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CHAPTER 10
CELL DIFFERENTIATION
AND REGULATION
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I. Fertilization and embryogenesis
The structures of sperm and ovum:
Animal spermatocyte can be differentiated as four spermatids by meiosis, and the
spermatid can be further differentiated as sperm by spermiogenesis. Matured sperm
looks like a tadpole composed of head and tail. Two important structures are included
in head: nucleus and acrosome. Acrosome is a big lysosome actually and contains
many types of hydrolases inside. When an ovum is fertilized, the acrosome releases out
a lot of acrosomal enzymes to digest ovum membrane to have sperm entered ovum. The
sperm tail is also called as flagellum that is the moving structure for sperm. The tail
can be described as 4 regions: neck, middle (mid-piece), trunk (tail), and terminal
(end-piece). The neck is very short and contains two centrioles overlapped. The sperms
of some animals have no tail.
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A model fig of sperm
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The section slide of sperm of some duck. L: vertical section of head. R: cross
section. A: acrosome; AS: acrosome spine; N: nucleus
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Zona pellucida
Yolk
Cortex
Embryonic
cells
Cortical
granules
Ovum cell
Plasma
Corona radiata
and cumulus
cell layer
Egg of bird
Ovum of mammalian
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Fertilization:
Fertilization procedures: Capacitation of sperm
Cortical reaction
Formation of original nucleus
Acrosome reaction
Fusion
During the capacitation, the activity of sperm is obviously increased by
the reactions as the follows: 1. Ca+ channel is activated; 2. Both consumption
of oxygen and glycolysis are increased significantly, and the value of pH is
raided up; 3. The activation of adenylate cyclase results in the intracellular
cAMP concentration increased, and protein kinase A activated; 4. Acrosomal
enzymes are activated.
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Sperms
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Sperms are competing each other to bind and enter the ovum
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Only one sperm that is most strong and fast ovum receives usually
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Only one sperm
can enter the ovum
usually
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The early events of
fertilization (From
Janice P. Evans 2002)
a: Acrosome
reaction
b: Cortical
reaction
c: Formation of
original nucleus
d: Fusion
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The fertilized ovum will
move to uterus,
implant and develop
there.
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The capacitated sperm can pass through the corona radiata and
cumulus cell layer of ovocyte, and reach at zona pellucida via its powerful
catalytic activity of hyaluronidase from plasma PH-20 of sperm. The
sperm can recognize the zona pellucida of ovocyte by the ZP binding
protein located on its acrosome, then, the acrosomal reaction is started.
During this reaction, the hydrolases are released out from acrosome and
the zona pellucida is degenerated to form a channel as an entrance for
sperm.
When the sperm enters the ovum, it cause the depolarization of
ovum cell membrane, then, via a serial complicated reactions, the
fertilization membrane (the sclerosed or hardened zona pellucida) is
formed in the perivitelline space (between the ovum cell membrane and
zona pellucida) where the sperm will be fused to ovum.
Sperm can excite the dormant ovum to finish meiosis and release
polar bodies. The nuclei of sperm and ovum are called as male
pronucleus and female pronucleus at this time. The both pronuclei move
to the center of the ovum and touch each other to be fused as a diploid
fertilized ovum with their chromosomes mixed. The fertilized ovum can be
called as zygote.
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Usually, one ovum can be combined with one sperm only. If more than one
sperms penetrate into an ovum, many polar sites and spindles will be formed
and the fertilized ovum will be proliferated abnormally that causes the death of
the embryo usually. But, in case of monozygotic twins (uniovular twins,
monovular twins) or multifetuses, the combination of one ovum and more than
one sperms can be developed normally, but it is very rarely to be seen. In many
cases of multiple infants delivery, it is the combination of multiple ova vs multiple
sperms.
After fertilization, the fertilized ovum inhibit the penetration of other sperms
by two ways as the follows: 1. The membrane of fertilized ovum is depolarized; 2.
The cortical reaction can damage sperm receptor and inhibit the formation of
fertilization membrane.
In some insects, molluscs, chondrichthyes, birds, amphibians, and reptilians,
the fertilization can be carried out with the combination of one ovum vs multiple
sperms. But, only one male pronucleus can be fused to female pronucleus, and
other sperm nuclei will be degenerated. The polyspermous fertilization described
as above is called as physiologic polyspermous fertilization.
During the fertilization, the nucleus, centromere, and mitochondria of sperm
are exported into ovum. But, only the maternal mitochondria can survive in
fertilized ovum. That is why mitochondrion follows matrilinear inheritance way.
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The recognition between sperm and ovum:
Heterogenous sperm can not bind to ovum because the ovum binding protein
located on the surface of the sperm can not recognize and bind to the sperm
receptor located on the membrane of ovocyte. The sperm receptor is located in
the zona pellucida, and called as ZP protein. The ovum binding protein
recognizes the ZP protein that is from a same species only.
There are 3 types of glycoprotein in the mouse zona pellucida: ZP1 (200KD),
ZP2 (120KD), and ZP3 (83KD). ZP3 is the first sperm receptor that can start
acrosomal reaction. If the sperms are treated with ZP3, the sperms will lose the
ability to fertilize ovum.
β1，4-galactosyltransferase (GalTase-I) is the protein on sperm surface that
can bind to ZP3. GalTase-I can bind to the N-acetylglucosamine at the terminal of
ZP3 glucose chain. Experimental data proved that the purified GalTase-I and its
antibody can inhibit the combination of sperm and ovum. GalTase-I can activate
G protein to start acrosomal reaction. The sperms of GalTase-I gene knockout
mice lost the ability to cause acrosomal reaction and pass through zona pellucida.
After pass through zona pellucida, the sperm goes into the perivitelline
space, and its fertilin (called as ADAM also) recognizes and binds to the integrin
located on the membrane of ovocyte. The combination of fertilin and integrin can
activate the fusion system for the sperm and ovum to finish the fertilization.
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ZP3 and GalTase-I (from D. J. Miller 2000)
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The interaction between sperm and the membrane of ovum
(By Janice P. Evans 2002)
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Ovum cleavage and embryonic development:
Fertilization can create a new life with a serial steps of ovum cleavage. A
fertilized ovum can be cleaved or developed as an embryo or degenerated.
Which final result will be taken is just depended if the fertilized ovum can take
nidation (implantation) or not in uterus. Ovum cleavage means a fertilized ovum
(Zygote) is cleaved (developed) as new cells to form an embryo. New cleaved
cells are called as blastomere. Ovum cleavage is similar to mitosis but without
gap phase. So, during the cleavage, the number of cell is increased without cell
growth. When the ratio of nucleus and plasma become normal, the cleavage is
changed as real mitosis.
The way selection of ovum cleavage is depended on yolk quantity and
distribution. The meridional cleavage means the cleavage section is parallel with
ovum axis. The latitudinal cleavage means the cleavage section is vertical with
ovum axis. The cleavage direction is associated with the direction of spindle that
is depended on the components of plasma and the signals from environment.
The fertilized ovum of amphibian can start cleavage at 2hrs post fertilization.
The fertilized ovum of mammalian starts cleavage at one day after fertilization.
The cleavage will take 2 cells stage, 4 cells stage, 8 cells stage, 16 cells stage
and more stages. But, at 16 cells stage, 1 – 2 cells in center of the cell mass will
be developed as embryo, and other cells will be developed as trophocytes to
form chorionic membrane.
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Usually, the embryo of animal is a mass at 64 cells stage called as morula,
and a big mass containing a blastocoel in center at 128 cells stage called as
blastocyst.
Some cells located on surface can move or fold into inside of embryo to form
gastrula. The migration of cells to form gastrula is called as gastrulation. The
cells remained outside are called as ectoderm, and the cells migrated into inside
are called as endoderm and mesoderm. The blastocoel will disappear when the
gastrula is formed.
Ectoderm will form nerve system, skin, hair, nails, and teeth. Mesoderm will
form bone system, muscle system, urogenital system, lymph or lymphoid tissue,
connective tissue, and blood system. Endoderm will form pulmonary system,
digestion system, liver, and others.
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From a fertilized ovum (zygote) to an embryo
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II. The major ways of cell differentiation
The cell differentiation is the procedure by that the new generated cells are
different from their parental cells in morphology and functions. By differentiation,
cell can choose some genes to be expressed, and some genes to be blocked,
finally, the expressed structural proteins form different structures and take
different functions. So, differentiation is the fundamental bio-protocol to form the
complicated and perfect organ system and individual that is a highly orderly and
exactly regulated “cell community”. Therefore, differentiation is genes’ differential
expression. Cell differentiation is operated with morphogenesis together.
Morphogenesis means the procedure by that the appearances of tissues, organ,
and individual will be formed by cell proliferation, differentiation, migration,
adhesion, apoptosis, and other behaviors.
The direction of cell differentiation is determined during the cell development.
When the direction is determined irreversibly, the position and future of cell have
been determined meantime. So, the procedure above is called as determination.
For example, the central cells and peripheral cells of a monrula of mammalian
have already got their future development irreversibly, the former will be
developed as embryo, and the latter as chorionic layer.
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Different animal species has different determination time point. For
mammalian embryo, each cell at 8 cells stage or even 16 cells stage can be
developed as an individual.
The mechanism of cell differentiation is very complicated. Briefly, a cell
determination is depended the internal features of cell and the environment in that
the cell exists. The former is associated with asymmetric division, and the latter
means the cell receives the external signals and responses to them.
Asymmetric division
Animal fertilized ovum is not a homogeneous in structures. The nucleus is
located at a site closed to membrane, and the centroles are formed in this site.
We call this site as northern polar or animal polar, and the opposite site as
southern polar or plant polar. The distribution of proteins and mRNA is not
homogeneous also. There are 20,000 – 50,000 types of mRNA in animal ovum to
the differentiation and development of the fertilized ovum. The homogeneity
makes ovum cleavage asymmetrically, that means each new cell will get different
genetic legacy or inheritance from their parental cell. During the embryonic
development, asymmetric division is carried out very often.
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Pathways of induction
Induction means that a cell’s development direction can be induced by
adjacent or distant cells. We call this induction as embryonic induction, and the
cells from that the inducing signals are released out as inductor or inducer. We
call a same type of cells that can response to signals as morphogenetic field.
There are many morphogenetic fields in an embryo.
Other inducing pathways include cascade signaling, gradient signaling,
antagonistic signaling, combinatorial signaling, and lateral signaling.
Some induction signals
Pathways
Ligands
Receptors
Antagonistics
Receptor Tyrosine
Kinase
EGF
EGF
Argos
FGF（branchless）
FGF
ephrins
Eph
TGFβ
TGFβ
BMP（Dpp）
BMP
Chordin（Sog）,noggin
Frizzled
Dikkopf，sFRP，Cerberus
Notch
Fringe
TGFβ Family
Nodal
WNT
WNT
Hedgehog
Hedgehog
Notch
Delta
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The pathways of induction signals
(From Thomas Edlund and Thomas M. Jessell 1999)
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Cascade signaling：Cascade signaling means the cells or tissue that were
generated by the primary induction can induct the differentiation for other cells or
tissues grade by grade. For example, the original visual cell can induct the
ectoderm cells rounding it to be differentiated as the lithocysts for crystalloid lens,
and the cells of crystalloid lens can induct the ectoderm cells to be differentiated
as cornea.
Gradient signaling：The external signals are distributed in a gradient. Each cell
has a threshold concentration of signal to response to the signal. The different
threshold concentration will cause the different spatiotemporal differentiations. We
call the signals in a cell or morphogenetic field that can regulate the cell
differentiation as morphogen.
Antagonistic signaling：The cell secrets can bind to the receptor or ligand of
some signal pathways to block the signaling. Many morphogenetic events are
caused by this pathway.
Combinatorial signaling：One type of signal can determine a differentiation for a
cell, but two types of signals can cause another differentiation way.
Lateral signaling：A little bit difference between the signals can be identified. But,
this difference can be obviously enlarged by the feedback regulation to determine
the differentiation. For example, in fruit fly, Notch signal pathway can activate the
lateral inhibition to inhibit the pre-neuron to be differentiated as neuron.
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It is not true that all differentiation events are caused by signal induction.
Some differentiations are associated with the cell internal features. We call this
differentiation regulation as autonomous mechanism. For example, the
asymmetric division is associated with homogeneity of ovum.
The regulation of cell quantity
It is easy to answer the question, why human body is large than mouse?
You can say because human body contains more cells than mouse body. But, it
is not easy to answer these questions: Why there are more cells contained in
human body than in mouse body? Why the cell quantity in each type of organ is
consistent almost?
The regulation of cell quantity is important not only for the tissue or organ
structures construction but also for the maintenance of an individual. The
regulation of cell quantity depends on cell proliferation and cell death.
Enhancement of cell division:
Most of cell proliferations are started by the excitation of external signals
of the induction ways. So, in a cell community, a cell proliferation should be
started or not, that depends on the request or permission from other cells or
community. The signals for cell proliferation include cytokines (growth factor),
hormone, and extracellular matrix (ECM).
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1．Peptide growth factors:
Peptide growth factor excites target cells by paracrine way. But, the selfcrine
way probably exists among the cells of same type.
If you culture one or several cells in a dish, they will not grow or grow very
slowly because there is no excitation from other cells. If you culture cells with a
medium containing no growth factor inside, the cells will stop to grow at G1/S
and turn to G0 phase.
Same event can happen in tumor development. Fisher（1967）injected a
big number of tumor cells into mouse portal vein and resulted in broad
metastasis in liver. The mouse died soon. But, when he injected 50 tumor cells
into the mouse portal vein, nothing happened to the mouse. He took a surgery
operation to the mouse to check the liver, and no any metastasis could be found
in the liver. But, after his performance, the mouse died from tumor metastasis in
liver soon because the level of growth factor was raised during the surgery
wound healing. The described above indicates that tumor development and
metastasis are depended on paracrine way and selfcrine way.
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2．Hormone:
Hormone stimulation can be considered as distant signaling excitation.
Hormone can be distributed at any where in body by the blood circulation system.
But, hormone stimulates the specific target cells only. For example, sex hormone
can put powerful effect on the sex differentiation. Androgen can promote the
development of penis and male germ organs, and estrogen can promote the
development of vagina, uterus and other female germ channels or cavities. If the
embryonic testes were removed, the embryo will be developed as female
individual.
3．Extracellular matrix (ECM):
ECM can interact with the integrin on cell surface to activate the focal
adhesion kinase (FAK), and by the growth receptor bound protein 2 (Grb2), FAK
can start the Ras signal pathway to cause cell proliferation.
In vitro, ECM can induct the differentiation direction of stem cells. If the stem
cells are cultured on a collagen IV coated dish, the stem cells will be differentiated
as epidermal cells. If on a collagen I or laminin coated dish, the stem cells will be
differentiated as fibroblasts. If on a collagen II coated dish, the stem cells will be
differentiated as chondrocytes.
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Inhibition of cell division:
There are two ways at least to inhibit cell division in embryo: 1. Regulate
down the level of stimulation signals. 2. Inhibit cell cycle promoter. During
myogenesis, the myostatin, a member of TGF-β super family, is a negative
regulator for muscle growth. The myostatin mutated animal can present the
double-muscled phenotype. If this gene is knockout, the weight of muscle tissue
of the mouse will be increased by 2 – 3 folds. Myostatin inhibits the proliferation of
muscle by up-regulating the level of p21, an inhibitor to CDK2, and downregulating the level of CDK2. The inefficiency of activity of CDK2 causes that the
transcription factor, E2F can not be released out from Rb, so, cell can not enter S
phase from G1 phase. Myostatin is secreted by muscle cells. The level of
myostatin is high in embryo, but low in adult. The excessive expression of
myostatin will cause muscular atrophy.
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Myostatin
downregulates
the muscle
growth
(from Heather
Arnold, 2001)
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Apoptosis:
Cell
death
Apoptosis: Disorder of programmed cell death (PCD) caused
by apoptosis genes and other inducers.
PCD: Cell death follows spatiotemporal sequence of cell
proliferation regulation.
Necrosis: Cell death caused accidentally by external pathogens,
stimulations or wounds.
We can regulate apoptosis partially for some special aims.
Apoptosis research is very hot for many years. The detail about it will be
presented in next chapter.
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The behaviors of cell
Cell behaviors are depended on the external signals and the features of cell.
The cell behaviors include directed mitosis, differential growth, apoptosis,
migration, differential adhesion, cell contraction, Matrix swelling, gap junction,
and fusion.
Directed mitosis: External signal can regulate the direction of spindle and
have the division directed. The new generated cells can be located on a specific
area during the directed mitosis. Directed mitosis is associated with asymmetric
division.
Differential growth: The original pattern will be changed during the
individual development. Different parts will form different organ or tissue.
Different growth follows spatiotemporal sequence.
Migration: Migration means that a mass of cells migrates to a place from
another place. Migration is important in the development of organ and tissue,
and wound healing. Migration rate is a useful parameter to the cell mobility or
motility.
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Differential adhesion: Differential adhesion means the cells form junction
by the glycoprotein on their surface or ECM temporarily or consistently. The
functions of differential adhesion include: (1) the cells in same type or relative
type are combined together to form tissue or organ; (2) the spine, folds, cavity,
and others will be formed by differential adhesion; (3) adhesion and release are
the basic procedures of migration.
Contraction: Contraction is driven by myosin and actin.
Fusion: Muscle cell is a plasmodium fused by myoblasts.
Gap junction: Cell communication can regulates differentiation by gap
junction. This is a popular way to differentiation regulation.
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Behaviors of developing cells (From Salazar-Ciudad, 2003）
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Myotube is
fused by
myoblasts
(From Harvey
Lodish, 1999)
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The changes of cell structure
The changes of chromosome structure
The deletion of genes: It means the lost of some parts of chromosome during
the cell differentiation in some animals, such as protozoa and insect.
The amplification of genes: It means that the copy quantity of some special
genes was specifically increased in a cell of some animals, such as in some cells
of fruit fly, the DNA was replicated without any cell division, so, the polytene
nucleus was formed.
The rearrangement of genes: It is a very important way to regulate the gene
expressions. For example, 106 – 108 antibodies can be generated in mammalian
body, but that does not means there are so many genes for antibodies in body.
The variety of antibody is just depended on the rearrangement of antibody gene
fragments.
The methylation of DNA: The activities of some genes of vertebrates are
inactivated by methylating them. Of course, demethylation can activate them. All
genes in cell can be sorted as two types: house-keeping genes and luxury
genes. The former means some genes are important for cell survival. The latter
means some genes are associated with cell differentiation, and expressed
specifically in some tissues only. Luxury genes keep demethylated in some tissues
and methylated in other tissues. All methylation are almost located on the “C” of 5'CG-3’, and makes cytimidine becomes methyl cytimidine.
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The replacement of isoform:
The one of the most popularly happened events in the development is the
serial replacements with spatiotemporal sequence. We call this replacement as
isoform replacement. It means that during a special stage, a molecule, cell, or
organ is replaced by another cell, molecule or organ that is very similar to original
one with same function but better to match development needs than original one
in a new environment. The unit in the serial replacement is called as isoform. We
have a lot of instances for it: the primary embryonic red blood cells are nucleated
erythrocyte, they will be replaced by non-nucleated (acaryote) erythrocytes
generated by liver. The embryonic hemoglobin molecules will be replaced by the
fetus hemoglobin molecules that will be replaced by adult those. The teeth will be
replaced by their isoforms when a baby is developed as a boy or girl. The
replacement of kidneys by their isoforms is the most complicated isoform
replacement during embryonic development.
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III. The potential differentiation ability of cells
Totipotence, pluripotence, and unipotence:
A fertilized ovum can be differentiated as any cell, tissue, organ, and
individual, so, it is totipotent cell. With the development, cells will lose their
potential differentiation ability from totipotent cells to pluripotent cells, finally to
unipotent cells (adult cells or somatic cells). For example, pluripotent
hematopoietic stem cells can be developed as any blood cells (unipotent cells).
Totipotence of fertilized ovum
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Totipotence of plant cell
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Matured animal cells are not of totipotence or pluripotence
because of plasma, not nucleus. A nucleus contains the repertoire of
genome DNA (Complete set of genes) that means a nucleus keeps
totipotence. That is why many scientists could clone the animal
individuals from a differentiated (adult) cell, such as goat, pig, and
others. Of course, a human adult cell should have the totipotence to be
developed as a human individual, but, laws do not allow you to do it!
For a person, his/her skin, blood cells, and mucosal cells need to
be continuously replaced by new cells generated from stem cells. Stem
cells are the cells with pluripotence that can be developed as many
types of cells, but not as a new individual naturally. Unipotent stem
cells are developed from pluripotent cells, and they can be developed
as some specific cells only. Unipotent stem cells is also called as
progenitor.
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Potential differentiation ability of stem cell
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The features of stem cells
The features of stem cells are as the follows: ① Keep non-differentiated or
low differentiated status in whole life; ② The number and location in body are not
variable usually; ③ Be of the recruiting themselves; ④ Can be proliferated
unlimitedly; ⑤ Pluripotence; ⑥ Most of stem cells are G0 phase cells; ⑦ Be
cleaved by two ways: symmetric division and asymmetric division. Former will
form two new stem cells, and latter will form one stem cell and one progenitor.
Totipotent stem cells: These cells keep totipotence to form any cells in body,
but not form an individual naturally.
Pluripotent stem cells: These cells keep pluripotence to form many types of
cell, or the cells of some tissue type, such as blood cells.
Unipotent stem cells: These cells keep unipotence to form one type of cell only,
and they are of the limited ability of recruiting themselves.
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The embryonic stem cells (ESC)
By the spatiotemporal sequence, stem cells can be sorted as ESC and adult
stem cells. ESC is the totipotent or pluripotent cell isolated from embryonic cell
mass or generated by the transplantation of the nucleus of adult cell.
ESC can be used to:
① clone animals. The cloning generation of animals by replacing an ovum
nucleus by a nucleus of adult cell is difficult. The cloned animals by this high
technology are of some genetic deficiencies, such as immunodeficiency.
② generate transgenic animals. ESC is the best vector to this aim
because the success rate can be obviously increased for the generation.
③ cell or tissue engineering. ESC can be artificially and directorially
differentiated as some special tissue or organ for the clinic needs.
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Somatic (Adult)
cell
Ovum or
embryonic cell
Generating an ESC by nucleus transplantation
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Regeneration
Regeneration means specially that a wounded organ can repair itself by
generating the wounded part with same form and function, and generally that a
molecule, cell, or organ can be replaced or regenerated.
The types of regeneration:
Physiological regeneration: It means cell replacement of isoform. For
example, 6 million old erythrocytes are replaced by new generated erythrocytes
per second in human body.
Repairing regeneration: It means the regeneration of wounded organ.
Many invertebrates have powerful ability for repairing regeneration.
Reconstruction: It means some special regeneration under experimental
conditions.
Asexual reproduction: Low grade living things, such as some parasites,
can take asexual reproduction.
There are some interested questions about regeneration as the follows:
1．How is the body informed for that which organ or part was lost or
damaged, and how much was lost? In other words, how to regulate the
regeneration?
2．Where are the new replaced part from? Is it from stem cells or the
remained differentiated cells adjacent the wound? Many experimental results
show that the differentiated (adult) cells can be dedifferentiated, migrated, or
proliferated for the wound healing.
3．Is it reconstruction or cell proliferation?
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