DATE CREATED: 18 Dec 2008
ACTION PLAN
PROBLEMS IDENTIFIED (PI) / OUTSIDE SKILL REQUIRED (OSR) / RESOLVED (R)
PAPER TITLE : Embryoid Body Size Dependent Neurogenesis Within Micro-Sized PEG Hydrogel Array With Different Well Size
A) BACKGROUND:
Embryonic stem cell fate could be affected by the size of EBs, supposing the fact that EB size is increasing with EB culture time. In this paper, microfabricated PEG hydrogel microwells with various diameters (d:150, 300, 450 um) were developed for the precise control of homogeneous EB size. EBs formed within microwell arrays and differentiated cells were analyzed and characterized by functional assay, molecular expression, and gene expression profile.
B) HYPOTHESIS
C) SPECIFIC AIMS
AIM 1 - Establish spontaneous neurogenesis from EBs of 150, 300, and 450 um.
AIM 2 - Distinguish gene level expression of neurogenesis from EBs of 150, 300, and 450 um.
D) Materials and Methods
1. Fabrication of Hydrogel Microwell Platforms
2. ES Cell culture and Embryoid Body Formation
3. Morphological Observation
4. Immunocytochemical Staining
5. Reverse Transcription - Polymerase Chain Reaction (PCR)
6. Inhibition and Activation Assay
E) DESIGN PITFALLS AND ALTERNATIVES
F) ANTICIPATED FIGURES FOR PAPER
Figures
Figure 1. ES cells were cultured in the microwell for 5 days for embryoid body formation (EB). Retinoic Acid was added on day 3 of EB formation. Then, EBs were replated on matrigel, fibronectin, and non coated well. (A) Schematic representation of the micromolding process to generate microwell array from and photocrosslinkable PEG-DA prepolymer solution (blue). (B) Phase contrast images of embryoid body cultured in the microwell at day 0, 3, and 5. (C) Schematic view of the differentiation protocol.
Figure 2. Phase contrast images of EBs cultured in 150, 300, 450 um microwell at Day 5, and 8. The expression of goosecoid (Gsc) gene locus that carry Green Fluorescent Protein (GFP) for the mesendormal marker.
Figure 3. Neuronal Differentiation of EBs cultured from PEG microwell (150, 300, 450 um). Type III beta-tubulin /PI immunostained images.
Figure 4. (A) RT-PCR analysis of ectoderm layer. hes (Ectoderm) and GAPDH at day 5.
G) FUTURE DIRECTIONS