Team:MIT mammalian Bone

From 2010.igem.org

(Difference between revisions)

Revision as of 13:50, 26 October 2010

Bacteria

Phage

Mammalian

Bone Formation

Background

The goal of this portion of the project was to synthetically differentiate stem cells into bone; this would act as the 'output' for our final system. Since stem cells can by tricky to genetically engineer, we chose to build our circuit in human endothelial kidney (HEK) cells. In the final system, HEK cells will secrete a diffusible morphogen to differentiate co-cultured stem cells. We used Bone Morphogenetic Protein 2 (BMP2) as our osteogenic signaling molecule; it is one of the central regulators of osteoblast differentiation in mammalian cells (1) and has been shown to induce transdifferentiation in multiple stem cell types (2, 3, 4). Here, we work with myoblastic progenitor (C2C12) and mesenchymal 2 (C3HT101/) stem cells, two cell lines which have been shown capable of osteoblast differentiation (5, 6).

In Vitro Osteoblast Differentiation

Our first task was to see if we could induce osteogenic differentiation in our stem cell lines. We obtained a stock of human recombinant BMP2 protein and added it to the supernatant of stem cell cultures at 100ng/ml and 300ng/ml, based on a review of concentrations used in the literature. We tested the response to both constitutive (all 5 days) and transient (induction stopped on day 2) BMP2 signaling. Micrographs were taken on day 5 to assay for changes morphology. Click here for a detailed experimental writeup. Morphology Results

References

1. Hogan, B. L. (1996) Harvey Lect. 92, 83-98

2. Katagiri, T., Yamaguchi, A., Komaki, M., Ab, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., Suda, T. (1994) J. Cell Biol. 127, 1755-1766

3. Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H, Omura S, Suda T (1990) . Biochem Biophy Res Commun 172:295–299

4. Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S (1991) . J Cell Biol 113:681–

5. Nishimura, R, Kato Y, Chen D, Harris SE, Mundy GR, and Yoneda T (1998) J Biol Chem 273: 1872-1879

6. Richard S, Torabi N, Franco GV, Tremblay GA, Chen T, et al. (2005). PLoS Genet 1(6): e74. doi:10.1371/journal.pgen.0010074

@@ Line 72: / Line 72: @@
 Our first task was to see if we could induce osteogenic differentiation in our stem cell lines. We obtained a stock of human recombinant BMP2 protein and added it to the supernatant of stem cell cultures at 100ng/ml and 300ng/ml, based on a review of concentrations used in the literature. We tested the response to both constitutive (all 5 days) and transient (induction stopped on day 2) BMP2 signaling. Micrographs were taken on day 5 to assay for changes morphology.
 <a href="https://static.igem.org/mediawiki/2010/1/1e/BMP2_Differentiation_Experimental_Writeup.pdf" target="_blank"> Click here for a detailed experimental writeup. </a>
+<b>Morphology Results </b>
+<a href="https://static.igem.org/mediawiki/2010/9/9d/Bone_morphology_1.jpg" class="thickbox" title="Bone Morphology"><img src="https://static.igem.org/mediawiki/2010/9/9d/Bone_morphology_1.jpg" width=400px></a>
 <a href="https://static.igem.org/mediawiki/2010/e/ed/Bone_ALP_Assay_7_6_2010-2.jpg" target="_blank"> <img width=600px src="https://static.igem.org/mediawiki/2010/e/ed/Bone_ALP_Assay_7_6_2010-2.jpg"> </a>