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From Embryo to Beating Heart Directed Differentiation of hESCs into Mesoderm Assignment 1 Assignment 1: Planning an experimental procedure to direct differentiation of human ES cells into cardiomyocytes. Please fill out the worksheet on your own (TOTAL = 50 points). Student Name: _________________________________________ 1) The following diagram represents the differentiation of ES cells into cardiomyocytes in culture. Think about the following questions and fill out Table 1 (review the lectures, primers and additional reading materials to fill in Table 1 and answer the questions) (10 points). Which experimental procedures or conditions do you use to generate each cell type during the differentiation depicted in this diagram? When do these cell types appear in the dish during the directed differentiation protocol? What do you expect the frequency for each cell type to be at the end of your protocol? TABLE 1 Human ES cell Embryoid Body What conditions do you use to promote each cell type in the dish? When do these cell types appear in the dish? What is the frequency for each cell type that you expect at the end of your experiment? 1 Cardiomyocyte From Embryo to Beating Heart Directed Differentiation of hESCs into Mesoderm Assignment 1 2) a) What possible experimental procedures can you use to differentiate human ES cells into cardiomyocytes? (Please list at least two different procedures) (5 points) ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ b) What are some advantages and disadvantages for each procedure? (5 points) Experimental procedure #1 (name) :__________________________________________ Advantages Disadvantages 1. 2. 3. 4. Experimental procedure #2 (name) :__________________________________________ Advantages Disadvantages 1. 2. 3. 4. 3) a) Briefly describe which method you would use for your directed differentiation experiment and why? (5 points) ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ _____________________________________________________________________________ 2 From Embryo to Beating Heart Directed Differentiation of hESCs into Mesoderm Assignment 1 b) What reagents and concentrations will you use in your media, to promote differentiation of human ES cells into cardiomyocytes with your method of choice? (5 points) Reagents Concentrations 1. 2. 3. 4. 5. 6. e) What fraction of EBs do you expect to become beating heart cells at the end of the differentiation procedure? Why? Justify your answer based on your readings and research (10 points). ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ h) Why do you think cardiomyocytes beat at different rates in the dish? Justify your answer based on your readings or research (10 points) ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 3
