Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity
At the end of this exercise, students will be able to:
1) Work as a group to identify a question and plan an experimental approach to address that question.
2) Identify and discuss about the advantages and disadvantages of potential experimental methods.
3) Determine which experimental approach will be the most appropriate to address the specific question.
4) Plan and prepare a written protocol for the experimental procedure to be used in the laboratory for this purpose.
The process of scientific research involves multiple steps that range from: a) identifying an important question, b) planning the experiments that would address the questions, c) gathering and sorting out the data, d) synthesizing and evaluating the information and e) reporting about the scientific findings.
Understanding how to approach each of these steps is essential not only for the success of the experiments but also for building a logical framework about everyday work in a research laboratory. Teamwork is an essential component of the scientific research since it engages us in discussions that may help to solve scientific problems. Various members of the team bring different perspectives on either how to solve a problem, or what experimental procedures to use to answer a specific question, that you may have not thought about on your own. Therefore, a group discussion about planning your experimental procedures is vary valuable before you embark on the wrong procedure that will not give you an answer and waste your time, resources and energy.
1) You should bring in the class your completed individual worksheet from Assignment
1 since this will serve as a guideline for group discussion and for planning your experimental procedure in the laboratory.
2) The instructor will arrange each student in groups of three before the beginning of this activity. The instructor will assign one student to present the opinion of the group in front of the class. The other members of the group will take notes and prepare to
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Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity answer questions from the instructor or other groups about their proposed experimental procedure or why they think that this is the most useful method to address the question.
3) You will discuss for 30 minutes and fill in a group worksheet that has identical questions to the individual worksheet from the Assignment 1 about experimental procedures that you are planning to use to determine the efficiency of the directed differentiation protocol. However this group worksheet should represent the consensus for the group and it should address the following questions:
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Which markers define each cell population (e.g. ES cells, EBs, immature endoderm or definitive endoderm) and when do these markers appear during differentiation?
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How can you use this information to design an experiment?
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What is the best experimental procedure to analyze the outcome of this experiment? You should prepare a list of advantages and disadvantages for your experimental approach to help you answer this question.
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What types of controls are you going to use for your experiments?
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What is the proportion of various cell types that you should expect at various days of the directed differentiation procedure?
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How are you going to determine the proportion of each cell type in your experiment?
4) At the end of the discussion, the selected representative from your group will present the group’s summary in front of the class for 5 minutes.
5) The other members of the group should be prepared to answer any questions from the instructor or other groups that will be asked at the end of the presentation (5 minutes).
6) Give your instructor the individual and group worksheets for evaluation.
7) The instructor will decide at the end of all presentation what is the best experimental approach to use in the laboratory for analyzing the efficiency of directed differentiation of human ESCs into endoderm.
Please refer to the reading material list from Assignment 1 in preparation for this assignment.
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Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity
Worksheet: Planning an experimental approach to answer a question.
Group #
Student 1:____________________________
Student 2:____________________________
Student 3:____________________________
Step 1: The following diagram represents the differentiation of ES cells into endoderm in culture. Think about the following small questions and fill in Table 1 (Please review the reading notes to answer the following questions and fill in Table 1).

What markers characterize each cell type in this diagram?

When are these markers expressed?
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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
Cell type
Molecular
Markers
Time (days) of appearance in the dish during
ES cell Embryonic
Body (EB)
Immature endoderm
Definitive endoderm
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Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity differentiation
Frequency at the end of the differentiation
Step 2: You should think how to use the information that you gathered in Table 1 to plan your experiment. a) What possible experimental procedures can you use to address the question?
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________________________________________________________________________ b) What are some advantages and disadvantages for each of the procedures?
Experimental procedure # 1 (name) :__________________________________________
Advantages Disadvantages
1.
2.
3.
4.
5.
Experimental procedure # 2 (name) :__________________________________________
Advantages Disadvantages
1.
2.
3.
4.
5. c) Briefly describe which method would be most appropriate for the experiment and why?
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Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity
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________________________________________________________________________ d) What positive and negative controls can you use for your experiment?
Positive control Negative control Additional controls e) What concentration are you going to use for the reagents?
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________________________________________________________________________ f) How much time do you need to complete the experiment and why?
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________________________________________________________________________ g) How are you going to gather your data?
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________________________________________________________________________ h) How are you going to analyze and organize your data?
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Module 2: From Embryo to Beating Heart
Section 4: Directed Differentiation of hESCs into Endoderm
2.4.2: In class group activity
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