Investigation into the mechanisms of mesendoderm specification during ES cell differentiation
Lead Research Organisation:
University of Edinburgh
Department Name: Inst of Stem Cell Research
Abstract
There are over two hundred different cell types in a human body. All cells are derived from one fertilized egg, therefore all cells have the same genome, which encode all information to make all different type of cells. However each type of cells has different character and different function to constitute individual tissues. This is caused by different usage of genes, different gene expression. In general, terminally differentiated cells do not divide, and therefore most of tissues are supplied cells to be maintained and renewed by lineage-committed stem cells and/or progenitor cells. The Lineage-committed stem cells continuously supply specific type of cells, but not cells in different lineage by keeping similar gene expression profiles, as hematopoietic stem cells do not give rise to muscles. During embryogenesis there are pluripotent cells, which can give rise to any type of cells in a body until periimplantation stage. However, during development those cells gradually lose the ability to give rise different type of cells. Some part of this loss of plasticity is explained by lineage specific transcription factors, which promote the lineage commitment or repress gene expression important for other lineages. In addition, there is a mechanism called ‘epigenetic modification‘, e. g. DNA methylation followed by accumulation of many other silencing molecules, and modification of histone, which acts as spools around which DNA winds, such as acetylation, methylation, phosphorylation. Epigenetic modification is inheritable active or silent marks on chromatin and function to restrict plasticity. These marks on chromatin are recognized by other molecules and/or change chromatin structure, and resulting in gene activation or silencing. Although these modifications do not introduce any changing in genomic sequence, they are copied in newly synthesized chromatin during replication with unknown mechanism and inherited by daughter cells when cells are divided. Therefore epigenetic modifications are important to maintain cell type specific gene expression and restrict cell fate.
Embryonic stem (ES) cells, which are derived from a pluripotent population, inner cell mass (ICM) of blastocyst, inherit the pluripotency and possess indefinite self-renewal ability in a certain condition. Because of their ability to give rise any type of cells in a body use of ES cells is expected as the most widely applicable strategy for regenerative medicine. To obtain certain type of cells from ES cells, specific culture conditions with specific cytokines, which allow ES cells go out from self-renewal and promote lineage commitment, are often used. Recently efficiency to generate endoderm, mesoderm and neuroectoderm has been improved, from which pancreas and hurt, muscle and cartridge, neuron for transplantation could be generated in future (1, 2). However, ES cells have a disadvantage in addition to this advantage, pluripotency, for the transplantation. Undifferentiated ES cells are tumorgenic. Little amount contamination of undifferentiated ES cells in transplanted tissue can be a high risk of tumor (3). To avoid this risk, understanding the mechanism of an initial step of ES cell differentiation and controlling undifferentiated-differentiated state is indispensable.
Mbd3 is a one of components of nucleosome remodeling and histone deacetylase (NuRD) complex, which possess activity to change chromatin structure and remove active epigenetic mark on histone, resulting in silence of target genes. Recently we found that ES cells disrupted Mbd3 gene by gene targeting have severe defect in their differentiation ability (4). Wild-type ES cells lose expression of undifferentiated cell specific genes and generate lineage committed differentiated cells in the absence of LIF, a cytokine which promotes self-renewal and prevents ES cell differentiation, therefore supplied in a culture medium to maintain undifferentiated ES cells. However, Mbd3-null ES cells stop to
Embryonic stem (ES) cells, which are derived from a pluripotent population, inner cell mass (ICM) of blastocyst, inherit the pluripotency and possess indefinite self-renewal ability in a certain condition. Because of their ability to give rise any type of cells in a body use of ES cells is expected as the most widely applicable strategy for regenerative medicine. To obtain certain type of cells from ES cells, specific culture conditions with specific cytokines, which allow ES cells go out from self-renewal and promote lineage commitment, are often used. Recently efficiency to generate endoderm, mesoderm and neuroectoderm has been improved, from which pancreas and hurt, muscle and cartridge, neuron for transplantation could be generated in future (1, 2). However, ES cells have a disadvantage in addition to this advantage, pluripotency, for the transplantation. Undifferentiated ES cells are tumorgenic. Little amount contamination of undifferentiated ES cells in transplanted tissue can be a high risk of tumor (3). To avoid this risk, understanding the mechanism of an initial step of ES cell differentiation and controlling undifferentiated-differentiated state is indispensable.
Mbd3 is a one of components of nucleosome remodeling and histone deacetylase (NuRD) complex, which possess activity to change chromatin structure and remove active epigenetic mark on histone, resulting in silence of target genes. Recently we found that ES cells disrupted Mbd3 gene by gene targeting have severe defect in their differentiation ability (4). Wild-type ES cells lose expression of undifferentiated cell specific genes and generate lineage committed differentiated cells in the absence of LIF, a cytokine which promotes self-renewal and prevents ES cell differentiation, therefore supplied in a culture medium to maintain undifferentiated ES cells. However, Mbd3-null ES cells stop to
Technical Summary
Recently some culture conditions for efficient production of neuroectoderm and mesoendoderm lineages from ES cells have been developed. However, even a small amount of remaining undifferentiated ES cells in these cultures would be a problem for transplantation because ES cells are tumorigenic. This proposal aims to understand the molecular mechanisms and importance of epigenetic modifications in ES cell self-renewal and in the initial steps of ES cell-mesoderm differentiation. Knowledge gained will then be applied to achieve tight regulation of transition between pluripotent cells and the mesoderm lineage in culture.
For this purpose, Mbd3 deficient ES cells will be used. Mbd3 is a component of the nucleosome remodeling and histone deacetylase (NuRD) complex and Mbd3 deficient ES cells have severe differentiation defects. In the absence of LIF Mbd3 deficient ES cells begin to express Fgf5, an early ectoderm marker, but maintain expression of the stem cell marker Oct4, without inducing the early mesoendoderm marker, Brachyury. Fgf5 expression can be reversed by culturing in the presence of LIF, therefore although Mbd3 deficient ES cells appear to initiate to differentiation they cannot progress beyond the point from where cells cannot return to undifferentiated ES cells. Interestingly Mbd3 deficient ES cells expressing an Mbd3ER fusion protein, which can be either nuclear or cytoplasmic depending upon the addition of 4-hydroxy tamoxifen (4OHT), can successfully differentiate and begin to express Brachyury in a 4OHT-dependent manner. I will use this system to dissect the gene expression events that occur during these early differentiation steps. To further dissect the initial events of ES cell differentiation I will use a Nanog-reporter gene construct to isolate ES cells expressing low levels of Nanog, which have recently been shown to be poised to differentiate. Gene expression patterns of Nanog-low expressing cells in wild-type and Mbd3 deficient ES cells will be compared in order to provide molecular snapshots of gene expression patterns immediately prior to, and immediately following the initiation of differentiation. From these experiments and subsequent functional analyses, I will be able to identify genes playing important roles in the early stages of ES cell differentiation, particularly towards the mesendoderm lineages. The results of this study will be used to identify conditions under which tight control of ES cell differentiation towards the mesoendoderm lineage can be achieved.
For this purpose, Mbd3 deficient ES cells will be used. Mbd3 is a component of the nucleosome remodeling and histone deacetylase (NuRD) complex and Mbd3 deficient ES cells have severe differentiation defects. In the absence of LIF Mbd3 deficient ES cells begin to express Fgf5, an early ectoderm marker, but maintain expression of the stem cell marker Oct4, without inducing the early mesoendoderm marker, Brachyury. Fgf5 expression can be reversed by culturing in the presence of LIF, therefore although Mbd3 deficient ES cells appear to initiate to differentiation they cannot progress beyond the point from where cells cannot return to undifferentiated ES cells. Interestingly Mbd3 deficient ES cells expressing an Mbd3ER fusion protein, which can be either nuclear or cytoplasmic depending upon the addition of 4-hydroxy tamoxifen (4OHT), can successfully differentiate and begin to express Brachyury in a 4OHT-dependent manner. I will use this system to dissect the gene expression events that occur during these early differentiation steps. To further dissect the initial events of ES cell differentiation I will use a Nanog-reporter gene construct to isolate ES cells expressing low levels of Nanog, which have recently been shown to be poised to differentiate. Gene expression patterns of Nanog-low expressing cells in wild-type and Mbd3 deficient ES cells will be compared in order to provide molecular snapshots of gene expression patterns immediately prior to, and immediately following the initiation of differentiation. From these experiments and subsequent functional analyses, I will be able to identify genes playing important roles in the early stages of ES cell differentiation, particularly towards the mesendoderm lineages. The results of this study will be used to identify conditions under which tight control of ES cell differentiation towards the mesoendoderm lineage can be achieved.