Adult Human Heart Extracellular Matrix Improves Human iPSC-CM Function via Mitochondrial and Metabolic Maturation

2025-06-24 Tue
extracellular matrix iPSC-derived cardiomyocyte functional maturation mitochondrial maturation metabolic maturation cell therapy
1. Academic BackgroundCardiovascular diseases, especially myocardial infarction (MI), are among the leading causes of death and disability worldwide. After a myocardial infarction, up to one billion cardiomyocytes (CM) can be lost in just a few hours. However, adult myocardial tissue itself has extremely low regenerative capacity, which means the heart cannot rely on self-repair to reverse cell loss, leading to heart failure and a series of severe consequences. As a result, over the past decade, researchers have actively explored novel cell replacement therapies, hoping to “seed” exogenous cardiomyocytes to reconstruct damaged heart structure and function.
Compared with direct somatic cell transplantation, induced pluripotent stem cells (iPSC) and their derived cardiomyocytes (iPSC-derived cardiomyocytes, iCM) have become highly promising strategies for cardiac regenerative medicine due to their theoretically unlimited amplification, low risk of immune rejection, and abundant sources. However, existing iPSC differentiation protocols mainly yield “immature-type” iCMs similar to fetal cardiomyocytes, which are significantly inferior to adult cardiomyocytes in terms of structure, metabolism, and function, severely limiting their clinical translation in regenerative therapies, disease modeling, and drug screening.
Previous attempts to promote iCM maturation have included long-term culture, mechanical and electrical stimulation, and chemical induction factors, but improvements in maturity have been limited and the methods are complex and time-consuming. In recent years, some scholars have proposed a new approach of using “extracellular matrix (ECM) memory” to guide stem cell differentiation and functional maturation. It has been found that the ECM is not just a passive scaffold for cells, but contains “memory” of specific developmental stages and tissue types, capable of influencing cell fate through integrated signaling, molecular regulation, and physical properties.
Based on this, the authors of this paper focused on the following: if decellularized ECM (dECM) derived from adult human hearts can be used as a “preconditioning” environment prior to iPSC differentiation, it may more effectively “induce” iPSCs towards the cardiac lineage and drive them towards functional and metabolic maturation closer to adult levels. This innovative idea directly addresses the biggest “bottleneck” in current iCM applications—immature function and metabolism—which is a significant scientific challenge in the field of cardiac regenerative medicine.
2. Introduction to the Source of the PaperThis study is titled “adult human heart extracellular matrix improves human ipsc-cm function via mitochondrial and metabolic maturation.” The paper was completed by S. Gulberk Ozcebe, Mateo Tristan, and Pinar Zorlutuna et al., with all authors primarily affiliated with the University of Notre Dame, USA (Departments of Bioengineering, Chemical and Biomolecular Engineering, and Aerospace/Mechanical Engineering), and some from the National Institute of Environmental Health Sciences (NIEHS), USA. The paper was published in 2025 in Stem Cells (Vol. 43, No. 5, Article ID sxaf005, DOI: 10.1093/stmcls/sxaf005), representing the latest original scientific research in this field.
3. Detailed Research Process and Technical Route1. Research Subjects and Overall DesignThis study used left ventricular myocardial tissue from three adult donors aged 30-50 years (hearts unsuitable for transplantation, identity information removed) as ECM raw material. The main research process included the following core steps:
(1) Preparation and characterization of adult cardiac ECM
(2) Human iPSC culture, ECM preconditioning, and cardiac differentiation
(3) Evaluation of iCM maturity (function, metabolism, gene expression, etc.)
(4) Investigation of possible molecular mechanisms/components underlying ECM-promoted effects
(5) Data analysis and statistical testing
2. Detailed Experimental Methods2.1 Preparation and Characterization of Adult Cardiac ECMLeft ventricular muscle samples were obtained from three adult heart donors aged 30-50 years, sliced ( mm), and decellularized through the following steps:
Delipidation + Decellularization: 1% sodium dodecyl sulfate (SDS) water bath for 24 hours + 1% Triton X-100 for 30 minutes to remove cellular structures and fat.
DNA Removal: 50 U/mL DNase water bath for 8 hours, finally washed with deionized water.
Post-treatment ECM was lyophilized, ground into powder in liquid nitrogen, hydrolyzed with pepsin, centrifuged to collect the supernatant, then neutralized to obtain solubilized ECM. The total ECM protein was quantified by Rapid Gold BCA, then diluted to 0.01 mg/ml (1×) and 0.05 mg/ml (5×) experimental concentrations.
Additionally, there was a decellularization protocol designed to “protect the extracellular vesicle (EV) integrity,” for later control groups.
Component Analysis: Mass spectrometry (MS) was used to identify and quantify ECM protein composition, specifically comparing the abundance of ECM proteins (such as collagens, glycoproteins, proteoglycans, etc.) in hearts of different ages (young, adult, aged), with a focus on those related to function.
2.2 iPSC Culture, ECM Preconditioning, and Cardiac DifferentiationThe dips 1016 seva human fibroblast iPSC line from the Harvard Stem Cell Institute was selected, maintained on 1% Geltrex-coated plates. When iPSCs reached 80%-85% confluence, batches were subjected to the following experimental treatments:
ECM Preconditioning: For five days prior to iPSC differentiation, 0.01 mg/ml or 0.05 mg/ml ECM solution (or equivalent amount of EV) was added to the culture medium, with daily medium changes, compared to the control group (no ECM added).
Classical Cardiac Induction: The canonical WNT pathway inhibitor protocol (CHIR99021 + IWP-4) was used to induce iPSC differentiation toward the cardiac lineage, then switched to B-27+RPMI medium for 30 days to obtain iCM populations.
2.3 Maturity and Functional EvaluationCell Functional Phenotype: Spontaneous contraction of iCMs was quantified by microscopic imaging and custom Matlab algorithms (video recording, image block tracking, speed/amplitude/area statistics); calcium transient detected with Fluo-4 AM dye to assess electrophysiological properties and pharmacological (isoproterenol) sensitivity.
Mitochondrial Network Structure: MitoTracker staining with high-resolution confocal imaging, ImageJ mitochondria analyzer used to evaluate mitochondrial coverage area, branch number, network nodes, etc.
Cellular Energy Metabolic State: Agilent Seahorse XF96 metabolic analyzer measured oxygen consumption rate (OCR), extracellular acidification rate (ECAR), calculated ATP output under glycolytic and oxidative phosphorylation states, energy phenotype, etc.
Gene Expression Profiling: RT-qPCR and Nanostring technology were used to quantify cardiac structural and functional genes (MYH6, MYH7, TNNT2, ATP2A2, etc.), with GAPDH for normalization.
Statistical Analysis: All experiments repeated at least three times, data presented as mean ± standard deviation (SD), one-way ANOVA and Tukey’s multiple comparison test used, P<0.05 considered significant.
2.4 Mechanistic Exploration of ECM EfficacyECM Heat/Sonic Treatment Groups: ECM was denatured by 80°C water bath for 3 hours to eliminate protein structure/function, compared the effect of native ECM and heat-treated ECM on iPSC differentiation; sonication of ECM used to release associated EVs, comparing with direct supplementation of purified EVs. Two decellularization strategies (SDS, PAA) compared for EV preservation efficacy.
Multiple Group Comparison: The above measurements (function, structure, metabolism, etc.) were used systematically to determine which ECM components mainly contributed to enhanced iCM maturation.
3. Data Analysis MethodsAutomated acquisition and analysis of experimental data, including cell beating tracked by custom Matlab block matching algorithm, fluorescence time-courses automatically extracting Ca²⁺ peaks and delays, batch mitochondrial skeleton statistics by ImageJ plugin, resulting in quantitative outputs for all physiological and molecular indices, ensuring standardized and objective analysis.
4. Main Experimental Results and Logical Reasoning1. Quality Confirmation of Adult Decellularized Cardiac ECMAfter multistep decellularization, delipidation, and DNA removal, histological staining (H&E, Masson’s trichrome) showed that cellular components disappeared and ECM structure was intact. Residual DNA was <50 ng/µl, meeting the standard. Mass spectrometry identified that over 50% of adult ECM consisted of type I, III, and VI collagens; cardiac development and function related glycoproteins/proteoglycans including fibronectin, fibrillins, Perlecan (HSPG2), and Galectin-1 were also present.
2. ECM Preconditioning Significantly Enhances iPSC Cardiac Differentiation and Functional MaturityFor iCM groups preconditioned with adult ECM, by day 30 of differentiation (compared with controls):
Phenotype: Flow cytometry showed cardiac troponin T (cTnT)-positive rate increased from 80.9% to 92.2%; fibroblast marker Vimentin expression significantly decreased.
Contractile Function: High-dose ECM group displayed significantly higher spontaneous beating frequency and velocity, with heatmaps showing tissue-like synchronized contraction; with equivalent area jitter, ECM groups exhibited higher mechanical activity. After isoproterenol pharmacological stimulation, peak interval shortened and sensitivity improved.
Electrophysiological Maturity: The high-dose ECM group’s iCMs showed typical ventricular-type action potential curves (appearance of “shoulder”, prolonged late plateau phase), with APD90 significantly prolonged.
Mitochondrial Network: ECM group iCMs had increased mitochondrial area and coverage; mitochondria shifted from spherical/fragmented to elongated/branched/networked forms, with branching and junction points multiplied, indicating highly mature energy metabolism apparatus.
Metabolic State: Energy profiles showed ECM-group cells had higher basal ECAR and OCR, overall ATP yield and oxidative respiration improved, both glycolytic reserve and oxidative phosphorylation enhanced, indicating a shift from fetal low metabolism to adult high-energy cardiac metabolism.
Transcriptomic Profile: Cardiac maturation marker genes MYH7, TNNT2, ATP2A2 were significantly upregulated in the ECM group, the MYH7/MYH6 ratio increased (indicative of functional cardiac subtype transition); proliferation and fetal-type genes (such as CTGF, NKX2-5, ACTA1, NPPA, etc.) were downregulated, indicating enhanced maturity. Apoptosis-related genes (CASP3, CASP9, BAX, BCL2L1) were lowered, suggesting a protective role of ECM.
Mechanistic Analysis: In heat-denatured ECM group, beating and energy performance decreased and mitochondrial networking was lost, suggesting that abundant proteins (e.g., collagens, some growth factors) have greater effects on structure than on function. EV and sonication groups showed convergence in some indicators, suggesting that EV components bound in ECM contribute to cellular energy and maturity, but are not absolutely decisive.
3. Synergistic Effect of Multiple ECM Components, Not a Single Main FactorCombining mass spectrometry and results from each experimental group, the adult cardiac ECM is shown to consist of numerous components involved in multiple regulatory processes, including large-molecule scaffolds such as collagens/glycoproteins, as well as growth factors, glycosaminoglycans, vesicles, and carbohydrate-binding proteins. The authors noted that simple protein denaturation cannot completely inhibit the promoting effect, implying that decorative small molecules on ECM (such as GAGs, HSPGs) and their bound signaling molecules and exosomal substances play critical synergistic roles. Thus, “matrix memory” in promoting human iPSC differentiation and maturation involves much more than a single pathway.
5. Conclusions and SignificanceThis study proposes and validates that “adult heart-derived ECM preconditioning” can significantly improve both the differentiation and “maturity” of iPSC-derived cardiomyocytes, mainly reflected in:
Structurally, inducing more and more mature cardiomyocytes;
Functionally, spontaneous and pharmacologically stimulated contraction and electrophysiological behavior becoming more similar to adult hearts;
In terms of energy metabolism, well-developed mitochondrial networks, stronger energy output and structural support capacity, as well as enhanced glycolysis and oxidative phosphorylation adaptability;
At the molecular level, more “adult-like” profiles in structural and functional gene expression, with reduction in apoptosis and immature features;
Multicomponent, multilayered regulation, emphasizing that an intact matrix microenvironment yields greater synergistic effects than single-component proteins.
Scientific and Application ValueScientific Significance: For the first time, this study systematically reveals the function-structure-energy-molecule correlation network by which adult human heart ECM directly promotes iPSC cardiac differentiation and maturation, providing experimental evidence for the “tissue memory” theory and new mechanisms for exogenous induction of stem cells.
Application Prospects: The scheme is easy to operate and low cost (only 5-day preconditioning required), promising for widespread application in cardiac regenerative therapy, disease modeling, and drug screening, greatly alleviating the immaturity limitation of iCMs, and providing theoretical and technical support for individualized regenerative medicine for cardiovascular diseases such as MI.
Highlights and Innovation: 1) A novel integrated “preconditioning + induced differentiation” pipeline; 2) The first systematic comparison of the influence of different ECM components/modifications on cardiac differentiation and maturation; 3) Multi-omics evidence dissects the integrated “structure-function-energy” maturation, providing a template for related fields.
6. Supplement and ProspectsThe current study is based on a single human iPSC line; if validated in more lines in the future, generalization and efficacy would be stronger.
No direct comparison with other “xenogeneic” ECMs was made, but literature notes that human/pig cardiac matrix compositions are extremely similar, which could provide theoretical support for large-scale commercial development in the future.
The study has not systematically evaluated the role of fatty acid and TCA cycle lipid metabolism in ECM-induced differentiation. Combined with other reports, fatty acid supplementation may further enhance iCM maturation.
This new strategy also lays a foundation for 3D printing, organ-on-chip, and other fields to achieve more realistic and functional “in vitro hearts.”
7. SummaryThe study published by S. Gulberk Ozcebe et al. in Stem Cells provides a feasible new strategy for overcoming the global challenge of “immature cardiomyocytes” in iPSC-based cardiac regenerative medicine. Adult human heart ECM, through its complex and memory-laden molecular network, can significantly and efficiently induce iPSCs to differentiate into cardiomyocytes that are closer to “adult-like” in both function and metabolism; this effect is not limited to proteins—vesicles, polysaccharides, and glycoproteins also play key roles. This method will greatly accelerate the standardized maturity of iCMs in regenerative therapy, disease modeling, and drug screening, and establishes a valuable paradigm for future research into ECM-dominated cell fate determination, interspecies applications, and tissue engineering.