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. 2004 Jun 29;101(26):9607-11.
doi: 10.1073/pnas.0403100101. Epub 2004 Jun 21.

Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

Qi-Qun Tang  1 , Tamara C Otto, M Daniel Lane
Affiliations

Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

Qi-Qun Tang et al. Proc Natl Acad Sci U S A. .

Abstract

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.

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Figures

Fig. 1.
Fig. 1.
Effect of treatment of C3H10T1/2 stem cells with BMP4-conditioned medium on their capacity to differentiate into adipocytes. (A) The 2-day postconfluent C3H10T1/2 stem cells and 3T3-L1 preadipocytes were induced (or not) by using our standard differentiation protocol, and the expression of adipocyte markers (C/EBPα, PPARγ, and 422/aP2) were detected on days 0 and 2 by immunoblotting. (B) C3H10T1/2 stem cells cultured with or without BMP4-conditioned medium were induced (or not) to differentiate by using our differentiation protocol, and the accumulation of cytoplasmic triglyceride was detected with Oil Red O staining (Upper) on day 8. This was compared to 3T3-L1 cells (Lower). (C) C3H10T1/2 stem cells cultured with or without BMP4-conditioned medium were induced to differentiate with the standard 3T3-L1 differentiation protocol, and the expression of adipocyte markers (C/EBPα, PPARγ, and 422/aP2) was detected with cell extract made on day 6.
Fig. 2.
Fig. 2.
Effect of treatment of C3H10T1/2 stem cells with recombinant BMP4 on their capacity to differentiate into adipocytes. (A) C3H10T1/2 stem cells were cultured without or with purified recombinant BMP4 at 10, 50, and 100 ng/ml until postconfluent and then induced to differentiate with our standard differentiation protocol. The accumulation of cytoplasmic triglyceride was detected by Oil Red O staining on day 8, and at which point the cells were photographed. (B) C3H10T1/2 stem cells were treated as above and the expression of adipocyte markers (C/EBPα, PPARγ, and 422/aP2) was detected with cell extract made on day 6. (C) C3H10T1/2 stem cells were treated with 50 ng/ml BMP4 at ≈30% confluency; whole cell extracts were made at times indicated, and Western blotting was performed with Abs against Smad1 (Santa Cruz Biotechnology) or phospho-Smad1 (Cell Signaling Technology, Beverly, MA).
Fig. 3.
Fig. 3.
Effect of recombinant BMP2 on commitment of C3H10T1/2 stem cells to the adipocyte lineage. (A) C3H10T1/2 stem cells were treated with or without BMP2 or BMP4 until postconfluence, and then induced to differentiate with the standard differentiation protocol. The accumulation of cytoplasmic triglyceride was detected by Oil Red O staining on day 8 and photographed. (B) The expression of the adipocyte marker 422/aP2 was detected with cell extract made on day 6.
Fig. 4.
Fig. 4.
Mitotic clonal expansion of BMP4-committed C3H10T1/2 stem cells upon induction with differentiation inducers. (A) C3H10T1/2 stem cells were cultured with or without 50 ng/ml BMP4 until postconfluence and then induced to differentiate with 3T3-L1 standard differentiation protocol. Cells were counted on day 6 and plotted. (B) Committed C3H10T1/2 (by BMP4 at 50 ng/ml) stem cells and 3T3-L1 and 3T3-F442A preadipocytes were induced to differentiate, and the expression level of cyclin A was followed by Western blotting.
Fig. 5.
Fig. 5.
Effect of previous treatment of C3H10T1/2 stem cells in cell culture on their capacity to generate adipose tissue when s.c. implanted into athymic mice. C3H10T1/2 stem cells were treated with or without 50 ng/ml BMP4 until near confluency and implanted into athymic mice. Later (4 wk), the fat pads derived from the implanted cells were analyzed with hematoxylin/eosin staining and photographed. The 3T3-F442A preadipocytes serve as a positive control, and the development of adipose tissue from these implanted cells were compared to endogeneous epididymal fat pads.
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