1. BACKGROUND
• During
aging,
the
immune
system
becomes
less
ef5icient
in
deterring
invaders,
infections,
and
disease.
• It
is
currently
known
that
aging
of
hematopoietic
stem
cells
(HSCs)
leads
to
an
increased
production
of
myeloid
cells
and
a
decreased
production
of
T
and
B
cells.
How
this
clonal
phenotype
arises
remains
inconclusive.
• To
better
understand
the
aging
process
,
it
is
important
to
examine
the
clonal
behavior
of
the
heterogeneous
stem
cell
population
over
time.
Tracking
Hematopoietic
Stem
Cells
in
vivo
Over
Time
Keck
School
of
Medicine
of
USC
Mike
Chin,
Rong
Lu
METHODS
Figure
1.
Genetic
barcoding
and
tracking
of
HSC
clones
in
vivo.
Donor
HSCs
were
isolated
from
the
bone
marrow
of
mice.
HSCs
(ckit+/
lineage(CD3,CD4,
CD8,B220,Gr1,Mac1,Ter119)-­‐
Sac1+/CD34-­‐/CD150+/Flk2-­‐)
were
infected
with
a
lentiviral
vector
so
that
each
HSC
received
a
unique
barcode.
Mice
were
lethally
irradiated
and
transplanted
with
barcoded
HSCs.
Peripheral
blood
was
collected
from
recipient
mice
at
several
time
points
over
1
year.
Genomic
DNA
from
granulocytes,
B,
CD4T,
and
CD8T
cells
were
quanti5ied
with
high
throughput
sequencing.
Figure
2.
HSC
production
changes
over
time.
The
proportion
of
granulocytes,
B,
CD4T,
and
CD8T
cells
relative
to
total
number
of
white
blood
cells
was
examined
on
Day
61,
117,
297,
362.
Myeloid
production
is
seen
to
increase
while
lymphoid
production
is
seen
to
decrease
over
these
4
time
points.
BLOOD
PRODUCTION
OVER
TIME
REFERENCES
Lu
R,
Neff
NF,
Quake
SR,
Weissman
IL.
(2011)
Tracking
single
hematopoieCc
stem
cells
in
vivo
using
high-­‐throughput
sequencing
in
conjuncCon
with
viral
geneCc
barcoding.
Nat
Biotechnol.
29,
928-­‐33.
Leventhal
J,
Miller
J,
Abecassis
M,
Tollerud
DJ,
Ildstad
ST.
(2013)
Evolving
approachs
to
hematopoieCc
stem
cell-­‐based
therapies
to
induce
tolerance
to
organ
transplants:
the
long
road
to
tolerance.
Clin.
Pharmacol
Ther.
93,
36-­‐45.
Weissman
IL,
Shizuru
JA.
(2008)
The
origins
of
the
idenCficaCon
and
isolaCon
of
hematopoieCc
stem
cells,
and
their
capability
to
induce
donor-­‐specific
transplantaCon
tolerance
and
treat
autoimmune
diseases.
Blood.
112,
3543-­‐53.
F
Median
Percentage
Figure
3.
Lineage
bias
of
HSC
clones
varies
over
time.
(A-­‐D)
Proportion
of
granulocyte,
B,
CD4T,
CD8T
clones
from
irradiated
recipients
on
days
61,
117,
297,
362
were
examined
for
clonal
lineage
bias.
Lineage-­‐
biased
clones
are
de5ined
as
those
whose
relative
copy
numbers
in
one
lineage
are
more
than
2.4142
(cotangent
22.5
degree)
times
their
relative
copy
numbers
in
the
other
lineage.
(E-­‐H)
Histograms
indicate
clonal
lineage
bias
behavior
over
time.
Clones
are
assigned
a
score
from
-­‐1.0
to
1.0,
indicating
the
relative
bias.
Figure
4.
Dominant
clone
production
varies
with
cell
type
over
time.
(A-­‐B)
Clonal
dominance
is
de5ined
as
the
percentage
of
a
cell
population
contributed
by
the
top
3
or
top
5
most
abundant
clones.
Averages
for
top
clones
are
plotted
for
all
cell
types
over
days
61,
117,
297,
and
362.
(C)
Heat
map
examining
clonal
contribution
over
time
for
each
cell
type.
Each
horizontal
line
represents
a
clone.
Bright
red
indicates
high
clonal
production.
0
20
40
60
80
Granulocyte
Bias
Balance
CD8T
Bias
Clonal
ComposiCon
(%)
*
**
*
0
20
40
60
80
100
CD4T
Bias
Balance
CD8T
Bias
***
***
***
***
***
***
*
*
Gr
Bias
B
Bias
D61
D117
D297
D362
0
10
20
30
40
50
60
Granulocyte
B
CD4
T
CD8T
Blood
composiCon(%)
***
***
**
***
***
***
**
**
***
**
****
***
**
Median
Percentage
Median
Percentage
Median
Percentage
0
10
20
30
40
50
Granulocyte
Biased
Balance
B
Cell
Biased
Clonal
ComposiCon
(%)
0
10
20
30
40
50
60
70
Granulocyte
Bias
Balance
CD4T
Bias
*
*
Gr
Bias
B
Bias
Gr
Bias
B
Bias
Gr
Bias
B
Bias
C
CLONAL
LEVEL
LINEAGE
BIAS
CLONAL
DOMINANCE
H
G
E
A
D
C
B
Gr
Bias
CD4T
Bias
Gr
Bias
CD4T
Bias
Gr
Bias
CD4T
Bias
Gr
Bias
CD4T
Bias
Gr
Bias
CD8T
Bias
Gr
Bias
CD8T
Bias
Gr
Bias
CD8T
Bias
Gr
Bias
CD8T
Bias
CD4T
Bias
CD8T
Bias
CD4T
Bias
CD8T
Bias
CD4T
Bias
CD8T
Bias
CD4T
Bias
CD8T
Bias
A
cv
0
10
20
30
40
50
D61
D117
D297
D362
Clonal
ComposiCon
(%)
*
*
0
10
20
30
40
50
60
70
D61
D117
D297
D362
*
*
TOP
3
TOP
5
D61
D117
D297
D362
D61
D117
D297
D362
D61
D117
D297
D362
D61
D117
D297
D362
Granulocyte
CD4T
B
CD8T
D61
D117
D297
D362
D61
D117
D297
D362
D61
D117
D297
D362
B
cv