Conditional Immortalization of Human Cardiac Fibroblasts for Pro-Fibrotic and Anti-Fibrotic Drug Screening.

BACKGROUND

The development of anti-fibrotic therapies for heart failure is hindered by the absence of phenotypically stable and scalable human cardiac fibroblast (CFB) models suitable for pre-clinical drug screening. This study aimed to establish and validate a conditionally immortalized CFB (iCFB) cell line for screening cardiac fibrosis drugs.

METHODS

An iCFB line was generated using a doxycycline (dox)-inducible system. The cellular characteristics were evaluated using molecular, protein, and functional analyses. The utility of the model was assessed by exposure to multiple pro-fibrotic stimuli (transforming growth factor-β1 (TGF-β1), angiotensin II, and palmitic acid) and two anti-fibrotic compounds (N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine [NS8593] and pirfenidone).

RESULTS

iCFB proliferation was tightly regulated by dox. Upon dox withdrawal, the iCFBs reverted to a quiescent state and exhibited a molecular expression profile (collagen type I alpha 1 chain [COL1A1], periostin [POSTN], gap junction protein alpha 1 [GJA1], and T-box transcription factor 20 [TBX20]) comparable to that of primary CFBs. This phenotypic fidelity, along with a robust capacity for TGF-β1-induced myofibroblast differentiation, was maintained during long-term culture up to population doubling 60. The model responded consistently to diverse pro-fibrotic stimuli, confirming the anti-fibrotic efficacy of the transient receptor potential melastatin 7 (TRPM7) inhibitor, NS8593, and demonstrating the therapeutic potential of the repurposed drug pirfenidone.

CONCLUSIONS

We developed a novel iCFB model that integrates long-term expandability, high biological fidelity, and broad responsiveness to fibrotic signaling. This robust platform is well-suited for mechanistic studies and drug screening, thereby facilitating and accelerating the discovery of anti-fibrotic therapeutics.