V4: cellular reprogramming

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V4 cellular reprogramming
Embryonic development Stem cells Differentiation
iPS cells Oct4 Paper4
3D-Model of the human genome, Spiegel Online
Model of a human cell, Spiegel Online
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Some human cells
Astrocyte (nerve cell) Cardiomyocyte
(heart muscle) (wikipedia.org) (http//www.kcl.ac
.uk/content/1/c6/01/66/46/gautel3.jpeg
Fibroblast (connective tissue) (wikipedia.org)
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Zygotes - fertilization
In living organisms that reproduce sexually,
development starts from a single cell, the
zygote. Zygotes are usually produced by a
fertilization event between two haploid cells
an ovum from a female and a sperm cell from a
malewhich combine to form the single diploid
cell. Human sperm and egg (sex cells) have one
complete set of chromosomes from the male or
female parent. Sex cells, also called gametes,
combine to produce somatic cells. Somatic cells
therefore have twice as many chromosomes. The
haploidity number (n23 in humans) is the number
of chromosomes in a gamete. A somatic cell has
twice that many chromosomes (2n46).

www.wikipedia.org
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some terms from developmental biology
somatic cells cells forming the body of an
organism germ cells (dt. Keimzelle, Ovolum) are
part of the germline. germline (dt. Keimbahn)
line of germ cells that have genetic material
that may be passed to a child/embryo. Germline
cells are immortal. Gametocyte eukaryotic germ
cell includes spermatocytes (male) and oocytes
(female) primordial germ cells predecessors of
germ cells. They migrate to the gonadal ridge.
They may be detected from expression of
Stella gonad (dt. Keimdrüse)

www.wikipedia.org
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Germ line development
Germline cells are produced by embryonic
cleavage. Cleavage division of cells in the
early embryo. The zygotes of many species
undergo rapid cell cycles with no significant
growth. The different cells derived from
cleavage are called blastomeres and form a
compact mass called the morula. Cleavage ends
with the formation of the blastula. Cleavage in
mammals is slow. Cell division takes 12 24
hours and is asynchronous.

www.wikipedia.org
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Differentiation
Zygotes contain DNA derived from both the mother
and the father, and this provides all the genetic
information necessary to form a new
individual. This property is named totipotency
(latin totus all, potentia
power/ability). Continuous cell division
produces daughter cells that start to specialize
on individual functions. This developmental
process of cells and tissue from a less
specialized to a more specialized state is called
differentiation in developmental biology.

www.wikipedia.org
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Glossary I
Totipotency Ability of a cell to give rise to all
cells of an organism, including embryonic and
extraembryonic tissues. Zygotes are
totipotent. Pluripotency Ability of a cell to
give rise to all cells of the embryo. Cells of
the inner cell mass (ICM see below) and its
derivative, embryonic stem (ES) cells, are
pluripotent. Multipotency Ability of a cell to
give rise to different cell types of a given cell
lineage. These cells include most adult stem
cells, such as gut stem cells, skin stem cells,
hematopoietic stem cells and neural stem
cells. Unipotency Capacity of a cell to sustain
only one cell type or cell lineage. Examples are
terminally differentiated cells, certain adult
stem cells (testis stem cells) and committed
progenitors (erythroblasts).

Hochedlinger, Development 136, 509 (2009)
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Chromatin-remodelling enzymes
Inner cell mass (ICM) Cells of the blastocyst
embryo that appear transiently during development
and give rise to the three germ layers of the
developing embryo. Embryonic stem (ES) cells
Pluripotent cell line derived from the ICM upon
explantation in culture, which can differentiate
in vitro into many different lineages and cell
types, and, upon injection into blastocysts, can
give rise to all tissues including the
germline. Primordial germ cells (PGCs) PGCs
give rise to oocytes and sperm in vivo and to
embryonic germ (EG) cells when explanted in vitro.

Hochedlinger, Development 136, 509 (2009)
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Adult stem cells
Embryonic stem cells only exist in the early
embryo. We all possess adult stem cells, from
which new specialized cells are formed throughout
our life time. Adult cells exist predominantly
in bone marrow (dt. Knochenmark), but also in
skin, fat tissue, umbilical cord, brain, liver,
and in pancreas (dt. Bauchspeichel-drüse). Adult
cells in cell culture have a much reduced ability
of self regeneration and a reduced ability for
differentiation compared to embryonic stem
cells. For example, neural stem cells can
differentiate to all cell types of neural tissue
(neorons, glia), but likely not into liver or
muscle cells.

www.wikipedia.org
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• In this figure, the pluripotent cells of the
  embryo are tracked in green.
• From left to right, the morula-stage
• mouse embryo (embryonic day
• 2.5 E2.5) holds a core of
• pre-ICM (inner cell mass)
• cells that turn into ICM cells
• at cavitation/blastulation (E3E4).
• At this stage, embryonic stem cell (ESC) and
  Trophoblast Stem Cell (TSC)
• cell lines can be derived in vitro, and
  implantation occurs in vivo. ...
• As the blastocyst fully expands (and undergoes
  implantation in vivo),
• the ICM delaminates giving rise to a primitive
  ectoderm and a
• primitive endoderm layer.
• At this stage, pluripotent cell lines that are
  known as embryonal carcinoma cells (ECCs)
• can be derived from the primitive ectoderm ...
• At E6 and subsequent stages, the experimental
  ability to derive ESCs, TSCs and ECCs from the
  mouse embryo is progressively lost, and the in
  vivo embryo will start gastrulating. This process
  involves the formation of a mesoderm layer
  between ectoderm and endoderm, and the formation
  of the primordial germ cells (PGCs).

Boiani Schöler, Nat Rev Mol Cell Biol 6, 872
(2005)
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3 primary germ cell layers

• The ectoderm is the outer layer of the early
  embryo. It emerges first and forms from the outer
  layer of germ cells.
• The ectoderm differentiates to form the nervous
  system (spine, peripheral nerves and brain),
  tooth enamel and the epidermis. It also forms the
  lining of mouth, anus, nostrils, sweat glands,
  hair and nails.
• The endoderm develops at the inner layer. Its
  cells differentiate to form the gastrointestinal
  tract, the respiratory tract, endocrine glands
  and organs, auditory systems, and the urinary
  system.
• The mesoderm is the middle layer. It
  differentiates to give rise to a number of
  tissues and structures including bone, cartilage,
  muscle, connective tissue (including that of the
  dermis), the middle layer of the skin, blood
  vascular, reproductive, excretory and
  urinogenital systems and contributes to some
  glands.

www.wikipedia.org
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Chronology of stem cell research

• 1998 embryonic stem cells
• In 1998, James Thomson (US) isolated for the
  first time embryonic stem cells from surplus
  embryos left over in fertilization clinics.
• Since then, the research has progressed at an
  incredible speed.
• Ethics pro
• ESC have the potential to grow replacement tissue
  for patients with diabetes, Parkinson or other
  diseases.
• Ethics contra
• The technique requires destroying embryos. This
  has big ethical consequences.
• In Germany, experimentation with humans is
  considered problematic due to the medical
  experiments pursued during the Nazi time.
• Therefore, the above methods are forbidded by law
  in Germany!
• Researchers are looking for new ways to generate
  stem cells without ethical problems.
• Spiegel Online

Cellular Programs
WS 2010 lecture 4
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Chronology of stem cell research

• 2006 - Induced pluripotent stem cells (iPS)
• The first solution was presented in August 2006
  by the two Japanese Kazutoshi Takahashi and
  Shinya Yamanaka.
• Using 4 control genes, they reprogrammed cells
  from mouse tail into a sort of embryonic state.
  The product was termed induced pluripotent stem
  cells (iPS cells).
• Drawback if used for medical treatment later,
  the inserted genes could enhance the risk of
  cancer.
• 2007 human iPS cells
• In 2007, similar success was managed with human
  skin cells.
• Fewer and fewer control genes are necessary to
  generate iPS cells.
• Spiegel Online

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How can one show that iPS cells have stem cell
potential?

Kim et al. Cell 136, 411 (2009)
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Chronology of stem cell research

• February 2009 only one reprogramming gene
  required
• In February 2009, Hans Schöler presented iPS
  cells of mice that were reprogrammed using only a
  single control gene from neural stem cells (paper
  V9).
• March 2009 Reprogramming gene removed
• Begin of March 2009 2 teams of researchers
  present iPS cells that do not contain additional
  control genes in the genome.
• Control genes were first inserted into the genome
  of human skin cells, and later removed.
• March 2009 Reprogramming gene not in genome
• End of March 2009 James Thomsom showed that
  control genes do not need to be inserted into the
  genome of the cells. He introduced an additional
  plasmid (ring genome) into the cell that was
  later removed.
• Spiegel Online

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Chronology of stem cell research

• April 2009 Reprogramming of mouse cells
  without genes
• Ende of April 2009 Sheng Ding (US) and others
  succeed to reprogram skin cells of mice into iPS
  without gene manipulations using proteins only.
• This eliminates the risk of cancer due to
  insertion of genes.
• May 2009 Reprogramming of human cells without
  genes
• US-korean team around Robert Lanza manages to
  reprogram human cells into iPS cells using
  proteins (TFs) only.
• Spiegel Online

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Direct reprogramming of cells (November 2010)
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Central regulator Oct4
POU domain is a part of Oct1 and Oct4. (Left)
Atomic X-ray structure of the complexes of the
transcription factors POU and FGF4 with
DNA. (Right) Atomic X-ray structure of the
complexes of the transcription factors POU and
UTF1 with DNA. Note the slightly different
positions of the TFs which lead to recognition of
slightly different DNA motifs.

Remenyi et al. Genes Dev 15, 2048 (2003)
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Gene regulation network around Oct4
Oct4 is part of a tightly interconnected network
involving 9 TFs that keep ES cells in the
pluripotent state. The master regulator Oct4 as
well as Sox2 and Dax1 have autoregulatory
feed-forward feedback loops.

Kim et al. Cell 132, 1049 (2008)
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Gene regulation network around Oct4
In this complicated network, the concentration
levels of the various TFs affect eachother in a
balanced manner of mutual control. The
concentration of Oct4 inside ES cells must be
regulated within a narrow interval. Already a
two-fold increase of Oct4 concentration causes
differentiation into primitive endoderm and
mesoderm A 50 decrease leads to differentiation
into trophoectoderm.

Kellner, Kikyo, Histol Histopathol 25, 405 (2010)
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Complicated regulation of Oct4

Kellner, Kikyo, Histol Histopathol 25, 405 (2010)
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Gene regulation network around Oct4
Experiments showed that 6632 human genes contain
binding motifs in their promoter regions for at
least one out of the nine TFs. Interestingly,
many genes contain more than one binding motif.
800 genes bind four and more transcription
factors. Kim et al. suggested that multiple
transcription factors bind simultaneously as
protein complexes.

Kim et al. Cell 132, 1049 (2008)
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Detect target genes of Oct4

• Antibodies against Oct4
• fish all DNA, that binds Oct4
• sequence DNA pieces

Boyer et al. Cell 122, 947 (2005)
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Assign target genes of Oct4
Chavez et al. BMC Genomics 10, 314 (2009)
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Promotors of 2 Oct4 target genes

Chavez et al. BMC Genomics 10, 314 (2009)
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Chavez et al. BMC Genomics 10, 314 (2009)
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Was machen die Oct4-Targetgene in der Zelle?

Chavez et al. BMC Genomics 10, 314 (2009)
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Dynamic mathematical simulations
Chickarmane et al. PLoS Comp Biol 2, e123 (2006)
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Dynamic mathematical simulations
Chickarmane et al. PLoS Comp Biol 2, e123 (2006)
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Summary

• Stem cell therapy may(!) open immense
  possibilities for future medicine.
• Harvesting pluripotent embryonic stem cells is
  forbidden in Germany. Experimentation with adult
  stem cells is very restricted.
• Ideally pluripotent stem cells should be grown
  directly from patient tissue.
• Reprogramming of differentiated cells opens up
  completely new therapies.
• Role of bioinformatics
• statistical preparation of experimental raw data
  (signal/noise)
• functional annotation of findings
• integrate individual data into network model
• simulate and predict effects of perturbations