A diabetic milieu promotes OCT4 and NANOG production in human visceral-derived adipose stem cells.
OBJECTIVE
Successful outcomes have been obtained by exploiting adipose-derived stem cells (ASCs) in regenerative medicine. NADPH oxidase (NOX)-generated reactive oxygen species (ROS) are known to control stem cell self-renewal. Several high glucose (HG)-mediated effects depend on NOX-generated ROS. In this study, we investigated whether, and how mechanistically, HG concentrations control ASC fate in patients with diabetes.


METHODS
ASCs from the visceral adipose tissue of non-diabetic (N-ASCs) and diabetic participants (D-ASCs), identified by surface markers, were counted and evaluated for ROS generation and stem cell properties. Their ability to release soluble factors was assessed by BioPlex analysis. To reproduce an in vitro diabetic glucose milieu, N-ASCs were cultured in HG (25 mmol/l) or normal glucose (NG) concentration (5 mmol/l), as control. ASC pluripotency was assessed by in vitro study. The p47(phox) NOX subunit, AKT and octamer-binding transcription factor 4 (OCT4; also known as POU5F1) were knocked down by small-interfering RNA technology. Stem-cell features were evaluated by sphere cluster formation.


RESULTS
The ASC number was higher in diabetic patients than in non-diabetic controls. Production of OCT4 and NANOG, stem-cell-specific transcription factors, was upregulated in D-ASCs compared with N-ASCs. Moreover, we found that ROS production and AKT activation drove D-ASC, but not N-ASC, secretion. When N-ASCs were cultured in vitro in the presence of HG, they also expressed OCT4/NANOG and formed spheres. By knock-down of the p47(phox) NOX subunit, AKT and OCT4 we demonstrated that NOX-generated ROS and their downstream signals are crucial for HG-mediated ASC de-differentiation and proinflammatory cytokine production.


CONCLUSIONS
We herein provide a rationale for exploiting D-ASCs in regenerative medicine and/or exploiting HG preconditioning to increase ASCs ex vivo.
Department of Medical Sciences, University of Torino, Corso Dogliotti 14, 10126 Torino, Italy.
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Dentelli
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Barale
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Togliatto
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Trombetta
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Olgasi
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Gili
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Riganti
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Toppino
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Brizzi
D053687:Adult Stem Cells / Q000378:metabolism* / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000209:etiology*; D018189:Immunomagnetic Separation; D050152:Intra-Abdominal Fat / Q000378:metabolism* / Q000473:pathology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D015854:Up-Regulation
D015415:Biomarkers; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt