{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T05:12:58Z","timestamp":1792818778292,"version":"4.5.0"},"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T00:00:00Z","timestamp":1718323200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Parents' Association Grant of Kitasato University School of Medicine"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db\/db) C57BL\/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay\n                    <jats:italic>in vitro<\/jats:italic>\n                    showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate\n                    <jats:italic>in vivo<\/jats:italic>\n                    effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db\/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-\u03b21 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-\u03b21 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and \u03b1-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0304676","type":"journal-article","created":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T13:30:42Z","timestamp":1718371842000},"page":"e0304676","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db\/db mice"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-5087-4675","authenticated-orcid":true,"given":"Ayami","family":"Hasegawa-Haruki","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5235-3161","authenticated-orcid":true,"given":"Koya","family":"Obara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nanako","family":"Takaoka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyoumi","family":"Shirai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuko","family":"Hamada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuko","family":"Arakawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryoichi","family":"Aki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0049-0041","authenticated-orcid":true,"given":"Robert M.","family":"Hoffman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuyuki","family":"Amoh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,6,14]]},"reference":[{"key":"pone.0304676.ref001","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1007\/s12325-017-0478-y","article-title":"Chronic Wound Healing: A Review of Current Management and Treatments.","volume":"34:","author":"G Han","year":"2017","journal-title":"Advances in Therapy"},{"issue":"5","key":"pone.0304676.ref002","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1172\/JCI32169","article-title":"Cellular and molecular basis of wound healing in diabetes","volume":"117","author":"H Brem","year":"2007","journal-title":"The Journal of Clinical Investigation"},{"key":"pone.0304676.ref003","volume-title":"international diabetes federation.","author":"Diabetes Atlas","year":"2021","edition":"1"},{"issue":"24","key":"pone.0304676.ref004","doi-asserted-by":"crossref","first-page":"2367","DOI":"10.1056\/NEJMra1615439","article-title":"Diabetic Foot Ulcers and Their Recurrence.","volume":"376","author":"DG Armstrong","year":"2017","journal-title":"New England Journal of Medicine"},{"issue":"7","key":"pone.0304676.ref005","doi-asserted-by":"crossref","first-page":"936","DOI":"10.2337\/dc16-2189","article-title":"Health Care Service and Outcomes Among an Estimated 6.7 Million Ambulatory Care Diabetic Foot Cases in the U.S","volume":"40","author":"GH Skrepnek","year":"2017","journal-title":"Diabetes Care"},{"key":"pone.0304676.ref006","doi-asserted-by":"crossref","DOI":"10.3402\/dfa.v4i0.21847","article-title":"The system of care for the diabetic foot: objectives, outcomes, and opportunities.","volume":"4","author":"NR Barshes","year":"2013","journal-title":"Diabet Foot Ankle"},{"issue":"17","key":"pone.0304676.ref007","doi-asserted-by":"crossref","first-page":"9958","DOI":"10.1073\/pnas.1733025100","article-title":"Nestin expression in hair follicle sheath progenitor cells.","volume":"100","author":"L Li","year":"2003","journal-title":"Proceeding of National Academy Sciences of the United States of America"},{"issue":"15","key":"pone.0304676.ref008","doi-asserted-by":"crossref","first-page":"5530","DOI":"10.1073\/pnas.0501263102","article-title":"Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons","volume":"102","author":"Y Amoh","year":"2005","journal-title":"Proceeding of National Academy Sciences of the United States of America"},{"issue":"18","key":"pone.0304676.ref009","doi-asserted-by":"crossref","first-page":"3513","DOI":"10.4161\/cc.21803","article-title":"Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper, middle, and lower part of the vibrissae hair follicle","volume":"11","author":"Y Amoh","year":"2012","journal-title":"Cell Cycle"},{"issue":"4","key":"pone.0304676.ref010","doi-asserted-by":"crossref","first-page":"864","DOI":"10.3390\/cells10040864","article-title":"Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Extensively Differentiate to Tyrosine-Hydroxylase-Expressing Dopamine-Secreting Neurons.","volume":"10","author":"M Yamane","year":"2021","journal-title":"Cells"},{"issue":"14","key":"pone.0304676.ref011","doi-asserted-by":"crossref","first-page":"2362","DOI":"10.1080\/15384101.2015.1042633","article-title":"From hair to heart: nestin expressing hair follicle associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells.","volume":"14","author":"M Yashiro","year":"2015","journal-title":"Cell Cycle"},{"issue":"5","key":"pone.0304676.ref012","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1538\/expanim.60.481","article-title":"Effect of three types of mixed anesthetic agents alternate to ketamine in mice","volume":"60","author":"S Kawai","year":"2011","journal-title":"Experimental Animals"},{"key":"pone.0304676.ref013","doi-asserted-by":"crossref","first-page":"1454","DOI":"10.1038\/sj.bjc.6600610","article-title":"Induction of vasculogenesis in breast cancer models","volume":"87","author":"K Shirakawa","year":"2002","journal-title":"British Journal of Cancer"},{"issue":"5","key":"pone.0304676.ref014","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1080\/15384101.2016.1146837","article-title":"Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart muscle tissue sheets.","volume":"15","author":"A Yamazaki","year":"2016","journal-title":"Cell Cycle"},{"key":"pone.0304676.ref015","doi-asserted-by":"crossref","first-page":"107","DOI":"10.3389\/fcell.2019.00107","article-title":"In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.","volume":"7","author":"P Jordi","year":"2019","journal-title":"Front Cell Dev Biol"},{"issue":"7","key":"pone.0304676.ref016","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"J Schindelin","year":"2012","journal-title":"Nature methods"},{"issue":"7142","key":"pone.0304676.ref017","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1038\/nature05766","article-title":"Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding","volume":"17447","author":"M Ito","year":"2007","journal-title":"Nature"},{"issue":"2","key":"pone.0304676.ref018","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1111\/iwj.13061","article-title":"Allogeneic keratinocyte for intractable chronic diabetic foot ulcers: A prospective observational study","volume":"16","author":"YG Hwang","year":"2019","journal-title":"International Wound Journal"},{"issue":"7193","key":"pone.0304676.ref019","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1038\/nature07039","article-title":"Wound repair and regeneration","volume":"453","author":"GC Gurtner","year":"2008","journal-title":"Nature"},{"issue":"5","key":"pone.0304676.ref020","doi-asserted-by":"crossref","first-page":"866","DOI":"10.1016\/j.jaad.2009.10.048","article-title":"Wound healing in the 21st century","volume":"63","author":"S Schreml","year":"2010","journal-title":"J Am Acad Dermatol"},{"issue":"2","key":"pone.0304676.ref021","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1177\/0192623308329280","article-title":"Differential evaluation of excisional non-occluded wound healing in db\/db mice.","volume":"37","author":"VI Tkalcevi\u0107","year":"2009","journal-title":"Comparative Study Toxicol Pathol"},{"key":"pone.0304676.ref022","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1016\/j.jss.2019.01.048","article-title":"Dynamic Histological Events and Molecular Changes in Excisional Wound Healing of Diabetic DB\/DB Mice","volume":"238","author":"XT Wang","year":"2019","journal-title":"J Surg Res"},{"issue":"1","key":"pone.0304676.ref023","doi-asserted-by":"crossref","first-page":"98","DOI":"10.2353\/ajpath.2010.090283","article-title":"Dermal transforming growth factor-beta responsiveness mediates wound contraction and epithelial closure","volume":"176","author":"M Martinez-Ferrer","year":"2010","journal-title":"Am J Pathol"},{"issue":"10","key":"pone.0304676.ref024","first-page":"1072","article-title":"Diabetic Wound-Healing Science","volume":"57","author":"JL Burgess","year":"2021"},{"issue":"4","key":"pone.0304676.ref025","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0231962","article-title":"Compromised angiogenesis and vascular Integrity in impaired diabetic wound healing.","volume":"2315","author":"UA Okonkwo","year":"2020","journal-title":"PLOS ONE"},{"issue":"4","key":"pone.0304676.ref026","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1007\/s00125-003-1064-1","article-title":"Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse","volume":"46","author":"M Galeano","year":"2003","journal-title":"Diabetologia"},{"issue":"1","key":"pone.0304676.ref027","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/S0002-9440(10)64522-1","article-title":"In vivo detection of human vascular endothelial growth factor promoter activity in transgenic mouse skin","volume":"157","author":"J Kishimoto","year":"2000","journal-title":"The American Journal of PATHOROGY"},{"issue":"3","key":"pone.0304676.ref028","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1182\/blood-2012-01-403386","article-title":"CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair","volume":"120","author":"S Willenborg","year":"2012","journal-title":"Blood"},{"issue":"36","key":"pone.0304676.ref029","doi-asserted-by":"crossref","first-page":"13291","DOI":"10.1073\/pnas.0405250101","article-title":"Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells","volume":"101","author":"Y Amoh","year":"2004","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"6","key":"pone.0304676.ref030","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1002\/stem.1661","article-title":"Concise review: evidence for CD34 as a common marker for diverse progenitors","volume":"32","author":"LE Sidney","year":"2014","journal-title":"Stem Cells"},{"issue":"4","key":"pone.0304676.ref031","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1002\/ccd.21292","article-title":"Role of progenitor endothelial cells in cardiovascular disease and upcoming therapies.","volume":"70","author":"A Kawamoto","year":"2007","journal-title":"Catheter Cardiovasc Interv"},{"issue":"16","key":"pone.0304676.ref032","doi-asserted-by":"crossref","first-page":"9010","DOI":"10.3390\/ijms23169010","article-title":"Comparison of the Behavior of Perivascular Cells (Pericytes and CD34+ Stromal Cell\/Telocytes) in Sprouting and Intussusceptive Angiogenesis.","volume":"23","author":"L D\u00edaz-Flores","year":"2022","journal-title":"Int J Mol Sci."},{"key":"pone.0304676.ref033","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.trsl.2021.05.006","article-title":"Macrophage polarization and diabetic wound healing.","volume":"236","author":"EL Amanda","year":"2021","journal-title":"Translational research."},{"issue":"1","key":"pone.0304676.ref034","doi-asserted-by":"crossref","first-page":"98","DOI":"10.2353\/ajpath.2010.090283","article-title":"The Dermal transforming growth factor-beta responsiveness mediates wound contraction and epithelial closure","volume":"176","author":"M Martinez-Ferrer","year":"2010","journal-title":"American Journal of Pathology"},{"issue":"6","key":"pone.0304676.ref035","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1046\/j.1464-5491.2002.00692.x","article-title":"Transforming growth factor-beta 1, 2, 3 and receptor type I and II in diabetic foot ulcers","volume":"19","author":"EB Jude","year":"2002","journal-title":"DIABETIC Medicine"},{"issue":"2","key":"pone.0304676.ref036","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.cyto.2011.06.016","article-title":"Dysregulation of monocyte\/macrophage phenotype in wounds of diabetic mice","volume":"56","author":"R Mirza","year":"2011","journal-title":"Cytokine"},{"issue":"10","key":"pone.0304676.ref037","doi-asserted-by":"crossref","first-page":"165850","DOI":"10.1016\/j.bbadis.2020.165850","article-title":"Mutual inter-regulation between iNOS and TGF-\u03b21: Possible molecular and cellular mechanisms of iNOS in wound healing","volume":"1866","author":"SA Abd El-Aleem","year":"2020","journal-title":"Biochim Biophys Acta Mol Basis Dis"},{"issue":"6","key":"pone.0304676.ref038","doi-asserted-by":"crossref","first-page":"594","DOI":"10.1111\/j.1464-5491.2006.01773.x","article-title":"The molecular biology of chronic wounds and delayed healing in diabetes","volume":"23","author":"R Blakytny","year":"2006","journal-title":"DIABETIC Medicine"},{"key":"pone.0304676.ref039","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1093\/eurheartj\/ehr304","article-title":"Nitric oxide synthases: regulation and function","volume":"33","author":"U F\u00f6rstermann","year":"2012","journal-title":"European Hert Journal"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0304676","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T13:31:16Z","timestamp":1718371876000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0304676"}},"subtitle":[],"editor":[{"given":"Jimin","family":"Han","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,6,14]]},"references-count":39,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6,14]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0304676","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,14]]}}}