Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

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 medicine^1,2. 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 deriv....

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.....

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 and .......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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