A novel preadipocyte cell line established from mouse adult mature adipocytes

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Abstract

We have established a novel preadipocyte cell line from mouse adult mature adipocytes. The mature adipocytes were isolated from fat tissues by taking only the floating population of mature fat cells. The isolated mature adipocytes were de-differentiated into fibroblast-like cells. The in vitro studies showed that the cells could re-differentiate into mature adipocytes after over 20 passages. The in vivo transplantation study also demonstrated that the cells had the full potential to differentiate into mature adipocytes, which has not been shown for the 3T3-L1 preadipocyte cell line derived from mouse embryo. We have further analyzed the expression profile of key fat regulatory genes such as the peroxisome proliferator-activated receptorγ or CCAAT/enhancer-binding protein gene families. We conclude that our cell line could be used as a preferred alternative to 3T3-L1, potentially reflecting the characteristics of mature adipocytes more, since the cell line is actually derived from adult mature adipocytes.

Section snippets

Materials and methods

Cell culture. Isolation of primary mouse adipocytes was performed using a modified method as described by Sugihara et al. [8]. Ten-week-old male ddY mice were used to obtain inguinal fat pads. The fat tissues were minced and digested using 0.1% (w/v) collagenase at 37 °C for 1 h [10]. After filtration and centrifugation (135g, for 3 min) of the digestion fluid, the floating primary adipocytes at the top layer and the SVF cells at the bottom of the tube were collected separately and washed. The

Establishment of fibroblast-like de-differentiated cells from mature adipose cells and characterization of these cells as preadipocytes

In order to obtain pure unilocular adipocytes and to avoid contamination with preadipocytes, fibroblasts or other stromal cells, we completely digested the adipose tissues with collagenase and thoroughly repeated pipetting [8], [14]. Subsequently, the dissociated adipose cells were washed and centrifuged three times. In culture flasks completely filled with medium, these cells floated to the top and adhered to the top inner surface of the flask (Fig. 1A). More than 98% of the isolated cells on

Discussion

In this study we have established a preadipose cell line, DFAT-D1, from mouse inguinal fat pads. DFAT-D1 cells originated from the unilocular fat cells and were thought to be de-differentiated into fibroblast-like cells. The DFAT-D1 cells are similar in shape to the established preadipocytes of the 3T3-L1 cells. We have confirmed that the DFAT-D1 cells have most of the characteristics of preadipocytes which have been observed in the 3T3-L1 cells in vitro. Our intriguing observation that DFAT-D1

Acknowledgments

We thank Shino Okumura, Hiroyuki Nobusue, Hidemasa Bono, Itoshi Nikaido, and Yutaka Nakachi for technical assistance and discussion. We also thank Julian Gough for helpful comments and English Edition. This work was supported by Grant-in-Aid for Development of New Technology from The Promotion and Mutual Aid Corporation for Private Schools of Japan and by “Nihon University Multidisciplinary Research Grant (2003, 2004).”

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    1

    These authors contributed equally to this work.

    2

    Present address: Graduate School of Science and Technology, Kobe University, Japan.

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