Bangladesh Journal of Pharmacology Volume: 19; Number 2; Year 2024 Cite this article as: Tran TT, Tran KT, Kim D, Nguyen NT. Bioactive peptides SL-13R and KS-13 enhance hu- man adipose-derived mesenchymal stem cell proliferation in vitro. Bangladesh J Pharmacol. 2024; 19: 69-71. Bioactive peptides SL-13R and KS-13 enhance human adipose-derived mesenchymal stem cell proliferation in vitro Dear Editor, The importance of peptide therapeutics in pharmaceu- tical research is increasing (Akbarian et al., 2022). The use of bioactive peptides to expand stem cells is urgent- ly needed to increase the number of these cells for clinical applications (Wang et al., 2022). It has been reported that the SL-13R peptide enhances the expansion of hematopoietic stem cells (HSCs) in vitro (Nii et al., 2021). However, the bioactivity of the SL-13R peptide in adipose-derived mesenchymal stem cells (AD-MSCs) is not known. Therefore, we aimed to examine whether the SL-13R peptide enhances AD-MSC proliferation in vitro. Recent studies have shown that some peptides can also promote the proliferation of both HSCs and MSCs (Dayem et al., 2023). Bioactive compounds such as genistein were shown to be potent stimulants of human AD-MSC proliferation in vitro (Han et al., 2014). In this study, AD-MSCs from donors were prepared as described in registered patents (Kim et al., 2013). The concentration of the SL-13R peptide was determined as previously described (Nii et al., 2021). AD-MSCs were extracted and cultured at 5 x 104 cells per plate in DMEM supplemented with or without SL-13R (10 µg/ mL) and another novel peptide, KS-13 (10 µg/mL). On days 1, 3, 5, and 7, the expansion of cells was assessed by MTT assay. Here, we demonstrated that both bio- active peptides SL-13R and KS-13 were able to enhance AD-MSC proliferation in vitro (Figure 1). As shown in Figure 1A, on day 1, the number of cells in the control, SL-13R-treated, KS-13-treated and genistein-treated samples reached 8214  814, 9475  462 (p=0.07 vs. con- trol), 10770  2307 (p=0.14 vs. control), and 10314  1193 (p=0.06 vs. control), respectively. On day 3, the number of cells in the control, SL-13R-treated, KS-13-treated and genistein-treated samples reached 27310  606, 31680  218 (p=0.0002 vs. control), 32827  1122 (p=0.001 vs. control), and 28463  693 (p=0.09 vs. control), respec- tively. On day 5, the number of cells in the control, SL- 13R-treated, KS-13-treated and genistein-treated sam- ples reached 29853  1272, 29793  691 (p= 0.94 vs. con- trol), 25840  4600 (p=0.21 vs. control), and 22830  845 (p=0.001 vs. control), respectively. On day 7, the number of cells in the control, SL-13R-treated, KS-13- treated and genistein-treated samples reached 15780  690, 21253  240 (p=0.0002 vs. control), 16800  1596 (p=0.36 vs. control), and 9958  1073 (p=0.001 vs. con- trol), respectively. These results demonstrated that both the SL-13R and KS-13 peptides enhanced AD-MSC proliferation compared to that of the control on day 3. Then, the gene expression of MSC stemness markers, including Nanog, Oct3/4, Sox2, Fgf2, Lif, and Myc, and MSC cell surface markers, including CD73, CD105, CD34, CD44, CD146, and HLA-DR in peptide-treated and genistein-treated AD-MSCs on day 3 were exami- ned by RT-qPCR. Table I shows that there were several significant differences in MSC stemness or surface marker gene expression between the peptide– and genistein-treated AD-MSCs.Karyotyping analysis revealed that the cellular numbers of the AD-MSCs were not affected by the peptides SL-13R and KS-13 (Figure 1B). Further investigation is needed to understand the effect of peptide treatment to characteristics of AD-MSCs. These results suggested that the peptides SL-13R and KS-13 maintained the characteristics of AD-MSCs in culture. The AHNAK, ANXA2, PLEC, and ERLIN2 genes are considered to interact with the SL-13R peptide during HSC expansion (Nii et al., 2021). Therefore, these genes were examined by RT‒qPCR in cultured AD-MSCs with or without SL-13R, KS-13; and genistein to deter- mine whether they are involved in AD-MSC expansion. As shown in Figure 2, the expression of AHNAK and ERLIN2 increased approximately 1.2-fold in SL-13R- treated cells but not in control cells, while the expre- ssion of ANXA2 was significantly increased in KS-13- treated cells compared to control cells. Besides, PLEC did not significantly differ between peptide- and genis- tein-treated cells and control cells. These results suggest that both peptides SL-13R and KS-13 have different functions and mechanisms in AD-MSCs than in HSCs. The statistical significance of differences was determi- ned by two-tailed Student’s t test and p-value <0.05 was considered statistically significant. In addition, the aryl hydrocarbon receptor (Ahr) has recently become a promising target for not only immune cell regulation but also stem cell proliferation (Han et al., 2020; Nguyen et al., 2010, Tran et al., 2022). Small molecules such as StemRegenin 1 (SR1), an Ahr antagonist, induce ex vivo expansion of CD34+ cells A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2024; 19: 69-71 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.v19i2.72739 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 from primary human HSCs by activating Ahr (Boitano et al., 2010). Furthermore, it was recently demonstrated that SR1 induces the expansion of CD34+ cells (Nii et al., 2021). This preliminary data showed that Ahr ligands such as 6-formylindolo[3,2-b]carbazole (FICZ) and kynurenic acid (KA) can significantly induce the 70 Bangladesh J Pharmacol 2024; 19: 69-71 C e ll n u m b e r Control (µg/mL) - - - - - - - - - - - - - - - - Figure 1: The expansion of human AD-MSCs by SL-13R, KS-13 and genistein in vitro. A) The number of AD-MSCs after days 1, 3, 5, and 7 in the control, SL-13R-treated, KS-13-treated and genistein-treated samples was examined by MTT. B) After day 2, peptide-treated AD-MSCs were harvested for karyotyping analysis (n=3). The results are the representative of three independent experiments A SL-13R (µg/mL) - 10 - - - 10 - - - 10 - - - 10 - - KS-13 (µg/mL) - - 10 - - - 10 - - - 10 - - - 10 - Genistein (µM) - - - 0.1 - - - 0.1 - - - 0.1 - - - 0.1 Day 1 Day 3 Day 5 Day 7 SL-13R KS-13 B Table I Expression of different parameters after 3 days Genistein SL-13R KS-13 Nanoga 1.2 ± 0.3 0.7 ± 0.1 (p=0.046 vs. KS-13) 1.0 ± 0.2 Oct3/4a 1.0 ± 0.1 0.8 ± 0.2 0.9 ± 0.1 Sox2a 1.1 ± 0.2 1.0 ± 0.2 1.1 ± 0.2 Lifa 0.8 ± 0.0 0.9 ± 0.3 1.4 ± 0.3 (p=0.038 vs. Genistein) Fgf2a 0.8 ± 0.1 1.1 ± 0.1 (p=0.021 vs. Genistein) 1.0 ± 0.1 (p=0.037 vs. Genistein) Myca 0.8 ± 0.1 0.8 ± 0.1 1.1 ± 0.2 CD73a 1.1 ± 0.3 0.8 ± 0.1 1.0 ± 0.3 CD105a 1.1 ± 0.1 1.0 ± 0.3 1.1 ± 0.3 CD34a 1.0 ± 0.1 1.3 ± 0.5 1.0 ± 0.1 CD44a 0.9 ± 0.2 0.8 ± 0.1 1.1 ± 0.2 CD146a 0.7 ± 0.1 0.8 ± 0.3 1.0 ± 0.3 HLA-DRa 0.9 ± 0.5 0.6 ± 0.4 (p=0.009 vs. KS-13) 1.7 ± 0.1 aWhen compared to control (GAPDH); Concentration of genistein (0.1 μM), SL-13R (10 μg/mL), KS-13 (10 μg/mL) were used; Data are mean ± S.D. expansion of AD-MSCs (unpublished data). Whether the bioactive peptides SL-13R and KS-13 regulate the functions of Ahr that promotes AD-MSC expansion in vitro is under investigation. Therefore, the use of bio- active peptides, natural compounds, and small mole- cules that interact with Ahr may potently improve the efficacy of AD-MSC-based therapies. Financial Issue: This work was funded by the projects QTJP01.01/20-22 and TDTBG0.02/21-23 from the Vietnam Academy of Science and Technology. Ethical Issue: The study was performed in accordance with protocols approved by the ethnics committee of the Hanoi Obstetrics and Gynecology Hospital [Ref. No. 2206 CN/PS] Conflict of interest: The authors declare that they have no conflicts of interest. Acknowledgements: The authors greatly appreciate Dr. Daisuke Sugiyama (Incubation Center for Advanced Medical Science, Kyushu University, Japan) for providing the SL-13R and KS-13 peptides. The authors also thank Drs. Le Xuan Hai, Duong Quoc Chinh and Tran Cong Hoang (National Institute of Hematology and Blood Transfusion, Vietnam), Drs. An Dang Pham, Duong Thi Thuy Le, Anh Hoang Phuong Le, Tien Vu Thi (Institute of Biotechnology, VAST) for their technical assistance. Thanh Trung Tran1, Kien Trung Tran2, Daeyong Kim3 and Nam Trung Nguyen1 1Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 2Hanoi Obstetrics and Gynecology Hospital, 929 La Thanh, Ba Dinh, Hanoi, Vietnam; 3N-Biotek, Inc., 402-803 Technopark, 655, Pyeongcheon-ro, Bucheon-si, Gyeonggi-do, 14502, Korea. Corresponding author: email: nam@ibt.ac.vn References Akbarian M, Khani A, Eghbalpour S, Uversky VN. Bioactive peptides: Synthesis, sources, applications, and proposed mechanisms of Action. Int J Mol Sci. 2022; 23: 1445. Boitano AE, Wang J, Romeo R, Bouchez LC, Parker AE, Sutton SE, Walker JR, Flaveny CA, Perdew GH, Denison MS, Schultz PG, Cooke MP. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 2010; 329: 1345-48. Dayem AA, Lee SB, Lim KM, Kim A, Shin HJ, Vellingiri B, Kim YB, Cho SG. Bioactive peptides for boosting stem cell cul- ture platform: Methods and applications. Biomed Pharma- cother. 2023; 160: 114376. Han H, Jayaraman A, Safe S, Chapkin RS. Targeting the aryl hydrocarbon receptor in stem cells to improve the use of food as medicine. Curr Stem Cell Rep. 2020; 6: 109-18. Han YS, Yun SP, Ko HS, Lee SH. Therapeutic effect of genistein-stimulated human mesenchymal stem cells in myocardial infarction. J Transplant Stem Cel Biol. 2014; 1: 7. Kim D. Method for culturing adiposed derived stem cell. 2013: Patent Numbers: 10-1624514, 10-1643315, 10-1590416. Nguyen NT, Hanieh H, Nakahama T, Kishimoto T. The roles of aryl hydrocarbon receptor in immune responses. Int Immunol. 2013; 25: 335–43. Nii T, Konno K, Matsumoto M, Bhukhai K, Borwornpinyo S, Sakai K, Hongeng S, Sugiyama D. The bioactive peptide SL- 13R expands human umbilical cord blood hematopoietic stem and progenitor cells in vitro. Molecules 2021; 26: 1995. Tran MD, Tran T, Nguyen N, Chu H, Nakahama T, Nguyen N. Anti-inflammatory activity of 9-hydroxycanthin-6-one extracted from hairy-root cultures of Eurycoma longifolia potentially via aryl hydrocarbon receptor induction. Bangladesh J Pharmacol. 2022; 17: 102-04. Wang L, Wang N, Zhang W, Cheng X, Yan Z, Shao G, Wang X, Wang R, Fu C. Therapeutic peptides: current applications and future directions. Sig Transduct Target Ther. 2022; 7: 48. Figure 2: Gene expression of AHNAK, ANXA2, PLEC and ERLIN2 with or without SL-13R, KS-13 and genistein in cultured AD- MSCs after day 3. The gene expression of AHNAK, ANXA2, PLEC and ERLIN2 in cultured AD-MSCs was measured by qPCR (n=3). The results are the representative of three independent experiments. Data are mean ± SD F o ld c h a n g e Control (µg/mL) - - - - - - - - - - - - - - - - SL-13R (µg/mL) - - 10 - - 10 - - - 10 - - - 10 - KS-13 (µg/mL) - - - 10 - - - 10 - - - 10 - - - 10 Genistein (µM) - 0.1 - - - 0.1 - - - 0.1 - - - 0.1 - - AHNAK ERLIN2 ANXA2 PLEC p=0.046 Bangladesh J Pharmacol 2024; 19: 69-71 71 mailto:nam@ibt.ac.vn