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H2 A-level Biology by Ms Yap

Syllabus 9744: Stem Cells


Definition/Unique Properties

Unspecialized
Long term self--renewal capability by reproducing itself for long period via mitosis
Can differentiate and give rise to specialized cells that make up tissues and organs of a body
-- Triggered by internal and external signals to undergo differential gene expression

-- Internal signals include activation of certain genes


-- External signals include chemicals secreted by other cells, cell--to--cell contact etc.

What is the difference between TOTIPOTENT, PLURIPOTENT, and MULTIPOTENT cells?

Totipotent:
These are stem cells capable of forming all cell
lineages of the three germ layers (endoderm,
ectoderm, and mesoderm) that exist in a human
body as well as extraembryonic tissue such as the
trophoectoderm and can give rise to a complete
human being.
Totipotency is a property maintained by the
fertilized egg (zygote) to at least the 4-cell stage of
the morula. The 8-cell morula is totipotent, and can
form everything.

Pluripotent:
These are stem cells that are capable of differentiating into all cell lineages of the three germ layers (endoderm, ectoderm,
and mesoderm) that exist in the human body but cannot give rise to a complete human being because they lack the
potential to form trophoectoderm that is needed for the development of extraembryonic tissues such as the placenta
and umbilical cord. The best example of pluripotent stem cells is embryonic stem cells. At 16- cell morula stage, cells have
already undergone molecular differentiation to become parts of the ICM (which gives rise to embryos three germ layers)
or the trophoectoderm/ trophoblast (which gives rise to the extraembryonic tissue). The ICM cannot form the
trophoblast/ trophoectoderm, and ICM is thus only pluripotent.

Multipotent:
These are stem cells that can develop
into a restricted subset of cell lineages.
Hematopoietic stem cells are a good
example of multipotent stem cells. They
can develop into any cell type certainly
within the limits of the hematopoietic
system and possibly also a few other
lineages.

Unipotent:
These are stem cells that can only
differentiate into a single type of
specialized cells or cell lineage, for
example adult muscle stem cells.
http://hamadyaseen.com/stem-cells.html

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Figure 1. The totipotent zygote is formed from
the sperm and egg.
After several rounds of division, totipotent zygote-
derived cells form blastocysts that differentiate
into trophoblasts that become the placenta, and
inner cell mass that forms the embryo. The inner
cell mass can be collected and expanded in
vitro under appropriate conditions to serve as the
pluripotent embryonic stem cells. Cells from
the gonads of the embryo can be collected and
expanded in vitro under defined conditions and
represent another potential source of pluripotent
stem cells. The embryonic stem cells of the inner
cell mass or the gonads can be differentiated into
cells of every lineage in the body. The
intermediate stem cells that are restricted to the
lineage of a particular organ are called
multipotent stem cells.http://www.nature.com/nm/journal/v7/n4/fig_tab/nm0401_393_F1.html

A. Injection of foreign DNA at totipotent stage ie oocyte can give rise to transgenic organisms, where foreign genes are
introduced into every cell in the organism, and every cell has been modified.

B. Injection of modified ES cells containing foreign DNA cannot give rise to transgenic organisms directly, because ES cells are
pluripotent and not totipotent, and can only give rise to chimeras. Modified ES cells returned into the ICM produce chimeras.

(Note: Microinjection is not into a single cell anymore, is into different cells where some cells get XX, some do not. Some
blastocysts have XX, some blastocysts do not have XX. )

Mouse ES cells can be put back into a mouse blastocyst and this blastocyst can then be returned to the uterus of a female mouse to
develop into a foetus. The injected ES cells take part in the development of the foetus and the resulting pup is born with a
mixture of cells, (a) from the host blastocyst and (b) cells that came from the injected ES cells. This new mouse with cells
from two different origins is known as a chimera.

ES cells can give rise to all adult cell types, including germ line cells. They are however, not totipotent in the same sense as the
fertilised oocyte. If implanted in a uterus, they are unable to give rise to an embryo. ES cells can contribute to germ line in
chimeras. Mouse ES cells injected into blastocyst from a different strain and implanted into a pseudopregnant foster mother to
form a chimeric animal. The injected cells may form germ cells (germ line transmission) and later these cells will produce
oocytes or sperms.

Figure 8.5. Generation of transgenic and knock out mice (blastocyst injection)

(FYI) Inner cell mass blastomeres can be isolated from an


embryo and cultured in vitro; such cultured cells are
called embryonic stem cells (ES cells). ES cells are almost
totipotent, since each of them can contribute to all tissues
except the trophoblast if injected into a host embryo.
Moreover, once in culture, these cells can be treated as
described in the preceding section so that they will
incorporate new DNA. This added gene (the transgene) can
come from any organism. A treated ES cell (the entire cell,
not just the DNA) can then be injected into another
early-stage embryo, and will integrate into this host. The
result is a chimeric mouse. Some of the chimeras cells will be derived from the hosts own inner cell mass, but
some portion of its cells will be derived from the treated embryonic stem cell. If the treated cells become part of
the germ line of the mouse, some of its gametes will be derived from the donor cell. If such a chimeric mouse is
mated with a wild-type mouse, some of its progeny will carry one copy of the inserted gene. When these
heterozygous progeny are mated to one another, about 25 percent of the resulting offspring will be homozygous
for (i.e., carry two copies of) the inserted gene in every cell of their bodies. Thus a gene cloned from some other
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organism will be present in both copies of the chromosomes within these mouse genomes. Strains of such
transgenic mice have been particularly useful in determining how genes are regulated during development.
Human Molecular Genetics 3rd edition, pg 582, http://10e.devbio.com/article.php?id=290,
http://www.tankonyvtar.hu/hu/tartalom/tamop425/0011_1A_Molekularis_terapiak_en_book/ch08.html

Stem Cells and Commitment

Generation of committed stem cells from pluripotent stem cells usually involves the formation of an
intermediate progenitor cell (e.g. myeloid progenitor cell)
Committed cells are those that have a more limited pathway of development compared to pluripotent
cells and are destined to produce a specific group of cells
Progenitor cells are partly differentiated cells that divide and give rise to differentiated cells
Stem Cells Cancer Cells
Can differentiate Cannot differentiate
Contact inhibition No contact inhibition
Do not metastasize Metastasizes
Long--term self--renewal capability conferred by telomerase

Asymmetric division is preceded by localization of regulatory molecules or differential segregation of cell


membrane proteins
Mitosis preserves a population of stem cells while producing a stream of differentiating cells

Stem cells give rise to stem cells and differentiating cells (HCI 2016 notes pg 11) Nathan,
http://rstb.royalsocietypublishing.org/content/363/1489/39

Significance: Mitotic division preserves population of undifferentiated cells, steadily produces stream of differentiating
cells
Symmetric division (mitosis): Produces 2
genetically identical cells (to parental cell)
o Scenario 1: Daughter cells can have
different fates if exposed to diff. signals
o Scenario 2: Daughter cells may differ
from birth if inherit diff. parts of
parental cell
Asymmetric division (mitosis): Produces 2 daughter cells with different cellular fates. Preceded by localisation of
regulatory molecules (cytoplasmic factors) or differential segregation of cell membrane proteins (e.g. receptors) between
daughter cells Produces stem cell + progenitor cell (with limited self-renewal potential)
o Scenario 1: Daughter cells may differ in size/shape/composition
o Scenario 2: Daughter cells genes may be in diff. states of (potential) activity Diff. internal signals confer diff.
fates on cells

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Comparison of Embryonic Stem Cells vs Adult Stem Cells

Embryonic stem cells Adult stem cells


From inner cell mass of blastocyst of donated fertilised From adult tissues/organs (and multipotent cells
Source eggs (IVF) / donated eggs fertilised by researchers in foetus, younger individual)
ES cells are defined by their origin (ICM of blastocyst) Origin of ASCs in mature tissues is unknown
Pluripotent (early embryonic cells totipotent, Multipotent (some ASCs able to become
Potency
but not used to make stem cell lines) pluripotent, but difficult to replicate studies)
1. Can become most cells/tissues of body
2. Easier to culture in lab
1. Less ethically problematic No destruction
3. Great potential for developing
of blastocyst involved
Advantages future therapy to cure disease
2. Already used in therapy (bone marrow
transplant)
ESCs easier to obtain pure and can be cultivated in large
numbers cf to ASCs
1. Hard to culture in lab
2. Most limited to become specific tissue types

Ethically problematic: Blastocyst must be destroyed when ASCs are rare and stem cell numbers in the body
Disadvantages
cells removed + Egg donation also problematic are very low making ASCs very difficult to
identify and purify. ASCs do not grow well in
culture where they often lose their distinctive
cellular properties.

Embryonic Stem Cells Blood Stem Cells


Both are unspecialized as they do not have any tissue specific functions
Both are capable of long--term self--renewal by symmetrical division
Both are able to give rise to fully differentiated cells carrying out specialized functions
Found in the inner cell mass of the blastocyst Found in the adult bone marrow
Pluripotent Multipotent
Can give rise to differentiated cell types that form Can give rise to all blood cells such as red blood
cells and white blood cells
More developmental pathways Fewer developmental pathways
Easy to obtain pure as they exist as a population of Difficult to obtain pure as they are dispersed in
cells in the blastocyst blood marrow tissues

Example question: UCLES N13/ P3/ Q5a (a) Describe features of zygotic stem cells and embryonic stem cells that distinguish
them from each other. [5]

Zygotic Stem cells Embryonic stem cells


1a. ZSCs are derived from zygote; 1b. ESCs are derived from embryos/ inner cell mass of the
blastocyst;
2a. ZSCs are totipotent; 2b. ESCs are pluripotent;
3a. Have the ability to give rise to all cell types that make up 3b. Have the ability to develop into all fetal tissues/ give rise
an organism;; to 3 germ layer;;
4a. ZSCs have the potential to become any cell of the extra- 4b. ESCs do not have the potential to make differentiated
embryonic membranes;; cells that form the extra-embryonic membranes;;
Describe how stem cells are differentiated into specialized cells in vitro. [3]

Stem cells are grown in culture medium containing a mixture of hormones and nutrients
This ensures they are capable of long term self--renewal
Hormones induce differentiation
Differential gene expression for (specialized cells) is turned on

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(FYI) Two main techniques to convert one cell type into another:
1. Reprogramming somatic cells into pluripotent stem cells (iPS cells),
2. Transdifferentiation to convert somatic cells directly into other types of specialized cells

The first technique, developed by Yamanaka in 2006, makes it possible to convert essentially any cell type in the body
back into pluripotent stem cells that are almost identical to embryonic stem cells. This is done by adding a quartet of
proteins: the transcription factors Oct4, Sox2, Klf4 and Myc. The resulting iPS cells can be grown and multiplied
almost indefinitely without losing their potential to differentiate (or change) into a broad range of cell types. If a
clinician wanted to use this technology to treat a patient with say, Parkinson's disease, she/he would prepare a skin
biopsy, grow skin-derived cells called fibroblasts in the lab, introduce the Yamanaka combination of four proteins and
wait a couple of months for the formation of stable populations or iPS cell lines. Since iPS cells can proliferate
indefinitely, they can be isolated relatively easily and a small initial population can be used to produce a large
number of cells. In our hypothetical Parkinsons treatment, the multiplied iPS cells would then be made to differentiate
into dopaminergic neurons, the cell type that is deficient in Parkinson's patients. As a final step the neurons would be
purified and injected back into the patient.

Fig: Comparison of iPS reprogramming and transdifferentiation: These processes might eventually be applied in
the clinic for cell replacement therapies.

The second technique, transdifferentiation, uses transcription factors to convert a given cell type directly into
another specialized cell type, without first forcing the cells to go back to a pluripotent state. Research in the 1980s
and 90s showed that fibroblasts can be converted directly into muscle cells at very high efficiencies using the
transcription factor MyoD.
http://www.eurostemcell.org/cell-replacement-therapies-ips-technology-or-transdifferentiation

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