OBJECTIVETo induce adipocyte differentiation in vitro by adipose-derived stromal cells (ASCs) harvested from transgenic mice with green fluorescent protein (GFP)and assess the possibility of constructing adipose tissues via attachment of ASCs to type I collagen scaffolds.

METHODSInguinal fat pads from GFP transgenic mice were digested by enzymes for isolation of ASCs (primary culture). After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks, and the adipocyte differentiation by ASCs in vitro was assessed by morphological observation and Oil Red O staining. Then they were attached to collagen scaffolds and co-cultured for 12 hours, followed by hypodermic implantation to the dorsal skin of nude mice for 2 months. The newly-formed tissues were detected by HE staining.

RESULTSThe cultured primary stem cells were fibroblast-like and showed active proliferation. After being incubated in an adipocyte differentiation medium, the lipid droplets in the cytoplasm accumulated gradually and finally developed into mature adipocytes, which showed positive in Oil Red O staining. A 0.5-cm3 new tissue clot was found under the dorsal skin of the nude mice and it was confirmed as mature adipose tissues by fluorescent observation and HE staining.

CONCLUSIONSASCs can successfully differentiate adipose tissues into mature adipocytes, which exhibit an adipocyte-like morphology and express as intracytoplasmic lipid droplets. It is an efficient model of adipose tissues engineered with ASCs and type I collagen scaffolds.

Adipogenesis ; Adipose Tissue ; cytology ; Animals ; Cell Separation ; Cells, Cultured ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Stem Cells ; cytology ; Stromal Cells ; cytology ; Tissue Engineering ; methods

OBJECTIVETo identify the expression of protein which is characteristic of stem cell, induce the adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice, and to assess the possibility of constructing adipose tissue via the attachment of ASCs to type I collagen scaffold.

METHODSInguinal fat pads from GFP transgenic mice were used for the isolation of ASCs. Expression of CD29, CD34, CD45, CD133 and HLA-DR were detected by flow cytometry. After expansion to three passages, the ASCs were incubated in an adipogenic medium for two weeks. Then they were attached to collagen I scaffold and co-cultured for 12 hours, followed by transplantation under the dorsal skin of athymic mice for 2 months. Adipogenic differentiation of ASCs in vitro was assessed by morphological observation, Oil red O staining and newly formed tissue was detected by HE staining.

RESULTSThe cultured stem cells were fibroblast-like cells and showed highly homogeneous appearance with active proliferation capacity. Stem cells' characteristic CD expression was proved. After being incubated in an adipogenic medium, they could differentiate into mature adipocytes. Accumulation of lipid droplets in the cytoplasm was testified by Oil red O staining, morphological and biological observation. 0.5 ml new tissue was formed and was confirmed by fluorescent observation and HE staining to be mature adipose tissue.

CONCLUSIONSAdipose derived stem cells can successfully differentiate into mature adipocytes exhibiting an adipose-like morphology and expression of intracytoplasmic lipid droplet. It was an efficient model for adipose tissue engineering with ASCs and type I collagen scaffold.

Adipocytes ; cytology ; Adipose Tissue ; cytology ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Stem Cells ; cytology ; Tissue Engineering ; methods