In this study, we evaluated the influence of MSC-CM on the mechanical response of HaCaT keratinocytes, focusing on key parameters such as morphology and migration. These aspects are crucial for biological processes like embryogenesis, tissue morphogenesis, and wound healing [20]. Given the growing interest in cell therapies for cutaneous wound repair, HaCaT cells serve as an important model due to their distinct genetic characteristics [21]. They are capable of differentiating and forming an epidermal equivalent in vitro using three-dimensional methods [22] and, when transplanted into nude mice, do not exhibit tumorigenic potential, instead responding to skin wound control mechanisms [23, 24]. These characteristics make HaCaT cells widely accepted in preliminary dermatologic studies.

More than 30 years ago, Friedenstein and collaborators first identified fibroblast-like cells that could be isolated from bone marrow via adherence to plastic surfaces [25]. Today, it is well established that MSCs reside in virtually all postnatal human tissues, contributing to tissue homeostasis (Klimczak & Kozlowska 2016). Their ability to differentiate into specialized cells and produce key molecules for healing makes them highly relevant for cell-based therapies (Deng et al. 2024). MSC-conditioned medium (MSC-CM) has been widely studied to assess its effects on other specialized cells [26, 27] and can be generated under various conditions, including three-dimensional cultures, hypoxic environments, or differing fetal bovine serum (FBS) concentrations [28, 29]. In this study, MSC-CM was prepared under standard atmospheric conditions (humidified, 37ºC, 5% CO₂), adhered to plastic surfaces at semi-confluence, and under FBS deprivation, considering that serum-derived factors might overlap with MSC-secreted molecules.

Upon skin injury, MSCs are activated and exhibit tropism toward the lesion site, playing a crucial role in wound healing. Keratinocytes, in response, may lose their epithelial characteristics and acquire a mesenchymal phenotype, becoming less adhesive and more migratory, thereby accelerating barrier restoration. TGF-β1 has been identified as a key regulator of this transition, promoting morphological changes in epithelial cells [30].

In our experiments, MSC-CM significantly increased keratinocyte migration and induced morphological alterations, even in short periods. The wound created via scratch assay in a HaCaT monolayer closed significantly faster under MSC-CM treatment. When HaCaT cells were co-cultured with MSCs in serum-free conditions, they exhibited directed migration toward MSCs, similar to responses observed when using TGF-β1 as a chemoattractant. This migratory effect was partially blocked by treatment with SB-431542, confirming that TGF-β1 is one of the soluble factors secreted by MSCs and plays an essential role in cell migration [30].

The profound dependence of the migratory effect on the TGF-β signaling pathway, as evidenced by the significant ~ 76% reduction in migration following SB-431542 treatment, suggests that the underlying mechanism extends beyond a straightforward paracrine stimulus from the MSC-CM. It is plausible that the initial TGF-β1 signal supplied by the secretome induces the HaCaT keratinocytes to initiate an autocrine amplification loop. Activated keratinocytes are well-documented to produce and secrete their own TGF-β isoforms, which subsequently bind to cell-surface receptors to self-sustain the migratory and phenotypic activation [31, 32]. This autocrine circuit is crucial for efficient re-epithelialization and offers an explanation for the robust inhibitory effect observed with the SB-431542 antagonist, as it effectively targets both the exogenous MSC-CM signal and the endogenous, amplified autocrine component.

Supporting our findings, Xuan et al. (2017) demonstrated that SB-431542 can block TGF-β1 expression and function, inhibiting human umbilical cord MSC (hUC-MSC) proliferation and contributing to hepatic anti-fibrosis via the TGF-β1/Smad pathway [33]. Additionally, SB-431542 was reported to inhibit B16 melanoma cell migration and invasion, reinforcing its role in suppressing EMT induction [34]. Other studies also highlight the migratory effects of MSC-secreted factors on keratinocytes. Walter et al. (2010) observed significant wound closure in scratched HaCaT monolayers following 27 h of MSC-CM treatment, attributing this effect to MSC-derived TGF-β1, IL-6, and IL-8 [27]. Ruiz-Cañada et al. (2017) further demonstrated that HaCaT cells, either wild-type or knockout for TGF-β receptor, exhibited differential migration responses, suggesting an optimal threshold for TGF-β signaling activation [35].

TGF-β not only regulates gene expression but also modifies cytoskeletal dynamics, promoting both loss of epithelial markers and activation of mesenchymal traits [36]. EMT-induced phenotypic changes enable epithelial cells to detach from organized tissue structures and migrate through the extracellular matrix [37]. F-actin polymerization is crucial for cell protrusion, generating tensile forces that facilitate cell contractility [38].

Here, we demonstrated that MSC-CM modulates actin fiber formation and movement in HaCaT keratinocytes. Under standard conditions (DMEM 10% FBS), keratinocytes exhibit peripheral actin stress fibers, supporting cell–cell junctions and reinforcing their epithelial identity. Following MSC-CM treatment, we observed a redistribution of actin fibers, with a decrease in peripheral support structures and an increase in centrally localized stress fibers, correlating with enhanced migratory capacity. These effects were replicated upon TGF-β1 stimulation, but not when its receptors were blocked, further confirming TGF-β1’s role in cytoskeletal reorganization.

Despite the promising findings demonstrated in vitro, we acknowledge that the present study possesses limitations inherent to the two-dimensional (2D) model utilized. The in vivo wound environment is characterized by a complex microenvironment, including a three-dimensional (3D) extracellular matrix, oxygen gradients, and the presence of various immune cells. In particular, hypoxic conditions and 3D culture systems are known to drastically modulate the secretome of MSCs, enhancing the release of factors such as VEGF, FGF, and immunomodulatory cytokines, which may be more relevant for tissue regeneration. The preparation of our MSC-CM under standard 2D, normoxic conditions, while serving as an initial validation standard, may underestimate the full therapeutic potential of MSCs [39, 40]. Therefore, our results should be interpreted within the context of a simplified in vitro system. Future studies focusing on secretome optimization in 3D bioreactors or under hypoxic conditions, followed by validation in preclinical in vivo models, are necessary steps to fully determine the clinical efficacy of MSC-CM in wound healing.

In summary, this report provides evidence that TGF-β1 secreted by human adipose-derived MSCs is a key factor in regulating the morphology and promoting the spreading of human keratinocytes. Therefore, recognizing the critical role of the MSC secretome in wound healing is essential for establishing the safety and effectiveness of cell-based therapies. These findings reinforce the potential of utilizing MSC-CM as a promising cell-free therapeutic alternative, which could be clinically applied as a safe and effective adjunctive treatment for tissue regeneration in chronic wounds.