabilization Kit | BD Biosciences | 554714 | Fixation/Permeabilization solution, Perm/Wash buffer |
BD Vacutainer CPT tube | BD Biosciences | 362753 | Blood cell separation tube |
CHIR99021 | Selleck Chemicals | S2924 | |
CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo Fisher Scientific | A16517 | Sendai virus reprogramming kit |
Digidata 1200B | Axon Instruments | Acquisition board | |
Direct-zol RNA Miniprep kit | Zymo Research | R2050 | RNA extraction kit |
DMEM/F12 | Thermo Fisher Scientific | 11330057 | |
Essential 8 medium | Thermo Fisher Scientific | A1517001 | E8 media for iPSC culture |
GlutaMAX supplement | Thermo Fisher Scientific | 35050061 | L-glutamine alternative |
Growth factor reduced Matrigel | Corning | 356231 | Basement membrane matrix |
iScript cDNA Snythesis Kit | Bio-Rad | 1708891 | cDNA synthesis |
IWR-1-endo | Selleck Chemicals | S7086 | |
KnockOut Serum Replacement (KSR) | Thermo Fisher Scientific | 10828028 | |
pCLAMP 7.0 | Molecular Devices | Electrophysiology data acquisition & analysis software | |
Recombinant human EPO | Thermo Fisher Scientific | PHC9631 | |
Recombinant human FLT3 | Thermo Fisher Scientific | PHC9414 | |
Recombinant human IL3 | Peprotech | 200-03 | |
Recombinant human IL6 | Thermo Fisher Scientific | PHC0065 | |
Recombinant human SCF | Peprotech | 300-07 | |
RPMI 1640 medium | Thermo Fisher Scientific | 11875093 | |
RPMI 1640 medium, no glucose | Thermo Fisher Scientific | 11879020 | |
SlowFade Gold Antifade Mountant | Thermo Fisher Scientific | S36936 | Mounting media |
StemPro-34 SFM | Thermo Fisher Scientific | 10639011 | PBMC culture media |
TaqMan Fast Advanced Master Mix | Thermo Fisher Scientific | 4444964 | qPCR master mix |
TrypLE Select Enzyme 10x, no phenol red | Thermo Fisher Scientific | A1217703 | CM dissociation solution |
UltraPure 0.5 M EDTA | Thermo Fisher Scientific | 15575020 | iPSC dissociation solution |
Y-27632 2HCl | Selleck Chemicals | S1049 |