In this study, we evaluated the potential of iPSCs in modulating the expression of the key genes of the Wnt signaling pathway and BMP4 in a mouse model of IPF induced by bleomycin (Fig. 5). Herein, we used C57BL/6 mice as a multipurpose model for IPF research and chose the intratracheal route of Bleomycin administration as it produces a homogeneous distribution of fibrotic lesions among lung lobes. The rationale for using C57BL/6 mice is that they are susceptible to bleomycin-induced lung fibrosis, which is a widely used method to mimic IPF in animals [27,28,29].

Schematic representation of the study flow. After successful induction of the iPSCs and the IPF mouse model, the iPSCs were injected via the tail vein, two days post bleomycin administration; on day 21, the histologic and molecular assessments were performed, and it was observed that besides alleviating the histologic features, iPSCs modulated the expression of the WNT pathway components which are known to contribute to the fibroblast transdifferentiation into myofibroblasts, although they exerted no effect on the reduced expression of the Bmp4 gene. BMP4 usually affects the WNT pathway by enhancing the expression of Dkk1

We also confirmed the identity of iPSCs by real-time PCR for pluripotency markers. Despite the absence of the Nanog gene in the lentiviral construct that we employed to deliver the pluripotency factors, we found that iPSCs expressed the Nanog gene as well. This suggests that Nanog expression may be induced by other factors or mechanisms during the reprogramming process and may further confirm the successful induction of the iPSCs.

We administered iPSCs by tail vein injection 48 h after bleomycin instillation to evaluate their therapeutic effects on IPF. Subsequently, histological and molecular analyses were performed on lung tissues at 3 weeks after bleomycin administration; it was found that iPSCs significantly reduced the inflammation and fibrosis in the lung, as evidenced by H&E staining, Masson's trichrome staining, and hydroxyproline assay (Fig. 5). Consistently, several previous studies have reported the beneficial effects of IPSCs or their conditioned medium in animal models or cell cultures of pulmonary fibrosis. For instance, How et al. [30] showed that iPSC treatment mediated the release of interferon gamma-induced protein 10 (IP-10), in bronchoalveolar lavage fluid and serum from bleomycin-treated mice. Gazdhar et al. [31] have also demonstrated that the secretome of iPSCs (iPSC-Sec) treatment significantly improved lung function, and reduced lung inflammation and fibrosis, in bleomycin-treated mice; it was also shown that iPSC-Sec contained various growth factors and anti-inflammatory molecules. Furthermore, Zhou et al. [32], also have found that IPSCs suppressed inflammatory mediators involved in the initiation and progression of fibrosis, in lung tissues and fibroblasts from bleomycin-treated mice; also, they revealed that iPSC treatment suppressed the expression of pro-fibrotic factors and inhibited the activation of TGF-β1/Smad signaling. These studies suggest that IPSCs may exert their antifibrotic effects by modulating multiple molecular pathways that regulate fibrosis. However, the exact mechanisms and interactions of these pathways are still unclear and remain to be elucidated in future studies. Accordingly, our findings are in line with previous studies that reported the antifibrotic effects of iPSCs in IPF models. However, our study is novel in demonstrating that iPSCs can modulate the Wnt signaling pathway in IPF. The Wnt signaling pathway has been implicated in various aspects of IPF pathogenesis, such as epithelial-mesenchymal transition (EMT), fibroblast activation and differentiation into myofibroblasts, ECM remodeling, angiogenesis, inflammation, and apoptosis [33, 34].

We also assessed the expression of Wnt signaling pathway genes (Wnt, β-catenin, Lef, and Dkk1); it was subsequently found that Wnt, β-catenin, and Lef genes were upregulated while Dkk1 was downregulated in the bleomycin-induced IPF model and iPSCs treatment normalized their expression. These results indicate that iPSCs may modulate the Wnt signaling pathway in IPF by downregulating the expression of Wnt activators and upregulating the expression of Wnt inhibitors. In the current study, we have also shown that bleomycin treatment would downregulate the expression of the BMP4 gene which is consistent with the concurrent downregulation of the Dkk1 gene and previous data [35], although the treatment of the IPF mice with iPSCs did not affect restoring the expression of BMP4 gene. This could indicate that BMP4 may not be essential for the regulation of WNT signaling by iPSCs in IPF which could be explained by several possibilities: First, iPSCs may secrete other factors that can inhibit WNT signaling [36]. Second, iPSCs may alter the expression or activity of other components of the WNT signaling pathway, such as LRP5/6 or GSK3β [6]. Third, iPSCs may affect the cross-talk between WNT signaling and other pathways that are involved in IPF pathogenesis, such as transforming growth factor-β (TGF-β) [35]. Overall, our findings could provide new insights into the molecular mechanisms and therapeutic potential of iPSCs for IPF treatment.

Modulating the Wnt signaling pathway may have multiple beneficial effects on IPF which is further supported by previous studies similar to our study but different in some aspects such as the disease model, the intervention, or outcome measures. For instance, Konigshoff et al. [37] found that the expression of Wnt ligands and receptors, as well as β-catenin was increased in lung tissues from IPF patients; it was also shown that Wnt ligands stimulated the proliferation and collagen production of lung fibroblasts, which is consistent with our findings that Wnt signaling pathway genes were upregulated in the bleomycin-induced IPF model and iPSCs treatment normalized their expression. However, this study did not use iPSCs or any other stem cells as a therapeutic intervention, nor did it measure markers of inflammation and fibrosis in the lung. Besides, Several similar studies have addressed the potential of treatment of pulmonary fibrosis in different murine models by inhibiting the WNT pathway using various therapeutic compounds such as dehydrozingerone (DHZ), a natural compound derived from ginger, XAV-939, and ICG-001 which are small molecule inhibitors of the WNT/β-Catenin pathway; all of which have shown promising results in attenuating lung inflammation and fibrosis [38,39,40,41]. These data are in line with our results highlighting the importance of the WNT pathway in pulmonary fibrosis and supporting the theory of targeting it as a possible therapeutic approach. However, utilizing cell therapy would have multiple advantages over these compounds as it may provide a source of alveolar epithelial cells or other lung cell types that can replace the damaged or lost cells and restore the lung function and structure [42, 43]. Also, Cell therapy may provide anti-inflammatory and antifibrotic factors [30,31,32] and may modulate multiple molecular pathways [42,43,44].

In a recent 2023 study similar to ours, the effects of umbilical cord mesenchymal stem cells (UCMSCs) were investigated on acute respiratory distress syndrome (ARDS)-associated pulmonary fibrosis in mice; the authors have found that UCMSCs significantly improved lung function, and reduced inflammation and fibrosis, by modulating the Wnt/β-catenin pathway [42]. However, this study used a different type of stem cells, a different disease model, and different outcome measures (lung function) than our study. Furthermore, in 2018, another relevant study has shown that bone marrow mesenchymal stromal cells (BMSCs) significantly improved lung function, reduced lung inflammation and fibrosis, and increased survival rate in silica-exposed rats by attenuating the activation of Wnt/β-catenin signaling, and restoring the expression of Wnt inhibitors and antagonists, such as DKK1 [43]. Although these two individual studies are in line with our data showing the possible effects of cell therapy on pulmonary fibrosis, the bleomycin-induced model utilized in our study better recapitulates the human IPF, which is the focus of our study; also, iPSCs have multiple benefits over the MSCs, such as unlimited availability and accessibility, as they can be derived from various somatic cells of any individual [45]. Besides, iPSCs have high quality and potency, with equal and controlled effects, unlike their MSC counterparts with varying potencies and different inflammatory/anti-inflammatory properties [45]. Quite contrary to our data one study in 2023 found that DKK1 expression was increased in lung tissues and blood samples from patients with IPF, as well as in mice treated with bleomycin. It was also shown that DKK1 promoted fibroblast activation and differentiation, by suppressing the WNT/β-Catenin pathway and enhancing the TGF-β/Smad pathway [46]. This discrepancy may be due to different experimental conditions, such as the dose and time point of bleomycin administration, or the method of measuring DKK1 expression. Alternatively, it may reflect the complexity and diversity of the Wnt signaling pathway in different conditions.

These studies suggest that targeting the Wnt signaling pathway could be a viable therapeutic strategy for pulmonary fibrosis. However, further research is needed to fully understand the mechanisms of action of these compounds and to evaluate their safety and efficacy in human clinical trials. Additionally, pulmonary fibrosis is a complex disease with multiple contributing factors, so a combination of therapeutic approaches may be necessary for effective treatment.

Lung function data, such as forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1), can reflect the degree of lung fibrosis and inflammation in BLM-induced IPF mice, as well as the response to iPSC treatment. Lung function data are also easier and faster to measure than histological or molecular data, which require invasive procedures and sophisticated techniques. Therefore, lung function data may be more relevant and meaningful for assessing the therapeutic effect of iPSCs on IPF in BLM-induced IPF mice, as they can directly evaluate the functional improvement or deterioration of the lungs after iPSC injection.

The findings of our study and the previous studies that have been discussed so far are very promising for the treatment of IPF in animal models, but they need to be validated and translated to human patients with IPF. There are currently no clinical trials involving iPSCs for IPF in humans [47]. However, there are some ongoing or planned clinical trials using iPSCs for other diseases, such as macular degeneration, Parkinson's disease, spinal cord injury, and heart failure [48]. These trials may provide valuable insights and experience for the future application of iPSCs for IPF in humans. Some of the challenges and barriers that need to be overcome include the safety and efficacy of iPSCs, the immune compatibility of iPSCs, the ethical and regulatory issues of iPSCs, and the cost and scalability of iPSCs [47]. There are several ways to improve the safety and efficacy of iPSCs, such as: Using non-integrative methods to generate iPSCs, which avoid the risk of insertional mutagenesis and genomic instability caused by viral vectors, or using xeno-free and feeder-free culture conditions to maintain iPSCs, which reduce the risk of contamination and immunogenicity from animal-derived products [47, 48]. These methods may help to improve the safety and efficacy of iPSCs for various therapeutic applications. However, more research and optimization are needed to overcome the remaining challenges and limitations.