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Lecture
outline
Stem
cells
in
your
body
Maintaining
and
dieren.a.ng
stem
cells
Nuclear
reprogramming
Stem
cells
and
regenera.on
Stem cells: cells dened by their ability to self renew and dieren7ate into one or many mature cell types. Vertebrate stem cells: Embryonic stem cells Adult stem cells: Skin, hair follicles, Intes7ne, germ cells, hematopoei7c, heart, hippocampus Plant stem cells: Shoot apical meristem, root apical meristem ,oral mersitem Stem cell terminology: To7-potent, pluripotent, mul7-potent, etc. What do these terms mean?
Embryonic stem cells are derived from the inner cell mass of the blastocyst
Posterior growth and formation of the ! body! This embryo has already
nished gastrulation!
3-somite!
Clonal
analysis
indicates
that
the
classic
germ
layer
model
does
not
hold
true
Mice
transgenic
for
an
inac.ve
LacZ
gene
are
used
for
lineage
labeling
Rare
mito.c
recombina.on
events
create
ac.ve
LacZ
Based
on
known
cell
division
rate
and
clone
size,
developmental
.me
of
recombina.on
event
can
be
extrapolated
Neural
and
mesodermal
.ssue
are
more
closely
related
than
neural
and
epidermal
.ssue
Dev
Cell.
2009
Sep;17(3):365-76
During
body
forma.on,
Wnt
signaling
allocates
tailbud
stem
cells
to
proper
fates.
Wnt
signaling
ini.ally
species
mesoderm
over
spinal
cord.
Wnt
signaling
controls
a
second
fate
decision
within
the
mesoderm
causing
cells
to
become
muscle
rather
than
blood
vessels.
Developmental
Cell
22,
223232,
January
17,
2012
The
tradi.onal
model
of
germ
layer
forma.on
and
its
derivi.ves
does
not
hold
true
for
posterior
.ssues.
Paraxial
mesoderm
and
neural
plate
are
more
closely
related
than
neural
plate
and
epidermis.
Wnt
signaling
turns
on
Tbx6
expression
and
represses
Sox2
expression.
Current
Opinion
in
Gene.cs
&
Development
2012,
22:374380
Stem cells are found in niches in the ovary, tes7s, bone marrow, intes7ne, skin, hair follicles, the heart and hippocampus. [a] In a simple niche a stem cell has a permanent partner to which it is aKached by an adherens junc7on. The cells divide asymmetrically to give another stem cell and a dieren7ated cell. [b] In a complex niche two or more dierent stem cells [red and pink] are supported by one or more partner cells. [c] In a storage niche stem cells are stored un7l ac7vated by an external signal.
True pluripotent stem cells characteris7cs: 1. Pluripotent: capable of forming a mul7plicity of 7ssues. 2. If injected into an irradiated mouse [whose stem cells are destroyed]: A. Should migrate to bone marrow B. Should rescue mouse; stem cell proper7es restored. C. Cells from rescued mouse should rescue other irradiated mice. D. Should be capable of forming a mul7plicity of 7ssues in subsequent rescued mice.
xxxxxxxx
A true stem cell should rescue an irradiated mouse whose own bone marrow is destroyed.
Typical of aging stem cells, HSCs go from 100% cycling in early life to nearly 100% quiescent in late life. JCB vol. 195 no. 5 709-720
N. Lobo ,Y. Shimono ,D. Qian & M.Clarke. Annual Review Cell & Developmental Biology 22: 675-699.2007
Myosta.n is a TGF- family member that inhibits myoblast prolifera.on and dieren.a.on.
Growing root .p Experimental data calls in to ques7on the func7on of root 7p stem cell niche. Organ regenera7on does not require a func7onal stem cell niche in plants
Lecture
outline
Stem
cells
in
your
body
Maintaining
and
dieren.a.ng
stem
cells
Nuclear
reprogramming
Stem
cells
and
regenera.on
Lecture
outline
Stem
cells
in
your
body
Maintaining
and
dieren.a.ng
stem
cells
Nuclear
reprogramming
Stem
cells
and
regenera.on
Nuclear
reprogramming
Seminal
experiment
by
John
Gurdon
showing
that
dieren.ated
cells
s.ll
have
all
of
the
gene.c
material
necessary
to
create
a
whole
animal
AND
that
the
nucleus
can
be
reprogrammed
by
cytoplasmic
factors.
cause adverse conditions, birds may migrate to 2. Geiser, F. & Ruf, T. Physiol. Zool. 68, 935966 (1995). ary to avoid them. However, many birds are seden- 3. Cossins, A. R. & Barnes, B. M. Nature 382, 582583 (1996). 4. Brigham, R. M. Physiol. Zool. 65, 457472 (1992). (Tb). tary and often rely on ephemeral, weather- 5. Reintertsen, R. E. Polar Res. 1, 269284 (1983). stain- dependent food sources so how do they 6. Krtner, G. & Geiser, F. Oecologia 123, 350357 (2000). dverse overcome periodic energy bottlenecks? 7. Krtner, G. & Geiser, F. J. Zool. Lond. 248, 501507 (1999). To answer this question, we investigated small urvive whether the Australian tawny frogmouth udy of (Podargus strigoides ; Fig. 1), a sedentary ted to bird which feeds mainly on arthropods, Ageing Nuclear transfer uses torpor in the wild. The study was 2,4,5), was performed over conducted from the Australian autumn to come 6 genera.ons. summer in an open woodland of Eucalyptus brief com and Acacia at 1,000 m altitude in a cool Telomere length temperate area ice have been cloned by nuclear on sequentially was measure by near Armidale, New South telomere shorten 13 Wales. We captured seven frogmouths and transfer into enucleated oocytes , Clone come of the clo southern b lot. only 12% of r fitted them with temperature-sensitive and here we describe the reiterative live-born clones Telomeres do not for donor nuclei transmitters (calibrated to the nearest 0.1 cloning of mice to four and six generations cannot be exclud C) weighing 3 g. All birds received an in two independent lines. Successive generkb shorten amer serial required into th 48.5 4 transfer on telom external backpack-style transmitter ations showed no signs of premature (long cloning (they may Teruhiko Wakay kb Kellie L. K. Tama 48.5 by gross behavioural ageing, as judged range) to measure skin temperature (Tskin), 12.2 even g et l onger!). D. Caroline Blan 8.6 parameters, and there was no evidence of and three birds had a second internal transBlanchard||, Atsu Tanemura, Mak 12.2 mitter (short range), to measure core Tb shortening of telomeres at the ends of chroAnthony C. F. Pe Peter Mombaert 8.6 an indicator of cellular normally and to determine TbTskin differentials, mosomes, Line A Line B *Department of An implanted under general anaesthesia. senescence in fact, these appeared to John A. Burns Scho Figure 1 Telomere lengths in successive generations (G1G5) of mice cloned from cumulus cells. Southern-blot analysis of terminal Hawaii, Honolulu, increase slightly in length. This increase is Transmitter signals were recorded at 10restriction-enzyme-cut fragments in five sequential generations shows that telomeres do not undergo incremental erosion in successive Department of Ce clonal generations. Genomic DNA isolatedgiven from peripheral-blood lymphocytes taken from representative from each generation Research, Kyoto Un surprising, that the number of animals mitotic min intervals for up to nine months6. was digested with the restriction enzyme Hin fI, resolved on a pulse field gel, transferred to a solid support and probed with a 5- PKyoto 606-8507, Ja greatly exceeds that ofof mice sexually All individuals entered torpor in winter: labelled (T AGdivisions ) oligonucleotide. Peripheral blood lymphocytes were sampled on the same day. Ages (in months) were: in line A, Life Sciences Divis donor, 18; G1, 16; G2, 14; G3, 12; G4, 9; G5, 9; in line B, G1, 15.5; G2, 13; G3, 11; G4, 9; G5, 7. Suffix numbers (G4-1 and G4-2, for Laboratory, Univer produced animals and that any deleterious Tb fluctuated around 3840 C during example) identify different pups of each generation. Cailfornia 94720, U ||Bekesy Laboratory effects of cloning might be expected to be activity and fell to about 36 C during the NATURE | VOL 407 | 21 SEPTEMBER 2000 was repeated with cumulus cells from adult assays designed to monitor signs of prema- Hawaii, Honolulu, in cloned mice. Our rest phase, with a lower limit of 34 C. On such as a decline in activity in Department of Ve G1 mice amplified as nucleus donors to sequentially produce the ture ageing,
Serial
cloning
of
mice
does
not
reveal
shortening
of
telomeres
over
subsequent
genera.on
Cloning of mice to six generations
M
Donor G1 G2
G1-2
G2-2
G3-1
G3-2
G4-1
G2-1
G4-2
G1-1
G4-1
G3
G4-2
G5
32
3 3
Active Rb protein inhibits E2F protein. Mitogen-activated G1-Cdk leads to the phosphorylation of Rb which inactivates it and releasing active E2F protein which promotes progression through the cell cycle.
Cells can be fused to form heterokaryons (cells with gene.cally dis.nct nuclei). Using dierent species allows tracking of cell-type specic DNA. Specic factors required for reprogramming can be iden.ed and then tested in other stem cell systems.
Lecture
outline
Stem
cells
in
your
body
Maintaining
and
dieren.a.ng
stem
cells
Nuclear
reprogramming
Stem
cells
and
regenera.on
Dedieren.a.on
Transdieren.a.on
Hydra
Hydra
Regenera.on
Is
Accomplished
with
Three
Dierent
Stem
Cell
Popula.ons(A)
Hydra
are
cnidarians
with
a
primary
body
axis
containing
a
hypostome
(or
head)
at
one
end
and
a
foot
at
the
other.
Cell
prolifera.on
in
the
body
column
con.nually
pushes
cells
to
the
poles
of
the
body.
Asexual
reproduc.on
is
accomplished
by
budding.(B)
The
body
wall
contains
two
epithelial
cell
layers,
ectodermal
and
endodermal
epithelial
cells.
Inters..al
stem
cells
exist
within
the
ectodermal
epithelial
cell
layer.(C)
The
ectodermal
and
endodermal
epithelial
cells
proliferate
con.nuously
to
maintain
these
.ssue
layers,
producing
dieren.ated
epithelial
cells,
and
are
therefore
considered
to
be
dis.nct
stem
cells.
A
third
stem
cell
type,
the
mul.potent,
inters..al
stem
cell
can
self-renew
and
produce
neurons,
nematocytes,
secretory
cells,
and
gametes.
Planaria
Planarian
Regenera.on
Is
Accomplished
with
Pluripotent
Stem
Cells
Called
cNeoblasts(A)
Neoblasts
(blue)
are
the
soma.c
dividing
cells
of
planarians
and
are
depicted
in
blue.
Dividing
cells
are
scasered
throughout
the
body,
but
restricted
to
behind
the
eyes
and
absent
from
the
pharynx
(centrally
located). (B)
Irradia.on
with
1750
rad
can
result
in
animals
with
a
single
surviving
dividing
cell.
This
single
cell,
a
clonogenic
neoblast
(cNeoblast),
can
divide
and
produce
a
colony
of
dividing
cells,
ul.mately
producing
dieren.ated
cells
spanning
germ
layers
(Wagner
et
al.,
2011).
For
example,
individual
cNeoblasts
can
generate
both
neurons
and
intes.ne
cells,
as
well
as
dened
dividing
cell
progeny
popula.ons.(C)
Irradia.on
with
6000
rad
eliminates
all
dividing
cells.
Transplant
of
a
single
cNeoblast
from
a
donor
strain
(red)
results
in
clonogenic
growth
and,
ul.mately,
the
restored
capacity
for
regenera.on.
Four phases of self-driven morphogenesis of the op.c cup in embryonic stem cell culture. The re.nal epithelium forms the op.c cup by evagina.on (phase 1), the distal epithelium becomes relaxed and asened (phase 2), the epithelial junc.on undergoes a strong apical constric.on (phase 3), and the neural re.na expands and invaginates to form an op.c-cup shape (phase 4). Three local rules for the .ssue mechanics involved have been elucidated40, 81: rule 1, mechanical relaxa.on of the neural re.na by local reduc.on of ac.ve myosin (loss of the phosphorylated regulatory subunit of myosin pMLC2 results in a loss of ac.ve myosin); rule 2, strong apical constric.on of the hinge epithelium (making this por.on of the epithelium wedge-shaped); and rule 3, rapid tangen.al expansion of the neural re.na, which causes inward bucking of the .ssue by genera.ng compression. Compression in the neural re.na was demonstrated by three-dimensional (3D) specic cell abla.on through pinpointed irradia.on with a mul.photon (MP) laser40, in which the gap lem by the abalated cell was quickly lled by strong lateral compression. How local mechanical rules for .ssue s.ness, strain and stress are self-controlled in a phase-specic manner is a fundamental ques.on for future studies in four-dimensional (4D) force biology.
H. OK, T. MaKhiesen, S-K Goh, L. Black, S. Kren, T.Netho & D. Taylor. Nature Medicine 14:213-221. Feb. 2008