Stage-associated differences in injury and stress responses of hiPSC-derived cardiomyocytes under macrophage-derived inflammatory stimulation

Q: What are the key findings of this study?

Early-stage day 7 human induced pluripotent stem cell-derived cardiomyocytes exhibited significantly greater injury and stress responses than more mature day 21 cells under macrophage-derived inflammatory stimulation.

Q: What is the clinical evidence from this study?

Population: Pediatric myocarditis (in vitro model). Intervention: THP-1 macrophage-derived conditioned media (M1-CM) vs. Control media (Vehicle or M0-CM) and between-stage comparison (D21 cells). Primary outcome: Cell viability, lactate dehydrogenase (LDH) release, and cardiac troponin I (cTnI) levels under inflammatory stimulation (p=<0.001).

Q: What does this research mean for the field?

Early-stage human induced pluripotent stem cell-derived cardiomyocytes exhibit significantly greater injury and stress responses than more mature cardiomyocytes when exposed to macrophage-derived inflammatory stimulation, partly mediated by CHOP-associated endoplasmic reticulum stress. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To examine stage-associated differences in human induced pluripotent stem cell-derived cardiomyocytes exposed to macrophage-derived inflammatory conditioned media and evaluate stress-response pathways.

Jing Wei; Qingbin Liu; Xiaoyi Chang; Yuna Zhang

doi:10.21037/tp-2026-0505

August 1, 2026Translational PediatricsOpen Access

Stage-associated differences in injury and stress responses of hiPSC-derived cardiomyocytes under macrophage-derived inflammatory stimulation

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Key result

Immature hiPSC-cardiomyocytes suffer significantly greater inflammatory injury and stress than mature cells.

P<0.001

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Why the study?

The influence of cardiomyocyte maturation status on responses to macrophage-driven inflammatory stimulation in pediatric myocarditis remains unclear.

Key Points

To examine stage-associated differences in human induced pluripotent stem cell-derived cardiomyocytes exposed to macrophage-derived inflammatory conditioned media and evaluate stress-response pathways.
Modeled inflammatory microenvironments using THP-1-derived macrophage conditioned media (M0-CM and M1-CM) on day 7 (D7, early-stage) and day 21 (D21, more mature) hiPSC-CMs.
Assessed cell viability by CCK-8, membrane injury by LDH release, and myocardial damage by cTnI ELISA.
Evaluated CHOP expression via Western blot and mitochondrial membrane potential (ΔΨm) via JC-1 staining following siRNA-mediated CHOP knockdown.
M1-CM significantly elevated TNF-α, IL-6, and iNOS levels compared with M0 and control groups (P<0.001).
D7 hiPSC-CMs displayed greater viability loss, higher LDH release, and increased cTnI release under inflammatory stimulation compared with D21 hiPSC-CMs.
CHOP expression was upregulated by M1-CM (P<0.001), while CHOP knockdown significantly decreased LDH release (P<0.001) and partially restored mitochondrial membrane potential (P<0.05).

Structured PICO

P

Population

In vitro study utilizing human induced pluripotent stem cell-derived cardiomyocytes at day 7 and day 21 to evaluate maturation-stage-associated responses to macrophage-derived inflammatory stimulation.

E

Exposure

Exposure to THP-1 macrophage-derived conditioned media (M0-CM and M1-CM) for 24 hours; small interfering RNA (siRNA)-mediated knockdown of C/EBP homologous protein (CHOP).

C

Comparator

Vehicle control medium without conditioned media; non-targeting negative control siRNA (si-NC).

O

Outcome

Cell viability (measured by CCK-8), lactate dehydrogenase (LDH) release, and cardiac troponin I (cTnI) levels to evaluate cellular injury.surrogate

Main Result

p-value: p=<0.001

This study suggests that early-stage cardiomyocytes are intrinsically more susceptible to macrophage-derived inflammatory injury than more mature cells, with damage partially mediated by CHOP-associated endoplasmic reticulum stress.

Limitations

The THP-1-derived conditioned media model does not fully capture the complexity of pediatric myocarditis, lacking patient-derived immune cells and viral triggers.
In vitro maturation of hiPSC-CMs at day 21 remains incomplete compared with adult cardiomyocytes.
The sample size was limited to a restricted in vitro system without independent hiPSC lines or independent differentiations.
CHOP knockdown provided functional evidence but did not establish CHOP as the central mediator, as additional ER stress markers were not assessed.
Simplified in vitro model does not fully reproduce the cellular, viral, and patient-specific context of pediatric myocarditis.
Day 21 hiPSC-CMs remain developmentally immature compared with adult cardiomyocytes and lack extensive electrophysiological or metabolic assessments.
The macrophage conditioned media may contain multiple cytokines, metabolites, and residual inducing agents.

Implication

Early hiPSC-CMs warrant caution in inflammatory models; leaves open maturation effects on myocarditis in vivo.

Abstract

Background: Macrophage-driven inflammation contributes to myocardial injury in pediatric myocarditis, but the extent to which cardiomyocyte maturation status influences responses to inflammatory stimulation remains unclear. This study aimed to investigate stage-associated differences in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to THP-1 macrophage-derived conditioned media and to explore the involvement of stress-response pathways. Methods: A macrophage-derived inflammatory microenvironment was modeled using THP-1-derived macrophage conditioned media (M0-CM and M1-CM). hiPSC-CMs at day 7 (D7) and day 21 (D21) were used as early-stage and relatively more mature in vitro stages, respectively. Cell viability was assessed using Cell Counting Kit-8 (CCK-8), and lactate dehydrogenase (LDH) release and cardiac troponin I (cTnI) levels were measured to evaluate injury. C/EBP homologous protein (CHOP) expression was analyzed by Western blot, and mitochondrial membrane potential (ΔΨm) was determined using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining following small interfering RNA (siRNA)-mediated CHOP knockdown. Results: M1-CM significantly increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels compared with M0 and control groups (P<0.001), along with upregulation of inducible nitric oxide synthase (iNOS) (P<0.001). Under inflammatory stimulation, D7 hiPSC-CMs showed greater decreases in viability and greater LDH and cTnI release than D21 hiPSC-CMs in the stage-stratified analyses. CHOP expression was induced by M1-CM (P<0.001), and CHOP knockdown decreased LDH release (P<0.001) and partially restored ΔΨm (P<0.05). Conclusions: model. CHOP-associated endoplasmic reticulum (ER) stress contributed partly to mitochondrial dysfunction, but this system does not fully recapitulate pediatric myocarditis and requires validation in more physiologically complex models.

Discussion

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Cite This Study

Wei et al. (2026) studied Pediatric myocarditis (in vitro model). THP-1 macrophage-derived conditioned media (M1-CM) vs. Control media (Vehicle or M0-CM) and between-stage comparison (D21 cells) was evaluated on Cell viability, lactate dehydrogenase (LDH) release, and cardiac troponin I (cTnI) levels under inflammatory stimulation (p=<0.001). Early-stage day 7 human induced pluripotent stem cell-derived cardiomyocytes exhibited significantly greater injury and stress responses than more mature day 21 cells under macrophage-derived inflammatory stimulation.

synapsesocial.com/papers/6ad0fcc53afa093ea9acffe7 https://doi.org/10.21037/tp-2026-0505

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Stage-associated differences in injury and stress responses of hiPSC-derived cardiomyocytes under macrophage-derived inflammatory stimulation

Figures

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

Stage-associated differences in injury and stress responses of hiPSC-derived cardiomyocytes under macrophage-derived inflammatory stimulation

Figures

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

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