Team:MIT mammalian Bone
From 2010.igem.org
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; we also considered using chinese hamster ovary (CHO) cells, which adhere better to the seeding substrate and might be more robust to mechanical stimulation. 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). Upon binding BMP2, the BMPRI receptor activates a downstream signaling cascade involving Smad proteins, which results in activation of the Runt-related transcription factor 2 (RUNX2), the main transcription factor controlling osteoblast differentiation. BMP2 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. HEK cells were also tested as a negative control for differentiation. Micrographs were taken on day 5 to assay for changes morphology. Click here for a detailed experimental writeup. Morphology Results ![]() Figure 1. Brightfield Microscopy Taken Prior to ALP Assay. Both C2C12 and C3HT101/2 cell lines showed significant morphological change in response to BMP2.
Morphology Results Summary
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