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Lecture 2: Introduction to
Stem Cells
Why do we need stem cells?
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DEVELOPMENT
• The development of a multicellular organism
begins from a single cell that generates all tissues.
Stem cells play a central role in the developing
organism.
REGENERATION
• We lose millions of cells every second
• Without a supply of cells we will lack:
– Intestine: 2 days
– Skin: 3 weeks
– Red blood cells: 4 months
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Functional properties of stem cells
1. Self-renew
2. Proliferate
Stem cell
Daughter
progenitor cell
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3. Progeny can
differentiate
neuron
cardiomyocytes
Daughter stem
cell
blood cells
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Introduction to Stem Cells:
Embedded Assessment
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The Unique Properties of Stem Cells
• Discuss with a person next to you:
Define a stem cell. What makes stem cells different from other cells?
Why are these differences important to science and medicine?
Embryonic stem (ES) cells
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• Present in the inner cell mass
(ICM)
Zygote
Blastocyst
ICM
Hatching
Feeders
Expansion
• Pluripotent (can generate all
embryonic tissues but NOT
extra-embryonic tissues)
• Proliferate rapidly in culture
• Give rise to an entire organism
when transplanted into a
blastocyst
• Produce tumors when
transplanted into the adult
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Secreted factors maintain
pluripotency of ES cells in culture
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Secreted factors (proteins):
Feeders
ES cells
Colonies of mouse ES cells in culture
•
Feeder layer (fibroblasts) secretes proteins that
interact with receptors on the ES cell membrane
to maintain its pluripotency, either by promoting
self-renewal and/or suppressing differentiation.
•
LIF (Leukemia Inhibitory Factor) or basic
Fibroblast Growth Factor (bFGF) present in the
media binds the LIF receptor on the ES cell
plasma membrane, in order to maintain both
pluripotency and the rate of cell proliferation.
•
Serum contains BMPs (bone morphogenetic
proteins) that maintain pluripotency of mouse
ES cells but induce differentiation of human ES
cells.
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Intracellular factors that maintain
pluripotency of ES cells
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Transcription factors (Oct4, Nanog, Sox2)
• Proteins that are found in the nucleus
• Bind DNA and regulate gene expression
• Maintain pluripotency of ES cells by:
LI
F
Trophoblast
Sox2
Hypoblast
ES
cells
A) promoting acquisition of ES cell fate
and self-renewal ability
B) preventing ES cells from acquiring
other cell fates (extra-embryonic lineages
that are required for embryo
implantation into the uterus)
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Adult stem cells
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• Multipotent stem cells found in the adult animal
• Committed to a particular lineage (e.g. skin, blood,
nervous system or gut)
• Can generate several types of differentiated cells that
belong to a particular lineage
• Divide slowly and are difficult to grow in the lab
• If the tissue regenerates quickly (blood or gut) or is
injured, they divide rapidly
• They are self-renewing, committed to a tissue and used
throughout life
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Tissues maintained by
adult stem cells in the body
Blood
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Skin
Intestinal epithelium
Sperm
(Margaret Fuller)
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Relationship between ES cells and
adult stem cells
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Lineage restrictions
Pluripotent
Multipotent
Multipotent
differentiation
Neural stem
cell
brain
Ectodermal cell
ES cell
Skin stem cell
Mesodermal cell
Endodermal cell
skin
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Differences between ES cells and
adult stem cells
ES cells
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Adult stem cells
• Proliferate very rapidly in the
embryo
• Quiescent or proliferate very slowly
• Pluripotent stem cells (generate
all tissues of the embryo)
• Multipotent cells (committed to a
particular lineage such as skin)
• Occur only in the embryo
• Found in fetal and fully developed
tissues
• Completely undifferentiated
• Have partially differentiated into a
more mature type of cell
• Found in the inner cell mass
(ICM) at the blastocyst stage of
the embryo
• Present in small numbers
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Theurapeutic potential of human ES cells
versus adult stem cells
ES cells
• Can produce all tissues of the
embryo and have unlimited
therapeutic potential
• Their isolation and use is
controversial due to ethical issues
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Adult stem cells
• Can produce a limited range of
differentiated cells
• Can be harvested from patients
and have therapeutic potential
• Can cause tumors (teratomas) if
transplanted into adults.
• Hematopoietic stem cells have
been used successfully to treat
leukemias and other bone/blood
cancers
• Can be differentiated in culture to
make progenitors for various
lineages (directed differentiation)
and used for therapies.
• In some cases, are difficult to
isolate
• The directed differentiation
protocol has been used
successfully for many cell types
• Limited therapeutic use
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• Difficult to grow in the laboratory
Relationship between adult stem cells
and transit amplifying cells
Transit amplifying progenitors
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REPAIR
Differentiation
INJURY
Skin stem cell
skin
Skin stem cell
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References
Stem cell niche
Loss of
contact with
the niche
niche
stem cell
stem cell
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Differentiation
daughter
progenitor cell
differentiated
cells
• Local microenvironment where adult stem cells reside in the organism.
• Influences many properties of stem cells:
– Number of stem cells
– Stem cell cycle progression and type of division (symmetric versus
asymmetric)
– Self-renewal properties of stem cells
– Differentiation into specific lineages
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How do niche cells control the
properties of stem cell?
I. Secreted local factors
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• Niche cells secrete several factors that
are important for stem cell
maintenance.
Niche cells
• Niche cells secrete unique factors for
their respective stem cell.
• Hematopoietic stem cells are
maintained by several factors in their
niche:
Stem cell
a) Stem cell factor (c-Kit receptor)
b) Wnt (Frizzled/LRP receptor)
c) Angiopoietin-1 (Tie2 receptor)
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How do niche cells control the
properties of stem cell?
II.
Cell-cell communication
Cell-ECM communication
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•
Cell-cell communication at the niche occurs
through a variety of molecules.
•
Adhesion is important for holding stem cells in
the niche.
•
Adherens junctions are specialized intercellular
contacts important for cell-cell communication
and adhesion.
•
Cadherins and ß-catenin are components of
adherens junctions.
•
Integrins are a second class of molecules
responsible for adhering stem cells to the
extracellular matrix.
Niche cells
Stem cell
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Stem cell niche
in response to injury
Normal state
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INJURY
niche cells
Disruption of
cell contact
Upregulation of stem cell
stem cell factors differentiation
stem cell
quiescent state:
stem cells
divide slowly
increased stem
cell proliferation
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Somatic-derived stem cells via
nuclear transfer
Nucleus
Fibroblasts from
patients
Enucleated
oocyte
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directed differentiation
ES cell
Progenitor
Neuron
• Create ES cells that match the donor’s genetic makeup for therapeutic purposes.
• Currently, no human ES stem cell lines have been derived from this method.
• ES cells derived from patients can be directed to differentiate into specific lineages
(e.g. dopaminergic neurons) to study a particular disease (e.g. Parkinson’s disease).
• This method may be used for cell-based therapies that would circumvent immune
rejection.
• Not extensively used at present, because: 1) iPS strategies are more feasible, 2)
stress to the egg causes a reduced efficiency for ES cell generation.
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Induced pluripotent stem cells
(iPS cells)
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• A type of pluripotent stem cell artificially derived from an adult
somatic cell by "forcing" expression of specific genes.
• iPS cells are believed to be similar to ES cells with respect to:
– A) stem cell gene and protein expression
– B) ability to differentiate into all lineages in vitro
– C) forming viable chimeras after injection into blastocysts or
tumors when transplanted into adult tissues
– D) potential to form an entire organism, such as a mouse
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iPS cells - a recent advance
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• IPS cells were first produced in 2006 from mouse tissue, and in 2007
from human.
• This is an groundbreaking advance in stem cell research, because it
allows researchers to obtain pluripotent stem cells, which are
important in research and potentially have therapeutic uses, without
the controversial use of embryos.
• Reprogramming adult cells to obtain iPS cells may pose significant
risks that could limit their use in humans. If viruses are used to alter
the cells’ genome, the expression of cancer-causing genes or
oncogenes may potentially be triggered after these cells are introduced
into animals.
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References
Production of iPS cells
iPS reprogramming
factors
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directed
differentiation
ectodermal cell
iPS cell
mesodermal cell
brain
heart
fibroblasts from
patients
endodermal cell
pancreas
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iPS reprogramming factors
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• Retroviruses (viruses that contain RNA, and convert RNA into DNA)
that infect fibroblast cells are commonly used.
• Virus encodes four transcription factors: Oct4, Sox2, Klf-4 and c-Myc.
C-Myc is a tumor-inducing gene (oncogene).
• Oct4 and Sox2 are necessary to induce pluripotency of fibroblasts.
• Transcription factors increase the efficiency of iPS production.
• Currently, reprogramming is inefficient and slow.
• Transcription factors modify gene expression in infected cells.
• Factors turn OFF genes that are part of the differentiated phenotype.
• Factors turn ON genes that both maintain pluripotency and the ability to
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self-renew.
How do we identify stem cells in
the adult organism?
Mouse bearing a blue
marker in every cell (lacZ)
Isolate “blue” mammary
stem cells by expression of
cell surface proteins
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Transplant into
recipient mouse
Stem cells from the donor mouse
populate mammary tissue in the recipient mouse
and form lactating ducts
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How do we identify stem cells
in an adult?
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1. A single cell that resides in the stem cell niche is genetically marked in vivo
with green fluorescent protein (GFP).
2. Progenitors derived from the original green cell will be also green.
3. Differentiated cells that are born from green progenitors will also be green.
Therefore one can determine that the labeled cell is a stem cell or progenitor.
Red blood cell
white blood cell
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Introduction to Stem Cells:
Embedded Assessment
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Compare and Contrast Stem Cell Types
Expected lifespan in
tissue culture
Human Embryonic
Stem Cells (hESCs)
Adult Stem Cells
(Tissue-specific stem
cells: hematopoietic,
neural, pancreatic,
etc.)
Induced Pluripotent
Stem Cells (iPSCs)
Potency
Source
Developmental Stage
Introduction to Stem Cells:
Embedded Assessment Answer Key
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Compare and Contrast Stem Cell Types
Expected lifespan in
tissue culture
Potency
Source
Developmental
Stage
Human Embryonic Immortal: they divide Pluripotent, capable Made from the inner The blastocyst forms
Stem Cells (hESCs) endlessly in culture.
of making any cell or cell mass of a
very early in
tissue in the body.
blastocyst.
development, between
2 and 4 days.
Adult Stem Cells
Life span is limited, and Multipotent: They are Found in organs and
(Tissue-specific stemdepends on the type of “lineage restricted”, tissues of the body,
cells: hematopoietic, adult stem cell.
and make only specificsuch as heart, bone,
neural, pancreatic,
types of cells.
fat, brain, and liver.
etc.)
Develop during the
fetal stage, and persist
throughout
adulthood.
Induced Pluripotent Immortal: they appear iPS cells are thought In theory, any
Any somatic or body
Stem Cells (iPSCs) to divide endlessly in to be pluripotent.
somatic or body cell cell can be used, such
culture.
can be
as a skin cell.
reprogrammed to an
embryonic state.
Summary
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TYPES OF STEM CELLS:
• Embryonic stem (ES) cells
• Adult stem cells
• Somatic-derived stem cells via nuclear transfer (SCNT)
• Induced pluripotent stem (iPS) cells
STEM CELL NICHE:
• Secreted factors
• Cell-cell interactions
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Introduction to Stem Cells:
Concept Mapping Terms
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Add the key terms/concepts from today’s lecture to your
previous concept map. You should include (but are not limited
to) the following terms/concepts:
• Embryonic stem cell
• Adult stem cell
• Induced pluripotent stem cell
• Nuclear transfer
• Stem cell niche
• Transcription factor
• Self-renewal
• Lineage restriction
Due by Thursday, April 7
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