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Comparative Study
. 2009 Jan 13:10:3.
doi: 10.1186/1471-2121-10-3.

Comparative characterization of mesenchymal stem cells from eGFP transgenic and non-transgenic mice

Cynthia B Ripoll  1 , Bruce A Bunnell
Affiliations
Comparative Study

Comparative characterization of mesenchymal stem cells from eGFP transgenic and non-transgenic mice

Cynthia B Ripoll et al. BMC Cell Biol. .

Abstract

Background: Adipose derived- and bone marrow-derived murine mesenchymal stem cells (mMSCs) may be used to study stem cell properties in an in vivo setting for the purposes of evaluating therapeutic strategies that may have clinical applications in the future. If these cells are to be used for transplantation, the question arises of how to track the administered cells. One solution to this problem is to transplant cells with an easily identifiable genetic marker such as enhanced green fluorescent protein (eGFP). This protein is fluorescent and therefore does not require a chemical substrate for identification and can be visualized in living cells. This study seeks to characterize and compare adipose derived- and bone marrow-derived stem cells from C57Bl/6 mice and eGFP transgenic C57Bl/6 mice.

Results: The expression of eGFP does not appear to affect the ability to differentiate along adipogenic or osteogenic lineages; however it appears that the tissue of origin can influence differentiation capabilities. The presence of eGFP had no effect on cell surface marker expression, and mMSCs derived from both bone marrow and adipose tissue had similar surface marker profiles. There were no significant differences between transgenic and non-transgenic mMSCs.

Conclusion: Murine adipose derived and bone marrow derived mesenchymal stem cells from non-transgenic and eGFP transgenic C57Bl/6 mice have very similar characterization profiles. The availability of mesenchymal stem cells stably expressing a genetic reporter has important applications for the advancement of stem cell research.

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Figures

Figure 1
Figure 1
Morphology and fluorescence of GFPTgASCs and GFPTgBMSCs. Top: ASCs derived from eGFP+ transgenic C57Bl/6 mice (P4), Bottom: BMSCs derived from eGFP+ transgenic C57Bl/6 mice (P3). 20× magnification.
Figure 2
Figure 2
A. Differentiation of mMSCs and GFPTgMSCs along mesodermal lineages. Cells were incubated in CEM and then transferred to adipogenic or osteogenic media for 14 days. All cells were passage 9 or lower. Cells which formed lipid vacuoles were stained with Oil Red-O while mineralization in osteogenic-differentiated cells was revealed with Alizarin Red staining.B. GFPTgMSCs retain fluorescence after differentiation. MSCs derived from eGFP+ transgenic C57Bl/6 mice (P6) retain fluorescence even after stimulated to undergo adipogenic differentiation. 20× magnification.
Figure 3
Figure 3
Quantitative differentiation of mMSCs and GFPTgMSCs along osteogenic and adipogenic lineages. A. Cells were incubated in CEM and then transferred to osteogenic media for 14 days. The cells were then stained with Alizarin Red and de-stained with 10% cetylpyridinium chloride. Optical density (OD) was measured at 560 nm and normalized to protein content. Graphs represent the ratio of normalized OD of differentiated cells and normalized OD of control cells. B. Cells were incubated in CEM and then transferred to adipogenic media for 14 days. The cells were then stained with Oil Red O and de-stained with isopropanol. OD was measured at 520 nm and normalized to protein content. Graphs represent the ratio of normalized OD of differentiated cells and normalized OD of control cells.
Figure 4
Figure 4
Immunophenotypic profile of mMSCs and GFPTgMSCs. A. Stem cells (P10 or lower) were incubated with antibodies for CD106, Sca-1, CD34, CD11b, or CD45 and assayed by FACS. Each antibody was tested individually and with isotype controls. B. Representative plots are shown with percentages corresponding to the average profile of all cell types. Red plot lines: isotype control; green plot: stem cells.
Figure 5
Figure 5
A. Growth Curves of mASCs and GFPTgASCs. Cells were plated at an original density of 100 cells/cm2 and cultured for 12 days. The difference in growth rate between mASCs and GFPTgASCs is not statistically significant (p > 0.05). B. Growth Curves of mBMSCs and GFPTgBMSCs. Cells were plated at an original density of 100 cells/cm2 and cultured for 12 days. The difference in growth rate between mBMSCs and GFPTgBMSCs is not statistically significant (p > 0.05).
Figure 6
Figure 6
Colony Forming Unit Assay. 100 cells were plated in a 56.7 cm2 culture dish and incubated for 14 days. Cells were stained with 3% Crystal Violet, and colonies 2 mm or larger were counted. The ability to form colonies was similar between all cell types (p > 0.05).
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