Induced pluripotent stem cell-derived cardiomyocyte in vitro models: tissue fabrication protocols, assessment methods, and quantitative maturation metrics for benchmarking progress
Data files
Jan 10, 2024 version files 89.18 KB
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Benchmarking_cleanup01052024.xlsx
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README.md
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May 13, 2024 version files 189.81 KB
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Benchmarking_v7.xlsx
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README.md
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Jul 19, 2024 version files 192.54 KB
Abstract
The advent of human induced pluripotent stem cells (hiPSCs) and techniques to differentiate cardiomyocytes from them has opened a viable path to creating in vitro models of normal and diseased hearts, accelerating more predictive drug screening and therapeutic strategies for cardiac pathologies. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) are more similar to fetal than adult cardiomyocytes, leading many in the field to explore approaches to enhance cell and tissue maturation. There are over 2,000 studies utilizing hiPSC-CMs in models composed of various combinations of cell and extracellular matrix components, using a plethora of differentiation protocols, culture formats, and methods for quantifying cardiomyocyte function. To assess the current state of this rapidly growing area, we systematically analyzed 300 studies using hiPSC-CM models for their selection of hiPSC lines, hiPSC-CM differentiation protocols, types of in vitro models, maturation techniques, and metrics used to assess cardiomyocyte functionality and maturity. Here, we provide the data compiled from our analysis of these papers so others in the field can utilize it to inform their research. Based on this analysis, we highlight the diversity of, and current trends in, in vitro model designs and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology, and gene expression and noted field-wide improvements over time. Finally, we observe that a persistent lack of coordination amongst investigators is limiting the field’s ability to benchmark and advance hiPSC-CM function against previous studies. We discuss opportunities to collectively pursue the common goal of hiPSC-CM model development, maturation, and assessment that we believe are critical to driving the entire community forward in engineering mature cardiac tissue.
README: Induced pluripotent stem cell-derived cardiomyocyte in vitro models: tissue fabrication protocols, assessment methods, and quantitative maturation metrics for benchmarking progress
https://doi.org/10.5061/dryad.ksn02v7bh
The advent of human induced pluripotent stem cells (hiPSCs) and techniques to differentiate cardiomyocytes from them has opened a viable path to creating in vitro models of normal and diseased hearts, accelerating more predictive drug screening and therapeutic strategies for cardiac pathologies. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) are more similar to fetal than adult cardiomyocytes, leading many in the field to explore approaches to enhance cell and tissue maturation. There are over 2,000 studies utilizing hiPSC-CMs in models composed of various combinations of cell and extracellular matrix components, using a plethora of differentiation protocols, culture formats, and methods for quantifying cardiomyocyte function. To assess the current state of this rapidly growing area, we systematically analyzed 300 studies using hiPSC-CM models for their selection of hiPSC lines, hiPSC-CM differentiation protocols, types of in vitro models, maturation techniques, and metrics used to assess cardiomyocyte functionality and maturity. Here, we provide the data compiled from our analysis of these papers so others in the field can utilize it to inform their research.
Additionally, we hope to encourage other researchers to contribute to these benchmarking efforts and support the collective advancement of the field. If you would like to contribute new data to be included in the database, please fill out the form linked here, and a group of researchers within the CELL-MET Engineering Research Center will periodically review each new entry and update the database.
Data and file structure
All data from our analysis has been deposited here into a spreadsheet. Rows indicate individual studies analyzed and columns are populated with specific metrics of interest, such as hiPSC line, hiPSC-CM differentiation protocol information, commonly used analysis metrics, etc. Column headers are listed and further defined, where applicable, below. If a specific box has been left blank, the information was either not explicitly stated in the manuscript or does not apply to the study being analyzed.
Publication year: Year paper was published in journal issue
Publication Category: Indicates how papers were selected for analysis (see methods)
hiPSC Line (Healthy Control): Healthy hiPSC lines used in the study (all diseased lines were excluded from analysis)
Number of Healthy Control hiPSC-CM Lines:
hiPSC Matrix Coating: Matrix coating applied to tissue culture plastic during hiPSC culture and expansion
hiPSC Backbone Media: Culture media used for hiPSC culture and expansion
Confluency at the Start of Differentiation (%): Published confluency of hiPSCs at start of CM differentiation
hiPSC-CM Backbone Media: Culture media used for hiPSC-CM culture
hiPSC-CM Media Supplements: Supplements added to hiPSC-CM backbone media for hiPSC-CM culture
Wnt Activator: Wnt signaling pathway activator used to initiate CM differentiation
Time of Wnt Activator: Start and end day of Wnt activator treatment
Wnt Inhibitor: Wnt signaling pathway inhibitor used in CM differentiation
Time of Wnt Inhibitor: Start and end day of Wnt inhibitor treatment
Day Insulin Added:
Purification Protocol: Method used to purify CM differentiation
Time of hiPSC-CM Purification: Days in the differentiation in which purification was conducted
Method to Assess Differentiation Purity: Method to assess CM purity
Differentiation Purity (%): Precent of CMs in differentiation product
hiPSC-CM Maintenance/Maturation Media: Media formulation used for maintain/maturing hiPSC-CMs listed if different than hiPSC-CM backbone media
hiPSC-CM Matrix Coating if Replated: Matrix coating applied to tissue culture plastic if hiPSC-CMs replated as a part of CM differentiation or maturation protocol
Maturation Techniques: “Yes” indicates that a specific maturation technique was used. Individual columns are used to indicate the use of the following maturation techniques: metabolic maturation, electrical stimulation, passive tension, the inclusion of other cell types besides CMs, mechanical stimulation, cell alignment, elastomeric surface, and cell-derived ECM.
2D Surface: Indicates the types of 2D surface CMs were cultured in a given study
3D: Platform Type: Describes the type of 3D culture system used in a given study
3D: Cell Types: Indicates the types of cells used in studies that use 3D culture systems
3D: Types of Stromal Cells (SC): Further specifies the types of stromal cells used in studies that use 3D culture systems
3D: Types of Endothelial Cells (EC): Further specifies the types of endothelial cells used in studies that use 3D culture systems
3D: Cell Composition (CM-EC-SC): Indicates the composition of each tissue by cell type (e.g., ”75, 0, 25” would indicate that the tissues used in the study were composed of 75% CMs, 0% ECs, and 25% SCs)
3D: Estimated Tissue Cell Density: Tissue cell density in units of 106 cells/mL as estimated from the data available in each study
3D: Estimated Tissue Size: Tissue size in units of mm^2 as estimated by multiplying the tissue length by the tissue width
Immunofluorescent Imaging?: “Yes” indicates that immunofluorescent imaging was used to analyze tissues in a given study
SEM or TEM: Indicates whether scanning or transmission electron microscopy was used
Sarcomere/Cellular Alignment Analysis?: “Yes” indicates that sarcomere/cellular alignment analysis was conducted
Sarcomere Length: Sarcomere length in μm
Cell Area: Cardiomyocyte area in μm^2
T-tubules Present?: “Yes” indicates the presence of T-tubule structures in CMs studied
Contractile Analysis Method: Describes how contractile force/stress/strain of CMs was quantified
Force: Measured contractile force (mN) of CMs
Stress: Measured contractile stress (mN/mm2) of CMs
Upstroke velocity: Measured upstroke velocity (μm/s) of contracting CMs
Calcium Handling Analyses Method: Describes the method used to visualize calcium flux dynamics in CMs
Amplitude: Amplitude of calcium fluxes quantified using ratiometric calcium dyes or genetically encoded sensors
Beat Rate: Contraction rate (in beats per minute) of CMs calculated from calcium imaging
Time to Peak: Measured time from the start of calcium flux to the peak (ms)
Time to Relaxation: Measured time from peak of calcium to full relaxation (ms). Percentage values in some boxes indicate the time to the indicated percent relaxation as opposed to full relaxation.
Conduction velocity: Calculated conduction velocity (cm/s) quantified from calcium imaging
Electrophysiology Analysis Method: Describes the method for assessing electrophysiological properties of CMs.
AP Conduction Velocity: Calculated conduction velocity (cm/s) quantified from electrophysiological measurements
AP Amplitude: Action potential amplitude (mV)
Resting membrane potential: Resting membrane potential of CMs (mV)
Max Capture Rate: Maximum frequency (Hz) that CMs were able to be paced
Metabolic Analyses Method: Describes the method used to assess the metabolic maturity of CMs
Fatty Acid Metabolism Measured?: “Yes” indicates the study provided a measure of fatty acid metabolism
Gene Analysis Method: Describes the method used to assess gene expression
Pharmacological Interventions: Pharmacologic agents used to test hiPSC-CM maturation or hiPSC-CM model potential for drug screening
Disease: Indicates if the study used hiPSC-CMs to model a specific cardiac disease
Acronyms and Abbreviations:
hiPSC: human induced pluripotent stem cell
hiPSC-CM: human induced pluripotent stem cell-derived cardiomyocytes
cTnT: cardiac troponin T
IHC: immunohistochemistry
ECM: extracellular matrix
PDMS: polydimethylsiloxane
PAA: polyacrylamide
PEG: polyethylene glycol
gelMA: gelatin methacrylate
MEA: multielectrode arrays
EC: endothelial cells
SC: stromal cells
hLF: human lung fibroblasts
hDF: human dermal fibroblasts
hCF: human adult cardiac fibroblasts
heCF: human embryonic cardiac fibroblasts
hFF: human foreskin fibroblasts
hMSC: human mesenchymal stem cells
hiPSC-SC: human induced pluripotent stem cell-derived stromal cells (typically derived from impure CM differentiations)
hiPSC-CF: human induced pluripotent stem cell-derived cardiac fibroblasts
hiPSC-MC: human induced pluripotent stem cell-derived mural cells
hiPSC-SMC: human induced pluripotent stem cell-derived smooth muscle cells
hiPSC-EC: human induced pluripotent stem cell-derived endothelial cells
HUVEC: human umbilical vein endothelial cells
CMVEC: cardiac microvascular endothelial cells
BOEC: human blood outgrowth endothelial cells
HCAEC: human coronary artery endothelial cells
SEM: scanning electron microscopy
TEM: transmission electron microscopy
TFM: traction force microscopy
AP: action potential
PCR: polymerase chain reaction
RNA-seq: RNA sequencing (utilizing next-generation sequencing technologies)
Version Changes:
May 13, 2024: Added additional columns to the Excel file containing new data points describing hiPSC-CM culture, differentiation, and maturation. These new columns include hiPSC Matrix Coating, hiPSC Backbone Media, hiPSC-CM Backbone Media, hiPSC-CM Media Supplements, hiPSC-CM Maintenance/Maturation Media, hiPSC-CM Matrix Coating, Tissue Culture Media, and whether a study conducted -omics analysis.
July 18, 2024: Added a column to the Excel file containing a new analysis describing whether each publication measured fatty acid metabolism.
Methods
Across over 2,000 studies utilizing hiPSC-CMs from 2016-2022, there exists high variability in differentiation protocols, maturation strategies, and assessment of cell or tissue functionality. To understand the nature of this variability, we systematically analyzed 300 studies using hiPSC-CM models and compiled data on hiPSC lines, hiPSC-CM differentiation protocols, types of 2D and 3D in vitro models, maturation techniques, and metrics used to assess hiPSC-CM functionality and maturity. Using a PubMed search for “(((induced pluripotent stem cells) OR (iPSC)) AND ((cardiomyocyte) OR (cardiomyocytes) OR (iPSC-CM) OR (iPSC-CMs) OR (induced pluripotent stem cell-cardiomyocytes) OR (induced pluripotent stem cell-CMs))) AND ((engineered heart tissue) OR (cardiac microtissue) OR (maturation) OR (mature))” we identified 846 potential publications for analysis. We eliminated publications focusing on atrial differentiation due to relatively limited published work in this space and focused our benchmarking on ventricular cardiomyocytes, which are of particular interest in the development of cardiac regenerative therapies. We also eliminated publications that were specifically focused on disease modeling or mechanistic studies unless maturation techniques (the primary focus of our analysis) were utilized in the study. To capture overall trends in the field, we focused on 300 publications. This included the top 100 cited publications from the 846 potential publications based on citation numbers reported by the Web of Science database as of 2022, 100 publications from the PubMed 846 potential publications (that were not the 100 top-cited publications) selected randomly, and 100 additional hiPSC-CM publications that were manually selected from references in recent high impact reviews on cardiac tissue engineering or top search results for “iPSC-cardiomyocytes” on Google Scholar that utilized unique culture platforms, maturation techniques, and/or assessments.