DOI: 10.5061/dryad.ksn02v7bh
title: Induced pluripotent stem cell-derived cardiomyocyte in vitro models: tissue fabrication protocols, assessment methods, and quantitative maturation metrics for benchmarking progress

description: 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.
description: 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 <em>in vitro
 </em>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.
description: # 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](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,](https://forms.gle/d9DXAEWqN1fpPQYb8) and a group of researchers within the [CELL-MET Engineering Research Center](https://www.bu.edu/cell-met/) 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.