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Comparative study of mouse induced pluripotent stem cells derived cardiomyocytes obtained from three different fibroblast sources

Castro Zalis, Marina (2012) MOBT15 20112
Degree Projects in Molecular Biology
Abstract
Abstract

Background – The ability to generate induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) has raised much expectation for treatment of myocardial infarction as a stem cell based therapy. In the present study we aimed to evaluate the ability of iPSC-CM production in vitro from three fibroblast sources and verify if there is inter-clonal or inter-source variance.
Methods and Results – Cardiomyocytes differentiation of iPSCs was induced through the embryoid body (EB) differentiation method. Gene expression analysis of different stage during embryoid bodies differentiation showed expression of pluripotency genes, cardiac related genes and endothelial genes. This confirms the presence of stem cells, mesodermal derived... (More)
Abstract

Background – The ability to generate induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) has raised much expectation for treatment of myocardial infarction as a stem cell based therapy. In the present study we aimed to evaluate the ability of iPSC-CM production in vitro from three fibroblast sources and verify if there is inter-clonal or inter-source variance.
Methods and Results – Cardiomyocytes differentiation of iPSCs was induced through the embryoid body (EB) differentiation method. Gene expression analysis of different stage during embryoid bodies differentiation showed expression of pluripotency genes, cardiac related genes and endothelial genes. This confirms the presence of stem cells, mesodermal derived cells, such as cardiac progenitor cells, and endothelial derived cells within EBs. qPCR analysis verifies that there is high variability on the expression profile between clones within each source (inter-clonal variance) rather than different source, suggesting that the efficiency of cardiomyocyte differentiation in vitro is source independent (p > 0.05).
Immunohistochemical analyses of EBs revealed the presence of cardiomyocyte-typical proteins such as GATA4 and MLC2v. The other cardiac related proteins could not be detected, requiring further procedure optimization.
Conclusions– Induced pluripotent stem cells can successfully differentiate into cardiomyocytes. The cardiomyocyte differentiation efficiency is rather clone than source dependent.


Popular science summary:

Evaluation of the cardiac differential potential of iPSCs

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Heart attacks are the most common CVD and have as consequence the death of cardiomyocytes (cells of the heart). Cardiomyocytes (CM) loss leads to heart insufficiency. Heart transplants are done as end-point therapy but it is limited by insufficient number of compatible donors and would require lifelong immunosuppression. Currently, one of the most promising treatments that can lead to heart tissue regeneration and repair is cell-based therapy using stem cells. Stem cells are pluripotent cells that can differentiate into diverse specialized cell types and have the ability to self-renew indefinitely. However, when stem cells are transplanted into the heart they form teratomas (benign tumour containing differentiated cells from all three germ layers). Thus, it is believed that transplantation of more differentiated cells, like cardiomyocytes into the heart is more appropriate. With this in mind, there is a need to produce CMs in vitro. CMs can be produced in vitro by differentiation of induced pluripotent stem cells (iPSCs) through the embryoid body (EBs) method. iPSCs can be developed in vitro from any differentiated cell type by transduction of a cocktail of retroviruses containing pluripotency factors. Thus, iPSCc are pluripotent stem cells obtained in culture.

In the present study we aimed to evaluate the ability of iPSCs derived cardiomyocytes (iPSCS-CM) production in vitro from three murine fibroblast sources and verify if the cardiac differentiation potential is source dependent. In order to identify the production of CM during embryoid body differentiation, we recurred to the use of cardiac markers that are expressed early in cardiac development and others that can identify mature CMs. Gene expression analysis by PCR and qPCR were performed in EB clones derived from cardiac fibroblasts, tail tip fibroblasts and embryonic fibroblasts at 3 different time points (day 3, day 9 and day 12 of EBs differentiation). For gene expression, mRNA was extracted, transcribed into cDNA and transcripts of the genes of interest were amplified by PCR and qPCR. In order to identify cardiac proteins on EBs at day 12, these were fixated, and proteins detected by the use of antibodies (immunohistochemistry staining). Gene expression results of different stage during EBs differentiation showed expression of pluripotency genes (Oct4 and Nanog), confirming the presence of stem cells, thus fibroblasts were successfully reprogrammed and that some cells within the EBs remain in an undifferentiated state. Cardiac cells, epithelial cells and mesenchymal cells were also identified by expression of appropriate genes, proving that cardiac cells and other cell types can be produced in vitro. qPCR analysis verified that there is high variability on the expression profile between clones within each source (inter-clonal variance) rather than different source, suggesting that the efficiency of CM differentiation in vitro is clone rather than source dependent.

Supervisor: M.D, PhD Stefan Jovinge
Master´s Degree Project 60 ECTS in 2012
Department of Biology., Lund University

Master´s Degree Project, Faculty of Medicine, Lund University (Less)
Please use this url to cite or link to this publication:
author
Castro Zalis, Marina
supervisor
organization
course
MOBT15 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3632698
date added to LUP
2013-04-10 15:14:44
date last changed
2013-04-10 15:14:44
@misc{3632698,
  abstract     = {Abstract

Background – The ability to generate induced pluripotent stem cells-derived cardiomyocytes (iPSC-CM) has raised much expectation for treatment of myocardial infarction as a stem cell based therapy. In the present study we aimed to evaluate the ability of iPSC-CM production in vitro from three fibroblast sources and verify if there is inter-clonal or inter-source variance.
Methods and Results – Cardiomyocytes differentiation of iPSCs was induced through the embryoid body (EB) differentiation method. Gene expression analysis of different stage during embryoid bodies differentiation showed expression of pluripotency genes, cardiac related genes and endothelial genes. This confirms the presence of stem cells, mesodermal derived cells, such as cardiac progenitor cells, and endothelial derived cells within EBs. qPCR analysis verifies that there is high variability on the expression profile between clones within each source (inter-clonal variance) rather than different source, suggesting that the efficiency of cardiomyocyte differentiation in vitro is source independent (p > 0.05).
Immunohistochemical analyses of EBs revealed the presence of cardiomyocyte-typical proteins such as GATA4 and MLC2v. The other cardiac related proteins could not be detected, requiring further procedure optimization.
Conclusions– Induced pluripotent stem cells can successfully differentiate into cardiomyocytes. The cardiomyocyte differentiation efficiency is rather clone than source dependent.


Popular science summary:

Evaluation of the cardiac differential potential of iPSCs

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Heart attacks are the most common CVD and have as consequence the death of cardiomyocytes (cells of the heart). Cardiomyocytes (CM) loss leads to heart insufficiency. Heart transplants are done as end-point therapy but it is limited by insufficient number of compatible donors and would require lifelong immunosuppression. Currently, one of the most promising treatments that can lead to heart tissue regeneration and repair is cell-based therapy using stem cells. Stem cells are pluripotent cells that can differentiate into diverse specialized cell types and have the ability to self-renew indefinitely. However, when stem cells are transplanted into the heart they form teratomas (benign tumour containing differentiated cells from all three germ layers). Thus, it is believed that transplantation of more differentiated cells, like cardiomyocytes into the heart is more appropriate. With this in mind, there is a need to produce CMs in vitro. CMs can be produced in vitro by differentiation of induced pluripotent stem cells (iPSCs) through the embryoid body (EBs) method. iPSCs can be developed in vitro from any differentiated cell type by transduction of a cocktail of retroviruses containing pluripotency factors. Thus, iPSCc are pluripotent stem cells obtained in culture.

In the present study we aimed to evaluate the ability of iPSCs derived cardiomyocytes (iPSCS-CM) production in vitro from three murine fibroblast sources and verify if the cardiac differentiation potential is source dependent. In order to identify the production of CM during embryoid body differentiation, we recurred to the use of cardiac markers that are expressed early in cardiac development and others that can identify mature CMs. Gene expression analysis by PCR and qPCR were performed in EB clones derived from cardiac fibroblasts, tail tip fibroblasts and embryonic fibroblasts at 3 different time points (day 3, day 9 and day 12 of EBs differentiation). For gene expression, mRNA was extracted, transcribed into cDNA and transcripts of the genes of interest were amplified by PCR and qPCR. In order to identify cardiac proteins on EBs at day 12, these were fixated, and proteins detected by the use of antibodies (immunohistochemistry staining). Gene expression results of different stage during EBs differentiation showed expression of pluripotency genes (Oct4 and Nanog), confirming the presence of stem cells, thus fibroblasts were successfully reprogrammed and that some cells within the EBs remain in an undifferentiated state. Cardiac cells, epithelial cells and mesenchymal cells were also identified by expression of appropriate genes, proving that cardiac cells and other cell types can be produced in vitro. qPCR analysis verified that there is high variability on the expression profile between clones within each source (inter-clonal variance) rather than different source, suggesting that the efficiency of CM differentiation in vitro is clone rather than source dependent. 

Supervisor: M.D, PhD Stefan Jovinge
Master´s Degree Project 60 ECTS in 2012
Department of Biology., Lund University

Master´s Degree Project, Faculty of Medicine, Lund University},
  author       = {Castro Zalis, Marina},
  language     = {eng},
  note         = {Student Paper},
  title        = {Comparative study of mouse induced pluripotent stem cells derived cardiomyocytes obtained from three different fibroblast sources},
  year         = {2012},
}