Stem cell and lineage

Stem cell in principle

The two defining characteristics of a stem cell are
perpetual self-renewal and
the ability to differentiate into a specialized adult
cell type.

Fig: The definition of a stem cell.

Each daughter produced when a stem cell divides can either remain a
stem cell or go on to become terminally differentiated.
In many cases, the daughter that opts for terminal differentiation
undergoes additional cell divisions before terminal differentiation is
completed; such cells are called transit amplifying cells.
 Stem Cells
 Stem cells are undifferentiated or partially differentiated cells that
can differentiate into various types of cells and proliferate
indefinitely to produce more of the same stem cell. 
 A stem cell is a relatively undifferentiated cell that when divides
produces
(i) one that retains its undifferentiated character;
(ii) a second cell that can undergo one or more paths of
differentiation
 Cells that are specialized to provide an indefinite supply of fresh
differentiated cells where these are lost, discarded, or needed in
greater numbers.
 Stem Cells
Thus, a stem cell has the potential to renew itself at each
division (so that there is always a supply of stem cells) while
also producing a daughter cell capable of responding to its
environment by differentiating in a particular manner. (This
potential is not always realized; in some instances, stem
cells divide symmetrically so that both daughter cells remain
stem cells.)
In many cases, the stem cell remains in the niche while its
sister leaves the niche and differentiates.
Why is there such an interest in stem cells?
• Increase understanding of how diseases occur- By watching stem cells
mature into cells in bones, heart muscle, nerves, and other organs and
tissue, researchers and doctors may better understand how diseases and
conditions develop.
• Generate healthy cells to replace diseased cells (regenerative
medicine). Stem cells can be guided into becoming specific cells that can
be used to regenerate and repair diseased or damaged tissues in people.
• Test new drugs for safety and effectiveness. Before using investigational
drugs in people, researchers can use some types of stem cells to test the
drugs for safety and quality. This type of testing will most likely first have
a direct impact on drug development first for cardiac toxicity testing.
Where do stem cells come from?
Embryonic stem cells- These stem cells come from embryos that are three to
five days old. At this stage, an embryo is called a blastocyst and has about 150
cells.
Adult stem cells- These stem cells are found in small numbers in most adult
tissues, such as bone marrow. Compared with embryonic stem cells, adult
stem cells have a more limited ability to give rise to various cells of the body.
Adult cells altered to have properties of embryonic stem cells (induced
pluripotent stem cells)- Scientists have successfully transformed regular adult
cells into stem cells using genetic reprogramming. By altering the genes in the
adult cells, researchers can reprogram the cells to act similarly to embryonic
stem cells.
Perinatal stem cells- Researchers have discovered stem cells in amniotic fluid as
well as umbilical cord blood. These stem cells also have the ability to change
into specialized cells.
 Where do these embryos come from in
embryonic stem cell research?
• The embryos being used in embryonic stem cell research
come from eggs that were fertilized at in vitro fertilization
clinics but never implanted in a woman's uterus.
• The stem cells are donated with informed consent from
donors.
• The stem cells can live and grow in special solutions in test
tubes or petridishes in laboratories.
 Why is there a controversy about using
embryonic stem cells?
• Embryonic stem cells are obtained from early-stage embryos
— a group of cells that forms when a woman's egg is
fertilized with a man's sperm in an in vitro fertilization clinic.
• Because human embryonic stem cells are extracted from
human embryos, several questions and issues have been
raised about the ethics of embryonic stem cell research.
Why can't researchers use adult stem cells
instead?
• Adult stem cells may not be as versatile and durable as are
embryonic stem cells.
• Adult stem cells may not be able to be manipulated to
produce all cell types
• Adult stem cells also are more likely to contain abnormalities
due to environmental hazards, such as toxins, or from errors
acquired by the cells during replication.
• However, researchers have found that adult stem cells are more
adaptable than was first thought.
Two ways for a stem cell to produce daughters
with different fates
Two ways for a stem cell to produce daughters with different fates:
asymmetric division and independent choice
Two ways for a stem cell to produce daughters with different fates: asymmetric division and independent choice.
(A) The asymmetric-division strategy gives a clone consisting of precisely one stem cell plus a steadily increasing number of differentiating
cells, in proportion to the number of cell divisions.
(B) The independent-choice strategy is more variable in its outcome. With a choice made at random by each daughter and with a 50%
probability for each one to remain a stem cell or differentiate, there is, for example, a 25% chance at the first division that both daughters
will differentiate, so that the clone eventually goes extinct. Or, at this division or later, a preponderance of daughters may chance to retain
stem-cell character, creating a clone that persists and increases in size. With the help of some mathematics, the probability distribution of
clone sizes generated from a single stem cell at any given time can be predicted on this stochastic assumption.
The observations in the gut and elsewhere fit the stochastic independent-choice strategy, but not the asymmetric-division strategy.
Properties of somatic stem cell
• When a stem cell divides at least one daughter cell will be a
stem cell, the other can terminally differentiate
• Stem cells can divide indefinitely
• A daughter cell can differentiate in at least one cell type
• In some organs, such as the gut, epidermis, and bone
marrow, stem cells regularly divide to replace worn-out cells
and repair damaged tissues.
• In other organs, such as the prostate and heart, stem cells
divide only under special physiological conditions, usually in
response to stress or the need to repair the organ.
Blood-forming (hematopoietic) stem cells and
the bone marrow niche
Blood-forming (hematopoietic) stem cells and
the bone marrow niche
The hematopoietic stem cell is located in
the bone marrow and generates a second
stem cell that is capable of becoming
either a lymphocyte progenitor cell (which
divides to form the cells of the immune
system) or a myeloid stem cell (which
forms the blood cell precursors).

The type of lineage that the cells take is regulated by the niche, which involves
contact between the stem cells and the matrices of bone cells, paracrine factors
from stromal cells and the pericytes surrounding the blood vessels, and systemic
hormones and neural signals. Mesenchymal stem cells also use the bone marrow as
a niche.
Stem Cell Vocabulary
The names of the two major divisions of stem cells are based on their
sources.
 Embryonic stem cells are derived from the inner cell mass of
mammalian blastocysts or from fetal gamete progenitor (germ) cells.
These cells are capable of producing all the cells of the embryo (i.e., a
complete organism).
 Adult stem cells are found in the tissues of organs after the organ
has matured. These stem cells, which are usually involved in replacing
and repairing tissues of that particular organ, can form only a subset
of cell types.
 Production and pluripotency of ES cells

Production and pluripotency of ES cells. ES cells are derived from the inner cell mass (ICM) of the early embryo. The
ICM cells are transferred to a culture dish containing an appropriate medium, where they become converted to ES
cells and can be kept proliferating indefinitely without differentiating. The ES cells can be taken at any time—after
genetic manipulation, if desired—and injected back into the inner cell mass of another early embryo. There they
take part in formation of a well-formed chimeric animal that is a mixture of ordinary and ES-derived cells. The ES-
derived cells can differentiate into any of the cell types in the body, including germ cells from which a new
generation of mice can be produced. These next-generation progeny are no longer chimeric, but consist of cells that
all inherit half their genes from the cultured ES cell line.
 Embryonic stem cells
Adult stem cells

Totipotent cells
Pluripotent stem cells
Multipotent stem cells
Unipotent stem cells

Progenitor cells/ Transit-amplifying cells

Fig: Example of the maturational series of stem cells

Stem Cell Potency
The ability of a particular stem cell to generate numerous different types of
differentiated cells is its potency.
 In mammals, totipotent cells are capable of forming every cell in the
embryo and, in addition, the trophoblast cells of the placenta. The only
totipotent cells are the zygote and (probably) the first 4-8 blastomeres to
form prior to compaction.
 Pluripotent stem cells have the ability to become all the cell types of
the embryo except trophoblast. Usually these embryonic stem cells are
derived from the inner cell mass of the mammalian blastocyst. However,
germ cells and germ cell tumors (such as teratocarcinomas) can also form
pluripotent stem cells.
Stem Cell Potency
 Multipotent stem cells are stem cells whose commitment is limited to a
relatively small subset of all the possible cells of the body. These are
usually adult stem cells. The hematopoietic stem cell, for instance, can
form the granulocyte, platelet, and red blood cell lineages. Similarly, the
mammary stem cell can form all the different cell types of the mammary
gland.
 Some adult stem cells are unipotent stem cells, which are found in
particular tissues and are involved in regenerating a particular type of
cell. Spermatogonia, for example, are stem cells that give rise only to
sperm.
Whereas pluripotent stem cells can produce cells of all three germ layers (as well as producing
germ cells), the multipotent and unipotent stem cells are often grouped together as
committed stem cells, since they have the potential to become relatively few cell types.
PROGENITOR CELLS
Although they are related to stem cells, progenitor cells are not capable of
unlimited self-renewal; they have the capacity to divide only a few times
before differentiating.
They are sometimes called transit-amplifying cells, since they usually
divide while migrating away from the stem cell niche.
Both unipotent stem cells and progenitor cells have been called lineage
restricted cells.
Progenitor cells are usually more differentiated than stem cells and have
become committed to become a particular type of cell.
In many instances, stem cell division generates progeny that become
progenitor cells, as in the formation of the blood cells, sperm cells, and the
nervous system
Transient amplifying cells
Multipotent Hematopoietic cells
Fig: A tentative scheme of hematopoiesis.
The multipotent stem cell
normally divides infrequently to
generate either more
multipotent stem cells, which
are self-renewing, or committed
progenitor cells, which are
limited in the number of times
that they can divide before
differentiating to form mature
blood cells.
Multipotent Hematopoietic cells
Fig: A tentative scheme of hematopoiesis.
As they go through their divisions, the
progenitors become progressively
more specialized in the range of cell
types that they can give rise to, as
indicated by the branching of this cell-
lineage diagram.
In adult mammals, all of the cells
shown develop mainly in the bone
marrow—except for T lymphocytes,
which as indicated develop in the
thymus, and macrophages and
osteoclasts, which develop from blood
monocytes. Some dendritic cells may
also derive from monocytes.
Stem Cells Depend on Contact Signals From
Stromal Cells
 Like the stem cells of other
tissues, hematopoietic stem
cells depend on signals from
their niche, in this case created
by the specialized connective
tissue of the bone marrow.

When they lose contact with

their niche, the hematopoietic
stem cells tend to lose their
stem-cell potential.
Fig: Dependence of hematopoietic stem
cells on contact with stromal cells.
Stem Cells Depend on Contact Signals From
Stromal Cells
The contact-dependent interaction between
the Kit receptor and its ligand is one of several
signaling mechanisms thought to be involved in
hematopoietic stem-cell maintenance.
The real system is certainly more complex.
Moreover, the dependence of hematopoietic
cells on contact with stromal cells cannot be
absolute, since small numbers of the functional
stem cells can be found free in the circulation.
SCF, stem-cell factor.
Fig: Dependence of hematopoietic stem
cells on contact with stromal cells.
Factors That Regulate Hematopoiesis Can Be
Analyzed in Culture
• While the stem cells depend on contact with bone marrow
stromal cells for longterm maintenance, their committed
progeny do not, or at least not to the same degree.
• These cells can thus be dispersed and cultured in a semisolid
matrix of dilute agar or methylcellulose, and factors derived
from other cells can be added artificially to the medium.
• The semisolid matrix inhibits migration, so that the progeny
of each isolated precursor cell remain together as an easily
distinguishable colony.
Factors That Regulate Hematopoiesis Can Be
Analyzed in Culture
• A single committed neutrophil progenitor, for example, may give rise to a
clone of thousands of neutrophils.
• Such culture systems have provided a way to assay for the factors that
support hematopoiesis and hence to purify them and explore their
actions.
• These substances are glycoproteins and are usually called
colony-stimulating factors (CSFs). Some of these factors
circulate in the blood and act as hormones, while others act in
the bone marrow as secreted local mediators; still others take
the form of membrane-bound signals that act through cell– cell
contact.
Factors That Regulate Hematopoiesis Can Be
Analyzed in Culture
• An important example of the latter is a protein called Steel or Stem
Cell Factor (SCF).
• This is expressed both in the bone marrow stroma (where it helps to
define the stem-cell niche) and along pathways of migration, and it
occurs both in a membrane-bound and a soluble form.
• It binds to a receptor tyrosine kinase called Kit, and it is required
during development for guidance and survival not only of
hematopoietic cells but also of other migratory cell types—
specifically, germ cells and pigment cells.
Erythropoiesis Depends on the
Hormone Erythropoietin
The best understood of the Colony Stimulating
Factors (CSFs) that act as hormones is the
glycoprotein erythropoietin, which is produced
in the kidneys and regulates erythropoiesis, the
formation of red blood cells.

Fig: A developing red blood cell (erythroblast). The cell is shown extruding its nucleus to
become an immature erythrocyte (a reticulocyte), which then leaves the bone marrow and
passes into the bloodstream. The reticulocyte will lose its mitochondria and ribosomes
within a day or two to become a mature erythrocyte. Erythrocyte clones develop in the
bone marrow on the surface of a macrophage, which phagocytoses and digests the nuclei
discarded by the erythroblasts.
Erythropoiesis Depends on the
Hormone Erythropoietin
In an erythrocyte of an adult mammal, even the nucleus,
endoplasmic reticulum, mitochondria, and ribosomes are
absent, having been extruded from the cell in the course of its
development. The erythrocyte therefore cannot grow or divide,
and it has a limited life-span—about 120 days in humans.
A lack of oxygen or a shortage of erythrocytes stimulates
specialized cells in the kidney to synthesize and secrete
increased amounts of erythropoietin into the bloodstream. The
erythropoietin, in turn, boosts the production of erythrocytes.
The effect is rapid: the rate of release of new erythrocytes into
the bloodstream rises steeply 1–2 days after an increase in
erythropoietin levels in the bloodstream. Clearly, the hormone
must act on cells that are close precursors of the mature
erythrocytes.
Parameters through which the
production of blood cells of a
specific type might be regulated
CSFs are defined as factors that
promote the production of colonies of
differentiated blood cells.
The factor might control the rate of
cell division or the number of division
cycles that the progenitor cell undergoes
before differentiating;
it might act late in the hematopoietic
lineage to facilitate differentiation;
it might act early to influence
commitment; or Fig: Some of the parameters through which the production
it might simply increase the probability of blood cells of a specific type might be regulated. Studies
in culture suggest that various colony-stimulating factors
of cell survival (CSFs) can affect all of these aspects of hematopoiesis.
A Core Set of Transcription Regulators
Defines and Maintains the ES Cell State
What is it that gives ES cells and related types of pluripotent stem cells
their extraordinary capabilities?
What can they tell us about the fundamental mechanisms underlying
stemness, cell differentiation, and the stability of the differentiated
state?
1. ES cells must avoid senescence. ES cells express high levels of
active telomerase, allowing them to escape senescence and
continue dividing indefinitely.
2. Genes that seem to be essential in one way or another for
the peculiar character of ES cells.
A Core Set of Transcription Regulators
Defines and Maintains the ES Cell State
 For example: A gene called Oct4 is exclusively expressed in ES
cells and in related classes of cells in the intact organism—
specifically, in the germ-cell lineage and in the inner cell mass
and its precursors.
 Oct4 codes for a transcription regulator.
 When it is lost from ES cells, they lose their ES cell character; and
when it is missing in an embryo, the cells that should specialize as
inner cell mass are diverted into an extra embryonic pathway of
differentiation and their development is aborted.
 Creating induced Pluripotent Stem cells (iPS)
 Fibroblasts and some other cell types
can be driven to switch their character and
differentiate as muscle cells if the muscle-
specific transcription regulator MyoD is
artificially expressed in them.
Could the same technique be used
to convert adult cell types into ES
cells, through forced expression of
factors such as Oct4?
A total of 24 candidate ES-critical genes were
tested.
 Creating induced Pluripotent Stem cells (iPS)
A total of 24 candidate ES-critical genes were tested.
None of them was able by itself to cause the conversion; but
a core set of four factors, all of them transcription
regulators: Oct4, Sox2, Klf4, and Myc (OSKM
factors) when coexpressed, reprogram mouse
fibroblasts, permanently converting them into cells
closely similar to ES cells.
ES-like cells created in this way are called induced
pluripotent stem cells, or iPS cells.
Like ES cells, iPS cells can continue dividing
indefinitely in culture, and when incorporated into
a mouse blastocyst they can participate in creation
of a perfectly formed chimeric animal.
 Creating induced Pluripotent Stem cells (iPS)
Fig: Reprogramming fibroblasts to IPS cells with
the OSKM factors. As indicated, the master gene
regulator proteins Oct4, Sox2, and Klf4 (OSK)
induce both their own and each other's synthesis
(gray shading). This generates a self-sustaining
feedback loop that helps to maintain cells in an
embryonic stem cell-like state, even after all of the
experimentally added OSKM initiators have been
removed. Myc overexpression speeds up early
stages of the reprogramming process through the
mechanisms shown. Stable reprogramming also
involves the permanently induced expression of
the Nanog gene, which produces an additional
master transcription regulator.
Conversion to an iPS character by the OSKM
factors is not only inefficient but also slow
• Fibroblasts take ten days or more from introduction of the
conversion factors before they begin to express markers of the iPS
state.
• Only a few of the cells that receive the OSKM factors will actually
become iPS cells—one in several thousand in the original
experiments (efficiencies about 0.01–0.1%).
• This suggests that the transformation involves a long cascade of
changes.
• Changes are being extensively studied, and they affect both the
expression of individual genes and the state of the chromatin.
 Selection of cells that have converted to an iPS
character
The experiment makes use of a gene (Fbx15) that is present in all cells but is
normally expressed only in ES and early embryonic cells (although not required
for their survival).
A fibroblast cell line is
genetically engineered to
contain a gene that
produces an enzyme that
degrades G418 under the
control of the Fbx15
regulatory sequence.
G418 is an aminoglycoside
antibiotic that blocks protein
synthesis in both bacteria and
eukaryotic cells.
 Selection of cells that have converted to an iPS
character
When the OSKM factors are artificially expressed in this cell line, a small
proportion of the cells undergo a change of state and activate the Fbx15
regulatory sequence, driving expression of the G418-resistance gene.

When G418 is added to the

culture medium, these are
the only cells that survive
and proliferate.
When tested, they turn out
to have an iPS character.
 Events in reprogramming of fibroblasts into
iPS cells
Fig: A summary of some of the major events
that accompany the reprogramming of mouse
fibroblasts to iPS cells.
By sorting cells at various times
after the OSKM induction shown,
one can carry out detailed
biochemical analyses on the
different cell populations shown.
This led to the discovery that two
major waves of new gene
transcription are induced.
Major DNA methylation changes
are observed only after the
alteration of chromatin structures.
 Events in reprogramming of fibroblasts into
iPS cells
In the first transcription wave,
among the genes prominently
induced are those for cell
proliferation, metabolism, and
cytoskeletal organization; in
contrast, genes associated with
fibroblast development are
repressed.

In the second transcription wave,

genes required for embryonic
development and for stem cell
maintenance are induced.
ES and iPS Cells Can Be Guided to Generate
Specific Adult Cell Types
• In culture, by exposing the ES or iPS cell to an appropriate sequence of
signal proteins and growth factors, delivered with the right timing, it is
possible to guide the cell along a pathway that approximates a normal
developmental pathway, so as to convert it into one of the standard
specialized adult cell types (Success requires trial and error, but has now been achieved for
many different final specialized states, including neuronal, muscular, and intestinal cell types).
• If ES or iPS cells are implanted directly into an embryo at a later stage or
into an adult tissue, however, they fail to receive the appropriate
sequence of cues; their differentiation then is not properly controlled,
and they will often give rise to a tumor of the type known as a teratoma,
containing a mixture of cell types inappropriate to the site in the body.
 ES and iPS Cells Can Be Guided to Generate
Specific Adult Cell Types
These cells can be cultured indefinitely as pluripotent
cells when attached as a monolayer to a dish.
Alternatively they can be detached and allowed to
form aggregates called embryoid bodies, which
causes the cells to begin to specialize.

Cells from embryoid bodies, cultured in media

with different factors added, can then be
driven to differentiate in various ways
Fig: Production of differentiated cells from ES or iPS cells in culture
 Use of iPS cells for drug discovery and for
analysis and treatment of genetic disease
The left side of the The right side of the
diagram shows how diagram shows how
differentiated cells the genetic defect
that are generated might be repaired in
from iPS cells derived the iPS cells, which
from a patient with a could then be induced
genetic disease can be to differentiate in an
used for analysis of appropriate way and
the disease grafted back into the
mechanism and for patient without
discovery of danger of immune
therapeutic drugs. rejection.
 An example: Timothy syndrome
• In this rare genetic condition, there is a severe, life-threatening disorder in
the rhythm of the heart beat (as well as several other abnormalities), as a
result of a mutation in a specific type of Ca2+ channel.
• To study the underlying pathology, researchers took skin fibroblasts from
patients with the disorder, generated iPS cells from the fibroblasts, and drove
the iPS cells to differentiate into heart muscle cells.
• These cells, when compared with heart muscle cells prepared similarly from
normal control individuals, showed irregular contractions and abnormal
patterns of Ca2+ influx and electrical activity that could be characterized in
detail.
• From this finding, it is a small step to development of an in vitro assay for
drugs that might correct the misbehavior of the heart muscle cells.
 Fill in the blank
• With the advent of iPS cells and direct transdifferentiation, one major
hurdle has been surmounted: the problem of …………………………………..
ES cells, because they are created from early embryos that generally
come from unrelated donors, will never be genetically identical to the
cells of the patient receiving the transplant. The transplanted cells
and their progeny are therefore liable to rejection by the immune
system. Both iPS and transdifferentiated cells, in contrast, can be
generated from a small sample of the patient’s own tissue and so
should escape immune attack when transplanted back into the same
individual.