Yu J.Tu Y.-K.Tang Y.-B.NAI-CHEN CHENG2020-03-092020-03-0920140142-9612https://www.scopus.com/inward/record.uri?eid=2-s2.0-84895036154&doi=10.1016%2fj.biomaterials.2014.01.015&partnerID=40&md5=1a8fa40bc9ff2630ad546a0090c527f4https://scholars.lib.ntu.edu.tw/handle/123456789/474107Cell sheet technology has emerged as an important tissue engineering approach. Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but their stemness and differentiation capabilities in the cell sheet format have not been well investigated. In this study, we found that l-ascorbate 2-phosphate (A2-P), a stable form of ascorbic acid, significantly enhanced ASC proliferation and induced ASC sheet fabrication in 7 days with abundant extracellular matrix deposition. Importantly, A2-P treatment significantly enhanced expression of pluripotent markers Sox-2, Oct-4 and Nanog, but treating ASCs with antioxidants other than A2-P revealed no stemness enhancement. Moreover, ASC treatment with A2-P and a collagen synthesis inhibitor, L-2-azetidine carboxylic acid or cis-4-hydroxy- d-proline, significantly inhibited the A2-P-enhanced expression of stemness markers. These findings demonstrated that A2-P enhances stemness of ASCs through collagen synthesis and cell sheet formation. We also showed that A2-P-stimulated collagen synthesis in ASCs may be mediated through ERK1/2 pathway. By culturing the ASC sheets in proper induction media, ASC transdifferentiation capabilities into neuron and hepatocyte-like cells were significantly enhanced after cell sheet formation, while adipogenic and osteogenic differentiation capacities were still maintained. Using a murine model of healing-impaired cutaneous wound, faster wound healing was noted in the group that received ASC sheet treatment, and we observed significantly more engrafted ASCs with evidence of differentiation toward endothelial and epidermal lineages in the cutaneous wound tissue. Therefore, A2-P-mediated ASC sheet formation enhanced ASC stemness and transdifferentiation capabilities, thereby representing a promising approach for applications in regenerative medicine. ? 2014 Elsevier Ltd.[SDGs]SDG3Ascorbic acid; Collagen; Cytology; Differentiation (calculus); Regenerative Medicine; Stem cells; Tissue; Tissue regeneration; Adipose derived stem cells; Cell sheet; Cell sheet technologies; Extracellular matrix deposition; Osteogenic differentiation; Stemness; Trans differentiations; Wound healing; Cell engineering; 5' nucleotidase; albumin; ascorbic acid derivative; CD34 antigen; cytochrome P450 3A4; endoglin; fibronectin; laminin; levo ascorbic acid 2 phosphate; mitogen activated protein kinase 1; mitogen activated protein kinase 3; nestin; octamer transcription factor 4; Thy 1 antigen; transcription factor NANOG; transcription factor Sox2; unclassified drug; adipogenesis; adipose derived stem cell; adult; animal experiment; animal model; article; bone development; cell aging; cell count; cell proliferation; cell sheet formation; cell transdifferentiation; cellular parameters; collagen synthesis; controlled study; extracellular matrix; female; flow cytometry; human; human cell; immunofluorescence; immunohistochemistry; liver cell; middle aged; mouse; nerve cell; nonhuman; priority journal; protein expression; real time polymerase chain reaction; reverse transcription polymerase chain reaction; stemness; tissue engineering; upregulation; Western blotting; Murinae; Adipose-derived stem cells; Cell sheet; Collagen; Differentiation; Stemness; Wound healing; Adipose Tissue; Adult; Animals; Antioxidants; Ascorbic Acid; Biological Markers; Cell Culture Techniques; Cell Proliferation; Cell Transdifferentiation; Collagen; Disease Models, Animal; Female; Humans; Mice; Mice, Nude; Middle Aged; Neurogenesis; Phenotype; Stem Cells; Wound Healingjournal article10.1016/j.biomaterials.2014.01.015244623602-s2.0-84895036154