DATE CREATED: Dec 2008
TITLE
MMP-sensitive PEG hydrogel for induction of Embryonic Stem Cells differentiation
SUMMARY
MMP-sensitive PEG Hydrogel:
- is a synthetic biomaterial whose biochemical and mechanical properties can be easily tailored
- well mimics some key features of the natural cell environment such as progressive degradation of the extracellular matrices by the cells themselves or the incorporation of bio-active ligand, like RGD peptides to promote adhesion.
- has a crosslinking reaction by Michael-type addition (simple mixing at RT) => no cell damaging UV exposure is required
- have been used to encapsulate fibroblasts and then promote their migration and proliferation.
=> Here, my goal is to test for the first time the suitability of such a gel with embryonic stem cells (ESCs).
Question I plan to answer:
- Do the ESCs respond to this kind of hydrogel and to the incorporated adhesion ligand RGD?
- Will ESCs start secreting MMPs (in order to migrate through the gel)? If yes, is there a type of MMP more present than others? Which one?
- Could I reproduce in 3D the results Dr. Yu-Shik Hwang [add a link] obtained by culturing EBs on Matrigel (2D)?
- Could we observe significant differences in terms of how well ESCs can differentiate in response to the stiffness of the matrix?
- Which combination of signaling peptides (RGD, Wnt5a,...) are the more efficient to induce endothelial differentiation?
ACTION PLAN
PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)
PAPER TITLE => please see my outline document for all the following information : Celine's Project Outline (give me little time to find out how to create a link leading to my .doc file^^)
A) Background
B) Hypothesis
B) Specific Aims
C) General Experimental Approach (Design etc)
D) Pitfalls and alternatives
E) Potential Figures
F) Future Directions
A) BACKGROUND
B) HYPOTHESIS
C) SPECIFIC AIMS
AIM 1 -
AIM 2 -
D) GENERAL EXPERIMENTAL APPROACH
E) PITFALLS AND ALTERNATIVES
F) ANTICIPATED FIGURES FOR PAPER or when you have data, FIGURES FOR PAPER
Fig.0
Zymogram after ...days to demonstrate the presence of MMPs secreted by the ESCs and which type.
Fig.1
immunostaining of my gels after 2-3 weeks encapsulation. I plan to use CD31 and SMCactin antibodies which are directed against endothelial and smooth muscle cells marker, respectively. In addition, DAPI will stain the nucleus of every cell. According to the zymography results, I might use a fourth stain marking the type of MMP which is present in the highest quantity.
Fig.2
I will then confirm the immunostaining results with analyze of these named above gene by PCR.
Fig.3
...
G) FUTURE DIRECTIONS
H) MATERIALS AND METHODS
I) RESULTS
J) DISCUSSION
K) LITERATURE