Confirm that all markers are expressed and differentiation of all cell types is occurring; replace any antibodies that do not work; revise differentiation protocol if necessary
Time course
Each day, d0–d10
Determine the peak day for expression of each marker
Hypothesis
Nanog decreases by d2. Endoderm peaks at d3-d4. Mesoderm and ectoderm peak between d6-d7
Determine a day to be used as + and – control for flow cytometry
Possible problems and solutions
Markers are expressed in undifferentiated cells— they cannot use ES cells as negative control
Determine whether markers for each cell type are expressed at similar times or have varying temporal expression
Determine the peak day for differentiation of each cell type by cycling through iterations of data collection
Time course
Peak day ± SD as determined in step 1; 24 hour intervals for iteration 1; 12 hour intervals for iteration 2
After iteration 1, the time between intervals and the time course being analyzed will be narrowed, and the number of replicates conducted will be increased
Hypothesis
Peak day for each cell type will be different; endo = d3; ecto = d7; meso = d7
Generate a comprehensive probabilistic model of ES cell differentiation over the course of time
Possible problems and solutions
Differentiation of germ layers can’t be modeled—the experiment will be conducted with late markers of differentiated cell types
Use the model generated in Step 2 as a tool to analyze the effect of STAT3 on differentiation
Time course
Peak day ± SD from the model generated in Step 2
Determine the peak day for differentiation of each cell type in cells that express dominant negative STAT3
Hypothesis
The absence of STAT3 will prevent differentiation of mesoderm and will delay or decrease ectoderm differentiation
Use statistical analysis (repeated measure ANOVA) to determine whether the loss of STAT3 leads to a change in the progression of ES cell differentiation over time
Possible problems and solutions
STAT3 has no effect on differentiation—a new target for modification of differentiation will be chosen
Confirm our results using GFP ES cell line and survival analysis