Influence of human induced pluripotent stem cell derived skin organoid-conditioned culture medium on the function of human dermal fibroblasts induced by high glucose

Liu Zhixin; Qiu Kaizhen; He Jia; Wang Jingru; Liu Bilai; Xin Qi; Li Guiqiang; Chen Xiaodong

doi:10.3760/cma.j.cn501225-20241020-00404

Volume 41 Issue 3

Mar. 2025

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2025 > 41(3): 286-294

Liu ZX,Qiu KZ,He J,et al.Influence of human induced pluripotent stem cell derived skin organoid-conditioned culture medium on the function of human dermal fibroblasts induced by high glucose[J].Chin J Burns Wounds,2025,41(3):286-294.DOI: 10.3760/cma.j.cn501225-20241020-00404.

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Influence of human induced pluripotent stem cell derived skin organoid-conditioned culture medium on the function of human dermal fibroblasts induced by high glucose

doi: 10.3760/cma.j.cn501225-20241020-00404

1.
The First Clinical Medical College, Guangdong Medical University, Zhanjiang 524023, China
2.
The Institute of Translational Medicine, Foshan First People's Hospital, Foshan 528000, China
3.
Department of Burns and Plastic Surgery, Foshan First People's Hospital, Foshan 528000, China
4.
Department of Medical Cosmetology, Foshan First People's Hospital, Foshan 528000, China

Funds:

General Program of National Natural Science Foundation of China 82172205

Youth Science Foundation Program of National Natural Science Foundation of China 82402915

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Corresponding author: Chen Xiaodong, Email: cxd234@163.com
Received Date: 2024-10-20

Abstract

Abstract

Objective To explore the influence of human induced pluripotent stem cell (iPSC) derived skin organoid-conditioned culture medium (SO-CM) on the function of human dermal fibroblasts (Fbs) induced by high glucose, with the aim of providing treatment ideas for diabetic wounds. Methods This study was an experimental research. Human iPSCs were induced into skin organoids. Human iPSC-derived skin organoids and human dermal Fbs were seeded into skin organoid culture medium (SOM) and cultured for three days, respectively. Then the cell culture supernatants were collected as SO-CM and Fb-conditioned culture medium (Fb-CM), respectively. The content of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), IL-18, C-C motif chemokine ligand 2 (CCL-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and VEGF-β in SOM, Fb-CM, and SO-CM was detected by enzyme-linked immunosorbent assay. Human Fbs of passage 8 and 9 induced by high glucose were divided into SOM group, Fb-CM group, and SO-CM group according to the random number table method, and were cultured with SOM, Fb-CM, and SO-CM all containing glucose at final molarity of 35 mmol/L, respectively. After 24 hours of culture, the Ki67 positive ratio of cells was calculated after immunofluorescence staining, and the cell absorbance value was detected by using cell counting kit-8, representing cell proliferation activity. The cell scratch test was performed to calculate the cell migration rate at 13 hours after scratching. After 48 hours of culture, the expression of reactive oxygen species in cells was detected by fluorescent probe method, and the rate of β-galactosidase-positive staining of cells was detected by β-galactosidase staining kit, representing cellular senescence. The sample size was three. Results There was no statistically significant difference in the content of TNF-α, PDGF, and TGF-β among the three culture media (P>0.05). Compared with that in SOM, the content of IL-10 and EGF in Fb-CM and SO-CM was significantly decreased (P<0.05), while the content of CCL-2 in FB-CM and VEGF in SO-CM was significantly increased (P<0.05). After 24 hours of culture, the Ki67 positive ratio ((45.2±6.0)% and (57.4±4.0)%) and absorbance values (124±5 and 158±12) of cells in the Fb-CM group and the SO-CM group were significantly higher than those in the SOM group ((29.6±2.1)% and 100±6, P<0.05), and the Ki67 positive ratio and absorbance value of cells in the SO-CM group were significantly higher than those in the Fb-CM group (P<0.05). At 13 hours after scratching, the cell migration rates in the Fb-CM group and the SO-CM group were significantly higher than that in the SOM group (P<0.05). After 48 hours of culture, the level of reactive oxygen species in the SO-CM group was significantly higher than that in the SOM group and the Fb-CM group (with both P values <0.05). After 48 hours of culture, there was no statistically significant difference in the rate of β-galactosidase-positive staining of cells among the three groups (P>0.05). Conclusions The SO-CM has high content of VEGF and can significantly promote the proliferation, migration, and expression of reactive oxygen species in human dermal Fbs induced by high glucose, but has no significant effect on cell senescence.
- Culture media, conditioned,
- Fibroblasts,
- Cell proliferation,
- Reactive oxygen species,
- Cell aging,
- Cell migration,
- Skin organoid

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References

[1]	WilkinsonHN,HardmanMJ.Wound healing: cellular mechanisms and pathological outcomes[J].Open Biol,2020,10(9):200223.DOI: 10.1098/rsob.200223.
[2]	HollJ,KowalewskiC,ZimekZ,et al.Chronic diabetic wounds and their treatment with skin substitutes[J].Cells,2021,10(3):655.DOI: 10.3390/cells10030655.
[3]	desJardins-ParkHE,FosterDS,LongakerMT.Fibroblasts and wound healing: an update[J].Regen Med,2018,13(5):491-495.DOI: 10.2217/rme-2018-0073.
[4]	TalbottHE,MascharakS,GriffinM,et al.Wound healing, fibroblast heterogeneity, and fibrosis[J].Cell Stem Cell,2022,29(8):1161-1180.DOI: 10.1016/j.stem.2022.07.006.
[5]	Al-RikabiAHA, TobinDJ, Riches-SumanK,et al.Dermal fibroblasts cultured from donors with type 2 diabetes mellitus retain an epigenetic memory associated with poor wound healing responses[J].Sci Rep,2021,11(1):1474.DOI: 10.1038/s41598-020-80072-z.
[6]	LiuY,LiuY,HeW,et al.Fibroblasts: immunomodulatory factors in refractory diabetic wound healing[J].Front Immunol,2022,13:918223.DOI: 10.3389/fimmu.2022.918223.
[7]	RaiV,MoellmerR,AgrawalDK.Role of fibroblast plasticity and heterogeneity in modulating angiogenesis and healing in the diabetic foot ulcer[J].Mol Biol Rep,2023,50(2):1913-1929.DOI: 10.1007/s11033-022-08107-4.
[8]	DashBC,KorutlaL,VallabhajosyulaP,et al.Unlocking the potential of induced pluripotent stem cells for wound healing: the next frontier of regenerative medicine[J].Adv Wound Care (New Rochelle),2022,11(11):622-638.DOI: 10.1089/wound.2021.0049.
[9]	ShafieeA,SunJ,AhmedIA,et al.Development of physiologically relevant skin organoids from human induced pluripotent stem cells[J].Small,2024,20(16):e2304879.DOI: 10.1002/smll.202304879.
[10]	YiSA,ZhangY,RathnamC,et al.Bioengineering approaches for the advanced organoid research[J].Adv Mater,2021,33(45):e2007949.DOI: 10.1002/adma.202007949.
[11]	HoangP,MaZ.Biomaterial-guided stem cell organoid engineering for modeling development and diseases[J].Acta Biomater,2021,132:23-36.DOI: 10.1016/j.actbio.2021.01.026.
[12]	LeeJ,RabbaniCC,GaoH,et al.Hair-bearing human skin generated entirely from pluripotent stem cells[J].Nature,2020,582(7812):399-404.DOI: 10.1038/s41586-020-2352-3.
[13]	DaneshmandiL,ShahS,JafariT,et al.Emergence of the stem cell secretome in regenerative engineering[J].Trends Biotechnol,2020,38(12):1373-1384.DOI: 10.1016/j.tibtech.2020.04.013.
[14]	L PK,KandoiS,MisraR,et al.The mesenchymal stem cell secretome: a new paradigm towards cell-free therapeutic mode in regenerative medicine[J].Cytokine Growth Factor Rev,2019,46:1-9.DOI: 10.1016/j.cytogfr.2019.04.002.
[15]	BormannD,GugerellA,AnkersmitHJ,et al.Therapeutic application of cell secretomes in cutaneous wound healing[J].J Invest Dermatol,2023,143(6):893-912.DOI: 10.1016/j.jid.2023.02.019.
[16]	QianJ,LuE,XiangH,et al.GelMA loaded with exosomes from human minor salivary gland organoids enhances wound healing by inducing macrophage polarization[J].J Nanobiotechnology,2024,22(1):550.DOI: 10.1186/s12951-024-02811-y.
[17]	KwakS,SongCL,LeeJ,et al.Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration[J].Biomaterials,2024,307:122522.DOI: 10.1016/j.biomaterials.2024.122522.
[18]	边晓玮,张翠萍,李炳旻,等.胎盘间充质干细胞外泌体对高糖培养的成纤维细胞衰老的影响[J].第三军医大学学报,2020,42(12):1163-1173.DOI: 10.16016/j.1000-5404.202001190.
[19]	GantwerkerEA,HomDB.Skin: histology and physiology of wound healing[J].Facial Plast Surg Clin North Am,2011,19(3):441-453.DOI: 10.1016/j.fsc.2011.06.009.
[20]	ParkJW,HwangSR,YoonIS.Advanced growth factor delivery systems in wound management and skin regeneration[J].Molecules,2017,22(8):1259.DOI: 10.3390/molecules22081259.
[21]	XueM,ZhaoR,MarchL,et al.Dermal fibroblast heterogeneity and its contribution to the skin repair and regeneration[J].Adv Wound Care (New Rochelle),2022,11(2):87-107.DOI: 10.1089/wound.2020.1287.
[22]	LermanOZ,GalianoRD,ArmourM,et al.Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia[J].Am J Pathol,2003,162(1):303-312.DOI: 10.1016/S0002-9440(10)63821-7.
[23]	ZhangP,SongX,DongQ,et al.miR-27-3p inhibition restore fibroblasts viability in diabetic wound by targeting NOVA1[J].Aging (Albany NY),2020,12(13):12841-12849.DOI: 10.18632/aging.103266.
[24]	WangZ,ZhaoF,LangH,et al.Organoids in skin wound healing[J/OL].Burns Trauma,2025,13:tkae077[2025-02-09].http://www.ncbi.nlm.nih.gov/pubmed/39759541.DOI: 10.1093/burnst/tkae077.
[25]	WangW,LiuP,ZhuW,et al.Skin organoid transplantation promotes tissue repair with scarless in frostbite[J/OL].Protein Cell,2024:pwae055(2024-10-04)[2024-10-20]. https://pubmed.ncbi.nlm.nih.gov/39363875/.DOI:10.1093/procel/pwae055.[published online ahead of print].
[26]	BaoP,KodraA,Tomic-CanicM,et al.The role of vascular endothelial growth factor in wound healing[J].J Surg Res,2009,153(2):347-358.DOI: 10.1016/j.jss.2008.04.023.
[27]	GoswamiAG,BasuS,HudaF,et al.An appraisal of vascular endothelial growth factor (VEGF): the dynamic molecule of wound healing and its current clinical applications[J].Growth Factors,2022,40(3/4):73-88.DOI: 10.1080/08977194.2022.2074843.
[28]	ShamsF,MoravvejH,HosseinzadehS,et al.Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies[J].Sci Rep,2022,12(1):18529.DOI: 10.1038/s41598-022-23304-8.
[29]	RoyR,ZayasJ,MohamedMF,et al.IL-10 dysregulation underlies chemokine insufficiency, delayed macrophage response, and impaired healing in diabetic wounds[J].J Invest Dermatol,2022,142(3Pt A):692-704.e14.DOI: 10.1016/j.jid.2021.08.428.
[30]	SaraivaM,O'GarraA.The regulation of IL-10 production by immune cells[J].Nat Rev Immunol,2010,10(3):170-181.DOI: 10.1038/nri2711.
[31]	IshidaY,KuninakaY,NosakaM,et al.CCL2-mediated reversal of impaired skin wound healing in diabetic mice by normalization of neovascularization and collagen accumulation[J].J Invest Dermatol,2019,139(12):2517-2527.e5.DOI: 10.1016/j.jid.2019.05.022.
[32]	WoodS,JayaramanV,HuelsmannEJ,et al.Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response[J].PLoS One,2014,9(3):e91574.DOI: 10.1371/journal.pone.0091574.
[33]	OhM,KimYJ,SonYJ,et al. Promotive effects of human induced pluripotent stem cell-conditioned medium on the proliferation and migration of dermal fibroblasts[J]. Biotechnol Bioproc E, 2017, 22 (5): 561-568. DOI: 10.1007/s12257-017-0221-1.
[34]	YanY,WuR,BoY,et al.Induced pluripotent stem cells-derived microvesicles accelerate deep second-degree burn wound healing in mice through miR-16-5p-mediated promotion of keratinocytes migration[J].Theranostics,2020,10(22):9970-9983.DOI: 10.7150/thno.46639.
[35]	OhM,LeeJ,KimYJ,et al.Exosomes derived from human induced pluripotent stem cells ameliorate the aging of skin fibroblasts[J].Int J Mol Sci,2018,19(6):1715.DOI: 10.3390/ijms19061715.
[36]	ZhangJ,GuanJ,NiuX,et al.Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis[J].J Transl Med,2015,13:49.DOI: 10.1186/s12967-015-0417-0.
[37]	ZhouAK,JouE,LuV,et al.Using pre-clinical studies to explore the potential clinical uses of exosomes secreted from induced pluripotent stem cell-derived mesenchymal stem cells[J].Tissue Eng Regen Med,2023,20(6):793-809.DOI: 10.1007/s13770-023-00557-6.
[38]	KobayashiH,EbisawaK,KambeM,et al.<Editors' Choice> Effects of exosomes derived from the induced pluripotent stem cells on skin wound healing[J].Nagoya J Med Sci,2018,80(2):141-153.DOI: 10.18999/nagjms.80.2.141.
[39]	KimS,LeeSK,KimH,et al.Exosomes secreted from induced pluripotent stem cell-derived mesenchymal stem cells accelerate skin cell proliferation[J].Int J Mol Sci,2018,19(10):3119.DOI: 10.3390/ijms19103119.
[40]	DunnillC,PattonT,BrennanJ,et al.Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process[J].Int Wound J,2017,14(1):89-96.DOI: 10.1111/iwj.12557.
[41]	HuoYN,ChenW,ZhengXX.ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration[J].Cell Mol Biol (Noisy-le-grand),2015,61(7):6-11.