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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Abstract

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.

Introduction

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.

Protocol

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

  1. Prepare PBMC media
    1. Mix 20 mL of basal PBMC culture media (1x) and 0.52 mL of supplement. Add 20 μL of SCF and FLT3 each (stock concentration: 100 μg/mL), 4 μL of IL3, IL6 and EPO each (stock concentration: 100 μg/mL) and 200 μL of L-glutamine alternative (100x). Mix them thoroughly. Filter in a sterile hood using a 0.22-μm filter unit. Name this as Complete Blood Media.
    2. Mix 20 mL of basal PBMC culture media (1x) and 0.52 mL of the Supplement. Add 200 μL of L-glutamine alternative (100x). Mix them thoroughly. Filter using a 0.22-μm filter unit. Name this as Supplement Blood Media.
  2. Prepare complete E8 media
    1. Mix 500 mL of E8 basal media and 10 mL of E8 supplement (thawed overnight at 4 °C) to make complete E8 media. Equilibrate to room temperature (RT) before use.
  3. Prepare iPSC passaging media
    1. Add 40 μL of Y-27632 Rock inhibitor (1:5,000 dilution, stock concentration: 10 mM) to 200 mL of complete E8 media. Mix it thoroughly. Equilibrate to RT before use.
  4. Prepare cardiomyocyte differentiation media
    1. Media I: Mix 500 mL of RPMI1640 with 10 mL of B27 minus insulin supplement (50x).
    2. Media II: Add an appropriate volume of CHIR99021 (GSK3 inhibitor) stock to Media I (CHIR99021 final concentration of 6 μM). Mix thoroughly.
    3. Media III: Add an appropriate volume of IWR-1 (Wnt inhibitor) stock to Media I (IWR-1 final concentration of 5 μM). Mix thoroughly.
    4. Media IV: Mix 500 mL of RPMI1640 with 10 mL of B27 supplement (50x). Mix thoroughly.
    5. Media V: Mix 500 mL of RPMI1640 (no glucose) with 10 mL of B27 supplement (50x). Mix thoroughly.
    6. Media VI: Add an appropriate volume of CHIR99021 stock to Media IV (CHIR99021 final concentration of 2 μM). Mix thoroughly.
  5. Prepare iPSC-CM passaging media
    1. Add 10 mL of Knockout Serum Replacement (KSR) to 90 mL of Media IV (KSR final concentration: 10%). Mix well.
  6. Prepare iPSC-CM freezing media
    1. Add 1 mL of DMSO to 9 mL of KSR (final concentrations: 10% DMSO/90% KSR) and mix well.
  7. Prepare basement membrane matrix medium-coated plates
    1. Thaw basement membrane matrix medium at 4 °C overnight and aliquot in 1.5 mL tubes. Add 1 mL of this medium to 250 mL of DMEM/F12 media (1:250 dilution) and mix them thoroughly. Apply 2 mL of the diluted solution per well in a 6-well plate and incubate in 5% CO2 at 37 °C for 30 min before use.

2. iPSC reprogramming of PBMCs

  1. Separate PBMCs from blood samples.
    1. Collect patient blood samples (~5 mL) and transfer into blood cell separation tubes (see Table of Materials). Mix by inverting 10x.
    2. Centrifuge at 1,500 x g for 30 min at room temperature.
    3. Take the tubes out carefully and spray with 70% ethanol. Under a biosafety hood, remove the caps without disturbing the mononuclear cells (buffy layer). PBMCs will be in a whitish layer just under the plasma layer (Figure 1A). Collect the whole buffy layer using a 1000 μL pipette and transfer to a 15 mL conical tube.
    4. Count the cell number using an automated cell counter. Spin the tube at 300 x g for 25 min at RT.
    5. Discard the supernatant. Wash with 10 mL of DPBS (Ca2+/Mg2+ free).
    6. Spin the tube at 300 x g for 15 min at RT.
    7. Remove the supernatant. Resuspend cell pellets in 1 mL of the freezing media (KSR plus 10% DMSO). Adjust cell density to make 1 x 106 cells per vial.
    8. Place PBMC cryovials in a cell freezing container and keep at -80 °C overnight. Transfer to a liquid nitrogen tank for long-time storage the next day.
  2. iPSC reprogramming.
    1. Add 3 mL of Supplement Blood Media in a 15 mL conical tube. Thaw PBMCs in 37 °C water bath and transfer them to a conical tube. Spin at 300 x g for 7 min at RT.
    2. Discard the supernatant. Resuspend PBMCs with Complete Blood Media. Seed them into two wells of a 24 well tissue culture plates (no basement membrane matrix).
    3. Incubate in 5% CO2 at 37 ˚C overnight. The next day gently remove half of the old media (0.5 ml) and add 0.5 mL of fresh Complete Blood Media.
    4. Change media every other day by refreshing half of the old media.
    5. After a week, aggressively wash the well with 1 mL of Supplement Blood Media and transfer cells into a 15 mL centrifuge tube.
    6. Count the cell number. Take 2 x 105 cells and centrifuge at 300 x g for 7 min.
    7. Discard the supernatant. Resuspend cells with 300 μL of Complete Blood Media. Perform transfection by adding appropriate volume of Sendai virus reprogramming vectors according to the manufacturer's instructions. Transfer them into one well of a 24-well plate (no basement membrane matrix). Incubate in 5% CO2 at 37 °C overnight.
    8. The next day spin at 300 x g for 7 min. Remove the supernatant and resuspend in 2 mL of Complete Blood Media. Transfer into one well of a 6 well plate pre-coated with basement membrane matrix. This is Day 1 (D1).
    9. Don't touch the plate the next day.
    10. On D3, remove 1 mL of the old media. Add 1 mL of Supplement Blood Media.
    11. Repeat step 2.2.10 on D5.
    12. On D7, remove 1 mL of the old media. Add 1 mL of complete E8 media.
    13. On D8, Repeat step 2.2.12.
    14. On D9, remove the old media. Add 2 mL of complete E8 media. Completely reprogrammed cells are expected to attach and start forming colonies.
    15. Refresh with 2 mL of complete E8 media every day.
    16. Around 2 weeks after Sendai virus transduction, large iPSC colonies will appear and be ready for picking.
    17. Cut iPSC colonies under a stereomicroscope in the hood and transfer individual colonies to a basement membrane matrix-coated 24-well plate pre-loaded with 0.5 mL of iPSC passaging media.
    18. Refresh with 0.5 mL of complete E8 media every day until iPSC colonies grow large enough for passaging into a new basement membrane matrix-coated 6-well plate.

3. Human iPSC maintenance and passaging

  1. When human iPSCs reach over 90% confluency, remove old media. Rinse with 3 mL of DPBS once.
  2. Add 1 mL of 0.5 mM EDTA in DPBS solution. Incubate in 5% CO2 at 37 °C for 5-8 min.
  3. Remove EDTA by aspiration. Add 1 mL of iPSC passaging media. Manually dislodge iPSCs.
  4. Take 600-900 μL of single cell suspension and re-plate them onto a basement membrane matrix-coated 6-well plate (dilution: 1:6 to 1:10). Incubate in 5% CO2 at 37 °C overnight.
  5. Refresh with 2 mL of complete E8 media every day. The iPSC cultures usually reach confluency after 3-4 days.

4. Chemically defined cardiomyocyte differentiation

  1. Culture human iPSCs in complete E8 media until 95% confluent (3-4 days).
  2. Remove the old media. Add 2 mL of CM differentiation Media II (6 μM CHIR in RPMI1640 plus B27 minus insulin supplement) to each well of a 6-well plate. This is D0. Do not touch on D1.
  3. On D2, replace with 2 mL of CM differentiation Media I (RPMI1640 plus B27 minus insulin supplement).
  4. On D3, replace with 2 mL of CM differentiation Media III (5 μM IWR-1 in RPMI1640 plus B27 minus insulin supplement). Do not touch on D4.
  5. On D5, replace with 2 mL of CM differentiation Media I.
  6. On D7, replace with 2 mL of CM differentiation Media IV (RPMI1640 plus B27 supplement). Thereafter, refresh the media every other day.
  7. On D11 when contracting cells are observed, replace with 2 mL of CM differentiation Media V (no glucose).
  8. On D13, replace with 2 mL of CM differentiation Media V.
  9. On D15, replace with 2 mL of CM differentiation Media IV.
  10. On D17-D21, replace with 2 mL of CM differentiation Media IV every other day.

5. Passage human iPSC-CMs

  1. Remove old media and rinse cells with 3 mL of DPBS once.
  2. Apply 1 mL of CM dissociation solution (see Table of Materials) to each well of a 6-well plate. Incubate in 5% CO2 at 37 °C for 5-8 min.
  3. Mechanically dissociate iPSC-CMs into single cells by vigorous pipetting.
  4. Transfer cells into a 15 mL conical tube. Add 2 mL of CM passaging media (10% KSR in RMPI1640 plus B27 supplement) to neutralize CM dissociation solution.
  5. Spin at 300 x g for 5 min at RT.
  6. Discard the supernatant. Resuspend cells with a desired volume of CM passaging media. Seed them into a basement membrane matrix-coated plate/dish. Human iPSC-CMs resume beating 1-3 days after passaging.

6. Expansion of human iPSC-CMs

  1. Rinse D10-12 beating iPSC-CMs with 3 mL of DPBS for each well of a 6-well plate once. Add 1 mL of CM dissociation solution (Step 5.2). Incubate in 5% CO2 at 37 °C for 7-10 min.
  2. Mechanically dissociate iPSC-CMs into single cells by vigorous pipetting.
  3. Transfer cells into a 15 mL conical tube. Add 2 mL of CM passaging media to neutralize CM dissociation solution.
  4. Spin at 300 x g for 5 min at RT.
  5. Discard the supernatant. Resuspend cells with an appropriate volume of CM passaging media. Pipette up and down to make single cell suspension. Seed one million of iPSC-CMs into a basement membrane matrix-coated 10 cm dish.
  6. The next day remove old media. Add 10 mL of cardiomyocyte proliferation media (Media VI: 2 μM CHIR99021). Change the media every other day.
  7. When iPSC-CMs become confluent after 7-9 days' culture, repeat the passaging step for further expansion of iPSC-CMs.

7. Immunofluorescence

  1. Before immunofluorescence staining, seed iPSC-CMs onto basement membrane matrix-coated coverslips that are placed in a 24 well plate (seeding density: 0.5-1 x 106 cells/mL). Maintain iPSC-CMs in culture for at least 4 days.
  2. Wash cells using 1 mL of DPBS once.
  3. Add 0.5 mL of 4% paraformaldehyde (PFA) and incubate for 15 min at RT.
  4. Wash cells using 1 mL of DPBS. Repeat once.
  5. Add 0.5 mL of 0.1% Triton X-100 and incubate for 20 min at RT.
  6. Wash with 1 mL of DPBS twice.
  7. Add 0.5 mL of 0.2% BSA in DPBS (blocking solution). Incubate at RT for 1 h.
  8. Add 200 μL of primary antibody diluted with blocking solution (dilution: 1:400-1:1000). Incubate at 4 °C overnight.
  9. Wash cells using 0.5 mL of blocking solution for 3 min with shaking. Repeat twice.
  10. Add 200 μL of secondary antibody diluted in the blocking solution. Incubate at RT for 1 h.
  11. Rinse cells three times with 0.5 mL of DPBS, each for 3 min with shaking.
  12. Counterstain nuclei with DAPI (1:2000 dilution) and incubate for 5 min at RT.
  13. Rinse cells three times with 0.5 mL of DPBS.
  14. Mount cells on the coverslips onto a microscope slide using 5 μl of mounting media. Store at 4 °C and protect from light.

8. Flow cytometry sample preparation

  1. Wash human iPSC-CMs with 3 mL of DPBS once.
  2. Add 1 mL of CM dissociation solution and incubate in 5% CO2 at 37 °C for 7-10 min.
  3. Dislodge cells using a 1,000-μL pipette. Transfer cell suspension to a round FACS tube through a strainer cap. The FACS tube is pre-filled with 1 mL of iPSC-CM passaging media (10% KSR) to neutralize the enzyme activity.
  4. Spin at 300 x g for 5 min.
  5. Remove supernatant without disturbing cell pellet. Add 250 μL of Fixation/Permeabilization solution (see Table of Materials). Incubate for 20 min at 4 °C.
  6. Add 1 mL of Perm/Wash buffer. Vortex briefly and spin at 300 x g for 4 min.
  7. Discard the supernatant. Add 100 μL of diluted primary antibodies (1:200-1:500) in 1x Perm/Wash buffer. Vortex briefly and incubate overnight at 4 °C.
  8. Wash cells by adding 1 mL of Perm/Wash buffer. Vortex briefly and spin at 300 x g for 4 min.
  9. Discard the supernatant. Add 100 μL of diluted secondary antibodies (1:500-1:1,000). Vortex briefly and incubate at RT for 1 h. Protect from light if secondary antibodies are conjugated with light-sensitive fluorescence.
  10. Wash cells by adding 1 mL of Perm/wash buffer. Vortex briefly and spin at 300g for 4 min.
  11. Discard the supernatant. Resuspend cells with 400 μL of FACS staining buffer (PBS/4% FBS). Store at 4 °C until loading to a FACS instrument.

9. Real time qPCR

  1. Remove old media in human iPSC-CM culture. Add 500-700 μL of lysis buffer to lysate cells. Incubate for 3 min at RT. Scape cell lysate and transfer to a 1.5-ml RNase-free tube. Proceed to total RNA extraction immediately or store at -80 °C.
  2. Isolate total RNA using an RNA extraction kit following the manufacturer's instruction.
  3. Measure the RNA concentration and assess the quality of total RNA by a spectrophotometer.
  4. Perform reverse transcription reaction using a cDNA synthesis kit. Total volume of RT reaction is 20 μL including 4 μL of reaction mix (5x), 1 μL of reverse transcriptase,1 μg of total RNA and RNase-free water.
  5. Incubate the complete RT reaction mix in a thermal cycler using the following protocol: 25 °C for 5 min; 46 °C for 20 min; 95 °C for 1 min; hold at 4 °C.
  6. Dilute cDNA by 1:10 using nuclease-free water. Set up real time qPCR reaction by mixing 1 μL of cDNA template, 1 μL of primers/probe, 10 μL of qPCR master mix and 8 μL of nuclease-free water.
  7. Run in a real-time PCR system. The cycling protocol is 50 °C 2 min (hold), 95 ˚C 10 min (hold), 95 °C 15 sec, 60 °C 1 min, repeat for 40 cycles.
  8. Collect CT values for each gene in each sample. Relative mRNA abundance is calculated by subtracting the CT value of target gene from the CT value of a housekeeping gene. Relative gene expression is analyzed by the 2-ΔΔCT method.

10. Whole-cell patch clamp recording

  1. Dissociate iPSC-CMs into single cells using CM dissociation solution as previously described.
  2. Seed cells at a low density on basement membrane matrix-coated coverslips. Culture them for 3-4 days in Media IV.
  3. Pull pipettes (resistance 0.9-1.5 MΩ) from borosilicate glass capillaries using a horizontal microelectrode puller.
  4. Incubate cells in Tyrode's solution (pH=7.35).
  5. Fill pipettes with electrode solution (pH=7.3) composed of the following chemicals: 120 mM aspartic acid, 20 mM KCl, 2 mM MgCl2, 5 mM HEPES, 10 mM NaCl, 5 mM EGTA, 0.3 mM Na-GTP, 14 mM phosphocreatine, 4 mM K-ATP and 2mM creatine phosphokinase.
  6. Place cells in the current clamp mode using a 1.5-2 diastolic threshold 5 ms current pulse at 1 Hz.
  7. Record action potentials (APs) using a microelectrode amplifier and a software-driven acquisition board.

Results

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 an...

Discussion

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 wh...

Disclosures

The authors declare no competing financial interests.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
ABI 7300 Fast Real-Time PCR SystemThermo Fisher Scientific
Axon Axopatch 200B Microelectrode AmplifierMolecular DevicesMicroelectrode Amplifier
B27 supplementThermo Fisher Scientific17504044
B27 supplement minus insulinThermo Fisher ScientificA1895601
BD Cytofix/Cytoperm Fixation/Permeabilization KitBD Biosciences554714Fixation/Permeabilization solution, Perm/Wash buffer
BD Vacutainer CPT tubeBD Biosciences362753Blood cell separation tube
CHIR99021Selleck ChemicalsS2924
CytoTune-iPS 2.0 Sendai Reprogramming KitThermo Fisher ScientificA16517Sendai virus reprogramming kit
Digidata 1200BAxon InstrumentsAcquisition board
Direct-zol RNA Miniprep kitZymo ResearchR2050RNA extraction kit
DMEM/F12Thermo Fisher Scientific11330057
Essential 8 mediumThermo Fisher ScientificA1517001E8 media for iPSC culture
GlutaMAX supplementThermo Fisher Scientific35050061L-glutamine alternative
Growth factor reduced MatrigelCorning356231Basement membrane matrix
iScript cDNA Snythesis KitBio-Rad1708891cDNA synthesis
IWR-1-endoSelleck ChemicalsS7086
KnockOut Serum Replacement (KSR)Thermo Fisher Scientific10828028
pCLAMP 7.0Molecular DevicesElectrophysiology data acquisition & analysis software
Recombinant human EPOThermo Fisher ScientificPHC9631
Recombinant human FLT3Thermo Fisher ScientificPHC9414
Recombinant human IL3Peprotech200-03
Recombinant human IL6Thermo Fisher ScientificPHC0065
Recombinant human SCFPeprotech300-07
RPMI 1640 mediumThermo Fisher Scientific11875093
RPMI 1640 medium, no glucoseThermo Fisher Scientific11879020
SlowFade Gold Antifade MountantThermo Fisher ScientificS36936Mounting media
StemPro-34 SFMThermo Fisher Scientific10639011PBMC culture media
TaqMan Fast Advanced Master MixThermo Fisher Scientific4444964qPCR master mix
TrypLE Select Enzyme 10x, no phenol redThermo Fisher ScientificA1217703CM dissociation solution
UltraPure 0.5 M EDTAThermo Fisher Scientific15575020iPSC dissociation solution
Y-27632 2HClSelleck ChemicalsS1049

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