Cellular Biology

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Cellular Biology
School of Life Sciences Shaanxi Normal University
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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 Acrosome reaction
Cortical reaction Formation of
original nucleus 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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(No Transcript)
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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.
ß1,4-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 cells 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
Receptor Tyrosine Kinase FGF(branchless) FGF  
Receptor Tyrosine Kinase ephrins Eph  
TGFß Family TGFß TGFß  
TGFß Family BMP(Dpp) BMP Chordin(Sog),noggin
TGFß Family Nodal    
WNT WNT Frizzled Dikkopf,sFRP,Cerberus
Hedgehog Hedgehog    
Notch Delta  Notch Fringe
 
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The pathways of induction signals (From Thomas
Edlund and Thomas M. Jessell 1999)
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Cascade signalingCascade 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
signalingThe 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
signalingThe 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
signalingOne type of signal can determine a
differentiation for a cell, but two types of
signals can cause another differentiation
way. Lateral signalingA 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-ß
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 down-regulating 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
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.
Cell death
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 ? Keep
non-differentiated or low differentiated status
in whole life ? The number and location in body
are not variable usually ? Be of the recruiting
themselves ? Can be proliferated unlimitedly ?
Pluripotence ? Most of stem cells are G0 phase
cells ? 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 ? 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. ? generate
transgenic animals. ESC is the best vector to
this aim because the success rate can be
obviously increased for the generation.
? 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?