title: Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.
abstract: Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.
authors: IUCPQ Research Center.
authors: Julie
authors: J
authors: Lessard
authors: IUCPQ Research Center.
authors: Julie Anne
authors: JA
authors: Côté
authors: IUCPQ Research Center.
authors: Marc
authors: M
authors: Lapointe
authors: CHU de Québec Research Center.
authors: Mélissa
authors: M
authors: Pelletier
authors: IUCPQ Research Center.
authors: Mélanie
authors: M
authors: Nadeau
authors: IUCPQ Research Center.
authors: Simon
authors: S
authors: Marceau
authors: IUCPQ Research Center.
authors: Laurent
authors: L
authors: Biertho
authors: IUCPQ Research Center; CHU de Québec Research Center; Laval University; andre.tchernof@criucpq.ulaval.ca.
authors: André
authors: A
authors: Tchernof
mesh_terms: D017667:Adipocytes / Q000166:cytology*; D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D054337:Cell Dedifferentiation / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D017364:Collagenases / Q000737:chemistry; D003584:Cytological Techniques / Q000379:methods*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D039902:Multipotent Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods
chemical_list: D017364:Collagenases