Trends In Stem Cell Biology: Summary Exam
Lecture 3. Pluripotency and reprogramming.
Learning goals lecture:
Basic concept of cel potency, pluripotency and reprogramming: Waddington’s landscape.
Cell potency = the potential of a cell to differentiate into various specialized cell types.
➔ Totipotent cell = can become everything, also extra-embryonal tissues supporting the
embryo, e.g. the placenta (zygote stem cells until stage of morula: 16/32-cell stage).
➔ Pluripotent cell = can become almost everything, but only of the embryo itself
(embryonic stem cells, inner cell mass: ectoderm, endoderm, mesoderm).
➔ Multipotent cell = can differentiate into specific subtypes of cells. Hematopoietic stem
cells can become different types of blood cells, mesenchymal stem cells can become
fat or muscle cells etc. (adult stem cells).
➔ Unipotent cells = can further differentiate, but only to their own type of cells (e.g.
keratinocytes).
Waddington’s epigenetic landscape = represents cell potencies with marbles on a hill: a
marble on top of the hill can roll down into any valley (totipotent). Further down the valley,
marbles can only roll down into other small valleys (multipotent).
,Chromatin state: differentiation is controlled by epigenetics. Epigenetic regulation of stem cells:
the DNA in cells is carefully packed in a specific way in the nucleus. DNA is packed in chromatin,
which can be epigenetically modified, regulating gene expression.
- Euchromatin = loosely packed chromatin: lot of space between different nucleosomes.
Epigenetic factors or transcription factors can bind to open regions.
- Heterochromatin = densely packed chromatin. Epigenetic factors or transcription
factors cannot bind, since there are no open regions. Heterochromatic regions are
transcriptionally inactive.
In pluripotent cells, the chromatin is loosely packed (globally decondensed), contains loosely
bound chromatin proteins and is accessible for transcription factors and flexible in gene
transcription.
In differentiated cells, the chromatin is densely packed (condensed heterochromatin), contains
stably packed chromatin proteins and gene transcription is tightly regulated.
The pluripotency network (transcription factors) acts to maintain pluripotency: self-induction of
its own expression, and of other pluripotency genes, by binding in promoters, and repress genes
that induce differentiation.
➔ Oct4, Sox2 and Nanog are transcription factors that regulate themselves and the whole
cell state: they make sure that genes for pluripotency are expressed and that genes that
induce differentiation are repressed.
Reprogramming = change of cell fate: in the reprogramming of cells, cells are artificially set
back to a more potent state (marbles are pushed up the hill), e.g. reprogramming of somatic
cells into pluripotent cells.
➔ The process of reprogramming costs energy.
➔ Somatic Cell Nuclear Transfer (SCNT) or ectopic expression of transcription factors.
➔ Transdifferentiation = one type of somatic cell is changed into another type of somatic
cell (valley to valley).
➔ Transcriptional or epigenetic (chromatin state) change of cells (gene expression change).
,Characteristics of pluripotent cells (can be tested):
- Multilineage differentiation: they can differentiate into multiple lineages (e.g. blood or
skin cells).
- Unlimited proliferation in vivo: they can proliferate in principle unlimited: many rounds of
culturing.
- Pluripotency marker genes: they express pluripotency markers (Oct4, Nanog, Sox2).
Absolute proof for cells to be pluripotent:
- If the cells are injected into a mouse blastocyst, they can incorporate into the blastocyst
and contribute to any tissue in the mouse (ectoderm, endoderm, mesoderm).
o The cells contribute to all somatic lineages and produce germ line (chimerism).
- The formation of a teratoma, a benign tumor structure due to injection of pluripotent
stem cells under the skin.
Reprogramming methods: somatic cell nuclear transfer and ectopic expression of
transcription factors.
Somatic Cell Nuclear Transfer (SCNT) = the nuclei of somatic cells are taken out and put into
de-nucleated oocytes (oocyte without nuclei). The oocyte is further developed, contains a full
set of DNA. This process mimics the fertilized oocyte.
Ectopic expression of transcription factors to generate Induced pluripotent stem cells
(iPSCs) = transcription factors can change somatic cells to pluripotent stem cells. Ectopic
expression of transcription factors: a gene is being produced in a cell type where it is normally
not found.
- Oct3/4, Sox2, Klf4 and c-Myc were the transcription factors used in this study.
- Later research shows that c-Myc can be omitted.
, Identification of key transcription factors (OSKM) for reprogramming iPSCs: screening
methods and the key factors.
Key transcription factors / Yamanaka factors (OSKM) = transcription factors important for the
reprogramming of cells to a pluripotent state; to generate induced Pluripotent Stem Cells (Oct4,
Sox2, Klf4, c-Myc).
To identify these key transcription factors, a reporter assay can be performed: this assay tests
whether a specific DNA sequence or transcription factor can drive gene expression. The
expression of the key transcription factors can be induced, to see if these factors can induce the
formation of pluripotency.
Reporter essay = experiment to test whether a DNA sequence (e.g. promoter) or a transcription
factor can drive gene expression.
- If a DNA element or a promoter can be activated by specific transcription factors, the
GFP protein will be expressed, turning the cells green.
The transcription factors necessary for pluripotency are not present in somatic cells. GFP will
therefore not be turned on in somatic cells. If the cell enters state of pluripotency, transcription
factors will become expressed, and the reporter gene can be turned on.
Screening of transcription factors in reprogramming → colonies with GFP signal are pluripotent
because the pluripotency specific promoter is expressed.
Molecular principles of reprogramming: transcriptional/epigenetic regulation.
In somatic cells, the chromatin structure is strictly regulated. To change gene expression
(transcriptional regulation) patterns, the chromatin state has to change (epigenetic regulation).
Pioneer transcription factors = the first transcription factors to bind to chromatin. They can
engage with closed chromatin. After binding, they establish competence for gene expression.
Other transcription factors can now bind to change the gene expression.
- Oct4 is a pioneer transcription factor. It can engage the closed heterochromatic regions.
It brings an epigenetic modulator, which can modulate epigenetic signature and open
the chromatin. Afterwards, Klf4 and Sox2 will also bind to change cell state.
Lecture 3. Pluripotency and reprogramming.
Learning goals lecture:
Basic concept of cel potency, pluripotency and reprogramming: Waddington’s landscape.
Cell potency = the potential of a cell to differentiate into various specialized cell types.
➔ Totipotent cell = can become everything, also extra-embryonal tissues supporting the
embryo, e.g. the placenta (zygote stem cells until stage of morula: 16/32-cell stage).
➔ Pluripotent cell = can become almost everything, but only of the embryo itself
(embryonic stem cells, inner cell mass: ectoderm, endoderm, mesoderm).
➔ Multipotent cell = can differentiate into specific subtypes of cells. Hematopoietic stem
cells can become different types of blood cells, mesenchymal stem cells can become
fat or muscle cells etc. (adult stem cells).
➔ Unipotent cells = can further differentiate, but only to their own type of cells (e.g.
keratinocytes).
Waddington’s epigenetic landscape = represents cell potencies with marbles on a hill: a
marble on top of the hill can roll down into any valley (totipotent). Further down the valley,
marbles can only roll down into other small valleys (multipotent).
,Chromatin state: differentiation is controlled by epigenetics. Epigenetic regulation of stem cells:
the DNA in cells is carefully packed in a specific way in the nucleus. DNA is packed in chromatin,
which can be epigenetically modified, regulating gene expression.
- Euchromatin = loosely packed chromatin: lot of space between different nucleosomes.
Epigenetic factors or transcription factors can bind to open regions.
- Heterochromatin = densely packed chromatin. Epigenetic factors or transcription
factors cannot bind, since there are no open regions. Heterochromatic regions are
transcriptionally inactive.
In pluripotent cells, the chromatin is loosely packed (globally decondensed), contains loosely
bound chromatin proteins and is accessible for transcription factors and flexible in gene
transcription.
In differentiated cells, the chromatin is densely packed (condensed heterochromatin), contains
stably packed chromatin proteins and gene transcription is tightly regulated.
The pluripotency network (transcription factors) acts to maintain pluripotency: self-induction of
its own expression, and of other pluripotency genes, by binding in promoters, and repress genes
that induce differentiation.
➔ Oct4, Sox2 and Nanog are transcription factors that regulate themselves and the whole
cell state: they make sure that genes for pluripotency are expressed and that genes that
induce differentiation are repressed.
Reprogramming = change of cell fate: in the reprogramming of cells, cells are artificially set
back to a more potent state (marbles are pushed up the hill), e.g. reprogramming of somatic
cells into pluripotent cells.
➔ The process of reprogramming costs energy.
➔ Somatic Cell Nuclear Transfer (SCNT) or ectopic expression of transcription factors.
➔ Transdifferentiation = one type of somatic cell is changed into another type of somatic
cell (valley to valley).
➔ Transcriptional or epigenetic (chromatin state) change of cells (gene expression change).
,Characteristics of pluripotent cells (can be tested):
- Multilineage differentiation: they can differentiate into multiple lineages (e.g. blood or
skin cells).
- Unlimited proliferation in vivo: they can proliferate in principle unlimited: many rounds of
culturing.
- Pluripotency marker genes: they express pluripotency markers (Oct4, Nanog, Sox2).
Absolute proof for cells to be pluripotent:
- If the cells are injected into a mouse blastocyst, they can incorporate into the blastocyst
and contribute to any tissue in the mouse (ectoderm, endoderm, mesoderm).
o The cells contribute to all somatic lineages and produce germ line (chimerism).
- The formation of a teratoma, a benign tumor structure due to injection of pluripotent
stem cells under the skin.
Reprogramming methods: somatic cell nuclear transfer and ectopic expression of
transcription factors.
Somatic Cell Nuclear Transfer (SCNT) = the nuclei of somatic cells are taken out and put into
de-nucleated oocytes (oocyte without nuclei). The oocyte is further developed, contains a full
set of DNA. This process mimics the fertilized oocyte.
Ectopic expression of transcription factors to generate Induced pluripotent stem cells
(iPSCs) = transcription factors can change somatic cells to pluripotent stem cells. Ectopic
expression of transcription factors: a gene is being produced in a cell type where it is normally
not found.
- Oct3/4, Sox2, Klf4 and c-Myc were the transcription factors used in this study.
- Later research shows that c-Myc can be omitted.
, Identification of key transcription factors (OSKM) for reprogramming iPSCs: screening
methods and the key factors.
Key transcription factors / Yamanaka factors (OSKM) = transcription factors important for the
reprogramming of cells to a pluripotent state; to generate induced Pluripotent Stem Cells (Oct4,
Sox2, Klf4, c-Myc).
To identify these key transcription factors, a reporter assay can be performed: this assay tests
whether a specific DNA sequence or transcription factor can drive gene expression. The
expression of the key transcription factors can be induced, to see if these factors can induce the
formation of pluripotency.
Reporter essay = experiment to test whether a DNA sequence (e.g. promoter) or a transcription
factor can drive gene expression.
- If a DNA element or a promoter can be activated by specific transcription factors, the
GFP protein will be expressed, turning the cells green.
The transcription factors necessary for pluripotency are not present in somatic cells. GFP will
therefore not be turned on in somatic cells. If the cell enters state of pluripotency, transcription
factors will become expressed, and the reporter gene can be turned on.
Screening of transcription factors in reprogramming → colonies with GFP signal are pluripotent
because the pluripotency specific promoter is expressed.
Molecular principles of reprogramming: transcriptional/epigenetic regulation.
In somatic cells, the chromatin structure is strictly regulated. To change gene expression
(transcriptional regulation) patterns, the chromatin state has to change (epigenetic regulation).
Pioneer transcription factors = the first transcription factors to bind to chromatin. They can
engage with closed chromatin. After binding, they establish competence for gene expression.
Other transcription factors can now bind to change the gene expression.
- Oct4 is a pioneer transcription factor. It can engage the closed heterochromatic regions.
It brings an epigenetic modulator, which can modulate epigenetic signature and open
the chromatin. Afterwards, Klf4 and Sox2 will also bind to change cell state.
