Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

Q: What are the key findings of this study?

Mechanical conditioning of iPSC-derived cardiac fibroblasts on 15 kPa versus 2 MPa substrates modulates passage-dependent α-SMA expression, TGF-β1 responsiveness, and 3D tissue remodeling (p<0.05).

Q: What is the clinical evidence from this study?

Population: Cardiac fibrosis / Fibroblast-to-Myofibroblast Transition (FMT). Intervention: Mechanical conditioning on soft (15 kPa) PDMS substrates and subsequent TGF-β1 stimulation vs. Rigid (2 MPa) PDMS or conventional tissue-culture polystyrene (TCP) substrates; untreated control. Primary outcome: Fibroblast-to-myofibroblast transition (cell area and α-SMA fluorescence intensity) and 3D engineered tissue remodeling (tissue width, projected size, passive tension, and passive stress) (p=<0.05).

Q: What does this research mean for the field?

Substrate stiffness during culture expansion modulates passage-dependent α-SMA expression, TGF-β1 responsiveness, and 3D tissue remodeling in iPSC-derived cardiac fibroblasts without irreversibly locking their phenotypic state. Novelty: ClaimNovelty.INCREMENTAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

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

Investigate how differentiation protocol, mechanical culture history, and serial passage expansion influence phenotypic drift, TGF-beta1 responsiveness, and autonomous 3D tissue remodeling in human iPSC-derived cardiac fibroblasts.

Huanzhu Jiang; Ganesh Malayath; Austin C. Kellogg; Nongmaithem Debeni Devi; Charlotte Chen; Nathaniel Huebsch

doi:10.64898/2026.10.20.759862

October 25, 2026Open Access

Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

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Key result

Physiological substrate stiffness modulates iPSC-derived cardiac fibroblast activation and 3D engineered tissue remodeling.

P<0.05

Why the study?

Phenotypic drift during the expansion of human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-cFBs), particularly premature fibroblast-to-myofibroblast transition, remains a concern when generating large cell numbers for drug screening and tissue engineering.

Does mechanical culture history on physiologically soft (15 kPa) versus rigid (2 MPa) substrates during serial expansion modulate phenotypic drift, TGF-beta1 responsiveness, and 3D tissue remodeling in human iPSC-derived cardiac fibroblasts?

Key Points

Investigate how differentiation protocol, mechanical culture history, and serial passage expansion influence phenotypic drift, TGF-beta1 responsiveness, and autonomous 3D tissue remodeling in human iPSC-derived cardiac fibroblasts.
Human iPSC-derived cardiac fibroblasts generated through two distinct epicardial differentiation protocols (Whitehead and Zhang) were expanded on poly(dimethylsiloxane) (PDMS) substrates of physiological (15 kPa) or rigid (2 MPa) stiffness to passage 2 (P2) or passage 5 (P5).
Cells were replated onto common tissue-culture polystyrene to quantify morphology, alpha-SMA expression, and response to 10 ng/mL TGF-beta1 treatment for 48 hours.
P2 and P5 fibroblasts were encapsulated in 3D collagen I gels around PDMS microposts to evaluate tissue compaction geometry, passive tension, and passive stress.
Whitehead-derived cells cultured on 2 MPa PDMS showed significantly greater projected area and higher alpha-SMA intensity at P2 and P5 than 15 kPa-conditioned cells, whereas Zhang-derived cells exhibited greater area on 15 kPa PDMS across passages but higher alpha-SMA on 2 MPa PDMS at P2.
Culture on 15 kPa PDMS preserved TGF-beta1 inducibility through P5 in Whitehead-derived cells, while rigid 2 MPa conditioning resulted in no detectable alpha-SMA induction at P5; Zhang-derived cells showed attenuated alpha-SMA induction by P5 under both conditions.
In 3D microtissues at P2, cells expanded on rigid 2 MPa PDMS generated significantly narrower, smaller tissues with higher passive stress than 15 kPa-conditioned cells across both differentiation protocols, while passive tension did not differ significantly.

Structured PICO

Does mechanical culture history on physiologically soft (15 kPa) versus rigid (2 MPa) substrates during serial expansion modulate phenotypic drift, TGF-beta1 responsiveness, and 3D tissue remodeling in human iPSC-derived cardiac fibroblasts?

P

Population

In vitro preclinical study of human iPSC-derived cardiac fibroblasts (MYBPC3fs and isogenic control lines) and primary human ventricular cardiac fibroblasts expanded through passages 2 to 5 and assessed over up to 10 days in 2D and 3D engineered tissues.

I

Intervention

Derivation and serial expansion (to P2 or P5) on physiologically soft poly(dimethylsiloxane) (PDMS) substrates (15 kPa elastic modulus), followed by transfer to 2D tissue-culture polystyrene (TCP) or encapsulation into 3D collagen I gel microtissues, with subsequent exposure to transforming growth factor-beta 1 (TGF-beta1, 10 ng/mL for 48 hours in 2D or from day 2 through day 10 in 3D).

C

Comparator

Derivation and serial expansion on rigid PDMS substrates (2 MPa elastic modulus) or conventional tissue-culture polystyrene (TCP) substrates under identical differentiation, passaging, and TGF-beta1 stimulation conditions.

O

Outcome

Basal phenotype and TGF-beta1-induced fibroblast-to-myofibroblast transition (FMT) markers, specifically projected cell area, alpha-smooth muscle actin (alpha-SMA) fluorescence intensity, and cell eccentricity in 2D and 3D cultures.surrogate

Main Result

p-value: p=<0.05

Mechanical culture history and differentiation protocol jointly modulate the phenotypic plasticity, TGF-beta1 responsiveness, and 3D tissue-remodeling behavior of iPSC-derived cardiac fibroblasts during serial expansion without irreversibly fixing their state, providing critical guidance for scaling cells in cardiac disease modeling and tissue engineering.

Limitations

The 15 kPa and 2 MPa PDMS substrates represent only two discrete mechanical environments, with 2 MPa serving as a deliberately rigid condition rather than a direct mimic of fibrotic myocardium.
Fetal-heart-like soft substrates (<1 kPa) were avoided due to technical challenges in flasks used for cell passaging.
The Zhang and Whitehead differentiation protocols differ in multiple parameters, preventing protocol-dependent effects from being attributed to a single mechanism.
The 2D and 3D TGF-β experiments differed in treatment duration, ECM environment, cellular organization, and mechanical constraint.
Quantification of cell area and α-SMA expression represent only selected dimensions of the overall fibroblast phenotype and do not identify a specific molecular mechanism of mechanical memory.
Human iPSC-cFBs may not fully recapitulate mature adult fibroblasts.
It is possible that contact inhibition or changes in local cell density masked fibroblast spreading in the 2D studies.
The present experiments do not identify a specific molecular mechanism of mechanical memory.

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Implication

Mechanical conditioning may modulate iPSC-fibroblast phenotype in models; hypothesis-generating for fibrosis research and leaves open clinical translation.

Abstract

Human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-cFBs) offer a renewable, genetically defined stromal cell source for studying cardiac fibrosis and cardiomyocyte-fibroblast crosstalk during development and disease. Because cardiac fibroblasts are proliferative, expansion of iPSC-cFBs in culture is attractive for applications requiring large cell numbers, including drug screening and tissue engineering. However, phenotypic drift during expansion, particularly premature fibroblast-to-myofibroblast transition (FMT), remains a concern. Here, we investigated how differentiation protocol, mechanical culture history, and serial expansion shape iPSC-cFB phenotype, transforming growth factor beta 1 (TGF-beta1) responsiveness, and fibroblast-autonomous remodeling in three-dimensional (3D) tissues. Initial studies of iPSC-cFBs expanded on conventional tissue-culture polystyrene (TCP) revealed passage-associated increases in projected cell area and alpha-smooth muscle actin (alpha-SMA) expression, two markers associated with FMT. These findings prompted us to examine how the stiffness of poly(dimethylsiloxane) (PDMS) substrates (15 kPa or 2 MPa) used during iPSC-cFB derivation and expansion influences basal phenotype and response to TGF-beta1. Across two independent differentiation protocols, mechanical culture history shaped passage-dependent basal phenotypes and responses to TGF-beta1. Notably, its effects on collective TGF-beta1 responses in three-dimensional cultures differed from those observed after replating cells onto two-dimensional substrates. Together, these findings demonstrate that mechanical culture history modulates iPSC-cFB responsiveness and tissue-remodeling behavior without irreversibly determining fibroblast state. Subsequent passage and microenvironmental conditions can attenuate or re-express these history-dependent phenotypes. This work supports the use of iPSC-cFBs in basic biological discovery and future tissue engineering applications.

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Cite This Study

Jiang et al. (2026) studied Cardiac fibrosis / Fibroblast-to-Myofibroblast Transition (FMT). Mechanical conditioning on soft (15 kPa) PDMS substrates and subsequent TGF-β1 stimulation vs. Rigid (2 MPa) PDMS or conventional tissue-culture polystyrene (TCP) substrates; untreated control was evaluated on Fibroblast-to-myofibroblast transition (cell area and α-SMA fluorescence intensity) and 3D engineered tissue remodeling (tissue width, projected size, passive tension, and passive stress) (p=<0.05). Mechanical conditioning of iPSC-derived cardiac fibroblasts on 15 kPa versus 2 MPa substrates modulates passage-dependent α-SMA expression, TGF-β1 responsiveness, and 3D tissue remodeling (p<0.05).

synapsesocial.com/papers/6ae28f09cbadf70daf6c43f9 https://doi.org/10.64898/2026.10.20.759862

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Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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