Download Primer - Workforce Development in Stem Cell Research

Primer: Developmental Biology Review
~iPSCs and New Technologies Course~
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 you will acquire as a result of this lecture
Students will know:
 Key Terms: central dogma, transcription, translation, chromosome, genome,
gene, genetic code, cell signaling, signal transduction, stem cell, differentiated
cells, differentiation, gametogenesis, germ cells, gametes, fertilization, zygote,
blastocyst, inner cell mass, germ layers, ectoderm, mesoderm, endoderm, iPS
cells.
 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
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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 therapeutic 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 multi-trillion-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 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, which 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 that arose from a single cell
through the process of mitosis (slide 2) and differentiation. The majority of these cells
perform very specialized functions (slides 3 and 4). Each cell type has a unique
morphological feature that is adopted for its specialized function. Immune cells, which
circulate 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 (of CO2 and O2). 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
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movements of the heart for pumping blood (slide 4).
Multicellular organisms do not arise fully formed, but rather arise through a process
of progressive changes 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
(slides 3 and 4). This unfolding of cellular diversity is called differentiation. Therefore, a
cell that has acquired specialized features during development has undergone
differentiation or is differentiated, 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 4). Stem cells are specialized immature
cells that can give rise to a large number of differentiated cell types and are therefore
pluripotent (slide 5). 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 cells, 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. Besides being pluripotent,
another feature that distinguishes stem cells from other cell types is their ability to selfrenew, or give rise to additional stem cells (slide 5).
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 (discussed further below and in lecture 2).
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 2). 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 father, during
fertilization. The mitotic phase is a relatively short period of the cell cycle (slide 2). Mitosis
alternates with interphase, where the cell prepares itself for cell division. Interphase itself
is divided into three sub-phases: G1 (first gap or growth), S (synthesis) and G2 (second gap
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or growth). 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 (ie. the center of the
chromatid). At the end of prophase, the nuclear envelope disassembles and microtubules
invade the nucleus to form the mitotic spindle, which will attach to and move the
chromosomes. 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 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 have moved 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 (division of the cytoplasm).
Each daughter cell now has a complete copy of its parent cell’s genome.
Mitosis and stem cells
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 6). 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, 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
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progenitors, on the other hand, are characterized by limited number of rapid cell divisions
before they differentiate into mature cells.
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 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
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 (basepairs). 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 simpler organisms are easier to explore in the
laboratory and understand in general.
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. Gene expression is
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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 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 “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. While
RNA is generally transcribed from DNA in all biological cells, DNA can be made from RNA in
certain instances and is a notable exception to the central dogma (slide 10). In nature,
retroviruses such as HIV use an RNA-dependent DNA polymerase called reverse
transcriptase to produce DNA and replicate its RNA genome. Scientists can use this enzyme
in a laboratory setting to make mRNA into complementary DNA (cDNA) to study gene
expression in cells (see below).
The strand of mRNA travels from the nucleus to a structure in the cytoplasm 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 of amino
acids long. This process is called translation (slides 11 and 12).
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 (slides 8 and 13). 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 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.
Proteins
Proteins are responsible for the specific traits of a species (slides 12 and 14).
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
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all cells since it is provides structural support for the cell. On the other hand, the
hemoglobin that carries oxygen in the blood is only present in red blood cells, the
neurotransmitter-synthesizing enzyme ChAT is present in motor neurons, and myosin light
chain 2 is only present in heart muscle cells (slide 14). A class of nuclear proteins, termed
transcription factors, has the ability to bind DNA and regulate the process of transcription
(slide 16). Finally some proteins are used to allow communication between cells or cell-tocell communication in a process termed cell signaling (slides 17 and 18). Many secreted
signaling molecules (proteins) function by binding to specific proteins (receptors) on a
cell’s plasma membrane. These secreted molecules often form gradients when released
from cells, and therefore have greater effects on neighboring cells compared to cells further
away. The binding of signaling molecules to their receptors activates intracellular proteins
and signal transduction pathways that lead to different cellular responses, including
transcription of certain genes and expression of certain proteins. Both transcription factors
and signaling molecules are essential for the process of embryonic development 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
All cells in the body (with very few exceptions) contain the same set of basic
instructions, namely the genes, for performing their functions. 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 present only in some cells but not others represent genes that are differentially
regulated (slides 15 and 16). 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 turned
off in progenitors and differentiated cells. On the other hand, the gene that encodes
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.
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However, genes are not the sole players in 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 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 lectures.
The Gametes, Fertilization, and Early Embryo (refer to Lecture 1, Part B slides)
Gametogenesis
Gametogenesis refers to the process of producing gametes (the sperm and oocytes)
(slides 3–6), which are specialized cells that will create a new organism during fertilization
(slides 6 and 14). Early in development (~3 weeks post-conception), a small population of
founder cells, the primordial germ cells (PGCs), are specified and set aside from other
cells. They then migrate back into the embryo proper to the genital ridges of the embryo
around 6 weeks of development. There, they rapidly proliferate to about 5–7 million germ
cells. These PGCs, now termed gonocytes, subsequently begin meiosis and differentiate
within the developing gonads (slide 3). These early germ cells become the gametes.
Gametogenesis is essentially meiosis, development, and maturation of the gametes (slides
8, 9 and 11), and occurs in the gonads or sexual organs – testes in the male and ovaries in
the female (slide 4). The production of sperm in males is called spermatogenesis (slide 9),
while the generation of oocytes (eggs) in females is called oogenesis (slide 11).
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Sperm and eggs are highly differentiated, specialized cells, with very unique
morphology and physiology. The sperm has a flagellum (tail), midpiece (packed with
mitochondria for ATP/power), and head (packed with DNA – to deliver its genetic material
to the egg) (slide 7). It is programmed to find, reach and fertilize the egg. To gain the
necessary changes in motility and physiology necessary for ascending the female
reproductive tract and fertilizing the egg, sperm respond to chemical cues and signaling
molecules within the female reproductive tract (in a process defined as capacitation).
Until sperm undergo this process of capacitation they are unable to fertilize an egg. Once
deposited into the female tract, the average sperm can live for 3–5 days or longer. The egg,
on the other hand, has distinct morphology and cellular properties that are unique for its
distinct purpose – to give rise to the embryo. It is one of the largest cells of the body, and at
full maturity is about 125 m in diameter. The egg is packed full of proteins and mRNA to
sustain early development and once activated by sperm, give rise to all the cells of the new
individual.
In males, meiosis occurs in precursor cells known as spermatogonia that divide
twice to give rise to 4 sperm (slide 9). Sperm undergo meiosis within the seminiferous
tubules of the testis. As they develop and mature they are pushed from the periphery to the
lumen of the tubule, where they will be released and travel to the epididymus for
maturation and storage. Spermatogonial stem cells, positioned at the periphery of the
tubules, are the stem cells that allow the continual production of sperm throughout the
man’s entire life (slides 9 and 10). It takes about 75 days for sperm to develop in the male
reproductive tract. The average man can produce sperm his entire life, with roughly 4,000
sperm made per heartbeat. In females, meiosis occurs in cells known as oogonia (slides 11
and 12). Each oogonium that initiates meiosis will divide twice to form a single oocyte and
two or three polar bodies. The first meiotic division in oogonia occurs in the fetus.
However, fetal oogonia stop dividing at the diplotene stage of prophase of meiosis I and are
dormant in the ovaries within a protective shell of somatic cells (that make up the follicle)
until puberty. At puberty, with the proper hormonal signals, some oocytes begin to resume
meiosis I and arrest at meiosis II until fertilization. Therefore, oocytes can take many years
to complete meiosis, up to 51 years or longer, and will only do so when the sperm provides
the proper signaling pathway to initiate the completion of meiosis upon sperm fusion
(slides 11 and 12). During the menstrual and ovarian cycle, each month around 1000
oocytes are recruited and begin to resume meiosis and mature. However, only one oocyte
(within a growing follicle) will become the large dominant egg that will be ovulated. Unlike
the situation in males, there are no oogonial stem cells within the ovary. Hence, females
lose many oocytes (follicles) over the course of their lifetime in the processes of cell death
(vast majority of oocytes) and ovulation, until they are eventually depleted at menopause
(slides 12 and 13). Therefore, there is a distinct reproductive lifespan or window of
fertility, which is determined by the number and quality of eggs remaining in the ovaries
(defined as ovarian reserve).
Meiosis
Meiosis is a reduction division in which the number of chromosomes per cell is
halved to generate four haploid cells (n; half the number of chromosomes) from a single
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diploid parental cell (2n; the normal number of chromosomes) (slide 8 day 2). Meiosis
involves two successive divisions – meiosis I and II. 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. 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 this
independent assortment of chromosomes is the random orientation of each pair along
the metaphase plate with respect to the orientations of the other chromosome pairs at the
equator. In other words, each pair of chromosomes (from both mom and dad) align
randomly along the metaphase plate and segregate to the dividing cells. Therefore,
different sperm and eggs have different combinations of chromosomes coming from the
mother and father. Non-sister chromatids randomly exchange bits of genetic information
within regions of homology in a process called homologous recombination. The exchange
between the non-sister chromatids results in shuffling of genetic information. New
combinations of DNA created during these chromosomal crossovers or chiasmata are a
significant source of genetic variation. 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 and completes the creation of two daughter cells, each with only the
haploid number of chromosomes (n). 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 chromosome from each chromosome
pair to a gamete; and 2) physical exchange of chromosomal regions by homologous
recombination during metaphase I results in new combinations of DNA within a
chromosome. The meiotic division is essential for producing the gametes (the sperm and
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. There are chromosomal
segregation errors that sometimes occur during meiosis, resulting in a duplication,
triplication, or loss of one or more chromosomes (or pieces of chromosomes). An incorrect
number of chromosomes is referred to as aneuploidy. An example in the human where
this occurs is trisomy 21, where the individual is born with 3 copies of chromosome 21,
which results in Down Syndrome. This syndrome is characterized by specific physical
characteristics and developmental and cognitive deficits, and occurs in about 1 in 700
births in the United States. Its incidence increases with increasing age of the mother.
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Fertilization
Fertilization is the process by which sperm and egg fuse together to begin the
creation of a new individual that contains genetic information derived from both parents
(slides 6 and 14). Fertilization accomplishes sexual reproduction, namely the combination
of genes from both parents that results in a genetically unique individual. It stimulates the
egg to develop into a complete organism. Sexual reproduction promotes the evolution of
species because it creates genetic diversity that is useful for adapting to constantly
changing and challenging environments. Genetic diversity is achieved through random
shuffling of chromosomes (independent assortment) and crossing over (homologous
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 alleles within a gene.
Prior to fertilization, the mammalian oocyte is released from the ovary and
propelled into the oviduct (Fallopian tube) of the female reproductive system. Mammalian
fertilization occurs inside the oviduct of the female. Fertilization consists of several major
events (slides 14). First, the motile sperm migrate to the Fallopian tubes, by traveling
through the cervix and uterus. Many chemical cues in the female reproductive tract and
intracellular signaling pathways in sperm are closely involved in this process, and allow the
sperm to alter their physiology and motility to reach the egg. 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
the acrosome reaction whereby the vesicular acrosome inside of the sperm head undergoes
exocytosis and releases enzymes that allow the penetration of the sperm through the outer
vestments of the egg and the glycoprotein envelope of the egg (the zona pellucida).
Fourth, the fusion of the sperm and egg plasma membranes and subsequent sperm entry is
followed by chemical reactions to block entry of any other sperm into the egg (waves of
calcium in the egg and the cortical reaction). This block to polyspermy results in a
hardening of the zona pellucida. This prevents too many copies of the genetic information
being present in the new organism. It also creates a protective shell around the developing
embryo and prevents the embryo from hatching and implanting in the wrong location
within the female oviduct. Fifth, the genetic information contained within the oocyte and
sperm are combined to create a diploid cell called the zygote. Finally, the zygote activates
metabolism 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 (slides 14 and 20). The human zygote is among the smallest in
the animal kingdom, with a diameter of only ~125 m 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 solid ball of cells (a morula) that resembles a mulberry
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(slides 16 and 17). 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 Fallopian tube, 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 (meaning germ sac) that contains two types of cells and a fluid-filled cavity. The
blastocyst takes in fluid from the surrounding environment and grows in size until it
actually hatches out of the zona pellucida and is then ready to implant into the uterus. The
blastocyst is composed of:
A) An external cell layer that becomes the trophoblast. This is the first cell type to
differentiate in the developing embryo. Trophoblast cells adhere to and invade the
endometrium of 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), that gives rise to
the embryo proper and the associated yolk sac, allantois and amnion. If the ICM 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 (slide 18). 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 18). First,
they can be derived from the ICM found in human blastocysts that are left over and donated
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 transferred into an oocyte that lacks its own nucleus (ie. has been
enucleated). The oocyte can then be induced to undergo cleavage and form a blastocyst; ES
cells that contain the exact 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. Most recently, stem cells known as induced pluripotent stem
cells (iPSCs) have been generated in culture from a wide variety of fully differentiated
adult cells through a process of cellular reprogramming. With the use of 3–4 specific
pluripotency transcription factors, adult cells can now be transformed into pluripotent
stem cells capable of giving rise to all cell and tissue types. IPS cells, as this course will
explore in detail, hold tremendous promise for regenerative medicine as they are
genetically identical to the person from whom they were generated and do not require the
use of embryos to produce (slide 19).
The importance of pluripotent stem cells to medicine is enormous. The hope is that
human ES and/or iPS cells can be used to produce new neurons for people with
neurodegenerative diseases (such as Alzheimer’s or Parkinson’s disease) or spinal cord
injuries, produce insulin-secreting pancreatic cells to treat diabetes or produce new blood
cells for people with anemia or blood cancers.
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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 20), which arise
during week 3 of embryo development. These germ layers generate distinct cell types. The
ectoderm (outer layer) gives rise to epidermal and pigmented cells of the skin and the
neurons of the brain and spinal cord. The mesoderm (middle layer) generates muscle,
bone, the kidneys and blood cells. Finally, the endoderm (internal layer) produces the
digestive system (stomach, gut, liver), lungs and endocrine glands. The germ cells
(precursors of sperm and eggs) 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 leftright 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 (slide 21). 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 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 21).
The best example of this process is found in the hematopoietic system (slide 22 day
2), which gives rise to all cells of 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
these stem cells shows some degree of restriction in its fate, because it cannot generate all
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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 (oxygencarrying molecule) and produces enormous amounts of this protein. Finally, the
erythroblast expels its nucleus and becomes a mature 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. Notably, with the revolutionary discovery of iPS cells we are
uncovering ways to reverse the differentiation process and reprogram mature cells to an
embryonic-like pluripotent state, which may have profound implications for science and
medicine.
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]
 Additional movies (developing human embryos, motile sperm, and animations from
lecture slides can be downloaded here: [LINK]
 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)
Additional movies (developing embryos, motile sperm, and animations from lecture slides
can be downloaded here: [LINK]
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?
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