Human urine-derived stem cells rescue cutaneous manifestation and suppress inflammation and fibrosis in vitro and in a mouse model of recessive dystrophic epidermolysis bullosa

Abstract

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a life-threatening disease characterized by persistent wound inflammation, tissue fibrosis, and even tumorigenesis in later stages. Despite its significant impact on patient health worldwide, treatment of RDEB has limited options. There is a clear need for now interventions. The goal of this study was to explore the potential efficacy of urine-derived stem cells (USCs) in RDEB. Methods: We isolated human USCs from healthy donors, and assessed their therapeutic effects on RDEB both in vitro with tissue-engineered skin in a three-dimensional co-culture system and in vivo with COL7A1^−/− (RDEB) mice. Results: USCs showed suppressive effects on expression of inflammation-and fibrosis-related genes involved in RDEB in vitro and in vivo. USCs could also extend the median life span (from 3 to 5 days), improve the expression of C7, and migrate to various organs of RDEB mice after intrahepatic administration. Conclusions: In summary, these results suggest the potential effects of USCs on improving the expression of C7 and would repair of RDEB, which supports the future use of USCs for the treatment of RDEB patients.

Keywords

Recessive dystrophic epidermolysis bullosa / Urine-derived stem cell / COL7A1 / Collagen VII / Tissue-engineered skin

References

[1] Has C, Bauer JW, Bodemer C, Bolling MC, Bruckner-Tuderman L, Diem A., et al. Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility. Br J Dermatol. 2020; 183: 614-27. https://doi.org/10.1111/bjd.18921.

[2] Soro L, Bartus C, Purcell S. Recessive dystrophic epidermolysis bullosa: a review of disease pathogenesis and update on future therapies. J Clin Aesthet Dermatol. 2015; 8: 41-6.

[3] Woodley DT, Chen M. Recessive dystrophic epidermolysis bullosa: advances in the laboratory leading to new therapies. J Invest Dermatol. 2015; 135: 1705-7. https://doi.org/10.1038/jid.2015.149.

[4] Fine JD. Life with Epidermolysis Bullosa (EB) 2009. Springer Vienna, Vienna. https://doi.org/10.1007/978-3-211-79271-1.

[5] Kim M, Li M, Intong-Wheeler LRA, Tran K, Marucci D, Murrell DF. Epidemiology and outcome of squamous cell carcinoma in epidermolysis bullosa in Australia and New Zealand. Acta Derm Venereol. 2018; 98: 70-6. https://doi.org/10.2340/00015555-2781.

[6] Liao Y, Ivanova L, Zhu H, Plumer T, Hamby C, Mehta B., et al. Cord blood-derived stem cells suppress fibrosis and may prevent malignant progression in recessive dystrophic epidermolysis bullosa. Stem Cells. 2018; 36: 1839-50. https://doi.org/10.1002/stem.2907.

[7] Dayal JHS, Mason SM, Salas-Alanis JC, McGrath JA, Taylor RG, Mellerio JE., et al. Heterogeneous addiction to transforming growth factor-beta signalling in recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma. Br J Dermatol. 2021; 184: 697-708. https://doi.org/10.1111/bjd.19421.

[8] Nyström A, Thriene K, Mittapalli V, Kern JS, Kiritsi D, Dengjel J., et al. Losartan ameliorates dystrophic epidermolysis bullosa and uncovers new disease mechanisms. EMBO Mol Med. 2015; 7: 1211-28. https://doi.org/10.15252/emmm.201505061.

[9] Martins VL, Vyas JJ, Chen M, Purdie K, Mein CA, South AP., et al. Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen. J Cell Sci. 2009; 122: 1788-99. https://doi.org/10.1242/jcs.042895.

[10] Guerra L, Odorisio T, Zambruno G, Castiglia D. Stromal microenvironment in type VII collagen-deficient skin: the ground for squamous cell carcinoma development. Matrix Biol. 2017; 63: 1-10. https://doi.org/10.1016/j.matbio.2017.01.002.

[11] Lei L, Zhao C, Qin F, He ZY, Zhong X-N. Th17 cells and IL-17 promote the skin and lung inflammation and fibrosis process in a bleomycin-induced murine model of systemic sclerosis. Clin Exp Rheumatol. 2016; 34 Suppl 100: 14-22.

[12] Nomura K, Imaizumi T, Mikami H, Tamai K, Takahashi M, Hashimoto I. Interleukin-1 induces collagenase production by recessive dystrophic epidermolysis bullosa fibroblasts. Arch Dermatol Res. 1990; 282: 379-82. https://doi.org/10.1007/bf00372088.

[13] Esposito S, Guez S, Orenti A, Tadini G, Scuvera G, Corti L., et al. Autoimmunity and cytokine imbalance in inherited epidermolysis bullosa. Int J Mol Sci. 2016; 17: 1625. https://doi.org/10.3390/ijms17101625.

[14] Wagner JE, Ishida-Yamamoto A, McGrath JA, Hordinsky M, Keene DR, Woodley DT., et al. Bone marrow transplantation for recessive dystrophic epidermolysis bullosa. N Engl J Med. 2010; 363: 629-39. https://doi.org/10.1056/NEJMoa0910501.

[15] Kiritsi D, Dieter K, Niebergall-Roth E, Fluhr S, Daniele C, Esterlechner J., et al. Clinical trial of ABCB5+ mesenchymal stem cells for recessive dystrophic epidermolysis bullosa. JCI Insight. 2021; 6:e151922. https://doi.org/10.1172/jci.insight.151922.

[16] Lee SE, Lee SJ, Kim SE, Kim K, Cho B, Roh K., et al. Intravenous allogeneic umbilical cord blood-derived mesenchymal stem cell therapy in recessive dystrophic epidermolysis bullosa patients. JCI Insight. 2021; 6: e143606. https://doi.org/10.1172/jci.insight.143606.

[17] Rashidghamat E, Kadiyirire T, Ayis S, Petrof G, Liu L, Pullabhatla V., et al. Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa. J Am Acad Dermatol. 2020; 83: 447-54. https://doi.org/10.1016/j.jaad.2019.11.038.

[18] Ebens CL, McGrath JA, Tamai K, Hovnanian A, Wagner JE, Riddle MJ., et al. Bone marrow transplant with post-transplant cyclophosphamide for recessive dystrophic epidermolysis bullosa expands the related donor pool and permits tolerance of nonhaematopoietic cellular grafts. Br J Dermatol. 2019; 181: 1238-46. https://doi.org/10.1111/bjd.17858.

[19] Petrof G, Lwin SM, Martinez-Queipo M, Abdul-Wahab A, Tso S, Mellerio JE., et al. Potential of systemic allogeneic mesenchymal stromal cell therapy for children with recessive dystrophic epidermolysis bullosa. J Invest Dermatol. 2015; 135: 2319-21. https://doi.org/10.1038/jid.2015.158.

[20] Hou PC, Wang HT, Abhee S, Tu WT, McGrath JA, Hsu CK. Investigational treatments for epidermolysis bullosa. Am J Clin Dermatol. 2021; 22: 801-17. https://doi.org/10.1007/s40257-021-00626-3.

[21] Kühl T, Mezger M, Hausser I, Handgretinger R, Bruckner-Tuderman L, Nyström A. High local concentrations of intradermal MSCs restore skin integrity and facilitate wound healing in dystrophic epidermolysis bullosa. Mol Ther. 2015; 23: 1368-79. https://doi.org/10.1038/mt.2015.58.

[22] Wang M, Xu X, Lei X, Tan J, Xie H. Mesenchymal stem cell-based therapy for burn wound healing. Burns Trauma. 2021; 9: tkab002. https://doi.org/10.1093/burnst/tkab002.

[23] Liao Y, Ivanova L, Zhu H, Yahr A, Ayello J, van de Ven C., et al. Rescue of the mucocutaneous manifestations by human cord blood derived nonhematopoietic stem cells in a mouse model of recessive dystrophic epidermolysis bullosa. Stem Cells. 2015; 33: 1807-17. https://doi.org/10.1002/stem.1966.

[24] Zhang Y, McNeill E, Tian H, Soker S, Andersson KE, Yoo JJ., et al. Urine derived cells are a potential source for urological tissue reconstruction. J Urol. 2008; 180: 2226-33. https://doi.org/10.1016/j.juro.2008.07.023.

[25] Song YT, Li YQ, Tian MX, Hu JG, Zhang XR, Liu PC., et al. Application of antibody-conjugated small intestine submucosa to capture urine-derived stem cells for bladder repair in a rabbit model. Bioact Mater. 2022; 14: 443-55. https://doi.org/10.1016/j.bioactmat.2021.11.017.

[26] Zhang XR, Huang YZ, Gao HW, Jiang YL, Hu JG, Pi JK., et al. Hypoxic preconditioning of human urine-derived stem cell-laden small intestinal submucosa enhances wound healing potential. Stem Cell Res Ther. 2020; 11: 150. https://doi.org/10.1186/s13287-020-01662-2.

[27] Zhang W, Hu J, Huang Y, Wu C, Xie H. Urine-derived stem cells: applications in skin, bone and articular cartilage repair. Burns Trauma. 2021; 9:tkab039. https://doi.org/10.1093/burnst/tkab039.

[28] Chua AW, Khoo YC, Tan BK, Tan KC, Foo CL, Chong SJ. Skin tissue engineering advances in severe burns: review and therapeutic applications. Burns Trauma. 2016; 4: 3. https://doi.org/10.1186/s41038-016-0027-y.

[29] Sierra-Sánchez Á, Magne B, Savard E, Martel C, Ferland K, Barbier MA., et al. In vitro comparison of human plasma-based and self-assembled tissue-engineered skin substitutes: two different manufacturing processes for the treatment of deep and difficult to heal injuries. Burns Trauma. 2023; 11: tkad043. https://doi.org/10.1093/burnst/tkad043.

[30] Groeber F, Holeiter M, Hampel M, Hinderer S, Schenke-Layland K. Skin tissue engineering-in vivo and in vitro applications. Adv Drug Deliv Rev. 2011; 63: 352-66. https://doi.org/10.1016/j.addr.2011.01.005.

[31] Morris AH, Stamer DK, Kyriakides TR. The host response to naturally-derived extracellular matrix biomaterials. Semin Immunol. 2017; 29: 72-91. https://doi.org/10.1016/j.smim.2017.01.002.

[32] Zhang W, Zhu Y, Li J, Guo Q, Peng J, Liu S., et al. Cell-derived extracellular matrix: basic characteristics and current applications in Orthopedic tissue engineering. Tissue Eng Part B Rev. 2016; 22: 193-207. https://doi.org/10.1089/ten.TEB.2015.0290.

[33] Wu W, Lu Z, Li F, Wang W, Qian N, Duan J., et al. Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model. Proc Natl Acad Sci USA. 2017; 114: 1660-5. https://doi.org/10.1073/pnas.1614775114.

[34] Chen AJ, Pi JK, Hu JG, Huang YZ, Gao HW, Li SF., et al. Identification and characterization of two morphologically distinct stem cell subpopulations from human urine samples. Sci China Life Sci. 2020; 63: 712-23. https://doi.org/10.1007/s11427-018-9543-1.

[35] Bharadwaj S, Liu G, Shi Y, Markert C, Andersson KE, Atala A., et al. Characterization of urine-derived stem cells obtained from upper urinary tract for use in cell-based urological tissue engineering. Tissue Eng Part A. 2011; 17: 2123-32. https://doi.org/10.1089/ten.TEA.2010.0637.

[36] Schosserer M, Reynoso R, Wally V, Jug B, Kantner V, Weilner S., et al. Urine is a novel source of autologous mesenchymal stem cells for patients with epidermolysis bullosa. BMC Res Notes. 2015; 8: 767. https://doi.org/10.1186/s13104-015-1686-7.

[37] Liao Y, Ivanova L, Sivalenka R, Plumer T, Zhu H, Zhang X., et al. Efficacy of human placental-derived stem cells in collagen VII knockout (recessive dystrophic epidermolysis bullosa animal model). Stem Cells Transl Med. 2018; 7: 530-42. https://doi.org/10.1002/sctm.17-0182.

[38] Zhou Q, Cheng Y, Sun F, Shen J, Nasser MI, Zhu P., et al. A comprehensive review of the therapeutic value of urine-derived stem cells. Front Genet. 2021; 12: 781597. https://doi.org/10.3389/fgene.2021.781597.

[39] Wu R, Soland M, Liu G, Shi Y, Zhang C, Tang Y., et al. Functional characterization of the immunomodulatory properties of human urine-derived stem cells. Transl Androl Urol. 2021; 10: 3566-78. https://doi.org/10.21037/tau-21-506.

[40] Raoufinia R, Rahimi HR, Keyhanvar N, Moghbeli M, Abdyazdani N, Rostami M., et al. Advances in treatments for epidermolysis bullosa (EB): emphasis on stem cell-based therapy. Stem Cell Rev Rep. 2024; 20: 1200-12. https://doi.org/10.1007/s12015-024-10697-4.

[41] Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 2008; 180: 2581-7. https://doi.org/10.4049/jimmunol.180.4.2581.

[42] Bian D, Wu Y, Song G, Azizi R, Zamani A. The application of mesenchymal stromal cells (MSCs) and their derivative exosome in skin wound healing: a comprehensive review. Stem Cell Res Ther. 2022; 13: 24. https://doi.org/10.1186/s13287-021-02697-9.

[43] Compton CC, Gill JM, Bradford DA, Regauer S, Gallico GG, O’Connor NE. Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study. Lab Invest. 1989; 60: 600-12.

[44] Kühl T, Mezger M, Hausser I, Guey LT, Handgretinger R, Bruckner-Tuderman L., et al. Collagen VII half-life at the dermal-epidermal junction zone: implications for mechanisms and therapy of Genodermatoses. J Invest Dermatol. 2016; 136: 1116-23. https://doi.org/10.1016/j.jid.2016.02.002.

[45] Fine JD, Johnson LB, Weiner M, Li KP, Suchindran C. Epidermolysis bullosa and the risk of life-threatening cancers: the national EB registry experience, 1986-2006. J Am Acad Dermatol. 2009; 60: 203-11. https://doi.org/10.1016/j.jaad.2008.09.035.

[46] Akasaka E, Kleiser S, Sengle G, Bruckner-Tuderman L, Nyström A. Diversity of mechanisms underlying latent TGF-β activation in recessive dystrophic epidermolysis bullosa. J Invest Dermatol. 2021; 141: 1450-60.e9. https://doi.org/10.1016/j.jid.2020.10.024.

[47] Aguado T, García M, García A, Ferrer-Mayorga G, Martínez-Santamaría L, Del Río M., et al. Raloxifene and n-Acetylcysteine ameliorate TGF-signalling in fibroblasts from patients with recessive dominant epidermolysis bullosa. Cells. 2020; 9: 2108. https://doi.org/10.3390/cells9092108.

[48] Twaroski K, Chen W, Pickett-Leonard M, Tolar J. Role of transforming growth factor-β1 in recessive dystrophic epidermolysis bullosa squamous cell carcinoma. Exp Dermatol. 2021; 30: 664-75. https://doi.org/10.1111/exd.14304.

[49] Ebens CL. Deconstructing progressive inflammatory fibrosis in recessive dystrophic epidermolysis bullosa. EMBO Mol Med. 2021; 13: e14864. https://doi.org/10.15252/emmm.202114864.

[50] Albrengues J, Bertero T, Grasset E, Bonan S, Maiel M, Bourget I., et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat Commun. 2015; 6: 10204. https://doi.org/10.1038/ncomms10204.

[51] Kuzet SE, Gaggioli C. Fibroblast activation in cancer: when seed fertilizes soil. Cell Tissue Res. 2016; 365: 607-19. https://doi.org/10.1007/s00441-016-2467-x.

[52] Annicchiarico G, Morgese MG, Esposito S, Lopalco G, Lattarulo M, Tampoia M., et al. Proinflammatory cytokines and Antiskin autoantibodies in patients with inherited epidermolysis bullosa. Medicine (Baltimore). 2015; 94: e1528. https://doi.org/10.1097/md.0000000000001528.

[53] Tampoia M, Abbracciavento L, Morrone M, Fumarulo R. IL-6/IL-10 ratio as a prognostic and predictive marker of the severity of inherited epidermolysis bullosa. Iran J Immunol. 2017; 14: 340-9.

[54] Zhou C, Wu XR, Liu HS, Liu XH, Liu GH, Zheng XB., et al. Immunomodulatory effect of urine-derived stem cells on inflammatory bowel diseases via downregulating Th1/Th17 immune responses in a PGE2-dependent manner. J Crohns Colitis. 2020; 14: 654-68. https://doi.org/10.1093/ecco-jcc/jjz200.

[55] Kang HS, Choi SH, Kim BS, Choi JY, Park GB, Kwon TG., et al. Advanced properties of urine derived stem cells compared to adipose tissue derived stem cells in terms of cell proliferation, immune modulation and multi differentiation. J Korean Med Sci. 2015; 30: 1764-76. https://doi.org/10.3346/jkms.2015.30.12.1764.

[56] Fu Y, Guan J, Guo S, Guo F, Niu X, Liu Q., et al. Human urine-derived stem cells in combination with polycaprolactone/gelatin nanofibrous membranes enhance wound healing by promoting angiogenesis. J Transl Med. 2014; 12: 274. https://doi.org/10.1186/s12967-014-0274-2.

[57] Ouyang B, Xie Y, Zhang C, Deng C, Lv L, Yao J., et al. Extracellular vesicles from human urine-derived stem cells ameliorate erectile dysfunction in a diabetic rat model by delivering proangiogenic MicroRNA. Sex Med. 2019; 7: 241-50. https://doi.org/10.1016/j.esxm.2019.02.001.

[58] Odorisio T, Di Salvio M, Orecchia A, Di Zenzo G, Piccinni E, Cianfarani F., et al. Monozygotic twins discordant for recessive dystrophic epidermolysis bullosa phenotype highlight the role of TGF-β signalling in modifying disease severity. Hum Mol Genet. 2014; 23: 3907-22. https://doi.org/10.1093/hmg/ddu102.

[59] Li T, Zhang M, Li Y, Sun Y, Huang J, Zeng A., et al. Twist-related protein 1 promotes transforming growth factor β receptor 1 in keloid fibroblasts via regulating the stability of myocyte enhancer factor 2A. Burns Trauma. 2024; 12: tkae024. https://doi.org/10.1093/burnst/tkae024.

[60] Tian SF, Jiang ZZ, Liu YM, Niu X, Hu B, Guo SC., et al. Human urine-derived stem cells contribute to the repair of ischemic acute kidney injury in rats. Mol Med Rep. 2017; 16: 5541-8. https://doi.org/10.3892/mmr.2017.7240.

[61] Li J, Luo H, Dong X, Liu Q, Wu C, Zhang T., et al. Therapeutic effect of urine-derived stem cells for protamine/lipopolysaccharide-induced interstitial cystitis in a rat model. Stem Cell Res Ther. 2017; 8: 107. https://doi.org/10.1186/s13287-017-0547-9.

[62] Chen CY, Du W, Rao SS, Tan YJ, Hu XK, Luo MJ., et al. Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1. Acta Biomater. 2020; 111: 208-20. https://doi.org/10.1016/j.actbio.2020.05.020.

[63] Bormann T, Maus R, Stolper J, Tort Tarrés M, Brandenberger C, Wedekind D., et al. Role of matrix metalloprotease-2 and MMP-9 in experimental lung fibrosis in mice. Respir Res. 2022; 23: 180. https://doi.org/10.1186/s12931-022-02105-7.

[64] Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL., et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009; 5: 54-63. https://doi.org/10.1016/j.stem.2009.05.003.