Anat Cell Biol.  2015 Jun;48(2):85-94. 10.5115/acb.2015.48.2.85.

Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering

• Ghoniem AA
• Acil Y
• Wiltfang J
• Gierloff M

• KMID: 1845276
• DOI: http://doi.org/10.5115/acb.2015.48.2.85

Abstract

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARgamma2, C/EBPalpha, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

Keyword

Fat tissue engineering; PPARgamma2; C/EBPalpha; Real time RT-PCR; Adipogenic differentiation

MeSH Terms

Bone Marrow
Cell Culture Techniques*
Cell Lineage
Gene Expression
Humans
PPAR gamma
RNA-Directed DNA Polymerase
Tissue Donors
Tissue Engineering*
PPAR gamma
RNA-Directed DNA Polymerase

Figure

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  Fig. 1 Oil-Red staining after 21 days (×20). Staining after 10 days not shown. hADSC, human adipogenic-derived stroma cell; hMSC, human bone marrow mesenchymal stroma cell; CM, culture medium; NAM, normal adipogenic medium; SAM, specific adipogenic medium. Scale bars=50 µm.

• 

  Fig. 2 Comparision of relative gene expression levels between NAM and SAM after 10 and 21 days. The comparison between the NAM and the SAM is portrayed per tissue (hADSCs or hMSCs). Relative gene marker expression (mean±standard deviation; n=3; independent t test: P<0.05) obtained from the real time reverse transcriptase polymerase chain reaction. Only significant differences between SAM over NAM where detectable and are shown as P-value above the graphs. hADSC, human adipogenic-derived stroma cell; hMSC, human bone marrow mesenchymal stroma cell; NAM, normal adipogenic medium; SAM, specific adipogenic medium.

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  Fig. 3 Flowchart shows the allocation of adipogenic growth media (SAM and NAM) with each cell type (hADSC and hMSC). Arrows show up- and downregulation of the relative gene expression level. To get a further insight of relative expression level ratios please refer to Fig. 1. Significant differences where only detectable for SAM towards NAM. For further statistic exploration please refer to Table 4. hADSC, human adipogenic-derived stroma cell; hMSC, human bone marrow mesenchymal stroma cell; NAM, normal adipogenic medium; SAM, specific adipogenic medium.

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