Work Force Development: Stem Cell Primer
Lecture 1
Learning Items A, C, D, E, U, and T
Essential questions (U & T)
 What are the major differences between immature (undifferentiated) and
mature (differentiated) cells?
 What are the different types of cell divisions and why are they important for
stem cell biology?
 What is the central dogma?
 What role does gene expression play in the process of differentiation?
 What are the cues for differential gene expression?
 How do stem cells and differentiated cells arise in the embryo?
 What is the process of differentiation and why is it important for stem cell
biology?
Key knowledge and skills will you acquire as a result of this lecture
Students will know:
 Key Terms: central dogma, transcription, translation, chromosome, genome,
gene, genetic code, cellular components, plasma membrane, cell signaling, signal
transduction, stem cell, differentiated cell, differentiation, germ layers, ectoderm,
mesoderm, endoderm.
 The process of fertilization and formation of germ layers during early
embryogenesis, their importance for stem cell biology and the generation of
embryonic stem cells.
 The basic differences between stem cells and differentiated cells at both genetic
and cell biological levels.
 The basic concepts of cell differentiation and when it begins during embryonic
development.
Students will be able to:
 Identify and classify various cell types in terms of their state of differentiation.
 Diagram the processes of transcription and translation and how they relate to
the process of differentiation.
 Compare and contrast patterns of gene expression and cell surface proteins
between immature progenitor cells and mature differentiated cells.
 Place pluripotent stem cells in the context of early embryonic development
1
Introduction
Stem cells are the foundation for every organ, tissue and cell that is present in our body.
Today, there is a great interest in stem cell research because it may hold the key to the
treatment of a variety of human diseases. Although scientists hope to achieve relief of
human suffering with stem cell research, there are moral concerns whether some types of
stem cell research (e. g. embryonic stem cells) justify the means. The controversy is about
using donated embryos for research and whether the embryo is a living human being. The
majority of these views are not based on a scientific understanding of the embryo, the
origin of stem cells and various biological and therapeutical properties of different types of
stem cells. During the progress of this course, we will try to elucidate the properties of
various stem cells, from their basic cell biological properties to their potential for treating
human diseases. Once the basics of cell biology are familiar, the unique properties of stem
cells become abundantly clear.
The Cell
Cells are the basic structural and functional
units of all organisms, from a one-celled bacterium to
a multitrillion-celled human. Most animal cells have
common structural features (slide 1). The cell is
surrounded by a plasma membrane that regulates
what enters and leaves the cell. Small molecules can
enter through the membrane by simple diffusion,
whereas large or highly charged molecules require
specialized transport mechanisms. The plasma
membrane also plays the role of intermediary; some
proteins on the cell surface act as receptors, receiving Slide 1
extracellular signals and relaying that environmental cue to the inside of the cell, a process
termed signal transduction. In typical animal cells, the largest structure inside a cell is the
nucleus, where the genetic information is stored in the form of DNA. A notable exception is
the red blood cell, which lacks a nucleus. The processes of transcription and gene
regulation, that are important for the developing organisms and stem cells, occur in the
nucleus. The majority of the metabolic or specialized cell functions, which are important for
the survival and activity of the organism as a whole,
take place in the jelly-like cytoplasm.
The specialized function of a cell is a result of
differentiation
Humans have an estimated 100 trillion or 1014
cells but the majority of these cells perform very
specialized functions (slide 2). Each cell type has a
unique morphological feature that is adopted for its
specialized function. Immune cells, which circulate
2
Slide 2
throughout the body to protect us against pathogens, change shape rapidly to
accommodate movement from the blood stream to the outer limbs while fighting infection.
Red blood cells, which transport oxygen to all tissues, have a dimpled disc-like shape to
maximize cell surface area for gas exchange (CO2 in and O2 out). Nerve cells or neurons
form a network that can stretch over great distances as compared to other cells, and
conduct the electrical and chemical signals that allow us to think and feel by means of
specialized cellular extensions called dendrites and axons. Finally, muscle cells of the heart
exhibit a remarkable and absolute synchrony when contracting, to control the unceasing
rhythmic movements of the heart for pumping blood.
Multicellular organisms do not arise fully
formed, but rather arise through a process of
progressive change called development. The
development of all multicellular organisms begins
with a single cell: the fertilized egg or zygote (see
below) that divides repeatedly to give rise to
hundreds of different cell types that have defined
functions (slide 3). This unfolding of cellular diversity
is called differentiation. Therefore, a cell that has
acquired specialized features during development has
undergone differentiation or is differentiated, Slide 3
whereas cells that lack these specialized features are
said to be immature or undifferentiated cells. Immature cells that give rise to a diverse
variety of differentiated cells are called pluripotent cells, whereas cells that produce only
a single cell type are called unipotent cells (slide 2). Stem cells are specialized immature
cells that can give rise to a large number of differentiated cell types and are therefore
pluripotent. Progenitor cells on the other hand can produce either one specialized cell
type (e.g. neuron; unipotent cell) or a small number of cell types (e.g. neurons plus skin
cells plus pigment cells; multipotent cell). The process of differentiation is important for
the stem cell biologist because this is how a variety of cell types can be produced from stem
cells in a dish. By manipulating the culture conditions, or the microenvironments of stem
cell, researchers can instruct a cell via signal transduction to adopt a particular cell fate
(i.e. a particular phenotypic characteristic). Acquiring a particular fate (i.e.
differentiation) interferes with the ability of a cell to generate multiple cell types. Therefore
specialized differentiated cells such as neurons and muscles in the adult have lost their
inherent ability to produce other cell types.
Cell Cycle and Types of Cell Division
The most powerful feature of nearly all cells is their ability to create identical copies
of themselves, or replicate, in order to pass their genetic information on to the next
generation. The ability of cells to replicate is important for stem cell biology, because cell
division is not only the central mechanism for development and growth of living organisms
but also for tissues renewal and regeneration. There are two major types of division: a)
mitosis and b) meiosis.
3
Mitosis
Mitosis is the process of copying and dividing
chromosomes from the nucleus followed by cell
division. It produces two daughter cells that are
genetic clones (i.e. identical to their parent) (slide 4).
In humans, cell division takes about twelve hours. The
process begins in the nucleus, which contains the
chromosomes or threadlike structures that are the
collections of genes. In humans, each cell contains 46
chromosomes found in 23 pairs. One set was
inherited from the mother and the other from the Slide 4
father during fertilization. The mitotic phase is a
relatively short period of the cell cycle (slide 4). Mitosis alternates with interphase, where
the cell prepares itself for cell division. Interphase itself is divided into three sub-phases: G1
(first gap), S (synthesis) and G2 (second gap). During these three phases, the cell grows by
producing proteins and cytoplasmic organelles. Chromosomes are replicated only during
the S phase.
The genetic material in the nucleus is normally found loosely bundled in a protein
coil called chromatin. At the onset of prophase (the first stage of mitosis), chromatin
condenses into a highly ordered structure, the chromosome. Since the genetic material
had already been duplicated earlier in S phase, each replicated chromosome is present as
two sister chromatids, bound together at the centromere. At the end of prophase, the
nuclear envelope disassembles and microtubules invade the nucleus. The centromeres of
each chromosome assemble along the metaphase plate, an imaginary line like an equator
that is equidistant from the two poles. This signals the beginning of the metaphase (the
second stage). The chromosomes are aligned into an imaginary equatorial plane due to a
counterbalance between the pulling power of two opposing microtubule attachments in
each sister chromatid. During anaphase two events occur: a) proteins that hold sister
chromatids together are cleaved, allowing them to separate; and b) sister chromatids that
are now distinct sister chromosomes are separated by shortening the microtubules thus
pulling the paired centrosomes (along with their attached chromosomes) apart to opposite
ends of the cell. At the end of anaphase, the cell has separated identical copies of the genetic
material into two distinct groups. At telophase, corresponding sister chromosomes attach
to opposite ends of the cell. A new nuclear envelope forms around each set of sister
chromosomes. Both sets are now surrounded by new nuclei and unwind back into the
relaxed chromatin state. Mitosis is now complete but cell division is not. In animal cells, a
cleavage furrow (pinching) containing a contractile ring develops where the metaphase
plate used to be, coming between the separated nuclei in a process called cytokinesis. Each
daughter cell now has a complete copy of its parent cell’s genome.
Mitosis and stem cells
4
The ability to divide endows stem cells with the capacity to produce large quantities
of new cells, sometimes on rather short notice, during embryonic development or when
tissues are injured in the adult. They achieve this by performing two types of mitotic cell
divisions. The first type is an asymmetric mitotic cell division that generates two different
diploid cells, one being a stem cell identical to the
mother cell and the other being a progenitor cell that
has a limited proliferative ability before it undergoes
differentiation (slide 5). This type of division
maintains stem cell numbers and allows them to
persist in the adult organism. The second type of
division is a symmetric cell division that generates
two diploid cells, both of which have identical
properties to the mother cell (i.e. a simple mitosis). It
is used to expand both the numbers of stem cells and
proliferating progenitors during embryonic growth, Slide 5
before they undergo a final differentiation into distinct cell types. Stem cells replace and
replenish cells that are injured as well as those that have aged and died in the mature
organism. During development, cells lose the ability to divide as they begin differentiation
and acquire mature phenotypic characteristics. Neurons represent an extreme case of this
end state since they cannot proliferate. Immature cells or progenitors, on the other hand,
are characterized by limited number of rapid cell divisions before they differentiate into
mature cells.
Meiosis
Meiosis is a reduction division in which the
number of chromosomes per cell is halved to generate
four haploid cells (n) from a single diploid parental
cell (2n) (slide 6). Meiosis involves two successive
divisions. Before meiosis begins, the DNA in the
original cell is replicated during S phase of the cell
cycle. The first meiotic division consists of several
stages. During prophase I, duplicated homologous
chromosomes are paired and line up together. Nonsister chromatids randomly exchange bits of genetic
information within regions of homology in a process Slide 6
called homologous recombination. The exchange between the non-sister chromatids
results in shuffling of genetic information. New combinations of DNA created during
chromosomal crossovers are a significant source of genetic variation. At the end of
prophase I, the chromosomes are fully condensed. The two duplicated pairs of
chromosomes and the points of chromosomal exchanges are fully visible; this structure is
also called the tetrad (having four parts). At the end of prophase, the nuclear membrane
disintegrates and the meiotic spindle begins to form. During metaphase I, homologous
pairs align along the equatorial plane and each duplicated chromosome is pulled randomly
to one side or the other. The physical basis underlying independent assortment of
chromosomes is the random orientation of each pair along the metaphase plate with
5
respect to the orientations of the other chromosome pairs at the equator. During anaphase
I each chromosome containing a pair of sister chromatids is pulled to opposite poles of
the cell, severing the recombination nodules. Finally, in telophase I the chromosomes
uncoil back into chromatin, the cell membrane is pinched completing the creation of two
daughter cells, each with only the haploid number of chromosomes. During Meiosis II each
chromosome's sister strands (chromatids) decouple and segregate into daughter cells
without DNA duplication. Thus meiosis II resembles a simple mitotic division with half the
number of chromosomes.
Meiosis generates genetic diversity at two levels: 1) independent alignment and
subsequent separation of homologous chromosome pairs during the first meiotic division
causes a random and independent allocation of one from each chromosome pair to a
gamete; and 2) physical exchange of chromosomal regions by homologous
recombination during prophase I results in new combinations of DNA within a
chromosome. The meiotic division is essential for producing the gametes (the sperm and
the oocytes), which are specialized cells that combine to form a new organism during
fertilization (see below). Meiosis thus facilitates stable sexual reproduction. In the absence
of a chromosome reduction mechanism during meiosis, fertilization would yield zygotes
that have twice as many chromosomes as the previous generation. Successive generations
would have an exponential increase in chromosome number.
The Central Dogma and its Implications for Stem Cell Biology
Genes and genomes
The chromosomes in a nucleus are tangles of
DNA with associated proteins. DNA has the form of a
twisted ladder (called a helix) consisting of two
strands. Each strand contains a quartet of molecules
called nitrogenous bases, linked one after another and
abbreviated A, C, T, or G (for adenine, guanine,
cytosine, and thymine). The bases in each strand
join at the center of the twisted ladder, in one of only
two combinations: A is always paired with T while G
is found with C (slide 7). Genetic information is thus
arranged in an elegant linear fashion. The order of the Slide 7
bases A, C, T, and G varies along the length of the chromosome, and certain sequences of
bases form the genes. Collections of genes are linked in stretches to make up a
chromosome. All life—plants, fungi, animals, bacteria, and algae—uses this same system.
However, not all DNA in chromosomes belongs to genes. Over 70% of the genetic material
in human chromosomes serves other purposes, likely carried along as evolutionary “junk”
from our ancient origins.
A complete volume of DNA-containing material that encompasses all the cell’s genes
is called a genome. The genomes of different species vary in size. The nematode (a small
roundworm found in the soil) has a genome thirty-eight times smaller than a human
6
genome, a fruit fly’s DNA is eighteen times smaller and the mouse has about the same
amount of DNA as us. How many genes are present in the human chromosomes? Estimates
vary, but the Human Genome Project, a ten-year government-sponsored research effort,
lists about 25,000 genes. In total, the human genome contains over 3 billion chemical
letters. If all of the letters in the genome of a single human stem cell were put on paper, the
amount of information would fill one and a half million pages.
Although the sizes of genomes differ, many genes among genomes are similar. For
example, one out of every four genes in the nematode is also present in a human version,
and the mouse and rat share over 90 percent of their genes with us. Even a rice plant has
over 1,000 genes, or 11 percent of its genome, in common with humans. For decades
biologists have used animals like the nematode, fly, and mouse as genetic models for
human biology. Science enjoys a double benefit from this; our knowledge of genes and
genomes progresses not only because of the degree of similarity found between simple
organisms and humans, but also because in general simpler organisms are easier to explore
and understand.
The Central Dogma
How can genes determine all the
characteristics of living things? The answer lies in the
fact that each gene codes for a specific protein.
Proteins determine how cells work and they look —
in another words the phenotypic characteristics of
cells (slide 12). Gene expression is the term for the
multi-step process that deciphers genetic information
to produce a specific protein. In the cell, DNA rarely
leaves the nucleus whereas proteins are found mostly
in the cytoplasm. How is the genetic information Slide 8
transported from the genes out to the cytoplasm
where proteins are made? An intermediary molecule called RNA, found in both the nucleus
and the cytoplasm, functions as a messenger to
shuttle the genetic information from the nucleus to
the cytoplasm (slide 8). Gene expression begins in the
nucleus, when a single strand of DNA serves as a
template for a new strand of RNA. An enzyme moves
along this strand, building a chain of RNA by
sequentially adding the complementary chemical
bases but substituting the new base uracil (U) for
thymine (T). Once it reaches the end of the gene,
which can be thousands of bases long, the new single
strand of mRNA (“m” is an abbreviation for Slide 9
“messenger”) is released and moves into the
cytoplasm. This process is called transcription (slide 9). The end result is a version of the
gene in RNA form.
7
The strand of mRNA travels from the nucleus
to a structure in the cytoplams called the ribosome,
which is the site of protein manufacturing. At the
ribosome, sequences of three letters in the mRNA—
AAU, CGA, UGA and so on—are converted into one of
twenty different kinds of amino acids, which are the
building blocks of proteins. For example, GUU always
specifies the amino acid valine (abbreviated “Val”).
The amino acids are linked together in long chains
that fold up to form proteins, which can be thousands Slide 11
of amino acids long. This process is called translation
(slide 10).
The process of gene expression takes
information originally coded in DNA, decodes it into
RNA and ultimately assembles proteins, one amino
acid at a time. This is the Central Dogma of
information flow (slide 8, 11). These two steps form a
fundamental principle that is central to understanding
stem cells. The sequence of three-base units in each
gene specifies a specific protein. Even one shift in the
order of bases will change the protein and in some Slide 10
cases may render the gene useless and the protein
unavailable to the animal. As a result, the interplay
between genes and proteins lies behind many if not
most illnesses. Thousands of diseases such as cystic
fibrosis, sickle cell anemia and Huntington’s disease
result from a single defective or mutated gene making
a dysfunctional protein. Heart disease, arthritis,
cancer and diabetes are likely caused by a
combination of environmental effects and mutations
in many genes. Dozens of genes spread among seven
different chromosomes may be responsible for breast
cancer alone.
Slide 12
Proteins
Proteins are responsible for the specific traits
of a species (slide 12). Proteins are the complex
machinery of the animal, and they facilitate all aspects
of life. There are many different kinds of proteins,
some of which are present in all cells whereas others
are unique to specific cells. For example, the
cytoskeletal protein actin is present in all cells since it
is provides structural support for the cell. On the
other hand, the hemoglobin that carries oxygen in the
8
Slide 12
blood is only present in red blood cells, the
neurotransmitter-synthesizing enzyme ChAT is
present in motor neurons, and tropomyosin is only
present in heart muscle cells (slide 13). A class of
nuclear proteins, termed transcription actors, has
the ability to bind DNA and regulate the process of
transcription (slide 14). Finally some proteins are
used to allow communication between cells in a
process termed cell signaling (slide 15). Both
transcription factors and signaling molecules are
essential for the process of embryonic development Slide 14
and stem cells. The presence of unique proteins in
some but not all cells underlies the phenotypic
cellular diversity that is present in multicellular
organisms. The process of differentiation is in other
words the process by which some cells express the
small subset of proteins that gives them unique
characteristics.
The Central Dogma and stem cells
Slide 15
All cells in the body (with very few exceptions)
contain the same set of basic instructions for performing their functions, namely the genes.
Genes, the discrete units of inheritance, interact with environmental cues to produce every
characteristic of a living cell: how the cell looks, behaves, grows, matures, survives and
dies. The interaction of genes and proteins underlies the peculiar potency of stem cells and
holds the secrets to how an organism grows and develops normally. Since nearly all animal
cells contain the same DNA and set of genes (a few exceptions exist), why are cells different
and show distinct phenotypic characteristics? How can this identical set of instructions
present in the genome produce different cell types? The phenotypic differences among cells
are due to differences in gene expression: which genes are turned on, or expressed, and
which genes are turned off, or repressed, in some cells versus other cells. During normal
development, many genes are expressed immediately, only to shut down as new sets of
genes swing into action. Some genes named
housekeeping genes, which encode proteins that
form structural components of every cell or function
in fundamental metabolic pathways, are expressed in
all cells at similar levels. On the contrast, genes that
encode proteins that are present only in some cells
but not others represent genes that are differentially
regulated (slides 16, 17). These genes are turned on
in some cells and remain off in other cells. For
example, genes that control the ability of stem cells to
proliferate and renew themselves via asymmetric cell
division are present only in stem cells and they are Slide 16
turned off in progenitors and differentiated cells. On the other hand, the gene that encodes
9
hemoglobin, the protein that carries oxygen in the
blood, is turned on only in red blood cells (a
differentiated cell). Therefore, understanding how
the gene that encodes hemoglobin is turned only in
that cell type during differentiation can be useful to
guide differentiation of stem cells in a dish to become
a particular cell type like a red blood cell.
However, genes are not the sole players in
Slide 17
137
regulating cellular diversity during differentiation.
During development, immature cells or progenitors,
found at various locations in the developing embryo,
encounter distinct signaling molecules (slide 18). The
progenitors that will become motor neurons,
encounter a secreted molecule termed Sonic
Hedgehog (Shh). Shh binds to the receptors (Patched
and Smoothened) in the plasma membrane of the
progenitor cell. This triggers a signaling cascade
inside the progenitors that promotes its
differentiation into a motor neuron. The progenitors
that will become heart muscle cells express a different Slide 18
receptor called BMPRI. This receptor interacts with the secreted molecules called Activin
or TGF to induce trigger a signaling pathway that induces its differentiation into heart
muscle cells. Finally, the progenitors that become red blood cells express a third type of
receptor that binds to a hormone named erythropoietin to induce its differentiation into
red blood cells. Therefore, the activation of different signaling pathways into distinct
immature cells during development allows them to acquire different fates.
Precise cycles of gene expression and cell signaling over time control the changes
from egg to multicellular embryo to fetus and finally to the newborn animal at birth.
Abnormal gene expression or cell signaling, when this precision is disrupted, is responsible
for developmental problems like birth defects. Stem cells help us understand not only how
disease begins but also offer potential medical therapies by replenishing or replacing cells
and tissues destroyed by disease. Manipulating stem cells in the laboratory can actually
model the steps a disease undergoes during its progression, allowing scientists to
understand how diseases arise in the earliest stages and to experiment with genetic or
chemical therapies. Aberrant genes might be corrected in stem cells then given back to a
patient, resulting in a fully functional protein. Research and therapies using both
approaches will be explored in later classes.
Fertilization, Cleavage and Gastrulation
Gametogenesis
Gametogenesis refers to the process of
producing gametes (the sperm and oocytes) which
10
20
Slide 21
are specialized cells that will create a new organism during fertilization. Gametogenesis is
essentially meiosis and occurs in the gonads or sexual organs. The production of sperm
cells in males is called spermatogenesis while the generation of oocytes in the female is
called oogenesis. In males, meiosis occurs in precursor cells known as spermatogonia
that divide twice to become sperm (slide 20). These cells continuously divide in the
seminiferous tubules of the testicles. Sperm is produced continuously throughout the
human male’s lifespan. In females, meiosis occurs in cells known as oogonia (slide 21).
Each oogonium that initiates meiosis will divide twice to form a single oocyte and two polar
bodies. The first meiotic division in oogonia occurs in the fetus. However, fetal oogonia stop
dividing at the diplotene stage of meiosis I and are dormant in the ovaries within a
protective shell of somatic cells until puberty. At puberty, oocytes resume meiosis I and
arrest at meiosis II until fertilization. Therefore the oogonia can take many years to
complete meiosis, and will only do so when the sperm provides the proper signaling
pathway to initiate the completion of meiosis upon sperm fusion.
Fertilization
Fertilization is the process in which sperm
and egg fuse together to begin the creation of a new
individual that contains genetic information derived
from both parents (slide 22). Fertilization
accomplishes sexual reproduction, namely the
combination of genes from both parents with the
creation of a new individual, because it stimulates the
egg cytoplasm to develop into a complete organism.
Sexual reproduction promotes evolution because it
creates genetic diversity that is useful for adapting to Slide 22
constantly changing and challenging environments.
Genetic diversity is achieved through random shuffling of chromosomes (independent
assortment) and crossing over (recombination) during meiosis. Sexual species maintain a
"library" of genes unavailable to asexual species. This library is defined by two terms:
heterozygosity, when an organism carries two different forms of a gene, and
polymorphism, when a population contains multiple variants of a gene.
Prior to fertilization, the mammalian oocyte is released from the ovary and
propelled into the uterine tubes (oviducts) of the female reproductive system. Mammalian
fertilization occurs inside the oviducts of the female. Fertilization consists of five major
events. First, the motile sperm migrates to the Fallopian tubes inside the uterus, where the
oocyte has been released, via chemical cues. Second, once the sperm has arrived in the
vicinity of the oocyte, the sperm and oocyte recognize and contact each other via proteins
present on their cell surfaces to ensure that they are from the same species. Third, the
signal-receptor interactions trigger a signal transduction pathway that leads to sperm
entry followed by a chemical reaction to block entry into the egg of any other sperm cells.
This will prevent too many copies of the genetic information being present in the new
organism. Fourth, the genetic information contained within the oocyte and sperm are
combined to create a diploid cell called the zygote. Finally, the zygote activates metabolism
11
inside the cytoplasm to begin development of a multicellular organism. The zygote by
definition has the potential (i.e. ability) to generate all cell types in an organism and is
therefore totipotent. The human zygote is among the smallest in the animal kingdom, with
a diameter only 0.01 mm and less than 1/1000 the volume of a frog zygote.
Cleavage and the origin of embryonic stem cells
Once fertilization has occurred, the zygote
undergoes a series of mitotic cleavages until after 2-3
days it generates a ball of cells (morula) that
resembles a mulberry (slide X). The mammalian
mitotic cleavages are the slowest in the animal
kingdom, occurring 12-24 hours apart. During the
process of cell division the embryo moves along the
uterine tubes until at the morula stage it arrives in the
uterine cavity, which is engorged and ready for the
embryo to implant. The morula progresses to a
blastocyst (immature ball sac) that contains two types Slide 23
of cells (slide 23):
a) an external cell layer that becomes the trophoblast. These is the first cell type to
differentiate in the developing embryo. Trophoblast cells adhere to and invade the uterine
wall to implant the embryo in the uterus. They eventually give rise to the embryonic
portion of the placenta that feeds the embryo in utero during development.
b) the internal cell layer, also called the inner cell mass (ICM), gives rise to the
embryo proper and the associated yolk sac, allantois and amnion. If the ICM mass cells or
blastomeres are removed from the blastocyst and grown in culture, these cells divide and
become embryonic stem cells (ES cells). The inner cell mass or ES cells are able to
produce all tissues of the embryo proper. However, they cannot generate trophoblast cells.
Therefore, these cells are pluripotent. ES cells can be grown indefinitely in an
undifferentiated state and will retain their pluripotency after prolonged culture.
Currently, human ES cells are obtained by two
major techniques (slide 24). First, they can be derived
from blastomeres found in human blastocysts that are
left over from in vitro fertilization. Second, they can
be produced from germ cells present in aborted
fetuses. ES cells can also be generated from morulae,
but this process is less common. Finally, the nucleus of
a somatic differentiated cell, such as a neuron or
fibroblast, can be removed and transfered into an
oocyte that lacks its own nucleus. The oocyte can then
be induced to undergo cleavage and form a blastocyst; Slide 24
ES cells that contain the genetic information of the
original differentiated cell can thus be derived from this blastocyt. These ES cells can be
used to treat diseases in a process known as therapeutic cloning. The importance of
pluripotent stem cells to medicine is enormous. The hope is that human ES cells can be
12
used to produce new neurons for people with neurodegenerative diseases (such as
Alzheimer’s or Parkinson’s disease) or spinal cord injuries, produce pancreatic cells to treat
diabetes or produce new blood cells for people with anemia or blood cancers.
Gastrulation, Germ Layer Formation and Lineage Restriction
Gastrulation and germ layer formation
The blastomeres that are present within the developing blastocyst give rise to all
tissues in the developing embryo. However, this process does not happen instantaneously
but occurs gradually over the course of development.
The first segregation within the inner cell mass
produces two layers. The lower layer is the hypoblast
and the upper layer is the epiblast. The hypoblast
gives rise to extra-embryonic endoderm which
becomes the yolk sac. These cells do not produce any
part of the newborn organism. The epiblast cells
generate part of the amnion (extra-embryonic tissue)
and the three germ layers of the embryo proper,
namely the ectoderm, mesoderm and endoderm (slide
25). These germ layers generate distinct cell types.
The ectoderm (outer layer) gives rise to epidermal Slide 25
and pigmented cells of the skin and the neurons of the brain. The mesoderm (middle
layer) generates muscle, bone, kidney and blood cells. Finally, the endoderm (internal
layer) produces the digestive system (stomach, gut, liver), lungs and endocrine glands. The
germ cells (precursors of the sperm and egg) are set aside at early stages of development
and do not arise from the three germ layers. Formation of the germ layers occurs during a
process called gastrulation. Gastrulation ensures not only the specification of three germ
layers but also the establishment of the fundamental dorsal-ventral, anterior-posterior and
left-right axes of the developing embryo.
Lineage restriction
During development, the inner cell mass can form all three germ layers that give rise
to the cell types of the adult organism. These cells are called pluripotent stem cells. They
renew themselves and give rise to germ layer cells. Each germ layer cell retains the ability
to proliferate and generate various cells, but it is more restricted in the types of cells that
it can produce. These germ layer cells are multipotent. An ectodermal stem cell can give
rise to either a skin stem cell that produces only skin
cells or a neural stem cell that produces only
neurons. These later stem cell types are even more
restricted in their possible fates than the endodermal
stem cell. Let’s think of the zygote as a marble rolling
down a hill within multiple diverging grooves that
lead to the lowest elevation, like a ski slope. At the
beginning of this process, the marble can choose any
possible groove to reach to the end. However, once it
13
Slide 146
has entered a specific groove, it is very difficult to go back uphill and choose another
groove. This increasingly limited availability of fate choices is called lineage restriction
(slide 26).
The best example of this process is found in
the hematopoietic system (slide 27), which give rise
to all cells in the blood. The entire cell population of
the hematopoietic system is generated from a
pluripotent hematopoietic stem cell. This cell can
renew itself and generate two lineage-restricted
multipotent stem cells. One of these is the common
myeloid precursor (CMP) that produces the red
blood cells, platelets, macrophages and granulocytes,
while the other is the common lymphoid precursor
that generates lymphocytes (cells that recognize
disease pathogens and produce antibodies). Each of
Slide 27
these stem cells shows some degree of restriction in
its fate, because it cannot generate all blood cells like the hematopoietic stem cell.
Eventually these multipotent stem cells produce progenitors that undergo a limited
number of divisions and produce only one type of cell in addition to renewing themselves.
These are called committed progenitor cells. For instance, the erythroid progenitor cell
can only generate red blood cells. It is the only cell capable of responding to the red blood
cell-stimulating hormone erythropoietin by producing a differentiated cell called the
proerythroblast. As the proerythroblast matures into the erythroblast, it expresses genes
that regulate the synthesis of hemoglobin and produces enormous amount of this protein.
Finally, the erythroblast expels its nucleus and becomes a red blood cell.
Development proceeds in a similar fashion, whereby cells have progressive
restrictions in their possible fates as they begin to differentiate. The further along the
process of differentiation, the more difficult it is for a cell to choose a different fate.
Therefore, during differentiation cells become progressively committed or dedicated to a
particular cell fate. These cells progressively lose their potential or ability to generate
multiple and varied cell types.
Potential: the ability of an immature cell to generate a variety of cell types.
Commitment: the process by which a cell is irreversibly dedicated to assume a specific
fate.
Differentiation: the process of acquiring a specific cell fate.
Readings, Videos, and Slide Presentations for Lecture 1
 Download the slide presentation for this lecture here: [LINK]
14
 Harvard University and BioVision’s “Inner Life of the Cell”
http://www.studiodaily.com/main/technique/tprojects/6850.html (requires Adobe
Flash Player 8 or above)
Activities and Assignments for Lecture 1
1) Which cellular processes covered in the Lecture 1 reading materials do you recognize in
the video “The Inner Life of a Cell”? How accurately are they depicted?
15