Bangladesh Journal of Pharmacology Volume: 20; Number 4; Year 2025 Cite this article as: Tran TT, Vu TT, Tran KT, Nakahama T, Nguyen NT. 6-Formylindolo[3,2-b]carbazole pro- motes proliferation of adipose-derived mesenchymal stem cells via activation of the Ahr–IL-6 signaling axis. Bangladesh J Pharmacol. 2025; 20: 169-72. 6-Formylindolo[3,2-b]carbazole promotes prolifera- tion of adipose-derived mesenchymal stem cells via activation of the Ahr–IL-6 signaling axis Dear Editor, The tryptophan photoproduct 6-formylindolo[3,2-b]car- bazole (FICZ) is a high-affinity endogenous ligand of the aryl hydrocarbon receptor (Ahr). Ahr has emerged as a promising target in stem cell research — including both mesenchymal stem/stromal cells (MSCs) and hematopoietic stem cells (HSCs) — due to its critical roles in regulating self-renewal, proliferation, differen- tiation, and immunomodulation, mediated through the regulation of metabolism-related enzymes (e.g., Cyp1a1, Cyp1b1) and cytokines (e.g., TGF-β, IL-6) (Yin et al., 2016; Seo et al., 2025). However, the contribution of the FICZ–Ahr–IL-6 network to MSC proliferation re-mains poorly understood. This study investigates the effect of FICZ on the proli- feration of adipose-derived MSCs (AD-MSCs) cultured in Dulbecco's Modified Eagle Medium (DMEM) supple- mented with 10%fetal bovine serum, 100 IU/mL peni- cillin, 100 μg/mL streptomycin, 0.25 μg/mL amphoteri- cin B, 1 ng/mL basic fibroblast growth factor, and 1 ng/ mL epidermal growth factor. We compare the fold- change in proliferation and expression of related genes — including Ahr, Cyp1a1, Cyp1b1, and Il-6 — in AD- MSCs treated with or without FICZ across different cell passages. Adipose tissues were obtained from healthy female donors (aged 25–30 years) at Hanoi Obstetrics and Gynecology Hospital. AD-MSCs were isolated as des- cribed elsewhere (Kim et al., 2013). The concentration of FICZ used (100 nM) was selected based on established protocols from previous reports (Huang et al., 2022; Vrzal et al., 2023). AD-MSCs at passages 1, 2, and 3 (P1, P2, P3) were seeded at a density of 5 × 10⁴ cells/well in 24-well plates and cultured in DMEM supplemented with 10%fetal bovine serum, with or without 100 nM FICZ. Morphological evaluation was performed on day 1, 3, 5, and 7 under an inverted phase-contrast microscope. Representative images from P2 cells (Figure 1, upper row) revealed no visible alterations in fibroblast-like morphology between treated and untreated groups. To assess multipotency, AD-MSCs at P2 were cultured for 21 days in StemMACS differentiation media: AdipoDiff (1 × 10⁵ cells/well in 12-well plates), OsteoDiff (5 × 10⁴ cells/well in 12-well plates), and ChondroDiff (2.5 × 10⁵ cells/tube in 15-mL conical tubes), with or without FICZ. Adipogenic, osteogenic, and chondrogenic differ- entiation was confirmed by oil red O, alizarin red, and alcian blue staining, respectively. FICZ-treated cells maintained their differentiation capa- city, evidenced by the accumulation of lipid droplets, calcium deposits, and glycosaminoglycans (Figure 1, lower row). Flow cytometry (MACSQuant® VYB, Mil- tenyi Biotec, Germany) analysis further confirmed that AD-MSCs retained their characteristic surface marker expression following FICZ treatment, including high levels of CD90, CD73, and CD105, and a lack of HLA- DR expression (Table I), indicating preserved immuno- phenotypic identity. There were no statistically signifi- cant differences in the expression of surface markers between untreated and FICZ-treated cells. Specifically, CD90 expression remained high (99.5% ± 0.6 in untreated vs. 99.0% ± 0.5 in treated cells; p>0.05), as did CD73 (99.7% ± 0.3 vs. 99.5% ± 0.4; p>0.05) and CD105 (95.5% ± 0.5 vs. 95.1% ± 0.5; p>0.05). Both groups showed negligible expression of the hematopoietic mar- ker HLA-DR (0.01% ± 0.01 vs. 0.02% ± 0.01; p>0.05). These findings indicate that FICZ treatment does not alter the characteristic surface marker expression of AD -MSCs. Next, to evaluate proliferation, AD-MSCs were harvested at P1, P2, and P3, and viable cells were coun- ted by using the trypan blue exclusion method with a hemocytometer. FICZ promoted the proliferation rate of AD-MSCs, with fold changes of 1.1 ± 0.0, 1.1 ± 0.0, and 1.2 ± 0.0 at P1, P2, and P3, respectively, compared to untreated controls (p<0.05) (Figure 2). These results suggest that FICZ significantly promotes the prolifera- tion of AD-MSCs without compromising their pheno- type. It has been reported that Ahr plays a role in stem cell proliferation in a ligand- and context-dependent man- ner (Larigot et al., 2018). Pharmacologically, FICZ is recognized as a potent, transient agonist of Ahr. To investigate the effect of FICZ on Ahr signaling and downstream targets, the expression of Ahr, Cyp1a1, and Cyp1b1 was evaluated in AD-MSCs. Passage 2 AD- MSCs were treated with 10–100 nM FICZ and harvested at 8, 16, 24, and 48 hours for qRT-PCR analysis. At 100 nM, FICZ induced peak gene expression at 24 A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2025; 20: 169-172 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v20i4.83467 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor hours, with Ahr upregulated ~3.8-fold (Figure 3A), Cyp1a1 ~7.1-fold (Figure 3B), and Cyp1b1 ~9.6-fold (Figure 3C), compared to untreated controls. Given the known role of IL-6 in promoting mesenchymal stromal cell proliferation (Dorronsoro et al., 2020), Il-6 gene expression was also assessed. FICZ treatment induced a ~3.2-fold increase in Il-6 expression at 24 hours (Figure 3D), coinciding with the transcriptional peaks of Ahr and its downstream genes. This finding supports a potential link between Ahr activation and IL-6 upregulation in AD-MSCs. Interestingly, we observed significantly higher Ahr expression in AD-MSCs cultur- ed in StemMACS MSC expansion medium compared to those in standard DMEM (unpublished data), sugges- ting the presence of an Ahr ligand on the proprietary formulation. Further studies are required to identify the 170 Bangladesh J Pharmacol 2025; 20: 169-172 A D -M S C A D -M S C + F IC Z A D -M S C A D -M S C + F IC Z Day 1 Day 3 Day 5 Day 7 Adipogenesis Osteogenesis Chondrogenesis Figure 1: Morphological observation and characterization of AD-MSCs cultured with or without FICZ (100 nM) at passage 2. Phase- contrast microscopy images of AD-MSCs cultured without or with FICZ at day, 3, 5, and 7 (A, magnification 4x). Scale bar: 50 μm. Adipogenesis, osteogenesis, and chondrogenesis differentiation of AD-MSCs. Following adipogenesis, osteogenesis and chondrogenesis differentiation, lipid droplets, calcium deposits and glycosaminoglycans are revealed, respectively (B, magnification 20x) 1.5 1.0 0.5 0 a F o ld c h a n g e A D -M S C A D -M S C + F IC Z A D -M S C A D -M S C + F IC Z A D -M S C A D -M S C + F IC Z P1 cells P2 cells P3 cells Figure 2: Proliferation of AD-MSCs treated with or without FICZ. Fold change in cell proliferation of FICZ-treated cells compared to untreated cells at passage 1 (P1), P2, and P3. Data are mean ± SD from three independent experiments. ap<0.05 vs. untreated control a a A B Table I %Expression of MSC surface markers and human leucocyte antigen-DR treated with FICZ CD marker untreated AD -MSC (%) FICZ (100 nM)- treated AD- MSC (%) p val- ue CD90 99.5 ± 0.6 99.0 ± 0.5 >0.05 CD73 99.7 ± 0.3 99.5 ± 0.4 >0.05 CD105 95.5 ± 0.5 95.1 ± 0.5 >0.05 HLA-DR 0.0 ± 0.0 0.0 ± 0.0 >0.05 Values are mean ± SD; n=3 animals in each group; treatment period was 7 days specific factors responsible for Ahr activation in this me- dium. A hypothetical model summarizing the proposed interplay between FICZ and Ahr-IL-6 signaling axis in promoting AD-MSC proliferation (Figure 4). Taken together, present findings suggest that FICZ acti- vates the Ahr–IL-6 signaling axis to enhance the proli- feration of AD-MSCs while preserving their multipo- tency, including the ability to differentiate into adipo- genic, osteogenic, and chondrogenic lineages. Impor- tantly, Ahr is also expressed in epidermal keratinocytes, where it regulates cutaneous immune responses and homeostasis (Tsuji et al., 2012; Nguyen et al., 2013; Tanaka et al., 2018). Given that FICZ is a photoproduct of tryptophan generated upon UV exposure, its regula- tory role in skin-resident cells—including keratinocy- tes and hypodermal adipose-derived MSCs—warrants further investigation. Elucidating the role of the FICZ– Ahr–IL-6 signaling axis in the skin micro-environment under physiological or light-induced conditions may provide novel insights into tissue regeneration and the treatment of inflammatory and degenerative skin disor- ders. Financial support: This work was funded by the VAST project TDTBG0.01/21-23 from the Vietnam Academy of Science and Technology. Ethical Issue: Adipose tissues were obtained in accordance with protocol approved by the hospital’s ethics committee (Ref. No. 2206 CN/PS). Conflict of interest: The authors declare that they have no conflicts of interest. Acknowledgments: The authors greatly appreciate Dr. Daeyong Kim, N-Biotek Inc. (Korea) for providing a training course on AD-MSC preparation and MSc. Pham Dang An for his technical assistance. Thanh Trung Tran1, Tien Thi Vu1 , Kien Trung Tran2, Taisuke Nakahama3, and Nam Trung Nguyen1 1Institute of Biology, Vietnam Academy of Science and Technology, Hanoi, Vietnam; 2Hanoi Obstetrics and Gynecology Hospital, Hanoi, Vietnam; 3Department of RNA Biology and Neuroscience, Osaka University, Suita, Osaka, Japan. Corresponding authors: Email: nam@ibt.ac.vn R e la ti v e e x p re s s io n R e la ti v e e x p re s s io n R e la ti v e e x p re s s io n R e la ti v e e x p re s s io n 4 3 2 1 0 8 6 4 2 0 10 8 6 4 2 0 4 3 2 1 0 Ahr Cyp1a1 Cyp1b1 IL-6 0 10 50 100 FICZ concentration (nM) Figure 3: Expression of Ahr, Cyp1a1, Cyp1b1, and Il-6 in FICZ- treated and untreated AD-MSCs. Gene expression levels were measured by qPCR at 8, 16, 24, and 48 hours. Data are mean ± SD from three independent experiments. GAPDH housekeeping genes were used for normalization. ap<0.01, bp<0.005 vs. untreated control Figure 4: Hypothetical model illustrating the potential role of FICZ and Ahr-IL-6 signaling axis in promoting AD-MSC prolif- eration while preserving stemness and differentiation potential. FICZ formed by the action of light on tryptophan also known as an endogenous ligand of Ahr can activate Ahr signaling in AD-MSCs and lead to the upregulation of downstream target genes such as Cyp1a1, Cyp1b1, and Il-6 that may contribute to the maintenance of AD-MSC stemness and differentiation potential, while supporting their proliferation capacity. AD-MSCs FICZ FICZ Ahr Cytoplasm Tryptophan Nucleus Cyp1a1 Cyp1b1 IL-6  Proliferation a a a a a b b b b b b b b b b b b b b Bangladesh J Pharmacol 2025; 20: 169-172 171 A B C D mailto:nam@ibt.ac.vn References Dorronsoro A, Lang V, Ferrin I, Fernandez-Rueda J, Zabaleta L, Perez-Ruiz E, Sepulveda P, Trigueros C. Intracellular role of IL-6 in mesenchymal stromal cell immunosuppression and proliferation. Sci Rep. 2020; 10: 21853. Huang J, Wang Y, Zhou Y. Beneficial roles of the AhR ligand FICZ on the regenerative potentials of BMSCs and primed cartilage templates. RSC Adv. 2022; 12: 11505-16. Kim D. 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