Abstract: Induced pluripotent stem cell-derived fibroblasts (iFBs) are increasingly used in autologous disease models, including cardiac 3D systems, due to their accessibility and compatibility. However, whether iFBs can adapt to tissue-specific characteristics within these models is underexplored, raising questions about their ability to fully recapitulate the specialised properties of native fibroblasts. Fibroblasts are highly heterogeneous, with distinct subtypes exhibiting tissue-specific functions critical for organ integrity and cellular interactions. For example, dermal and cardiac fibroblasts perform unique roles, and primary skin and lung models show improved performance when cultured with tissue-specific fibroblasts. In this study, we developed iFBs using a modified differentiation protocol and examined their capacity to acquire tissue-specific molecular phenotypes upon co-culture with primary cells. Early-stage iFBs were co-cultured with keratinocytes (ectoderm), cardiomyocytes (mesoderm), and bronchial epithelial cells (endoderm) to assess whether interactions could induce tissue-specific transcriptional changes across all three germ layers. Transcriptomic analyses revealed that the molecular profiles of iFBs shifted context-dependently, suggesting they exhibit functional plasticity and highlighting both their potential and limitations in mimicking native fibroblast behaviour. Additionally, targeted experiments with iFBs indirectly co-cultured with keratinocytes, cardiomyocytes, and bronchial epithelial cells showed that paracrine signalling could induce tissue-specific molecular and morphological changes. However, these adaptations were lost when co-culture was removed, indicating the phenotype is not fully stable without continued interaction. While autologous systems are a key goal for in vitro disease modelling, variability in iPSC differentiation and maturity can influence the performance of multi-cellular models relative to human physiology. Our findings emphasise the importance of validating iFBs' ability to integrate into more complex tissue models. This study demonstrates that iFBs can adopt tissue-specific transcriptional profiles, bringing them closer to their primary counterparts.
