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 downregulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering. Video LinkThe video component of this article can be found at

Role of the TGF‐β pathway in dedifferentiation of human mature adipocytes

“…Cells floated up and adhered to the top inner ceiling surface of the flask. After seven days, the medium was removed and the flasks were inverted so that the cells were on the bottom until day 12 in the same medium . The medium was changed every three days.…”

“…After 5 days, the filter is removed and the DFAT cells sink through the filter to the bottom of the dish . We have recently described a modified version of the ceiling culture approach in six‐well plates, allowing us to decrease the number of cells used and test a larger number of culture conditions .…”

“…Although many groups have used the ceiling culture approach, many aspects of this unique cellular process remain to be characterized. Our previous reports suggest that the TGF‐β pathway may be involved in the dedifferentiation process and contribute to the generation of a proliferative cell population that could be useful in cellular therapy or tissue engineering. From the technical standpoint, previous culture systems limited our ability to test various incubation conditions during the process.…”

Morphological Changes Induced By Extremely Low‐Frequency Electric Fields

“…In another study, dedifferentiation of astrocyte in the central nervous system has been proven to be successful in both in‐vivo and in‐vitro studies [Fu et al, ], along with peripheral nerve [Fu et al, ], musculoskeletal [Fu et al, ], and skin [Fu et al, ] regeneration caused by dedifferentiation. The possibility of dedifferentiation was also studied in human adipocytes [Lessard et al, ; Corsa and MacDougald, ; Fu et al, ] and mice (in‐vivo study). Dedifferentiation and its relation to regenerative medicine are well‐known today [Fu et al, ; Pesaresi et al, ] and new tools to achieve regeneration without chemical or genetic manipulations can be quite promising.…”

TCF7L2 plays a complex role in human adipose progenitor biology which may contribute to genetic susceptibility to type 2 diabetes

“…DFAT cells were generated by selection and de-differentiation of lipid-laden, in vitro differentiated immortalised APs (23) with modifications (See supplemental information).…”