Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

Abstract

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation

“…The mouse preadipocyte cell line DFAT was established from dedifferentiated mature adipocytes of GFP transgenic mice by ceiling culture (a method to culture lipid-containing adipocytes based on their lipid buoyancy) 12 .…”

“…To investigate the function of MKL1 in adipocyte differentiation in vivo, we transfected DFAT cells (which are derived from green fluorescent protein (GFP) transgenic mice) 12 with MKL1 siRNA for 48 h (resulting in a significant decrease in Mkl1 expression but no change in Pparg expression at this time ( Supplementary Fig. 5d)) and then injected the cells subcutaneously into mice.…”

“…12). Unlike 3T3-L1 cells, these DFAT cells do not undergo spontaneous adipogenesis, with the adipocytic differentiation of DFAT cells thus being more tightly controlled than that of 3T3-L1 cells 12,13 . Here, we examine the roles of actin cytoskeleton remodelling in adipocytic differentiation by using DFAT cells.…”

Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues

“…According to the different studies [14, 16-17, 19, 23], in the following 2 to 3 weeks of uninterrupted culture, the fat drops within the adipocytes will gradually disappear and the cells shift into fibroblast-like morphology, reestablishing a strong proliferation ability as well. Distinct to the ASCs, DFAT cells derived from ceiling culture method were identified as a more homogenous cell group in most reports [16][17][18][19][23][24][25][26]. Although, this simple technique needs no complex procedures such as transfection or chemical induction, several key points should still be paid attention to.…”

“…First, a thorough digestion and disperse is required to obtain purified adipocytes. Fat tissue has complex cellular composition, including adipocytes, ASCs, preadipocyte, fibroblasts, blood cells, endothelial cells, pericytes, smooth muscle cells and so on [3,9,25,[27][28][29][30][31][32]. The purity of derived DFAT cells might be compromised by the contamination of those cells above when they're attached to the floating adipocytes after incomplete enzymatic treatment.…”