Download Immune response evaluation after transplantation of induced

Differentiation of induced pluripotent stem cells (iPSC) into functional cardiomyocytes (CMs), characterization of
iPSC-CMs and evaluation of the immune response after transplantation of iPSC-CMs into mice hearts.
Or
Immune response evaluation after transplantation of induced pluripotent stem cell derived cardiomyocytes into
mice.
Instructor: Kovalev Katya Department of Biotechnology
Abstract:
Cardiovascular diseases are one of the leading death causes in the western world today. An example for a
cardiovascular disease is myocardial infarction; this is a condition in the heart caused after a heart attack, in which part
of the cardiac cells do not receive oxygen and as a result they die, causing a permanent damage to the heart.
Accumulation of heart damage highly raises the risk of death for patients that had a heart attack. Some tissues in our
body, such as skin, are able to undergo regeneration; the ability to recreate new cells after the tissue is damaged.
Cardiac heart cells are unable to do so, which means that damage caused to the heart is irreversible and may lead to
development of progressive heart failure.
Heart transplantation is an established procedure for heart failure patients, but the main problems of this method is the
limited amount of donors and the lifelong uptake of immunosuppressive drugs in order to avoid organ rejection after
the transplantation.
One of the recent solutions proposed to prevent heart failure is myocardial cell replacement; which means the
replacement of damaged cardiac cells with new functional cells. Transplantation of human cardiomyocytes (CMs) can
result in optimal recovery of the scar, created after the apoptosis of damaged cardiac cells. The transplanted cardiac
cells are generated from induced pluripotent stem cells (iPSC) which were taken from the patient who is in need of this
treatment, which means there is no need for immune suppression after transplantation since the cells are autologous
(i.e. their origin is from the same patient to whom they will be transplanted). If the cells are taken from a patient with
identical DNA (such as the patient's identical twin), these cells are called syngeneic.
On the contrary, if we transplant iPSC generated from another patient, they will be rejected; this model is an allogeneic
cells transplantation model.
Research regarding immunogenicity of iPSCs is inconsistent; while some articles describe iPSC as immune privileged
others show that iPSC and iPSC derivatives (other types of cells generated from iPSC, such as cardiomyocytes) can
elicit immune response. In our lab we research the potential immunogenicity of iPSC derived CMs (iPSC-CMs).
The techniques we are using in the lab:
The main mouse type we use in our lab is C57BL/6 mice (black mice, fig 1.A) since this is a common mouse type
used in scientific research. This is the process used for generation of mouse iPSC: first we isolated skin cells (also
called skin fibroblasts) from black mice (fig 1.B), these skin cells were reprogrammed into iPSC (fig 1.C) by inserting
a gene cassette of pluripotent genes. Furthermore, mouse iPSC were differentiated into cardiomyocytes (fig 1.D) using
the hanging drop method. After generating functional cardiomyocytes, these cells were injected into mice hearts
(fig 1.E) in order to examine immune response after injection.
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Figure 1
B
A
Isolation of skin
fibroblasts
C
Reprograming to
induced
pluripotent stem
cells
E
D
Differentiation
into
cardiomyocytes
Injection of
CMs to the
heart
Immunogenicity is investigated in two models (fig. 2);
1. Syngeneic model: in these model iPSC-CMs transplanted into genetically identical mice. iPSC are generated from
black (C57BL/6) mice and transplanted later on to other black mice which have identical genetic information (same
DNA). In this model we expect minimal immune response after transplantation.
2. Allogeneic model: Transplantation of iPSC-CMs generated from black mice into genetically different mice, in our
case ICR mice. We chose to work with white mice (this type is called ICR mice) since they are genetically different
from black mice and easy to recognize between the two mouse types. In this model we expect immune rejection after
transplantation.
Syngeneic
Figure 2
Allogeneic
Immune rejection
Immune tolerance
Student mission / objective:
The main objective of this project is to give an overview of the research done in the medical and biotechnology field,
and let the students gain knowledge in the field of stem cells and at the same time experience lab work; plan, execute
and analyze experiments. This experience will hopefully raise students' curiosity to science and encourage them to
proceed to higher education.
The students will learn about the process of creating induced pluripotent stem cells (iPSC) and the differentiation of
iPSC into functional cardiomyocytes (CMs). The students will differentiate the cells using the hanging drop method,
and then characterize the cell by immunostaining. We will use fluorescent microscopy and confocal microscopy to
observe the cells and take pictures of the staining. The students will experience and learn about the following
techniques: hematoxylin and eosin staining, freeze and cut tissues embedded in optical cutting temperature compound
(OCT), and time laps microscopy. In case we will have extra time, there is a possibility to learn and practice some
molecular biology techniques.
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Requirements:
Must have 5 units in biology. Basic knowledge in physics and chemistry is an advantage for this project.
Please read the following paper in order to get a sense of the project:
Paper name:
Zhao, T., Zhang, Z. N., Rong, Z., & Xu, Y. 2011. Immunogenicity of induced pluripotent stem cells. Nature, 474(7350):
212-215.
Questions about the paper:
1. What are induced pluripotent stem cells (iPSC) and how are they generated?
2. Which methods are used to examine immune response?
We will discuss the answers when we meet at the dinner in the opening ceremony.
Please feel free to contact me with questions regarding the project at [email protected]
Recommended reading material:
Some of these articles are not available online. You can contact me and I'll email you the pdf files of the articles.
Zwi-Dantsis, L. and L. Gepstein, Induced pluripotent stem cells for cardiac repair. Cell Mol Life Sci, 2012. 69(19): p.
3285-99.
Jeremy I. Pearl, L.S.K., Mark M. Davis, Joseph C. Wu. , Science Translational Magazine 2013.
Rong, Z., et al., An effective approach to prevent immune rejection of human ESC-derived allografts. Cell Stem Cell,
2014. 14(1): p. 121-30.
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