TRENDS IN STEM CELL BIOLOGY
PLURIPOTENCY AND REPROGRAMMING
What are stem cells?
Stem cells are cells with the potential to develop into many different types of cells in the body.
Totipotent cell can become everything. Pluripotent stem cells can become any part of the embryo itself. The difference
between totipotent and pluripotent is that totipotent cells can differentiate into any cell type in the entire organism,
including the extraembryonic tissues like the placenta, while pluripotent cells can differentiate into any cell type of the
embryo's three germ layers but not the extraembryonic tissues. Totipotent cells are found only in the very early stages of
development, such as the zygote, and transition to a pluripotent state shortly after fertilization. Embryonic stem cells are
pluripotent and they are capable of differentiating into all three germ layers (ecto-, endo, and mesoderm)
Multipotent stem cells are a type of adult stem cell that can
self-renew and develop into a limited range of specialized cell
types within a specific tissue or organ, but not every cell type
in the entire body. Unlike pluripotent stem cells, which can
become any cell type from all three embryonic germ layers,
multipotent cells are more restricted to the specific lineage or
tissue they belong to.
Unipotent stem cells are a type of adult stem cell that can only
differentiate into one specific cell type, but they possess the
important ability to self-renew to produce more of themselves.
Unlike pluripotent or multipotent stem cells, their
differentiation potential is very limited, making them vital for
the ongoing maintenance, repair, and regeneration of tissues
and organs within the body. Examples include epidermal stem
cells that produce new skin cells or spermatogonia that give
rise to sperm.
Oligopotent stem cells are a type of stem cell with a limited ability to differentiate into a few, closely related cell types within
a specific lineage. For example, hematopoietic stem cells (HSCs), found in the bone marrow, are a classic example of
oligopotent cells that can develop into all types of myeloid and lymphoid blood cells. Oligopotent cells are a type of stem
cell that has a more restricted differentiation potential compared to pluripotent or multipotent stem cells.
If the chromatin is very loosely packed then transcription factors and other
proteins can easily access the chromatin to regulate the genes, this is called
euchromatin. Heterochromatin are very compact and it is difficult for effectors to
enter and these heterochromatin regions are transcriptionally inactive.
In embryonic stem cells (pluritpotent) the chromatin is very loosely packed and
this makes them accessible so it is easier for different kind of proteins or effectors
to bind to the chromatin and activate transcription and they are flexible in gene
transcription so they can go in different directions of cell states.
In fully differentiated cells the chromatin structure is very condensed and some
parts are open and this is to ensure that genes that are important for the cell
structure are expressed and the genes that are not relevant they are tightly bound
so that they are not expressed. This is to tightly regulate gene transcription.
,In the pluripotency network in ESCs the gene regulatory network is very unique to keep the stem cells pluritpotent. Oct4,
Sox2, and Nanog are important transcription factors to keep the cells pluripotent, they can regulate themselves and
regulate the whole cell state. They make sure that the genes that are important for pluripotency are expressed and the genes
that should not be expressed because they can induce differentiation, are not expressed.
Reprogramming is the change of cell fates and this takes energy to do. Such as the generation of pluripotent stem cells or
transgeneration where one type of somatic cells will be changed to another type of somatic cells. The molecular principle
of reprogramming is the transcriptional or epigenetic change so the change in gene expression.
The principle of somatic cell nuclear transfer (SCNT) is to take the nucleus from a somatic (body) cell and insert it into an
enucleated (nucleus-removed) egg cell, which is then activated to divide and develop into an embryo. The egg's cytoplasm
contains factors that reprogram the somatic nucleus, resetting it to a pluripotent state.
Ectopic expression of transcription factors, here you change somatic cells to pluripotent stem cells using transcription
factors and this is easier to do in the lab compared to SCNT. These are cells induced pluripotent stem cells (IPSCs). The
Yamanaka transcription factors are necessary to induce the pluripotent state.
Reporter assay: If you put a piece of DNA (enhancer, regulatory element or protomer) in front of a reporter gene (GFP) and
if the DNA element can be activated by specific transcription factors then GFP will be expressed and this is one way to do
a screening to test the DNA sequence and its function etc. In somatic cells the transcription factors that are necessary for
pluripotency are not present that’s why the GFP can not be expressed, once the cells enter the state of pluripotency then
the transcription factors will be expressed and then the reporter gene (GFP) will also be expressed. This is an easy
screening to test how you can easily enhance pluripotency in cells.
Pluripotent stem cells can proliferate forever and in the screening of TFs in reprogramming researchers saw that the cells
kept growing and they were able to passage them for a very long number of days. In terms of gene expression the
pluripotent cells and the fibroblasts have totally different gene expressions.
Differentiation capacity is tested in vivo where teratomas, iPSCs are injected into nude mice. Differentiation capacity in
vitro are used in embryoid bodies, in EBs all three germlayers cells can be distinguished by western blotting or PCR by
looking at the different genes/proteins. The pluripotent stem cells are not differentiated into the three germ layers.
, To characterize iPSCs you can look at unlimited growth, morphology (tightly packed, compact, and relatively round
colonies with smooth borders), pluripotency markers (Oct4, Nanog, and Sox2), and differentiation capacity in vivo and in
vitro. You can also look at chimera/germline transmissions but not in human iPSCs because the genetic information of
iPSCs contribute to chimera and germ cells of chimera.
You can use iPSCs for fundamental research (developmental biology and early embryogenesis), disease modeling
(developmental diseases, not easily retrievable tissues), and regenerative medicine (compound testing and screening and
regeneration of tissues).
Sequential events of reprogramming
The transcription factors used in reprogramming are often called transgenes because they are not part of the cell before
you introduce them. So Oct4, Sox2, Klf4, and Myc (OSKM). Once these transcription factors are introduced the cells start
to change. The first process is called mesenchymal to epithelial transition (MET), this is specific for reprogramming using
fibroblasts because they have a mesenchymal cell signature. Here motile, spindle-shaped mesenchymal cells transform
into stable, polarized epithelial cells. This reversible process involves the loss of migratory and invasive mesenchymal
traits and the acquisition of epithelial characteristics like cell-cell junctions and apical-basal polarity. The next step is
maturation and once the cells are repgorammed they become transgene independent which means that if you take away
the transgene, the cells can stay pluripotent. These pluripotent genes are endogenously expressed at that point. The
reason for that the transgenes are not necessary anymore is that when the transgenes are continuously expressed, the
cells will never differentiate so when the transgenes are not expressed anymore the cells can differentiate.
Pioneer transcription factors means that the transcription factors can engage or bind to the closed chromatin and then
they will establish the competence for gene expression which means that they bring other factors such as epigenetic
factors to change the chromatin state. The reprogramming factors OSK function as pioneers. Oct4 engages closed
chromatin and it brings along Brg1 to open heterochromatin and Flf4 and Sox4 will bind to change the cell state.
PLURIPOTENCY AND REPROGRAMMING
What are stem cells?
Stem cells are cells with the potential to develop into many different types of cells in the body.
Totipotent cell can become everything. Pluripotent stem cells can become any part of the embryo itself. The difference
between totipotent and pluripotent is that totipotent cells can differentiate into any cell type in the entire organism,
including the extraembryonic tissues like the placenta, while pluripotent cells can differentiate into any cell type of the
embryo's three germ layers but not the extraembryonic tissues. Totipotent cells are found only in the very early stages of
development, such as the zygote, and transition to a pluripotent state shortly after fertilization. Embryonic stem cells are
pluripotent and they are capable of differentiating into all three germ layers (ecto-, endo, and mesoderm)
Multipotent stem cells are a type of adult stem cell that can
self-renew and develop into a limited range of specialized cell
types within a specific tissue or organ, but not every cell type
in the entire body. Unlike pluripotent stem cells, which can
become any cell type from all three embryonic germ layers,
multipotent cells are more restricted to the specific lineage or
tissue they belong to.
Unipotent stem cells are a type of adult stem cell that can only
differentiate into one specific cell type, but they possess the
important ability to self-renew to produce more of themselves.
Unlike pluripotent or multipotent stem cells, their
differentiation potential is very limited, making them vital for
the ongoing maintenance, repair, and regeneration of tissues
and organs within the body. Examples include epidermal stem
cells that produce new skin cells or spermatogonia that give
rise to sperm.
Oligopotent stem cells are a type of stem cell with a limited ability to differentiate into a few, closely related cell types within
a specific lineage. For example, hematopoietic stem cells (HSCs), found in the bone marrow, are a classic example of
oligopotent cells that can develop into all types of myeloid and lymphoid blood cells. Oligopotent cells are a type of stem
cell that has a more restricted differentiation potential compared to pluripotent or multipotent stem cells.
If the chromatin is very loosely packed then transcription factors and other
proteins can easily access the chromatin to regulate the genes, this is called
euchromatin. Heterochromatin are very compact and it is difficult for effectors to
enter and these heterochromatin regions are transcriptionally inactive.
In embryonic stem cells (pluritpotent) the chromatin is very loosely packed and
this makes them accessible so it is easier for different kind of proteins or effectors
to bind to the chromatin and activate transcription and they are flexible in gene
transcription so they can go in different directions of cell states.
In fully differentiated cells the chromatin structure is very condensed and some
parts are open and this is to ensure that genes that are important for the cell
structure are expressed and the genes that are not relevant they are tightly bound
so that they are not expressed. This is to tightly regulate gene transcription.
,In the pluripotency network in ESCs the gene regulatory network is very unique to keep the stem cells pluritpotent. Oct4,
Sox2, and Nanog are important transcription factors to keep the cells pluripotent, they can regulate themselves and
regulate the whole cell state. They make sure that the genes that are important for pluripotency are expressed and the genes
that should not be expressed because they can induce differentiation, are not expressed.
Reprogramming is the change of cell fates and this takes energy to do. Such as the generation of pluripotent stem cells or
transgeneration where one type of somatic cells will be changed to another type of somatic cells. The molecular principle
of reprogramming is the transcriptional or epigenetic change so the change in gene expression.
The principle of somatic cell nuclear transfer (SCNT) is to take the nucleus from a somatic (body) cell and insert it into an
enucleated (nucleus-removed) egg cell, which is then activated to divide and develop into an embryo. The egg's cytoplasm
contains factors that reprogram the somatic nucleus, resetting it to a pluripotent state.
Ectopic expression of transcription factors, here you change somatic cells to pluripotent stem cells using transcription
factors and this is easier to do in the lab compared to SCNT. These are cells induced pluripotent stem cells (IPSCs). The
Yamanaka transcription factors are necessary to induce the pluripotent state.
Reporter assay: If you put a piece of DNA (enhancer, regulatory element or protomer) in front of a reporter gene (GFP) and
if the DNA element can be activated by specific transcription factors then GFP will be expressed and this is one way to do
a screening to test the DNA sequence and its function etc. In somatic cells the transcription factors that are necessary for
pluripotency are not present that’s why the GFP can not be expressed, once the cells enter the state of pluripotency then
the transcription factors will be expressed and then the reporter gene (GFP) will also be expressed. This is an easy
screening to test how you can easily enhance pluripotency in cells.
Pluripotent stem cells can proliferate forever and in the screening of TFs in reprogramming researchers saw that the cells
kept growing and they were able to passage them for a very long number of days. In terms of gene expression the
pluripotent cells and the fibroblasts have totally different gene expressions.
Differentiation capacity is tested in vivo where teratomas, iPSCs are injected into nude mice. Differentiation capacity in
vitro are used in embryoid bodies, in EBs all three germlayers cells can be distinguished by western blotting or PCR by
looking at the different genes/proteins. The pluripotent stem cells are not differentiated into the three germ layers.
, To characterize iPSCs you can look at unlimited growth, morphology (tightly packed, compact, and relatively round
colonies with smooth borders), pluripotency markers (Oct4, Nanog, and Sox2), and differentiation capacity in vivo and in
vitro. You can also look at chimera/germline transmissions but not in human iPSCs because the genetic information of
iPSCs contribute to chimera and germ cells of chimera.
You can use iPSCs for fundamental research (developmental biology and early embryogenesis), disease modeling
(developmental diseases, not easily retrievable tissues), and regenerative medicine (compound testing and screening and
regeneration of tissues).
Sequential events of reprogramming
The transcription factors used in reprogramming are often called transgenes because they are not part of the cell before
you introduce them. So Oct4, Sox2, Klf4, and Myc (OSKM). Once these transcription factors are introduced the cells start
to change. The first process is called mesenchymal to epithelial transition (MET), this is specific for reprogramming using
fibroblasts because they have a mesenchymal cell signature. Here motile, spindle-shaped mesenchymal cells transform
into stable, polarized epithelial cells. This reversible process involves the loss of migratory and invasive mesenchymal
traits and the acquisition of epithelial characteristics like cell-cell junctions and apical-basal polarity. The next step is
maturation and once the cells are repgorammed they become transgene independent which means that if you take away
the transgene, the cells can stay pluripotent. These pluripotent genes are endogenously expressed at that point. The
reason for that the transgenes are not necessary anymore is that when the transgenes are continuously expressed, the
cells will never differentiate so when the transgenes are not expressed anymore the cells can differentiate.
Pioneer transcription factors means that the transcription factors can engage or bind to the closed chromatin and then
they will establish the competence for gene expression which means that they bring other factors such as epigenetic
factors to change the chromatin state. The reprogramming factors OSK function as pioneers. Oct4 engages closed
chromatin and it brings along Brg1 to open heterochromatin and Flf4 and Sox4 will bind to change the cell state.
