Work Force Development: Stem Cell Primer
Lecture 2
Learning Items B, C, D, E, R and V
Essential questions (R, B and V)
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What are embryonic stem cells and where do they come from?
What are adult stem cells and where are they found?
What are the major differences between embryonic and adult stem cells?
What are the major types of artificially-derived stem cells?
What is the stem cell niche and what role does it play?
How do we determine if a specific cell is a stem cell?
Key knowledge and skills will you acquire as a result of this lecture
Students will know:
 Key Terms: embryonic, adult, and induced pluripotent stem cells, nuclear
transfer, the niche, signaling molecule, transcription factor, self-renewal and
lineage restriction.
 The origin of embryonic stem (ES) cells, markers that define these cells and
factors that maintain their pluripotency.
 Various types of adult stem cells, their biological purposes and properties.
 The basic differences between embryonic and adult stem cells, at both the
biological and therapeutic level.
 Artificially derived stem cells (somatic-derived stem cells via nuclear transfer
and iPS cells) and their therapeutic potential.
 The role of the stem cell niche in maintaining pluripotency of stem cells and
controlling their proliferation.
Students will be able to:
 Compare and contrast various types of stem cells, their origins and their
biological and therapeutic purposes.
 Describe how secreted or intracellular factors are required to maintain the
pluripotency of embryonic stem cells.
 Describe how injury or damage can affect the stem cell niche and the biology of
stem cells.
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Why do we care about stem cells?
Stem cells have taken center stage in basic and medical research, as they provide a
great resource for clinical use in many diseases. In order to understand the full potential of
these cells for therapeutic purposes, it is essential to review their biological functions. Stem
cells are important cells during development and in adult organisms (slide 1). Tissues
undergo significant wear and tear during our lifetime. We lose millions of cells every
second, billions every hour. If we were unable to resupply these cells we would lose our
intestine in two days, our skin in three weeks and our red blood cells in four months. In
order to counteract these processes, cells and tissues must be rebuilt at a prolific rate every
day. Stem cell populations are reserves to replenish injured, aging or dead cells in
organisms.
Properties of stem cells
The term “stem cell” describes a biological function, and both embryonic and adult
stem cells have the following properties (slide 2): a) they are undifferentiated, immature
cells; b) they regenerate themselves in a process termed “self-renewal” (depicted with the
brown circular arrow); and c) they are able to differentiate or mature into other cell types.
The most incredible property of stem cells is their capacity for self-renewal. Stem cells are
also sometimes able to divide at different rates throughout our life. They can either selfrenew or differentiate because they undergo either symmetric or asymmetric cell division.
Stem cells can divide by a symmetric division to generate two cells with functional
properties identical to the parental cell, thus expanding the number of stem cells.
Alternatively stem cells can undergo asymmetric division to produce a daughter stem cell
identical to the parent stem cell, plus a progenitor cell that can differentiate into various
cell types.
Embryonic stem cells
Embryonic stem (ES) cells are generated from the developing embryo (blastocyst
stage) (slide 4). They are derived from cells within the inner cell mass (ICM) of the early,
pre-implantation embryo and are pluripotent (i.e. can generate all tissues and cells of the
body). ES cells do not normally generate extra-embryonic tissues (e.g. trophoblast or
hypoblast) that are necessary for implanting the embryo in the uterus or regulating the
process of gastrulation. Thus, though very primitive cells in a developmental sense, they
are already somewhat restricted in terms of what tissue types they may form. ES cells can
proliferate rapidly in culture to generate more pluripotent ES cells (i.e. they self-renew).
Experimentally, ES cells differentiate into other cell types depending on the environment in
which they are grown. When mouse ES cells are transplanted inside a blastocyst, they
contribute to the entire normal organism. The growth rate of ES cells also gives them some
cancer-like properties. If they are implanted under the skin of a mouse, they are able to
form certain types of tumors known as teratomas. These tumors contain cells representing
the three primary germ layers of the body: ectoderm (such as skin and nerves), mesoderm
(including muscle, bone, and cartilage), and endoderm (gut and lung epithelium).
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ES cells are one type of pluripotent stem cell, whereas other varieties can be
obtained from multiple sources. Embryonic-like cells can be generated from morula cells (a
pre-blastula embryo of 8 cells), from naturally-occurring tumors like teratomas (so-called
“embryonal carcinoma” or EC cells) or from primordial germ cells (which are termed
“embryonic germ” or EG cells). Embryonic and embryonic-like cells can be made by other
technologies such as somatic cell nuclear transfer or creating induced pluripotent stem
cells (iPSCs). Both of these will be discussed below.
Compared to other commonly-used tissue culture cells, ES cells are relatively
challenging to grow in culture due to their robust capacity to differentiate spontaneously
into other cell types. The ability of ES cells to maintain pluripotency depends on many
factors. These can be classified into two categories: secreted and intracellular factors. ES
cells are normally cultured on a mitotically inactivate mouse fibroblast “feeder” layer that
helps to maintains ES cell pluripotency (slide 5). Fibroblasts secrete several molecules that
bind receptors in the plasma membrane of ES cells to promote self-renewal and suppress
ES cell differentiation. Some of these factors remain unknown. One factor that is necessary
for mouse ES cell culture is LIF – leukemia inhibitory factor, and basic fibroblast growth
factor (bFGF) is required for human ES cells. Both LIF and bFGF are secreted proteins. Each
binds to its receptor on the ES cell surface and influences gene expression. These factors
control: a) the rate of ES cell proliferation, b) cell cycle progression and c) activation of
gene signals that maintain a pluripotent state. LIF and bFGF are commonly added to their
respective ES cell growth media. Serum (blood fluid with the cells removed) is another
important component of mouse ES cell growth medium. Serum contains a multitude of
factors, including a class of proteins termed bone morphogenetic proteins (BMPs) that
are important for mouse ES cells to self-renew and remain pluripotent.
Intracellular proteins are a second group of factors that influence the pluripotency
of ES cells (slide 6). We’ll focus on three examples of these proteins that are particularly
important: Oct4, Nanog and Sox2. These proteins are called transcription factors. They
are localized in the nucleus of ES cells, where they bind DNA and regulate (i.e. turn “ON” or
“OFF”) gene expression. These proteins maintain ES cell pluripotency by turning ON genes
that maintain the self-renewal properties of ES cells. These transcription factors also
prevent ES cells from acquiring other fates, such as the extra-embryonic tissues necessary
for in utero implantation or more mature cell types like blood or nerve (as just two
examples), by turning OFF genes that promote these alternative fates. These transcription
factors have also emerged as being crucial for iPSC technology (see below).
Adult stem cells
Adult stem cells (also called tissue-specific stem cells) are multipotent meaning
they are capable of generating many (but not all) cell types. They are found in several
tissues of the adult organism (slide 7). Not all tissues and organs appear to have stem cells
(such as the heart for example), although research seeking to identify and/or activate stem
cells in such non-regenerative tissues is ongoing. Adult stem cells are committed
(restricted) to a particular lineage (e.g. skin, blood, nervous system or gut), and each type
produces more differentiated cells that belong to that specific lineage. Adult stem cells are
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very rare in tissues (often 0.001% or lower), divide slowly unless the tissue turns over
rapidly (e.g. gut) or becomes injured (e.g. a skin wound) and are a source of self-renewing
cells for that tissue during life. They maintain their self-renewing properties by
interactions with their local microenvironment, called the niche (see below).
Adult stem cells are quiescent (meaning they are undividing or at rest) and don’t
differentiate unless there is a need to replace tissue that is damaged or dead (slide 10). In
response to injury (e.g. a skin burn or wound), adult skin stem cells exit their quiescent
state and begin to divide rapidly, producing a stem cell (self-renewal) and an immature
progenitor. This pool of rapidly proliferating progenitor cells is called transit amplifying
cells. The transit amplifying cells divide a few times before they undergo differentiation
into distinct cell types (e.g. skin).
There are several different types of adult stem cells (slide 8, Table 1): a)
hematopoietic stem cells are found in the bone marrow and produce red and white blood
cells; b) skin stem cells are localized in the bulge at the base of a hair shaft within the skin,
and regulate both skin regeneration and hair growth; c) stem cells of the gut are found in
the crypts, or involutions of the small and large intestine, and divide very rapidly; d)
spermatogonia, which generate sperm cells by meiosis, also are a type of adult stem cell
present in the testes and are also called germ stem cells; e) neural stem cells are found in
various regions of the brain and spinal cord, and produce the astrocytes, glial cells and
neurons that constitute neural networks underlying smell, memory and learning; f)
mammary stem cells are found in breast tissue and produce the mammary ducts and
milk-producing cells after pregnancy in females; and g) mesenchymal stem cells (MSCs)
are found in loose connective tissue and may mature into a variety of tissues. MSCs have
been found in the placenta, fat tissue, lung, bone marrow, umbilical cord and teeth. MSCs
can differentiate into osteoblasts, adipocytes, and chondrocytes as well as myocytes and
possibly neuron-like cells. However, it is not known whether MSCs are “true” stem cells
because it has been difficult to isolate MSCs individually. It is unclear whether the unique
properties of MSCs are due to a mixture of progenitor cells in the culture or a single
multipotent cell type. This situation highlights some of the difficulties arising in general
from experiments to isolate and characterize stem cells.
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Table 1
Type of adult
stem cell
Hematopoietic
(HSCs)
Skin
Gut
Germ
Neural (NSCs)
Mammary
Mesenchymal
(MSCs)
Location
Bone marrow
Hair follicle
bulge
Crypts
(involutions) of
the small and
large intestine
Testicles
Adult brain
Mammary
glands (female
breasts)
Placenta, fat
tissue, lung,
bone marrow,
umbilical cord,
teeth
Function
Additional information
Produce red and white blood
cells.
Regulate epithelial cell (skin)
regeneration and hair growth
Promote regeneration of the
gut tissue
Replaces the blood system every 4-6 weeks
Produce sperm cells
Produce neurons, astrocytes,
and glial cells
Produce mammary ducts and
milk-producing cells
Undergo meiosis to generate sperm
May differentiate into a variety
of tissues: osteoblasts,
adipocytes, chrondocytes,
myocytes and possibly
neuron-like cells
Unknown whether MSCs are true stem cells
Divide very rapidly. The lining of the gut
renews every 22-48 hours.
Stimulated by sexual and lactating hormones
after pregnancy
The first adult stem cells form early in embryonic and fetal life. The field of
developmental biology has studied the origins of adult stem cells and how they are formed
during the process of cellular lineage specification (i.e. where different types of cells come
from during development). This information has in turn provided clues regarding how to
generate different cell types from pluripotent cells grown in the laboratory. The schematic
diagram (slide 9) depicts a possible pathway from ES cells to generate adult skin stem cells
in culture. In this example, ES cells may generate three different, multipotent, tissuerestricted stem or progenitor-like cells that belong to the ectodermal, mesodermal or
endodermal lineage. The ectodermal lineage produces the nervous system and skin, and
generates either skin stem cells or neural stem cells depending on the culture conditions.
The skin stem cell is committed to the skin lineage (i.e. it will not generate cells
contributing to other tissues).
Although ES cells and adult stem cells are both self-renewing and immature cells,
there are major differences in their biological properties (slide 10). For example, ES cells
proliferate very rapidly whereas adult stem cells are mostly quiescent or divide very
slowly. Adult stem cells divide rapidly only when there is a need in their respective tissues,
such as during tissue replacement and repair. Also, ES cells are pluripotent stem cells that
are able to generate the three tissue layers of the embryo (the previously mentioned
ectoderm, mesoderm and endoderm). Adult stem cells are more restricted in terms of what
cell types they can produce and are therefore multipotent, meaning that they are
committed to a particular lineage such as skin, blood, nervous system, muscle or sperm.
However, adult stem cells are very rare and make up only a tiny percentage of cells within
their particular organ or tissue, making their isolation and use a challenge. At present,
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scientists are still investigating under which conditions adult stem cells are best grown in
the laboratory.
Both ES cells and adult stem cells have great potential for understanding and
treating many human diseases (slide 11). ES cells can produce all tissues of the embryo
and therefore have an unlimited potential to generate many types of cells in the laboratory.
ES cells can be differentiated into progenitors for various lineages, a process called
directed differentiation, and be used successfully to generate many cell types. Tissuespecific cells made from ES cells may then be injected into the relevant organs and used for
therapies. For example, skin tissue might be grown in the laboratory and then employed to
help burn victims. However, the major limitation to the use of ES cells specifically in
regenerative therapies concerns the low probability that cells derived from them will
immunologically “match” an intended patient. This is why efforts to use knowledge from ES
cell research to generate other, “patient-matched” pluripotent stem cells are so important
(as will be discussed below). Also, the source of human ES cells (i.e. the human embryo) is
controversial, due to ethical issues concerning use of human embryos for research
purposes. In addition, although pluripotent cell-based therapies are designed to direct
differentiation of cells into more mature, tissue-specific forms, any residual ES cells that
remain in the culture may cause tumors (teratomas) if introduced into the body.
In contrast, adult stem cells can be isolated directly from patients, avoiding the
problem of tissue rejection due to an immune response against cells from a different
genetic source. Some adult stem cells have already been used successfully for therapies.
Hematopoietic stem cells have been used successfully to treat leukemias, other bone/blood
cancers, immunodeficiency and even certain metabolic diseases. Other adult stem cell types
such as MSCs and neural stem cells remain very important resources that, following
additional research, also promise to make substantial contributions to regenerative
medicine.
Injury or damage disrupts the balance between stem cells and niche cells, and
stimulates proliferation of stem cells (slides 13-16). In the normal adult, stem cells are
quiescent. They divide very slowly due to cell-cell contacts and a limited supply of secreted
factors that promote proliferation. Upon injury, contacts between stem cells and either the
niche cells or extracellular matrix may be disrupted. Following injury there is increased
signal secretion that stimulates production of growth factors from the niche. These
mechanisms promote stem cell proliferation (exit from the quiescent state). Some stem
cells will then undergo differentiation because there is limited physical space in the niche.
Engineering Pluripotent Stem Cells
Pluripotent stem cells can be engineered from somatic cells via multiple methods,
including nuclear transfer or direct reprogramming with genetic factors. To produce
pluripotent cells using nuclear transfer, the nucleus of a somatic cell (e.g. fibroblast) is
removed (slide 17). At the same time, the nucleus of an egg cell (oocyte) is taken out and
discarded. The nucleus of the somatic cell is then inserted into the enucleated egg cell. After
insertion into the egg, the somatic cell nucleus is reprogrammed (i.e. has its gene
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expression altered, turning OFF genes marking its differentiated state and turning ON
genes that induce pluripotency) by unknown factors present within the oocyte. The egg,
now containing the nuclear DNA of the somatic cell, is stimulated with electricity and
begins dividing. After several mitotic divisions in culture, a blastocyst may form from which
pluripotent stem cells can be derived. This process is very inefficient in species where it
does work, and as of this writing has never been accomplished using human cells. The
technique of transfering a nucleus from a somatic cell into an oocyte is also referred to as
cloning. Importantly, these NT-ES cells contain the same genetic material (DNA) as the
original fibroblast cells (i.e. those taken from the patient’s skin) and may in theory be used
for therapeutic purposes in a manner that would avoid immune system rejection. As
described above, such cells made from patients may be differentiated into specific lineages
(e.g. dopaminergic neurons) to enable the study of a particular disease (e.g. Parkinson’s
disease). Currently no human ES stem cell lines have been derived using this method. This
technique is not very widely studied due to: 1) the considerable difficulty, both practically
and ethically, in obtaining human eggs, and 2) the advent of iPS technology (see below).
Another method to generate pluripotent stem cells is through a technique called
direct or cellular reprogramming. Induced pluripotent stem cells (iPS cells) are
reprogrammed cells made by a technique that “forces” expression of pluripotency-related
genes in a somatic differentiated cell (slide 18). iPS cells are similar to ES cells in many
respects, such as their pluripotency and matching expression of genes and proteins. iPS
cells were first produced in 2006 from mouse cells and in 2007 from human cells (slide
19). This was an incredibly important and monumental advance in biology, as it not only
opened the door to a much clearer understanding of how pluripotency is biologically
regulated but may also allow researchers to obtain pluripotent stem cells which are a
genetic match for patients, without the controversial use of human embryos.
iPS cells are typically derived by introducing specific pluripotent stem cellassociated genes into non-pluripotent cells such as adult fibroblasts, by way of a viral
vector in a process called transduction (slides 20, 21). This is often achieved using a type
of virus called a retrovirus. Retroviruses have an RNA genome that is converted to DNA
before inserting at random locations into the host cell’s own genome. The reprogramming
genes include the master transcription factors Oct-4 and Sox2. The Oct-4 and Sox2
transcriptional regulators and are necessary to induce somatic cells into an embryonic
state. Oct-4 plays a crucial role in maintaining ES cell pluripotency, and is primarily found
only in pluripotent cells such as ES cells. The absence of Oct-4 in blastomeres and ES cells
leads them to change into trophoblast cells. The Sox2 gene is also associated with
maintaining pluripotency. 3–4 weeks after the reprogramming genes are introduced to
cells, small numbers of cells become morphologically and biochemically similar to
pluripotent stem cells, and are isolated through morphological selection (formation of
colonies), the presence of a genetic marker or antibiotic selection.
However, reprogramming adult cells to obtain iPS cells may pose significant risks
that could also limit their use in humans. If viruses are used to alter the cells genetically,
the expression of cancer-causing genes or oncogenes may be activated after they are
injected into organisms. In February 2008, scientists developed a technique to remove
these reprogramming genes after inducing pluripotency, increasing the potential safety of
iPS cells for the treatment and study of human diseases. In April 2009, scientists produced
mouse iPS cells without any genetic alteration of adult cells. Here, a repeated treatment of
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the cells with critical proteins was enough to induce pluripotency. By such means, today’s
breakthroughs are refined further to create even more effective methods for eventual use
in cellular therapies.
Several other genes such as Klf4, c-Myc, Lin-28 and Nanog increase the production
efficiency of iPS cells. Klf4 was initially required for mouse iPS cell production but was later
shown not to be required for human iPS cells. The c-Myc proto-oncogene has been
implicated in cancer. Using the "myc" family of genes to induce iPS cells is troublesome
because 25% of mice transplanted with c-myc-induced iPS cells developed lethal tumors.
Klf-4 and c-Myc work by changing gene expression in differentiated cells, primarily by
pushing cells to proliferate, preventing cell death and making the genome more responsive
to changes in patterns of gene expression. Reprogramming factors turn OFF genes that are
active in differentiated cells and turn ON genes that maintain pluripotency. In ES cells,
Nanog is also required for pluripotency. However, Nanog is not necessary for iPS induction,
although human iPS cells are often produced using Nanog as one of the factors. Lin-28 is a
regulator of a specific class of factors known as microRNAs, that in turn regulate many
oncogenes. Though cellular reprogramming superficially appears “simple” considering that
very few genes are required to push mature cells “developmentally backwards” into
pluripotency, in reality the process is anything but simple when one considers the many
interactive regulatory networks affected by each of these factors during the
reprogramming process.
How can we determine if a cell is a stem cell?
Stem cells are very rare in adult tissues. Adult stem cells can be isolated by virtue of
their expression of characteristic cell surface proteins. The tissue is dissociated and cells
are identified and purified based on their expression of such markers. To test if the isolated
cells are true stem cells, one must perform a functional assay, namely introduce cells into a
tissue and see if they grow and provide differentiated cells to that tissue or organ. In order
to distinguish unambiguously the injected and host cells from one another, the injected
cells must be marked in some way. This underlies a type of study called a “lineage tracing
experiment”. In one example, adult stem cells can be isolated from a mouse expressing the
marker -galactosidase, an enzyme that turn cells blue in the presence of a specific
substrate. These cells are then transplanted into a recipient mouse (white mouse) to
determine what percentage of the newly formed tissue is blue (only the newly injected cells
are capable of turning blue) (slide 22). If the isolated cells are true stem cells, they will form
all cells and tissues of a particular organ (e.g. the mammary gland). Another common
method of identifying stem cells is by marking one cell with the green fluorescent protein
(GFP; a fluorescent protein isolated from the jellyfish) using genetic methods in living
animals (slide 23). The progeny of that green cell can be identified in vivo after
transplantation to determine which differentiated cell types it produces.
Readings, Videos, and Slide Presentations for Lecture 2
 Please watch a series of movies from the International Society for Stem Cells
(http://www.isscr.org/public/MakingSenseOfStemCells.htm) regarding human
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embryonic stem cells, adult stem cells and their therapeutic use and cloning and nuclear
transfer.
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