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From Embryo to Beating Heart Cell
Directed Differentiation of hESCs into Mesoderm
Module 1: From Embryo to Beating Heart Cell
Teacher’s information for specific activity: Analyze the outcome and problems for a
particular directed differentiation protocol.
This activity will ask students to think critically about experiments they have done
during the course (i.e. directed differentiation of stem cells into a particular lineage such
as cardiomyocyte or beating heart cell), to analyze and compare their results and to
discuss potential problems and outcomes.
Goals for this Activity:
 Students will learn how to grow human embryonic stem cells and direct their
differentiation to the cardiomyocyte lineage.
 Students will learn how to assess the efficiency of the protocol and discuss the
problems that arise, in order to improve their yield in the future.
Essential Questions: How do you evaluate the efficiency of a directed differentiation
protocol that drives embryonic stem cells to a particular lineage such as the
cardiomyocyte?
Student Outcomes for the Project: At the end of the exercise, students should be able
to:
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1. Identify the potential outcomes for a particular directed differentiation protocol
(e.g. types of undifferentiated and differentiated cells present in a dish at the end
of the experiment, such as cardiomyoblasts versus cardiomyocytes) and explain
how these cells can be distinguished using morphological, functional and
molecular criteria.
2. Assess the efficiency of the directed differentiation protocol. Students will
analyze the cell population they have obtained in the dish at the end of the
protocol, using laboratory techniques such as immunofluorescence with various
antibodies that label distinct cell populations. They will also determine the
proportion of undifferentiated (e.g. myoblast progenitors) versus differentiated
cells (e.g. myocytes) in their cultures.
3. Identify and discuss the potential problems with a particular directed
differentiation protocol. Students should be able to predict the outcome of the
experiment (directed differentiation) based on prior knowledge about these
processes that is derived from lectures or additional reading materials. They will
compare both the end results among student groups aand detailed descriptions of
how they performed the experiments. Finally, they will discuss potential sources
of variability (e.g. growth factors, handling and dissociation of stem cells or
progenitors, etc.) that could account for differences in the results/efficiencies
observed among groups.
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From Embryo to Beating Heart Cell
Directed Differentiation of hESCs into Mesoderm
Assessment:
Since the desired goals and outcomes for this activity are quite complex, student skills be
will assessed using the following activities:
i) Theoretical skills
 Students will understand: 1) the concept of stem cells versus differentiated cells,
2) the potential outcomes for a given directed differentiation protocol, and 3) how
these cell can be distinguished using morphological, functional and molecular
criteria. This will be achieved by writing an essay before the beginning of the
activity and participating in an in-class discussion at the beginning of the 1st class
session of the activity.
ii) Laboratory/Practical skills
 Students will learn how to use their theoretical skills in the lab, by devising a
protocol to determine the cell types in the dish (Part I is a take-home assay and
Part II a group discussion) of the academic prompt.
 Carry out the specific protocol (e.g. immunofluorescence) during the 1st and 2nd
days of the activity).
 Analyze numbers of various cell types that are present in the dish at the end of the
2nd lab day.
 Write a lab report with several components, such as a detailed experimental
protocol and the data obtained after analyzing the experiment.
 Compare their results with other groups.
iii) Analytical skills:
 Students will determine which is the best protocol to use for analyzing a particular
outcome, based on knowledge of the markers that define cells at various stages of
differentiation through Part I (take-home essay) and Part II (group discussion) of
the academic prompt.
 Students will compare the results (outcomes) that they have observed at the end of
the differentiation protocol in the classroom.
 Students will discuss potential problems that account for variability in the directed
differentiation of ES cells to cardiomyocytes at the end of the protocol, through a
discussion directed by the instructor on the 3rd day of the activity.
Learning activities: The specific goals require four assignments and three classes, to be
completed in the last week of the course. The instructor should aim to accomplish goal 1
and part of goal 2 at the beginning of the week and the rest of the goals more realistically
towards the end of that week.
The instructor will need to prepare the students at the end of class in the given week for
any activities that take place the following week. He/she will need to spend at least 15
minutes explaining the desired goals for the next week activity to the students and
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From Embryo to Beating Heart Cell
Directed Differentiation of hESCs into Mesoderm
outlining the take-home assignment that students need to work on and accomplish before
the following class.
Assignment # 1 and 2: This assignment assesses goal 1 and some components of goals 2
and 3. The assignment has been designed with three components. A prompt is given to
students at the end of the previous class so that they can think and write about the issues
that will be discussed during the following class (please read Activity1_hESCEndodermStudent.doc about the prompt). Two assignments will be built around this
prompt.
Assignment 1: Part 1 (take-home essay): This is an individual assignment where each
student will write a short (1-2 page) report, in the form of a summary, to answer question
1 of the prompt.
The instructor should look for the following points in order to give full credit to the
student:
a) The student should define and briefly describe how to distinguish each cell type
morphologically, functionally and molecularly. A schematic diagram is always useful in
this case, where students provide characteristics for each cell population such as the
names of genes/proteins that define each population and the factors that mediate the
transition from one state to another.
One potential schematic diagram that students can be expected to provide in their initial
written report about the stages of cardiomyocyte differentiation is shown below. Students
should be specific about the names of genes or proteins (A-C) that will be used to analyze
the state of cell differentiation (EB = embryoid body).
b) Students should describe possible outcomes (e.g. the normal range reported for the
fraction of cardiomyocytes) of the experiment, based on their reading of previously
reported results concerning this subject.
c) Students should provide a potential experimental approach that they will use to
determine the fraction of undifferentiated versus differentiated cells in their cultures. This
can be difficult and will depend on how much lab experience they have had previous to
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From Embryo to Beating Heart Cell
Directed Differentiation of hESCs into Mesoderm
the stem cell course. However, the suggested scientific papers should provide sufficient
information on which procedures students can use for the desired experiment.
This assignment is due on the first day of the class in that week. Students submit
individual reports at the end of class for evaluation by the instructor.
First day (Class 1) of the activity: The instructor begins by explaining again the desired
outcome of the activity and outlining the activities for the class. The instructor then
begins to describe the second part of the assignment.
Part 2 of Assignment 1 occurs on the 1st day of the class, in the form of a group
discussion. Students are placed in groups (3 students/group) on the first day of this
activity, in order to discuss their reports (30 minutes) and determine whether there is a
consensus on each component of the assignment:
a) markers that define each cell population
b) the fraction of differentiated cells that they should expect at the end of the analysis
c) a procedure (protocol) that they will use to analyze the outcome.
At the end of the discussion, one representative from each group presents the consensus
of the group. The instructor writes the main conclusions that each group has provided for
each component on the board. The instructor and/or other groups comment on the results
from each group, and together they come up with a consensus for which markers and
optimal procedure to use for the class. The instructor then provides and explains the
detailed protocol that will be used by the students during the class. This part of the
activity should take about 30 minutes.
Assignment 2: Students will assess the efficiency of the directed differentiation protocol
and will analyze the cell population obtained in the dish at the end of the protocol, using
laboratory techniques such as immunofluorescence with selected antibodies that label
distinct cell populations. They will also determine the proportion of undifferentiated (e.g.
myoblast progenitors) versus differentiated cells (e.g. cardiomyocytes) in their cultures.
This assignment will begin in this class and continue to the second class session of the
week. The laboratory section of the class should take about 90 minutes. Students spend
30 minutes feeding and examining cells in culture, and the rest of the time performing the
immunofluorescence procedure. The protocol will be provided to students. The instructor
should supervise distribution of 4% paraformaldehyde for the fixation step of the
immunofluorescence, because this compound is quite toxic. The protocol should be
continued until the incubation step with primary antibodies, which continues overnight
(and can last up to two days). During incubation with the blocking reagent, students will
prepare primary antibody dilutions and complete their lab notebooks.
Day 2 of the activity (second class session): This session begins with the
immunofluorescence technique (washing the primary antibody and incubating with the
secondary antibody). During the incubation step with the secondary antibody, students
can complete remaining laboratory procedures such as feeding cells and examining the
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From Embryo to Beating Heart Cell
Directed Differentiation of hESCs into Mesoderm
differentiation states of cells that are still in culture. At the end of the procedure, students
will analyze the number of cells that belong to each category and prepare a plot of their
findings using Excel (the instructor should provide a brief tutorial on how to prepare such
a graph). This activity can take about 90 minutes of the class, and therefore the second
day of the activity is heavily weighted towards lab procedures.
At the end of the second class, the instructor posts a summary of yields from each group
for the differentiation protocol.
Third day of the class
Assignment 3: This in-class activity aims to discuss potential problems with a given
directed differentiation protocol. Students will work in groups (those assigned at the
beginning of the activity) and compare results among groups as well as details regarding
how they performed the experiments. Finally, they will discuss potential sources of
variability (e.g. growth factors, handling and dissociation of stem cells or progenitors,
etc.) that could account for differences in the results/efficiencies observed among groups.
This activity will take about one hour during the third class session (last day of the
activity).
Students should return to the prompt given on the first class and think about Question 2.
What are some reasons that the yields are very different among groups?
Students are divided into their original groups and spend the first 30 minutes devising a
list of potential problems that might account for the low yield. During this time they will
also prepare a short (10 minute) PowerPoint presentation (3-4 slides) where they briefly
present their experimental approach, data and outcomes as well as discuss reasons that
they think contributed to a low yield. In preparation for this discussion, students should
mention: 1) variability in growth factors or other components of the media; 2) media
preparation; 3) the process of cell dissociation and how that can affect differentiation
efficiencies. The instructor writes the conclusions from each group on the board and
wraps up by summarizing the discussion.
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