Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND REAGENTS FOR THE DETERMINATION OF TELOMERE
LENGTH IN A SEMI-AUTOMATIC MANNER OF EVERY SINGLE CELL IN
A IMMOBILIZED CELL POPULATION
FIELD OF THE INVENTION
The invention relates to methods for determining telomere length within the
cells of an
immobilized cell population as well as to methods for the identification of
stem cells in
a cell population based on the telomere length. Both methods rely on detecting
the
fluorescent emission of telomere-specific probes which have been previously
contacted
with the population of cells wherein the telomere length of the individual
cells is to be
determined or wherein the stem cells are to be identified.
BACKGROUND OF THE INVENTION
Telomere length is a parameter of interest not only with respect to the study
of the
telomere biology but also as a marker for aging and cancer. Regarding aging,
it is
known that telomere length decreases with age because telomerase activity in
adult
tissue is not sufficient to prevent telomere shortening, thus compromising
cellular
viability (Harley et al., 1990 and Blasco et al., 1997). In the case of cancer
cells,
telomere length is maintained due to the over-expression of telomerase or due
to the
activation of alternative mechanisms which promote telomerase elongation (Kim
et al.,
1994 and Bryan et al., 1997).
Thus, telomere length can be used both in aging studies, as a marker of
biological
fitness of human populations (Cawthon et al., 2003; Epel et al., 2004, and
Valdes et al.,
2005, Lancet, 366:662-664), in cancer and in screening methods for the
identification of
compounds interfering with said biological fitness.
The most widely used method for determining telomeric length is the so-called
telomere
restriction fragment assay (Moyzis et al., 1988). This method is based on a
Southern
blot hybridisation of a telomeric restriction fragments derived from genomic
DNA using
probes specific for the telomere repeats. However, TRF is a time-consuming
technique
which requires plenty of cells and only provides an average telomeric length
of the cell
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population under study without giving an indication of telomere length in
individual
cells.
Another method for determining telomere length is quantitative fluorescent in
situ
hibridisation (FISH) based on the use of fluorescence microscopy on a
preparation of
metaphasic cells using a telomere-specific probe (Lansdorp et al., 1996;
Zijlmans et al.,
1997), (Martens et al., 1998). This technique is also cumbersome and time-
consuming
and requires cells in metaphase, which excludes from all those cells which can
not
proliferate in culture.
Another method for determining telomere length is flow fluorescent in situ
hybridisation (flow-FISH) based on the determination of telomeric fluorescence
in
interphase cells using flow cytometry wherein the cells are labelled with a
fluorescently-labelled telomere-specific probe (Rufer et al., 1998; Baerlocher
et al.,
2006). However, this method is only applicable to cells in suspension and the
results are
frequently biased due to auto-fluorescence of the cytoplasm.
The hybridization protection assay described by Nakamura et al., (Clinical
Chemistry,
1999, 45:1718-1724) is based on a chemo luminescence determination of the
amount of
telomere-specific probe and normalized to the signal obtained with an Alu
repeat-
specific probe. However, this method requires a constant number of Alu repeats
in the
genome.
Other methods for determining fluorescence length include the hybridization
assay
(Freulet-Marriere et al, 2004), primed in situ labeling (PRIMS) (Therkelsen et
al.,
1995), PCR-based methods such as STELA (Baird, D.M., et al., 2003) and
quantitative
PCR (Cawthon, R.M., 2002).
The identification of adult stem cell compartments is essential for studying
adult stem
cell properties and regulation, as well as for their potential use in
regenerative medicine.
The common approach to locate stem cell niches has been based on the different
expression of a protein marker, or more usually a complex set of protein
markers, in
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stem cell environments compared to more differentiated compartments, as well
as on
the general property that stem cells are long-term residents of a tissue and
have a low
proliferative rate (i.e. label-retaining techniques) (Fuchs et al., 2004,
Cell, 116:769-778;
Moore and Lemischka, 2006, Science, 311:1880-1885). These approaches are
limited
because each type of stem cell niche has its own specific set of markers.
Cotsarelis et al.(Cell, 1990, 61:1329-1337), Potten et al., (lnt. J. Exp.
Pathol., 1997, 78:
219-243) and Zhang et al. (Nature, 2003, 425:836-841) have relied on the
identification
of long-term retention cells (LRCs) for the identification of skin,
intestinal, and
hematopoietic stem cells. This assay is based in the identification, using DNA
labeling,
of cells in a given tissue that undergo slow cycling as measured by their
ability to retain
the labeled DNA for a much longer period than the rapid cycling progenitor
cells.
However, it is still not completely undisputed that LRCs are stem cells (Kiel
et al.
Nature, 2007, 449:238-42).
Doetsch et al., (Cell, 1999, 97:703-716), Ohlstein and Spradling, (Nature,
2006,
439:470-474), Palmer et al., (Mol. Cell Neurosci., 1997, 8:389-404) and Sanai
et al.,
(Nature, 2004, 427:740-744) have used in vivo lineage tracing to search for
cells that
give rise to the downstream lineages to identify neural stem cells and
Drosophila gut
stem cells were identified.
Kim et al., (Cell, 2005, 121:823-835) have relied on the identification of
multipotent
cells, as revealed by their co-expression of multiple downstream lineage
markers of
stem cells. In this way, lung stem cells were identified as bronchioalveolar
stem cells
(BASCs) based on their co-expression of two downstream Clara and Alveolar
lineage
markers, CCA and SP-C, and their ability to give rise to Clara and Alveolar
lineages.
The ability of stem cells to express certain types of multiple drug-resistant
genes and
display a unique pattern in flow cytometry assay has also been used to
identify the so-
called side population (SP). SP has been shown to be enriched with HSCs
(Goodell et
al., 1997, Nature Medicine, 3:1337-1345) and stem cells in other non-
hematopoietic
tissues (Goodell et al., Methods Mol. Biol., 2005, 290:343- 352).
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Additionally, several methods for the identification of adult stem cells have
been
developed based on functional characteristics of stem cells such as binding to
soybean
agglutinin (Reisner et al., 1982, Blood 59:360-363), resistance to the
treatment of either
5-fluorouracil (Gordon et al., 1985, Leukemia research 9:1017-1021 and Berardi
et al.,
Science 1995267:104-108) or alkylating agent (Sharkis et al., 1997, Stem cells
(Dayton,
Ohio) 15 Suppl 1, 41-44; discussion 44-45) and density-gradient (Juopperi et
al., 2007,
Experimental hematology, 35 :335-341).
W007124125 describes a method for the identification of stem cells wherein a
cell
population is treated with a DNA damaging agent, which results in the
quiescent stem
cells residing on the tissue become activated in order to replenish lost
cells. These cells
can be detected using a marker of DNA biosynthesis.
Thus, there is a need in the art for additional methods for the determination
of telomere
length and for methods for the identification of stem cell compartments within
adult
tissues which overcome the disadvantages of the methods known in the art as
well as
which are generally applicable to any tissue.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a method for the determination of
the telomere
length of a cell in an immobilised tridimensional test cell population
comprising
(i) contacting said test cell population and at least two homogeneous
immobilised control cell populations of known and stable telomere
length and having different average telomere lengths with a probe that
hybridises specifically to a repeat region within telomeric DNA and
which is labelled with a first fluorescent dye under conditions allowing
the probe to hybridise in situ to its complementary target sequences on
telomeres,
(ii) determining the average fluorescence intensities in the cell of said test
population and the average fluorescence intensity value of the cells
within each control cell population in response to a radiation adequate
to excite the fluorescent dye attached to said probe wherein the
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determination of the fluorescence signal in the cells is carried out on an
image of the cells obtained by fluorescence microscopy on a section of
said tissue sample or preparation of immobilised cells and
(iii) assigning to the cell within the test cell population an average
telomere
5 length
value, wherein said value is the average telomere length of a
cell within a control cell population showing an average cellular
fluorescence intensity value substantially identical to the fluorescence
intensity values of the cell within the cell population as determined by
interpolation.
In a second aspect, the invention provides a method for the identification of
stem cells
in a cell population which comprises
(0 contacting said cell population with a probe that hybridises
specifically to a repeat region within telomeric DNA and which is
labelled with a first fluorescent dye under conditions allowing the
probe to hybridise in situ to its complementary target sequences on
telomeres,
(ii) determining the average fluorescence intensities of each cell
within a
representative sample of in the cells of within said cell population in
response to a radiation adequate to excite the fluorescent dye
attached to said probe
wherein those cells within the sample showing the highest average fluorescence
intensity are identified as stem cells.
In a third aspect, the invention provides a method for the identification of
stem cells in a
test cell population which comprises
(0 contacting said test cell population and at least two homogeneous
control cell populations of known and stable telomere length and
having different average telomere lengths with a probe that
hybridises specifically to a repeat region within telomeric DNA and
which is labelled with a first fluorescent dye under conditions
allowing the probe to hybridise in situ to its complementary target
sequences on telomeres,
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(ii) determining the average fluorescence intensities in each cell of a
representative sample of cells within each cell of said test cell
population and the average fluorescence intensity value of the cells
within each control cell population in response to a radiation
adequate to excite the fluorescent dye attached to said probe and
(iii) assigning to each cell within the representative sample of the test
cell
population an average telomere length value, wherein said value is
the average telomere length of a cell within a control cell population
showing an average cellular fluorescence intensity value substantially
identical to the fluorescence intensity values of the cell within the
cell population as determined by interpolation
wherein those cells showing the highest telomere length value are identified
as stem
cells.
In a fourth aspect, the invention provides a method for the identification of
compounds
capable of triggering mobilisation of stem cells within a tissue having a
known spatial
distribution of stem cells comprising the steps of
(0 contacting said tissue sample with a candidate compound
under
conditions adequate for promoting mobilisation of the stem cells
within said tissue,
(ii) contacting a sample of said tissue with a probe that hybridises
specifically to a repeat region within telomeric DNA and which is
labelled with a first fluorescent dye under conditions allowing the
probe to hybridise in situ to its complementary target sequences on
telomeres and
(iii) determining the average fluorescence intensity of a representative
sample of cells the cells present in the region of the tissue sample
known to contain the stem cells
wherein a decrease in the average fluorescence intensity in the area which is
known to
comprise stem cells when compared to a sample which has not been treated with
the
candidate compound is indicative that the compound is capable of triggering
mobilisation of stem cells within the tissue sample.
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In a fifth aspect, the invention provides a method for the identification of
compounds
capable of triggering mobilisation of stem cells within a tissue having a
known spatial
distribution of stem cells comprising the steps of
(0 contacting said tissue with a candidate compound under
conditions
adequate for promoting mobilisation of the stem cells within said
tissue,
(ii) contacting said tissue and at least two homogeneous control cell
populations of known and stable telomere length and having different
average telomere lengths with a probe that hybridises specifically to
a repeat region within telomeric DNA and which is labelled with a
first fluorescent dye under conditions allowing the probe to hybridise
in situ to its complementary target sequences on telomeres,
(iii) determining the average fluorescence intensities of the cells a
representative sample of cells present in the region of the tissue
sample known to contain the stem cells and an average fluorescence
intensity value of the cells within each control cell population in
response to a radiation adequate to excite the fluorescent dye
attached to said probe and
(iv) assigning to each cell within the representative sample of cells
present the region of the tissue sample known to contain the stem
cells an average telomere length value, wherein said value is the
average telomere length of a cell within a control cell population
showing an average cellular fluorescence intensity value identical to
the fluorescence intensity values of the cell within the cell population
as determined by interpolation
wherein a decrease in the average telomere length in the area in the cells
within the
region of the tissue known to comprise stem cells when compared to a sample
which
has not been treated with the candidate compound is indicative that the
compound is
capable of triggering mobilisation of stem cells within the cell population.
In a sixth aspect, the invention relates to an array comprising at least two
immobilised
three-dimensional cell populations being each cell population physically
separated from
the other(s) and wherein each cell population has a stable and known telomere
length
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which is different to the average telomere length of the other cell
population(s) of the
array.
In a seventh aspect, the invention relates to a method for determining the
telomere
length of a cell within a tridimensional cell population from a collection of
at least two
fluorescence microscopy images obtained using a fluorescently-labeled telomere-
specific probe and corresponding to different focal planes of said cell
population
comprising the steps of:
(i) converting the at least two fluorescence microscopy images
corresponding to different focal planes into a single image by adding
up the fluorescence intensities at each position within the image,
(ii) determining the average fluorescence intensity of said cell within the
image of the cell population obtained in step (ii) and
(iii) assigning to said cell an average telomere length value, wherein said
value is obtained by interpolation of the average intensity of the cell
within a data set of telomere length values and corresponding
fluorescence intensity values obtained from different cell populations
of known and stable telomere length processed by fluorescence
microscopy in parallel to the cell of the test cell population.
In a eighth and ninth aspect, the invention relates to a computer program
including
encoded means to carry out the steps of the methods according to the invention
and a
computer-readable support comprising encoded means adapted to carry out the
steps of
the methods according to the methods of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Cells with the longest telomeres are enriched at the hair follicle
stem cell
compartment and show stem cell behaviour upon treatment with mitogenic
stimuli. Representative telomere length pseudo-color images of "resting" wild-
type (a)
and first generation telomerase-deficient (G1 Terc-/-) tail skin (b). Nuclei
are coloured
according to their average telomere fluorescence in arbitrary units (a.u.).
The different
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epidermal compartments are indicated and separated from the dermis (not
studied here)
by a dashed line. Asterisk indicates the sebaceous glands. Scale bars
correspond to 50
M. Note the specific enrichment of cells with the longest telomeres at the
hair bulge
area (the known hair follicle stem cell niche) in both wild-type (telomerase-
competent)
and telomerase-deficient mice. Bottom panels show the percentage of cells
showing a
given telomere fluorescence within the indicated epidermal compartment. A
total of 3
skin sections per mouse out of 2 mice per genotype were used for
quantification of
percentage of cells and standard deviation. n=total number of cells within the
indicated
compartment used for the analysis. c. Upon TPA treatment, wild-type epidermal
cells
showing the longest telomeres localized not only to the hair follicle stem
cell
compartment (hair bulge) but also to the transit-amplifying (TA) compartments
(hair
bulb and infundibulum). In contrast, this enrichment of cell with long
telomeres to the
TA compartments was abolished in TPA-treated G1 Terc-/- skin. d. Absolute
number of
cells showing an average telomere fluorescence between 1800-3000 a.u. per
indicated
skin compartment standard deviation (SD). A total of 3 skin sections per
mouse out of
a total of 2 mice per genotype were used for quantification the number of
cells per skin
compartment and standard deviation. n= total number of cells of each
compartment used
for the analysis (6 independent hair follicle images were counted). e.
Absolute total
number of epidermal cells per skin section with telomere fluorescence between
1800-
3000 a.u. SD. Note that TPA induces a net telomere elongation in wild-type
epidermis, which is abolished in the absence of telomerase activity in G1 Terc-
/- skin. A
total of 6 skin sections per genotype and condition were used for
quantification
purposes. n= total number of cells in the epidermis included for the analysis.
Statistical
significant is indicated for each comparison on top of the bars.
Figure 2. Cells with the longest telomeres are enriched at the hair follicle
stem cell
compartment in mice from a FVB genetic background. a. Representative telomere
length pseudo-color images of 2 month-old wild-type tail skin from a FVB
genetic
background. Nuclei are coloured according to their average telomere
fluorescence in
arbitrary units (a.u.). The different epidermal compartments are indicated and
separated
from the dermis (not studied here) by a dashed line. Asterisk indicates the
sebaceous
glands. Scale bars correspond to 50 M. Note the specific enrichment of cells
with the
longest telomeres at the hair bulge area (the known hair follicle stem cell
niche) Left
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panel shows the percentage of cells showing a given telomere fluorescence
within the
indicated epidermal compartment. A total of 3 skin sections per mouse out of 2
mice
were used for quantification of percentage of cells and standard deviation. n=
total
number of cells within the indicated compartment used for the analysis. b.
Telomere
5 length frequency histograms for cells located in the indicated skin
compartments.
n=number of nuclei per compartment analyzed for telomere FISH. Statistical
significance values are indicated.
Figure 3. Controls for the telomapping technique. a. Quantification of
centromere
10 fluorescence in different skin compartments with a PNA major satellite
probe.
Representative major satellite Cy3 fluorescence of wild-type tail skin. The
different
epidermal compartments are indicated and separated from the dermis by a dashed
line.
Asterisk indicates the sebaceous glands. Scale bars correspond to 50 M.
Quantification
of major satellite fluorescence signal in different compartments is shown in
the right
panel. No statistically significant differences in centromere fluorescence
were detected
between the different skin compartments, therefore ruling out that differences
in "probe
accessibility" or ploidy may explain the differences in telomere length
described here.
Five independent skin sections were used for the analysis. n=total number of
nuclei per
skin compartment used for the analysis. b. Nuclear size is shown for the
indicated skin
compartments. Note that no major differences are observed between the hair
bulge
(stem cell compartment) and the more differentiated interfollicular epidermis
and
infundibulum compartments, which cannot explain the telomere length
differences
described in Fig. lA
more marked decreased in nuclear size was detected in the hair bulge
compartment,
which did not reach statistical significance. A total of 8 independent skin
sections were
used for the analysis. n=total number of cells analysed for the indicated
compartment. c.
Quantification of centromere fluorescence in different testis compartments
with a PNA
major satellite probe. Representative major satellite Cy3 fluorescence of wild-
type
testis. The different testis compartments are indicated and separated by a
dashed line.
Scale bars correspond to 200 M. Quantification of major satellite
fluorescence signal
in different compartments is shown in the right panel. No statistically
significant
differences in centromere fluorescence were detected between the different
testis
compartments, therefore ruling out that differences in "probe accessibility"
or ploidy
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may explain the differences in telomere length described here. Five
independent testis
sections were used for the analysis. n= total number of nuclei per testis
compartment
used for the analysis.
Figure 4. Validation of telomapping results using conventional Q-FISH. a.
Telomere fluorescence obtained by telomapping or conventional Q-FISH in the
indicated skin compartments was represented relative to that of the hair bulge
(100%).
b. Note a very significant correlation between the telomere length values
obtained with
telomapping and conventional Q-FISH.
Figure 5. Calibration of telomapping technique using an array paraffin-
embedded
cell lines of known telomere length. a. Telomere fluorescence obtained by
telomapping of a paraffin-embedded array of the indicated human and mouse cell
lines.
In parenthesis is shown the known telomere length of these cell lines as
determined by
conventional Q-FISH on metaphases (Canela et al., 2007). Note that the
telomapping
technique is able to detect differences of telomere length of less than 1 Kb
(P<0.001
when comparing HeLa to HeLa2 cell line). b. Calibration curve to convert
telomapping
arbitrary units of fluorescence into kilobases. Note the linear correlation
between both
techniques. c. Average telomere length expressed in kilobases of the different
mouse
skin compartments as determined by telomapping and calibrated using the
calibration
curve shown in part (c). Note a decrease of telomere length of all the skin
compartments
compared to the hair bulge (stem cell compartment). n=total number of nuclei
analyzed
per compartment. Statistically significant differences between compartments
are also
indicated. To better assess differences in telomeric signal, maximum
projections of 16-
bits confocal images were obtained from cell line microarray and tail skin
paraffin
sections.
Figure 6. Telomere distribution pattern of wild type and GI Terc-/- epidermal
cells
before and after TPA treatment classified according to their location within
the
epidermis. Telomere fluorescence histograms for wild-type (left panels) and
telomerase-deficient G1 Terc4- mice (right panels) before (black columns) and
after
(red columns) TPA treatment. Epidermis has been subdivided in four different
compartments: hair bulge, hair bulb, infundibulum and interfollicular
epidermis. Total
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number of nuclei analyzed and average telomere length SD are indicated for
each
compartment, genotype and condition. A total of 6 skin sections per genotype
and
condition were used for quantification purposes. Statistical significance
comparisons
between the hair bulge and the different compartments are indicated for both
WT and
G1 Terc-/- skin. In addition, all telomere fluorescence comparisons between
untreated
and TPA-treated cases are significant (P<0.05), except P> 0.05 (not
significant) for hair
bulb, infundibulum and interfollicular epidermis of G1 Terc4- mice.
Figure 7. Clonogenic potential of GFP+ and GFP- K15-EGFP sorted cells. a.
Quantification of size and number of macroscopic colonies obtained from 1x104
GFP '
and GFP- purified keratinocytes from three independent 1-year-old K5-EGFP mice
and
cultured for 10 days on J2-3T3 mitomycin-C-treated feeder fibroblasts. Note
that GFP '
cells form 3 times more colonies than GFP- cells. b. Quantification of size
and number
of macroscopic colonies obtained from total keratinocytes (unsorted) from
three
independent 1-year-old K5-EGFP mice and cultured for 10 days on J2-3T3
mitomycin-
C-treated feeder fibroblasts. The percentage of colonies that are GFP-positive
within
each colony size range is indicated.
Figure 8. Isolated hair bulge stem cells from K15-EGFP mice show the longest
telomeres and telomerase activity. a. Representative DAPI and Cy3 images of
GFP+
and GFP- FACS-sorted keratinocytes from K15-EGFP mice. b. Histograms showing
telomere fluorescence frequencies on interphase nuclei as determined by Q-
FISH.
Average telomere fluorescence and standard deviation are indicated.
Differences in
telomere length between GPF+ and GFP- cells were highly significant (P<0.001).
n=number of nuclei used for the Q-FISH analysis from 2 independent K15-EGFP
mice.
The red lines highlight the increased frequency of long telomeres in GFP-
positive cells.
c. Number of telomere spots per nuclei in sorted GFP+ and GFP- cells, indicate
that
there are no differences in ploidy between these two populations. d.
Representative
DAPI and Cy3-centromeric images of GFP+ and GFP- FACS-sorted keratinocytes
from
K15-EGFP mice. e. Quantification of major satellite fluorescence signal in
sorted GFP+
and GFP- cells. No significant differences in centromere fluorescence were
detected
between both cells populations, therefore ruling out that differences in
"probe
accessibility" or ploidy may explain the differences in telomere length
described in Fig.
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13
2b. Two independent mice were used for the analysis. tr-- total number of
nuclei used
for the analysis. f. Average telomere length in kilobases of purified GFP-
positive and
GFP -negative cells from 0.5 and 1.5 year-old K l5-EGFP mice as determined by
Flow-
FISH. A previous immunostaining against GFP was performed to allow
identification of
cells according to their GFP expression (Experimental Procedures). Bars
indicate
standard errors, "n" is the number of cells analyzed per condition.
Statistical
significance calculations are indicated and were obtained with Kolmogorov-
Smirnov
tests. g. Telomere length as determined by TRF in sorted GFP- (a population
enriched
in differentiated cells) and GET+ tail skin keratinocytes (a population
enriched in stem
cells) from K1 5 -EGFP mice. Note increased TM; size in GIFP+ hair bulge cells
compared to GET- cells. h. Telomerase TRAP activity of purified GYP+ and GFP-
keratinocytes from K15-EGFP mice. The protein concentration tested is
indicated.
Samples were pretreated (+) or not (-) with RNase. An internal control (IC)
for PCR
efficiency was included (Experimental Procedures). HeLa cells are shown as a
positive
control for telomerase activity.
Figure 9. Telomapping maps the longest telomeres to the EGET+ cells in K15-
EGFP skin sections. a. Simultaneous detection of GFP and telomere fluorescence
in
K15-EGFP back skin. The different epidermal compartments are indicated and
separated -from the dermis (not studied here) by a dashed line. Right panels
show
confocal images corresponding to Alexarm488 fluorescence (GFP immunostaining)
and
the combined DAPI+GIT image. Note that UT-expressing cells localize to the
bulge
area of the hair follicle. the known putative stem cell niche. Left panels
show
topographic telomere length maps generated according to GIP status: all
nuclei, GFP
nuclei, and GFP+ nuclei. Nuclei are coloured according to their average
telomere
fluorescence in arbitrary units (a.u.). GFP-positive cells at the hair bulge
showed the
longest telomeres. Scale bars correspond to 50 nm. b. Telomere fluorescence
frequency
histograms according to GFP status in back and tail skin hair follicles from K
l5-EGFP
mice. Differences in telomere length between GPF+ and GFP- cells were highly
significant (P0.00 l) both in the back and tail skin. Average telomere
fluorescence and
standard deviation are indicated. n=number of nuclei analyzed. Four-to-six
skin sections
of either tail or back skin from a total of 2 mice were analyzed. The red
lines highlight
the increased frequency of long telomeres in GFP-positive cells. c. Percentage
of cells
CA 02723950 20150603
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with the longest telomeres (red cells after telomapping) or lvith the shortest
telomeres
(green colour atter telomapping) that are either GET+ or (FP-. Note that GFP+
cells are
enriched in the population of the cells with the longest telomeres.
Figure 10. Cells with the longest telomeres locate to stem cell compartments
in
different origin mouse tissues. a. Representative topographic telomere length
map of a
small intestine histolog,ical section generated from confbeal telomere Q-FlSH
images.
The different small intestine compartments are indicated. The dashed line
separates the
epithelial cells (ep) from other cell types not studied here: lamina propia
(LP),
muscularis mucosa (mm) and submucosa (subm). Scale bar corresponds to 70 pm.
Nuclei are coloured according to their average telomere fluorescence in
arbitrary units
(a.u.). Note that nuclei with the longest telomeres localized above the Paneth
cells at the
known stem cell niche (positions +4 to +5), as well as in the transient
amplifying (TA)
compartment (positions above +5). b. Scheme representing the small intestine
compartments of villi and 1..,ieberkiihn crypts. The crypts are further
divided in (i) the
Paneth cells at the bottom of the crypt between positions +1 and +3, (ii) the
stern cell
niche at position +4 to +5, right above the Paneth cells, and (iii) the TA
compartment at
positions >+5. c. Percentage of cells showing a given telomere fluorescence
within the
different compartments. The stem cell niche and the TA compartment are
enriched in
cells with the longest telomeres. while the villi are enriched in cells with
the shortest
telomeres. Average and standard deviation for the percentages are indicated. A
total of
39 crypts and 28 villi from 3 independent mice were quantified. n¨number of
nuclei per
compartment analyzed. Number in parenthesis indicates the cell position in the
crypt. d.
Telomere length frequency histograms for cells located in the indicated
compartments.
A remarkable increase in telotnere length is observed in stem cells and TA
cells
compared to Paneth cells and the villi. Average and standard deviation are
indicated. A
total of 39 crypts and 28 villi from 3 independent mice were quantified.
n=number of
nuclei per compartment analyzed for telomere FISH. Number in parenthesis
indicates
the cell position in the crypt. Vertical lines indicate the different
telomere
fluorescence ranges. All telomere fluorescence comparisons between the stem
cell compartment and the rest of the compartments are highly significant
(P<0.001), except significant (P<0.05) for comparison between the stem
cell compartment and the TA compartment. c, f, g. Right
CA 02723950 20150603
panels show representative telomere length pseudo-colour images of
histological
sections from cornea (e), testis (t) and brain hippocampus (g) of wild-type
mice. Nuclei
are coloured according to their average telomere fluorescence in arbitrary
units (a.u.).
Scale bars correspond to 200 Magnifications are shown of cornea and brain
5 sections for clarity purposes. Note that cells with the longest telomeres
localize
preferentially within the described stem cell compartment of each organ.
Middle panels
show the percentage of cells containing a given telomere fluorescence within
each
epidermal compartment. Right panels show telomere fluorescence histograms of
nuclei
in each compartment. Average telomere fluorescence and standard deviation are
10 indicated. The lines highlight the increased frequency,' of cells with
long telomeres in the
analyzed stem cell compartments. A total of b different images from each organ
from 3
independent mice were used for quantification purposes. CB: ciliary body. L:
lens,
SGZ: subgranular zone, GCL: granular cell layer. H: hilius, CA: pyramidal cell
layers.
All telomere fluorescence comparisons between each of the stem cell
compartments and
15 the corresponding differentiated compartment are highly significant
(P<0.001).
Figure 11. Telomere shortening with age in mouse stern cell compartments. a.
Representative telomere length pseudo-color images of different age wild-type
(a) and
third generation telomerase-deficient (G3 Terc-/-) tail skin (b). Nuclei are
coloured
according to their average telomere fluorescence in arbitrary units (a.u.).
The different
epidermal compartments are indicated and separated from the dermis (not
studied here)
by a dashed line. Asterisk indicates the sebaceous glands. Scale bars
correspond to 50
}Al. Note the decrease in cells with the longest telomeres at the hair bulge
area (the
known hair follicle stem cell niche) in wild-type mice with increasing age, as
well as in
6 month-old G3 telomerase-deficient mice. Right panels show the percentage of
cells
showing a given telomere fluorescence within the indicated epidermal
compartment. A
total of 2 skin sections per mouse out of 3 mice per genotype were used for
quantification of percentage of cells and standard deviation. n= total number
of cells
within the indicated compartment used for the analysis. c. Telomere length
frequency
histograms for cells located in the indicated compartments in mice of the
indicated age
and genotype. Notice statistically significant telomere shortening in wild-
type mice in
all the different skin compartments when comparing 2 month old to 2 year old
mice,
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16
including the hair bulge where the stem cells are located. A third generation
G3 Terc-
deficient mouse is shown for comparison. n=number of nuclei per compartment
analyzed for telomere FISH. Statistical significance values are indicated in
the Figure.
d. Average telomere fluorescence in the indicated stem cell compartments at
the
indicated age. Note a faster rate of telomere loss at > 1 year or age. e.
Average telomere
fluorescence in the indicated differentiated compartments of different tissues
at the
indicated age. Note a faster rate of telomere loss at > 1 year or age.
Figure 12. Telomere shortening with age in mouse small intestine stem cells a.
Representative topographic telomere length maps of small intestine
histological sections
from wild-type (a) or G3 Terc-deficient (b) mice of the indicated age with
confocal
telomere Q-FISH images. The different small intestine compartments are
indicated. The
dashed line separates the epithelial cells (ep) from other cell types from a
different
origin not studied here: lamina propia (LP), muscularis mucosa (mm) and
submucosa
(subm). Scale bar corresponds to 70 lam. Nuclei are coloured according to
their average
telomere fluorescence in arbitrary units (a.u.). Note that the percentage of
cells with the
longest telomeres decreases in the stem cell compartment when comparing 2
month-old
mice with 2 year old-mice. Telomapping of the small intestine of a 6-month old
G3
Terc-deficient mouse is shown for comparison c. Telomere length frequency
histograms
for cells located in the indicated compartments. A statistically significant
decrease
(P<0.001) in telomere lesngth is observed in all different compartments,
including the
stem cells, when comparing 2 month-old mice with 2 year-old mice. Average and
standard deviation are indicated. More than 20 crypts from at least 2
independent mice
per age group and genotype were quantified. n=number of nuclei per compartment
analyzed for telomere FISH. Statistical significance values are also
indicated.
Figure 13. Telomere shortening with age in cornea epithelium stem cells a.
Representative topographic telomere length maps of cornea epithelium sections
from
wild-type mice of the indicated age with confocal telomere Q-FISH images. The
different cornea compartments are indicated. CB, ciliary body. The dashed line
separates the epithelial cells from other cell types from a different origin
not studied
here. Nuclei are colored according to their average telomere fluorescence in
arbitrary
units (a.u.). Middle panels show the percentage of cells containing a given
telomere
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17
fluorescence within each epidermal compartment. Right panels show telomere
fluorescence histograms of nuclei in each compartment. Note that the
percentage of
cells with the longest telomeres decreases in the stem cell compartment when
comparing 2 month-old mice with 2 year old-mice. A total of 6 different cornea
images
from 3 independent mice were used for quantification. For telomere length
frequency
histograms, average telomere length and standard deviation are indicated.
Number of
nuclei analyzed is also indicated. Statistical significance values are also
indicated.
Figure 14. Telomere shortening with age in male germ line stem cells a.
Representative topographic telomere length maps of testis epithelium sections
from
wild-type mice of the indicated age with confocal telomere Q-FISH images. The
different cornea compartments are indicated. The dashed line separates the
highlights
the first cell layer of the seminiferous tubules, where the stem cells have
been located
(periphery). Nuclei are colored according to their average telomere
fluorescence in
arbitrary units (a.u.). Scale bars correspond to 200 gm. Middle panels show
the
percentage of cells containing a given telomere fluorescence within each
epidermal
compartment. Right panels show telomere fluorescence histograms of nuclei in
each
compartment. Note that the percentage of cells with the longest telomeres
decreases in
the stem cell compartment when comparing 2 month-old mice with 2 year old-
mice. A
total of 6 different testis images from 3 independent mice were used for
quantification.
For telomere length frequency histograms, average telomere length and standard
deviation are indicated. Number of nuclei analyzed is also indicated.
Statistical
significance values are also indicated.
Figure 15. Telomere shortening with age in brain stem cells. a. Representative
topographic telomere length maps of brain sections from wildtype mice of the
indicated
age with confocal telomere Q-FISH images. The different cornea compartments
are
indicated. SGZ: subgranular zone, GCL: granular cell layer, H: hilus. The
dashed line
highlights the basal layer of the epithelium. Nuclei are coloured according to
their
average telomere fluorescence in arbitrary units (a.u.). Scale bars correspond
to 200 gm.
Middle panels show the percentage of cells containing a given telomere
fluorescence
within each epidermal compartment. Right panels show telomere fluorescence
histograms of nuclei in each compartment. Note that the percentage of cells
with the
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longest telomeres decreases in the stem cell compartment when comparing 2
month-old
mice with 2 year old-mice. A total of 6 different brain images from 3
independent mice
were used for quantification. For telomere length frequency histograms,
average
telomere length and standard deviation are indicated. Number of nuclei
analyzed is also
indicated. Statistical significance values are also indicated.
Figure 16. Decreased clonogenic potential of epidermal stem cells with mouse
aging. Aging affects the proliferative potential of mouse keratinocytes stem
cells.
Quantification of size and number of macroscopic colonies obtained from
isolated
keratinocytes from 2-days-old, 2-months-old and 27-31-months old mice and
cultured
for 10 days on J2-3T3 mitomycin-C-treated feeder fibroblast. Note that colony
number
decreases with aging. In the case of keratinocytes obtained from adult tail
skin 10 times
more cells were seeded onto the feeder layer to better assess their low colony
formation
efficiency. Statistical significance values are indicated.
DETAILED DESCRIPTION OF THE INVENTION
Determination of telomere length on immobilised cell populations
The authors of the present invention have shown that, unexpectedly, it is
possible to
determine the telomere length of every single cell in an immobilized
tridimensional cell
population using fluorescence microscopy on said cell population followed by a
semi-
automatic image analyses. By processing in parallel the sample under study and
a
preparation of different cell types of known and stable telomere length which
are
different between them, it is possible to obtain a standard curve establishing
a
correspondence between arbitrary fluorescence units and telomere length. By
interpolating within the standard curve the arbitrary fluorescence values of
the cells of
the sample under study, it is possible to obtain an average telomere length
for each cell.
For instance, example 1 describes the determination of telomere length in a
cell
population and the ability of the method developed by the inventors to detect
differences of telomere length of less than 1 kb.
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Thus, in a first aspect, the invention relates to a method (hereinafter, first
method of the
invention) for the determination of the telomere length of a cell in an
immobilised
tridimensional test cell population comprising
(0
contacting said test cell population and at least two homogeneous
immobilised control cell populations of known and stable telomere length
and having different average telomere lengths with a probe that hybridises
specifically to a repeat region within telomeric DNA and which is labelled
with a first fluorescent dye under conditions allowing the probe to hybridise
in situ to its complementary target sequences on telomeres,
(ii) determining
the average fluorescence intensities in a cell of said test cell
population and the average fluorescence intensity value of the cells within
each control cell population in response to a radiation adequate to excite the
fluorescent dye attached to said probe wherein the determination of the
fluorescence signal in the cells is carried out on an image of the cells
obtained by fluorescence microscopy on a section of said tissue sample or
preparation of immobilised cells and
(iii) assigning to the cell within the cell population an average
telomere length
value, wherein said value is the average telomere length of a cell within a
control cell population showing an average cellular fluorescence intensity
value substantially identical to the fluorescence intensity values of the cell
within the cell population as determined by interpolation.
The expression "tridimensional cell population", as used herein, relates to a
group of
cells with characteristic proportions in particular stages of both the cell
cycle and the
differentiation program, and having characteristics in common and which are
organized
so that the cell population extends substantially in all three spatial
dimensions, thus
excluding dissociated cells in suspension as well as cells immobilized in a
two
dimensional support. The characteristics include without limitation the
presence and
level of one, two, three or more cell-associated molecules (e.g., cell-
surface antigens).
It is understood that a tridimensional cell population, as used herein,
includes tissues as
well as cells grown in a tridimensional scaffold. In principle, any cell
preparation can be
analysed using the first method of the invention. In a preferred embodiment,
the test cell
population is a tissue sample selected from the group of skin, small
intestine, testis,
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cornea and brain wherein said tissues can be normal non-transformed tissues or
tumours, either primary or metastatic isolated from each of said tissues.
Step (i) of the first method of the invention requires contacting the test
cell population
5 and at least two homogeneous immobilised control cell populations with a
probe that
binds specifically to the telomere. The probes useful in the present invention
are those
which are complementary to, or hybridise under stringent conditions, to the
DNA
sequences which appear in the telomeres. As such, the probes used in the
methods of the
invention do not substantially cross-react with sequences founds in other
regions of the
10 chromosome, including centromeric regions. Accordingly, telomere-specific
oligonucleotides may be designed using telomeric sequences that are well known
in the
art. For example, complete sets of telomeric probes for human chromosomes are
described in NIH/IMM Collaboration, (Nature Genetics, 1996, 14:86); Knight et
al.,
(Am. J. Hum. Genet., 2000, 67:320-332); Knight et al., (J.Med.Genet., 2000,
37:401-
15 409) and Veltman et al., (Am.J.Hum.Genet., 2002, 70:1269-1276). Further,
complete
sets of telomeric probes for human chromosomes may be purchased from Vysis
Inc.
(Downers Grove, Ill.; as ToTelVysion and TelVysion chromosome-specific
telomere
probes) and from Open Biosystems (Huntsville, Ala.; as Human Chromosome
Telomeric Region Probes).
Probes suitable for use in the present invention include probes specifically
directed to
the simple tandem repeat (TTAGGG)õ. Moreover, the telomeric probes suitable
for use
in the methods according to the present invention may further comprise a minor
groove
binder (MGB), a locked nucleic acid (LNA) and/or a peptide nucleic acid (PNA).
A
"minor groove binder" (MGB) moiety binds to the minor groove of DNA with high
affinity. When this minor groove binder is conjugated to one end (the 5'-end
or the 3'-
end) of short oligodeoxynucleotides, the conjugates form unusually stable
hybrids with
complementary DNA in which the tethered MGB group resides in the minor groove.
A
"locked nucleic acid" (LNA) is a RNA derivative in which the ribose ring is
constrained
by a methylene linkage between the 2'-oxygen and the 4'-carbon. This
conformation
restriction increases binding affinity for complementarity sequences. A
peptide nucleic
acid (PNA) is an oligonucleotide analogue in which the sugar phosphate
backbone is
replaced by a protein like backbone. In PNA, nucleobases are attached to the
uncharged
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polyamide backbone yielding a chimeric pseudopeptide-nucleic acid structure,
which is
homomorphous to nucleic acid forms. Chimeric DNA-PNA and pure PNA probes can
be used to provide stronger binding of the probe. A unique property of the
peptide
nucleic acid (PNA) probes is their discriminatory advantage due to their
increased
affinity for DNA, which allows the use of short PNA probes. Moreover, the
fluorescence yielded by probe staining is considered to be quantitative due to
the fact
that PNA binds preferentially to DNA at low ionic salt concentrations and in
the
presence of formamide, thus the DNA duplex may not reform once it has been
melted
and annealed to PNA probe, allowing the probe to saturate its target repeat
sequence (as
it is not displaced from the target DNA by competing anti sense DNA on the
complimentary strand), thus yielding a reliable and quantifiable readout of
the
frequency of PNA probe target at a given chromosomal site after washing away
of
unbound probe.
In a preferred embodiment, the probe that specifically binds to the telomere
is a probe
that comprises the sequence (CCCTAA)3. In a more preferred embodiment, the
telomeric probe is a PNA. In a still more preferred embodiment, the telomeric
probe is a
PNA that comprises the sequence (CCCTAA)3.
It will be understood that the test cell population which is to be studied
according to the
method of the present invention and the control cell populations used as
standard for
telomere length in the method of the present invention must be first
permeabilized so as
to render the probe accessible to the nuclei of the cells. Means for the
permeabilization
of cell membranes are known to the skilled person and include treatment with
non-ionic
detergents and treatment with hydrophobic solvents such a methanol.
Preferably,
permeabilization is carried out using 100% methanol. Moreover, the cell
preparation
may also be partially or totally fixed. Fixing can be carried out using any
method known
in the art such as formaldehyde, paraformaldehyde, acetic acid, acetic
acid/methanol
mixtures and the like. In addition, the cells may also be treated with
protease in order to
remove background signal resulting from non-specific binding of the probe to
proteinaceous compounds. By way of example, the cells may be treated with
pepsine at
pH 2 at 37 C.
CA 02723950 20150603
22
The hybridization probe is added to the cells into a hybridization medium. The
concentration of probe used in the methods described herein may be selected by
titrating increasing amounts of the probe and determining the concentrations
which
provide plateau hybridization. These concentrations are preferably used in the
method
of the invention. In a preferred embodiment of the invention for visualizing
and
optionally determining the length of telomere repeats in nucleic acid
molecules in
morphologically preserved materials, the amount of hybridization probe used is
between 0.1-10 ug/ml, preferably 0.3 pg/ml. The hybridization medium and
hybridization conditions are selected so as to favour hybridization ofthe
probe with the
denatured nucleic acid molecules in the preparation to be tested, and
disfavour
renaturation of the denatured nucleic acid molecules with their complementary
single
strand. Generally, a hybridization medium is selected which has a low ionic
strength
and typically contains a buffer, denaturing agent and blocking reagent.
Suitable buffers
include Iris and Flepes. Examples of suitable denaturing agents are formamide
and
DMSO. A blocking reagent is selected so that it substantially blocks non-
specific
binding of the probe. Examples of blocking reagents which may be used in the
method
of the invention are protein solutions such as BMP (Boehringer-Mannheim, Gmbh,
FRG). In a preferred embodiment of the method of the invention for detecting
and/or
determining the length of multiple copies of a telomeric repeat in a nucleic
acid
molecule, the hybridization medium contains a buffer (e.g. 10 mM TRIS, p1-
1=7.2),
formamide 50% -100%, most preferably- 70% formamide, BMP (1-5% W/V, most
preferably 0.25% W/V, and the labelled probe.
The hybridization medium containing the hybridization probe may be applied to
the
morphologically preserved biological materials. Generally, 5 to 50 i1.
preferably 30 tl
of the hybridization medium is applied per cell preparation. The hybridization
probe is
applied and the target nucleic acid molecules are denatured simultaneously by
heat or
pH treatment, preferably the mixture is treated for 0.1 to 1 hours at 70 to 80
C, most
preferably 3 minutes at 80 T. Hybridization is carried out for about 0.1 to 24
hours,
most preferably 2 hours, at 4 to 40 C, preferably 25 C. After hybridization,
the slides
are washed with buffer (e.g. formamide/TBS/TweenTm).
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In the methods of the invention for detecting and/or quantitating multiple
copies of a
repeat sequence in cell suspensions, about 10 to 1000 1, preferably 200 1 of
hybridization medium is added to the cells. The hybridization is carried out
for about 5
min to 24 hours, preferably 8 to 18 hours at room temperature. After
hybridization the
cells are washed with buffer (e.g. formamide/BSA/Tween; Tris/NaCl/Tween/BSA)
and
resuspended in buffer (e.g. PBS and 7AAD for FACSort; DAPI for FACStar).
The telomeric probe is labelled with a fluorescent dye which allows detecting
the
telomere-associated fluorescence. Any fluorescent dye known in the art can be
used for
the purposes of the present invention as long as suitable filters to select
excitation and
emission wavelengths are available. By way of example, Table 1 provides a list
of
possible fluorescence dyes that can be coupled to telomeric-specific probes.
Table 1: Commonly used fluorescente dyes
Molecula Excitacion (nm) Emision (nm)
FAM 488 518
HEX 488 556
TET 488 538
CY3 550 570
CY5.5 675 694
JOE 527 548
6-ROX 575 602
Cascade Blue 400 425
Fluoresceina 494 518
Texas Red 595 615
Rodamina 550 575
Rodamina Green 502 527
Rodamina Red 570 590
Rodamina 6G 525 555
6-TAMRA 555 580
5-TMRIA 543 567
Alexa 430 430 545
Alexa 488 493 516
Alexa 594 588 612
Bodipy R6G 528 550
Step (i) of the first method of the invention involves the simultaneous
contacting of the
telomeric and, optionally, the centromeric probe with the test cell population
and with a
series of control cell populations having stable and known telomere lengths.
The
number of control cell populations that can be used in step (i) varies
although it can be
appreciated that the highest number of control cell populations that are used,
the more
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accurate will be the correlation between arbitrary fluorescence units and
telomere
length. However, a minimum of two different control cell populations can be
used. It
will be appreciated by the person skilled in the art that any cell line which
is
homogenous, i.e, it consists of essentially a unique cell type, which shows
stable
telomere length (i.e. the telomere length does not substantially vary during
proliferation
cycles or in response to different culture conditions) and wherein the average
telomere
length is know can be used as control cell population in the second method of
the
invention. By way of example, the cell lines Hela 2, HeLa, MCF7, HeLa S3,
293T,
L5178Y-S, MEFs BL6 G3 Terc-/-, MEFs BL6 wild type, HeLa 1211, MEFs 129Sv/BL6
wild-type and L5178Y-R are suitable for the purposes of the method of the
invention
since they meet the requirements mentioned above (see Canela et al., 2007,
Proc.Natl.Acad.Sci.USA, 104 :5300-5305).
Thus, in a preferred embodiment, step (i) of the first method of the invention
is carried
out using at least two, at least three, at least four, at least five, at least
six, at least seven,
at least eight, at least nine, at least ten or at least eleven of the cell
populations
mentioned above. However, the skilled person will appreciate that the method
of the
invention is not limited to the use of the particular cell lines mentioned
above but that
any cell line can be used as long as the above requirements are met. The
average
telomere length of a cell line, if not previously known, can be determined
using
standard techniques known to the skilled person such as telomere restriction
fragment
assay (TRF) (Moyzis et al., 1988, Proc.Natl.Acad.Sci.USA, 85:6622-6626).
In a preferred embodiment, the control cell populations are processed in
parallel with
the cell population under study. Since the test cell population is a tissue or
a cell
preparation immobilized in a tridimensional matrix, the control cell
populations are also
be immobilized so as to process all samples in the same manner. Preferably,
the cell
populations may be fixed using formaldehyde, paraformaldehyde or acetic acid
and then
embedded in a proper support so as to form blocks. Media suitable to form
blocks
containing the control cell populations include gelatine, alginate, chitosan,
PLGA, and
the like. The blocks are then processed in the same manner as the tissue
samples, i.e. by
embedding in paraffin, sectioning and inspection by fluorescence microscopy.
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Step (ii) of the first method of the invention requires determining the
average
fluorescence intensity in the cells of the cell population, wherein said
fluorescence
reflect the number of telomeric repeats and hence, the length of the
telomeres. Several
ways can envisaged to determine fluorescence intensity in the cells associated
to the
5 telomere-specific probe.
Since the cell population is a tissue or a preparation of immobilized cells in
a
tridimensional matrix (e.g., cells embedded in gelatin), the determination of
the
fluorescence signal is preferably carried out on an image of the cells
obtained by
10 fluorescence microscopy on a section of said tissue sample or said
preparation of
immobilized cells. In case the sample to be analysed is embedded in paraffin,
the tissue
must be first deparaffinized prior to the analysis using the methods of the
invention.
Typically, deparaffinisation is carried out by applying sequential washes with
an
organic solvent (e.g. xylene) and rehydrated using ethanol/water mixtures of
decreasing
15 ethanol concentrations (100, 95 and 70%). The invention also
contemplates processing
in parallel a cell population which is a cell suspension or a monolayer, in
which case,
said cell preparation must be immobilized in a support which can then be
processed as
the tridimensional tissue sample. Agents suitable for immobilising cells
include
gelatine, alginate, agarosa, agar, inuline, carrageenan, polyacrilamide,
polystirene,
20 dextran, pectine, carboxymethylcellulose. In a preferred embodiment, the
cells are
embedded in gelatine. Once the cell population is immobilized in the solid
support, the
blocks can be processed in the same manner as the tissue sample (paraffin
embedding,
deparafinisation, rehydration and permeabilisation). In case the cell
population is a cell
monolayer grown on a support, the cells are first detached from the support
and then
25 treated as the cells in suspension. Tissues that can be analysed using
the method of the
invention include, without limitation, skin, small intestine, testis, cornea
and brain.
Image collection is carried out using wave-length filters that allow the
excitation of the
image using a wave-length specific for the fluorescent dye which is attached
to the
telomeric probe. Whenever a normalisation probe and/or a fluorescent DNA stain
must
be simultaneously detected, images from the same filed are captured
sequentially using
different filter sets for each dye. By way of example, when the cells are
labelled with a
FITC (telomeric probe), Cy3 (normalization probe) and DAPI (DNA stain), the
images
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are collected using an excitation filter 380/10 nm, dichroic: Fura/FITC and
emission
435LP for DAPI visualization, excitation filter 548/20 nm, dichroic:
Fura/TRITC and
emission: Fura/TRITC for Cy3 detection and excitation filter: 480/10 nm,
dichroic:
Fura/FITC and emision: Fura/FITC or 535/50 for FITC detection.
Preferably, when quantification of the telomere-associated fluorescence is to
be
determined, the images are collected using a confocal microscope. The use of a
confocal
microscopy allows the elimination of out-of-focus light or flare in specimens
that are
thicker than the focal plane by the use of a spatial pinhole. By way of a
example, in a
section of X gm of thickness, confocal microscopy allows collection of ten
X/10 gm
focal planes which are then combined in a single image adding the intensity of
every
focal plane.
Confocal microscopes suitable for use in the present invention include,
without
limitation, confocal laser scanning microscopes, spinning-disk (Nipkow disk)
confocal
microscopes and Programmable Array Microscopes (PAM).
In order to determine the fluorescence intensity at a single cellular level,
the method
requires first to define the regions within the image that correspond to cell
nuclei. These
regions are used then to define a mask which is applied to the fluorescence
image
derived from the telomere-specific probes to obtain a combined image with
telomere
fluorescence information for each nucleus. The average fluorescence in the
nuclear area
is then normalized to the nuclei area, thus providing a value of "average gray
values"
(total gray value/nuclei area) units (arbitrary units of fluorescence). This
method allows
the determination of the average fluorescence intensity for the total nuclear
area, thus
excluding that differences in nuclear size may influence telomere length
measurements.
Preferably, the regions within the image corresponding to the cell nuclei are
selected by
visualization with a fluorescent DNA dye. Exemplary nuclear stains include,
for
example, DAPI, Hoechst 33342 dye, 7-actinomycin-D, 7-Aminoactinomycin D,
Chromomycin A3, propidium iodide, Nuclear fast red or LDS751. The skilled
person
will appreciate that the DNA dye must emit at a wavelength which allows the
capturing
of the telomere fluorescence without interference from the DNA fluorescence.
Preferably, the telomere-specific probe is labeled with Cy3 and the DNA is
labeled with
DAPI.
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Once the fluorescence has been determined on the immobilized cell preparation,
the
fluorescence signal may be influenced by changes in nuclear size and
differences in
ploidy. Therefore, the signal obtained using telomeric-specific probes must be
normalized to an internal control signal control so as to rule out that
different values are
not due to differences in ploidy as well as in probe accessibility. For this
purpose, the
invention contemplates the labeling of the cell population with a second probe
(hereinafter "the normalization probe") that binds specifically to a region in
the cell
nuclei which is found in a constant copy number. Preferably, the normalization
probe is
a probe which hybridizes specifically to the centromeric DNA and, more in
particular,
to a unique repetitive sequences found in the centromeric regions of primate
chromosomes. For example, the centromeric oligonucleotides may correspond to
"alphoid" or "alpha-satellite" DNA, which is present at the centromeric region
of every
chromosome of an animal cell with a sequence that is different for each
chromosome
(see, e.g., Lee et al., Human Genet., 1997, 100:291-304 and Jabs et al., Am.
J. Hum.
Genet., 1987, 41:374-90).
Probes specific for each of the centromeres of all of the human chromosomes
may be
purchased as "CEP Probes" from Vysis Inc. (Downers Grove, Ill.), or as "Human
Chromosome-Specific Centromeric Probes", from Open Biosystems (Huntsville,
Ala.).
Alternatively chromosome specific centromeric oligonucleotides may be designed
using
known sequences. For example, the probes discussed in the following
publications may
be used to design suitable centromeric oligonucleotides for each of the human
chromosomes: chromosome 1: Waye et al., (Genomics (1987) 1:43-51); Hardas et
al.,
(Genomics (1994) 21:359-63); Solus et al., (Somat. Cell. Mol. Genet. (1988)
14:381-
91); chromosome 2: Ostroverkhova et al., (Am J Med Genet. (1999) 87:217-20;
Matera
et al., (Hum Mol Genet. (1992) 1:535-9); chromosome 3: Delattre et al., Hum
Hered.
(1988) 38:156-67; Varella-Garcia et al., (Cancer Res. (1998) 58:4701-7);
chromosome
4: Grimbacher et al., (Genet. Med. (1999) 1:213-8); chromosome 5: Matera et
al.,
(Genomics (1993) 18:729-31); Reichenbach et al., (Am. J. Med. Genet. (1999)
85:447-
51) chromosome 6: Lastowska et al., Cancer Genet. Cytogenet. (1994) 77:99-
105);
chromosome 7: Mark et al., (Exp Mol Pathol. (1999) 67:109-17); Zhao et al.
(Ann. Clin.
Lab. Sci. (1998) 28:51-6); Jenkins et al., (Cancer Res. (1998) 58:759-66);
chromosome
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
28
8: Zhao et al., (Ann. Clin. Lab. Sci. (1998) 28:51-6); Macoska et al.,
(Urology (2000)
55:776-82); Mark et al., (Exp. Mol. Pathol. (1999) 66:157-62); chromosome 9:
Rocchi
et al., (Genomics (1991) 9:517-23); Gutierrez-Angulo et al., (Genet Couns.
(2001)
12:359-62); chromosome 10: Wang et al., (Somat. Cell Mol. Genet. (1996) 22:241-
4);
Devilee et al., (Genomics (1988) 3:1-7); Howe et al., (Hum Genet. (1993)
91:199-204);
chromosome 11: Voorter et al., (Int. J. Cancer (1996) 65:301-7); Kraggerud et
al.,
(Cancer Genet. Cytogenet. (2003) 147:1-8); chromosome 12: Looijenga et al.,
(Cytogenet. Cell Genet. (1990) 53:216-8); Zhao et al., (Ann. Clin. Lab. Sci.
(1998)
28:51-6); chromosome 13: Warren et al., (Genomics (1990) 7:110-4); chromosome
14:
Earle et al., (Cytogenet Cell Genet. (1992) 61:78-80); chromosome 15:
Stergianou et al.,
(Hereditas (1993) 119:105-10); chromosome 16: Greig et al., (Am. J. Hum.
Genet.
(1989) 45:862-72); chromosome 17: Fink et al., (Hum Genet. (1992) 88:569-72);
chromosome 18: Verma et al., (Ann Genet. (1998) 41:154-6); chromosome 19:
Hulsebos et al., (Cytogenet. Cell Genet. (I 988) 47:144-8); chromosome 20:
Meloni-
Ehrig et al., Cancer Genet. Cytogenet. (1999) 109:81-5); chromosome 21:
chromosome
21: Maratou et al., (Genomics, 1999, 57:429-32); Verma et al., (Clin. Genet.
(1997)
51:91-3); X chromosome: Yang et al., (Proc. Natl. Acad. Sci. (1982) 79:6593-
7); Crolla
et al., (Hum. Genet. (1989) 81:269-72); and Y chromosome: Davalos et al., (Am.
J.
Med. Genet. (2002) 111:202-4); Rivera et al., (Ann. Genet., 1996, 39:236-9);
Tho et al.,
(Am. J. Obstet. Gynecol. (1988) 159:1553-7). In a preferred embodiment, the
centromeric probe comprises the sequence TCGCCATATTCCAGGTC (SEQ ID NO:3).
As the telomeric probe, the normalization probe may be an oligonucleotide, a
locked
nucleic acid (LNA) and/or a peptide nucleic acid (PNA) and may be attached to
a minor
groove binder (MGB). Centromeric probes suitable for use in the present
invention are
known in the art.
The skilled person will appreciate that the normalization probe must be
labeled with a
fluorescent dye so that the probes are suitable for normalization of the
fluorescence
emitted by the telomeric probe. It will be appreciated that dyes as described
in Table 1
are suitable for labeling the centromeric probe. However, since the
centromeric and the
telomeric probes are to be used in the same samples, the centromeric probe
must contain
a label which can be detected without interfering with the fluorescence
produced by the
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29
telomere probe. Suitable combinations of markers that can be applied to the
centromeric
and telomeric probes to allow individual detection of the fluorescence emitted
by each
of them include FITC and Cy3, Cy3 and rhodamine, FITC and rhodamine, AMCA and
FITC, AMCA and TRICT, FITC and TRITC, FITC and R-PE, R-PE and PE-Cy5, Cy2
and PE-Texas Red, Cy2 and PE-CY5.5, PE-Texas Red and PE-CY5.5, Alexa 488 and
Cy3, Alexa 488 and PE-A1exa647, Cy3 and PE-A1exa647, Cy3 and FITC, Cy3 and
Cy5, FITC and Cy5, FITC and coumarine and the like.
The second labeling can be carried out at the same time, prior or after the
labeling with
the telomeric-specific probes.
Step (iii) according to the first method of the invention requires converting
the average
fluorescence intensity obtained from the cells to an average telomere length
value. This
step is preferably carried out by interpolation. As used herein,
"interpolation" means the
process of calculating a new point between two existing data points. The
interpolation
process comprises comparing the average fluorescence intensity in a given cell
within
the population under study with a data set which contains at least two pairs
of
fluorescence/telomere length values obtained from the control cell populations
processed in parallel. The skilled person will appreciate that many methods
exist for the
interpolation of a given fluorescence value within a correspondence table
reflecting
telomere lengths as a function of fluorescence intensity. By way of example,
the
interpolation can be carried out using methods such as piecewise constant
interpolation
(also known as nearest neighbour interpolation), linear interpolation,
polynomial
interpolation, spline interpolation, rational interpolation, trigonometric
interpolation,
bilinear interpolation, bicubic interpolation and the like. As it will be
appreciated, the
accuracy of the interpolation method will depend on the number of values
included in
the standard data set, although it is possible to carry out an interpolation
with only a
single pair of values. Preferably, the interpolation is carried out using a
data set
comprising at least three, at least four, at least five, at least six, at
least seven, at least
eight, at least nine, at least ten, at least eleven pairs of values. The
results of the
interpolation applied to each cell within the cell population under study will
provide an
average telomere length of each cell.
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Identification of stem cells using telomere-dependent fluorescence intensity
values
Moreover, the authors of the present invention have shown that, surprisingly,
telomere
length can be used to identify those compartments within tissues that comprise
stem
5 cells.
Without wishing to be bound by any theory, it is believed that the possibility
of
identifying stem cells within a cell population lies in the fact that,
contrary to most
somatic cells, stem cells contain certain telomerase activity but rarely
divide and are
found in protected microenvironments or niches. Thus, despite the fact that
they suffer
telomere shortening, the presence of telomerase activity results in that the
telomere
10 length
decreases more slowly that the rest of somatic cells. Thus, in a given tissue,
there
are differences in telomere length between stem cells and the rest of somatic
cells, being
telomeres longer in stem cells than in somatic cells.
The assay is advantageous over the assays known to date because it is tissue-
15
independent, i.e. telomere length can be used to identify stem cell niches in
every tissue
when compared to the methods known in the prior art wherein stem cell niches
in each
tissue had to be carried out using markers specific for the tissue (e.g. CD34
and keratin
15 in hair follicle). As shown in the experimental part (example 2), telomere
length has
been used to identify stem cell compartments in the hair follicle, confirming
the known
20
location of the stem cell niche in the hair follicle bulge. Moreover, the
assay has been
validated studying other tissues wherein the location of the stem cell niche
is known.
For instance, telomere length according to the assay of the invention confirms
the
bottom of the intestinal crypts as the location wherein the stem cell niche is
found in
intestine (example 4), the limbus as the location wherein the stem cell niche
of the
25 cornea
is found (example 4) and the periphery of the seminiferous tubes as the
location
wherein the stem cell niche in testis is found (example 4).
Thus, in a second aspect, the invention relates to a method for the
identification of stem
cells in a cell population which comprises
30 (0
contacting said cell population with a probe that hybridises
specifically to a repeat region within telomeric DNA and which is
labelled with a first fluorescent dye under conditions allowing the
CA 02723950 2010-11-09
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31
probe to hybridise in situ to its complementary target sequences on
telomeres,
(ii)
determining the average fluorescence intensities of each cell within a
representative sample of cells within said cell population in response
to a radiation adequate to excite the fluorescent dye attached to said
probe
wherein those cells within the sample showing the highest average fluorescence
intensity are identified as stem cells.
"Stem cells", as used herein, relate to cells derived from adult tissues, i.e.
they are non
embryonic stem (ES) cells or non embryonal germ (EG) cells, that have
extensive
proliferation potential and are capable of differentiating into most
specialized cell types
present in the tissue wherein they are found, i.e. they are pluripotential. In
contrast to ES
or EG cells, which are able to differentiate into cells of the three major
lineages
(ectodermal, enodermal and mesodermal), adult stem cells are usually limited
in their
differentiation capabilities to the lineage of the tissue wherein they are
found. Typical
adult stem cells which can be identified using the methods of the present
invention
include, without limitation:
- Haematopoietic stem cells, giving rise to all the types of blood cells:
red
blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils,
basophils, eosinophils, monocytes, macrophages, and platelets,
- Bone marrow stromal cells (mesenchymal stem cells), giving rise to a
variety of cell types including osteocytes, chondrocytes, adipocytes as well
as other kinds of connective tissue cells such as those in tendons,
- Neural stem
cells in the brain give rise to its three major cell types:
neurons), astrocytes and oligodendrocytes,
- Intestine stem cells giving rise to absorptive cells, goblet cells,
Paneth cells,
and enteroendocrine cells and
- Epidermal stem cells which occur in the basal layer of the epidermis and
at
the base of hair follicles. The epidermal stem cells give rise to
keratinocytes.
The follicular stem cells can give rise to both the hair follicle and to the
epidermis,
- Testicular stem cells,
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32
- Mammary stem cells
- Cardiac stem cells
- Pituitary stem cells
- Cancer stem cells
The expression "cell population", as used herein, relates to a group of cells
with
characteristic proportions in particular stages of the cell cycle and having
characteristics
in common. The characteristics include without limitation the presence and
level of one,
two, three or more cell-associated molecules (e.g., cell- surface antigens).
It is
understood that cell population includes tissues, cells grown in culture as
well as
dissociated cells which may be either in suspension in an appropriate culture
medium as
well as immobilized in a two dimensional support or in a tridimensional
scaffold.
Step (i) according to the second method of the invention requires contacting
the cell
population with a probe that binds specifically to the telomere. This method
is carried
out essentially as described for the first method of the invention regarding
the telomeric
probes, fluorescent dyes, hybridization conditions, pre-treatment of the
cells.
Step (ii) of the second method of the invention requires determining the
average
fluorescence intensity of each cell in a representative sample of the cell
population,
wherein said fluorescence reflect the number of subtelomeric repeats and
hence, the
length of the telomeres. "Representative sample", as used herein, refers to a
sample of
the cell population which contains a subset of the cells of the sample which
is large
enough to provide a statistically significant representation of all the
different cell types
within the cell population and which allows to obtain statistically
significant
information of the whole cell population. The representative sample can
comprise as
low as 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%. The representative sample may be formed by the
totality of cells of the cell population.
Several ways can envisaged to determine fluorescence intensity in the cells
associated
to the telomere-specific probe. In case that the cell population is a tissue
or a
preparation of immobilized cells (e.g., cells embedded in gelatin), the
determination of
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33
the fluorescence signal is preferably carried out as described in the first
method of the
invention regarding treatment of the cells, image collection, type of
microscopy, region
of the cell used for image collection.
In case that the cell population to be analyzed is a cell suspension, the
measurement of
the fluorescence signal may be carried out using a fluorescence-activated cell
sorter, i.e.
a technique known as flow-FISH. Flow-FISH, as originally described by Rufer et
al
(Nature Biotechnol., 16:743-747), allows the use of a conventional cell sorter
to
determine telomere length based on the fluorescence emission of cells
previously
contacted with a telomeric-specific probe. Each flow-FISH experiment begins
with the
acquisition of the premixed calibration (MESF) beads. Several thousand events
are
collected, and the mean fluorescence and coefficient of variation (CV) of each
of the
five peaks is recorded and plotted against the MESF content provided by the
manufacturer to control for the linearity of the instrument. CV is defined as
the standard
deviation (s) of the fluorescent intensity of a population of beads expressed
as a
proportion or percentage of the mean (m) intensity (C1/45/m). The next steps
are
related to the selection of the optimal values for the detectors, amplifiers,
fluorescence
compensation setting and threshold values for analysis of flow FISH samples.
Once
appropriate instrument settings have been selected, these can be saved and
recalled for
future experiments, although minor day-to-day adjustments are typically
required
between experiments and between samples. The instrument settings are further
adjusted
to provide a good separation of the events of interest over the entire range
in the
selected channels. Various compensation settings are selected for the analysis
of cells
simultaneously labeled with fluorescein, phycoerythrin (PE), LDS751 and Cy-5.
Except
for the compensation setting for fluorescence 2 channel (F12; PE) fluorescence
detected
in the Fll (green fluorescence) channel, the setting for green fluorescence
detection is
typically not readjusted after the acquisition of the MESF bead data because
the range
of telomere fluorescence in test cells is typically known. In one embodiment,
the cells
population to be analyzed is mixed with a second population of cells whose
average
telomere length is known. The two cells populations can usually be
distinguished based
on forward light scatter (providing a measure of the cell size), side scatter
(providing a
measure of the cell complexity) and the intensity of the fluorescence due to
the DNA
dye since different cell populations usually uptake DNA dyes to different
extents. The
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34
cell suspension to be analyzed may be permeabilized so as to ensure access of
the
telomeric probe to the cell nuclei. Permeabilization may be carried out using.
e.g.
methanol. Moreover, the cell may also be partially or totally fixed. Fixing
can be carried
out using any method known in the art such as formaldehyde, paraformaldehyde,
acetic
acid, acetic acid/methanol mixtures and the like. Moreovoer, the cells may
also be
treated with protease in order to remove background signal resulting from non-
specific
binding of the probe to proteinaceous compounds.
Irrespective of whether the fluorescence determination of the cells within the
representative sample is carried out by fluorescence microscopy on tissue
sections or by
flow-FISH on cells on suspension, the fluorescence signal may be influenced by
changes in nuclear size and differences in ploidy. Therefore, the signal
obtained using
telomeric-specific probes must be normalized to an internal control signal
control so as
to rule out that different values are not due to differences in ploidy as well
as in probe
accessibility. Normalisation of the signal is carried out essentially as
described for the
first method of the invention regarding the type of probe that is used and the
type of dye
to be used.
Once the fluorescence intensity values in each cell of the representative
sample is
determined and, optionally normalised to the control fluorescence levels and
limited to
those areas corresponding to the cell nuclei, the cells which show the highest
fluorescence values are selected as candidate stem cells within the cell
population. The
term "highest", when referred to the fluorescence values, relates to those
absolute
values which are the highest among the cell population under study. The
assignation of
a cell as having high fluorescence value can be done using the percentile
method, which
reflects the value of the fluorescence intensity below which a certain percent
of
observations fall. Percentiles can be calculated as quartiles, wherein the
fluorescence
values of the whole cell population is divided in four intervals and wherein
high
fluorescence would correspond to those cells whose fluorescence is found in
the upper
quartile. Percentiles can also be calculated by dividing the fluorescence
values of the
whole cell population in two groups with respect to a threshold level (the
median) and
wherein the cells showing high fluorescence value would be those cells whose
fluorescence is above said median value.
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Identification of stem cells based on telomere length
The authors of the present invention have also improved the method for the
5
identification of the stem cells by including an additional step in the
identification
method wherein the arbitrary values of fluorescence intensity obtained from
the images
are correlated with average telomere lengths. In this way, the identification
of the stem
cell in a sample is carried out based on the average telomere length of the
cell rather
than on the fluorescence intensity. As it can be seen in the experimental
section, longer
10
telomere length values appear in cells present in compartments which were
known to
correspond to the stem cell niche in hair follicles (example 3), intestine
(example 4),
cornea (example 4) and testis (example 4). By processing in parallel the
sample under
study and at least two preparations of cells of known and stable telomere
length and
showing different telomere length, it is possible to obtain a standard curve
between
15
arbitrary fluorescence units and telomere length. By interpolating within the
standard
curve the arbitrary fluorescence values of individual cells of the sample
under study, it
is possible to obtain an average telomere length for each cell. The cells
showing the
largest telomere lengths are then the candidate stem cells in the tissue under
study. The
use of a standard curve allows the detection of small differences in telomere
length (1
20 kb). Thus, in another aspect, the invention relates to a method
(hereinafter "third
method of the invention") for the identification of stem cells in a test cell
population
which comprises
(0 contacting said test cell population and at least two homogeneous
control cell populations of known and stable telomere length and
25 having
different average telomere lengths with a probe that
hybridises specifically to a repeat region within telomeric DNA and
which is labelled with a first fluorescent dye under conditions
allowing the probe to hybridise in situ to its complementary target
sequences on telomeres,
30 (ii)
determining the average fluorescence intensities in each cell of a
representative sample of cells within said test cell population and the
average fluorescence intensity value of the cells within each control
CA 02723950 2010-11-09
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36
cell population in response to a radiation adequate to excite the
fluorescent dye attached to said probe and
(iii) assigning to each cell within the representative sample of the
test cell
population an average telomere length value, wherein said value is
the average telomere length of a cell within a control cell population
showing an average cellular fluorescence intensity value substantially
identical to the fluorescence intensity values of the cell within the
cell population as determined by interpolation
wherein those cells showing the highest telomere length value are identified
as stem
cells.
Step (i) of the third method of the invention is carried out essentially as
described in the
first method of the invention regarding the probe that hybridises specifically
to a repeat
region within telomeric DNA, the hybridisation conditions, the normalisation
(centromeric) probes and the labelling in parallel of a series of cell
populations of stable
and known telomere length.
Step (ii) of the third method of the invention is carried out essentially as
described in the
first method of the invention regarding the conditions suitable for excitation
of the
fluorescent probes and for determination of the average fluorescence intensity
in each
cell of the representative sample.
Step (iii) requires converting the average fluorescence intensity obtained
from the cells
to an average telomere length value and is carried out essentially as
described in the first
method of the invention.
Once the telomere lengths of the different cells within the representative
sample of the
test cell population have been determined by the interpolation method carried
out in step
(iii), the cells showing the highest telomere lengths will then be considered
as
candidates for being the stem cells within the cell population under study.
The telomere
length values (expressed in kb) can then be statistically analysed using the
same
methodology as with the fluorescence intensity values as explained above.
Preferably,
the distribution of telomere lengths within the cell population is divided in
quartiles and
CA 02723950 2010-11-09
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37
the cells whose telomere length is found in the upper quartile are then
considered as
candidate stem cells.
Screening methods
The authors of the present invention have also shown that the telomere mapping
methods developed in the present invention also allow to monitor the process
of
mobilization of stem cell from the stem cell niche into non stem cell
compartments by
comparing the telomere distribution before and after the application of a
signal known
to cause mobilization of stem cells from the stem cell niches. In particular,
example 2 of
the experimental part shows that animals treated with the phorbol ester TPA
undergo a
rearrangement of the stem cell compartment which manifests itself in a
decrease in the
average fluorescence and telomere lengths of the cells found in the stem cell
niche.
Thus, by providing a tissue with a known distribution of stem cells, it is
possible to
identify compounds which promote stem cell mobilization. Thus, in another
aspect, the
invention relates to a method (hereinafter "the first screening method of the
invention")
for the identification of compounds capable of triggering mobilisation of stem
cells
within a tissue having a known spatial distribution of stem cells comprising
the steps of
(0
contacting said tissue sample with a candidate compound under
conditions adequate for promoting mobilisation of the stem cells
within said tissue,
(ii) contacting a sample of said tissue with a probe that hybridises
specifically to a repeat region within telomeric DNA and which is
labelled with a first fluorescent dye under conditions allowing the
probe to hybridise in situ to its complementary target sequences on
telomeres and
(iii) determining the average fluorescence intensity of a representative
sample of cells present in the region of the tissue sample known to
contain the stem cells
wherein a decrease in the average fluorescence intensity in the area which is
known to
comprise stem cells when compared to a sample which has not been treated with
the
candidate compound is indicative that the compound is capable of triggering
mobilisation of stem cells within the tissue sample.
CA 02723950 2010-11-09
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38
In yet another aspect, the invention relates to a method (hereinafter "the
second
screening method of the invention") for the identification of compounds
capable of
triggering mobilisation of stem cells within a tissue having a known spatial
distribution
of stem cells comprising the steps of
(0 contacting said tissue with a candidate compound under
conditions
adequate for promoting mobilisation of the stem cells within said
tissue,
(ii) contacting said tissue and at least two homogeneous control
cell
populations of known and stable telomere length and having different
average telomere lengths with a probe that hybridises specifically to
a repeat region within telomeric DNA and which is labelled with a
first fluorescent dye under conditions allowing the probe to hybridise
in situ to its complementary target sequences on telomeres,
(iii) determining the average fluorescence intensities of a representative
sample of cells present in the region of the tissue sample known to
contain the stem cells and an average fluorescence intensity value of
the cells within each control cell population in response to a radiation
adequate to excite the fluorescent dye attached to said probe and
(iv) assigning to each cell within the representative sample of cells
present the region of the tissue sample known to contain the stem
cells an average telomere length value, wherein said value is the
average telomere length of a cell within a control cell population
showing an average cellular fluorescence intensity value identical to
the fluorescence intensity values of the cell within the cell population
as determined by interpolation
wherein a decrease in the average telomere length of the cells within the
region of the
tissue known to comprise stem cells when compared to a sample which has not
been
treated with the candidate compound is indicative that the compound is capable
of
triggering mobilisation of stem cells within the cell population.
The method requires the knowledge in advance of tissues wherein the stem cell
niche is
known. In principle, any tissue which is known to contain a population of stem
cells and
CA 02723950 2010-11-09
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39
whose spatial distribution is known may be used in the screening methods of
the
invention. By way of example, tissues that may be used in the present
invention include
skin, intestine, cornea, testis and central nervous system.
In the murine skin, a stem cell population is located in the hair follicle.
The epidermis
comprises four compartments, namely, the hair bulge, wherein the stem cells
are
located, the bulb, the infumdibulum, wherein the transit amplifying cells are
found and
the interfolicular epidermis.
In small intestine, the stem cells reside in the Lieberkuhn crypts, just above
the Paneth
cells and below the transit amplifying cells. The more differentiated cells
are found in
the epithelium forming the intestinal villi (see Gregorieff et al., 2005,
Gastroenterology,
129:626-638 and Marshman et al., 2002, Bioessays 24, 91-98).
In the cornea, the stem cells are located in the limb, corresponding to the
peripheral
cornea, just above the ciliated body (see Lavker and Kligman, 1988, J. Invest.
Dermatol., 90:325-330 and Lehrer et al., 1998, J. Cell Science., 111:2867-
2875).
In testis, the stem cells are located in the peripheral region of the
seminiferous tubes
(Guan et al., 2006, Nature 440, 1199-1203).
In the central nervous system, stem cells can be found in the subgranular zone
of the
hippocampus, localised between the granular cell layer and the hilio (see
Alvarez-
Buylla and Lim, 2004, Neuron 41, 683-686 and Sage et al., 2000, Genes &
Development 14: 3037-3050).
Once an adequate tissue has been selected for carrying out the method of the
invention,
the method involves in step (i) contacting a tissue with a compound whose
activity as
promoter of stem cell mobilization wants to be studied. It will be understood
that the
contacting step can be carried out in several different ways. In one
embodiment, the
contacting step is carried out in the living animal prior to isolating the
tissue sample by
administering to said animal the compound to be tested prior to removal of the
tissue for
telomapping studies. Any suitable means of administration of a compound to an
animal
CA 02723950 2010-11-09
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is possible within the present invention as long as the compound is able to
reach the
stem cell niche of the target tissue. By way of example, the compound may be
administered orally, intradermically, parenterally and the like. In another
embodiment,
the contacting step may be carried out on the isolated tissue maintained under
perfusion
5 by providing the compound to be tested to the perfusion media. In another
embodiment,
the tissue under study is isolated from the vasculature of the organism where
it is found
by using a catheter system as described e.g. in US6699231 and the compound
under
study is then provided directly to the vasculature of the isolated tissue.
10 Moreover, the invention contemplates no limitation as to the type of
compound that can
be tested. In case the candidate compound is a molecule with low molecular
weight, it is
enough to add said molecule to the culture medium. In the event that the
candidate
compound is a molecule with a high molecular weight (for example, biological
polymers such as a nucleic acid or a protein), it is necessary to provide the
means so
15 that this molecule can access the interior of the cells forming the
tissue. In the event that
the candidate molecule is a nucleic acid, conventional transfection means such
DNA
precipitation with calcium phosphate, DEAE-dextran, polybrene,
electroporation,
microinjection, liposome-mediated fusion, lipofection, infection by retrovirus
and
biolistic transfection can be used. In the event that the candidate compound
is a protein,
20 the cells can be put in contact with the protein directly or with the
nucleic acid encoding
it coupled to elements allowing its transcription / translation once they are
in the cell
interior. To that end, any of the aforementioned methods can be used to allow
its
entrance in the cell interior. Alternatively, it is possible to put the cell
in contact with a
variant of the protein to be studied which has been modified with a peptide
which can
25 promote the translocation of the protein to the cell interior, such as
the Tat peptide
derived from the HIV-1 TAT protein, the third helix of the Antennapedia
homeodomain
protein from D.melanogaster, the VP22 protein of the herpes simplex virus and
arginine
oligomers (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21:99-103,
Schwarze, S.R.
et al., 2000, Trends Pharmacol. Sci., 21:45-48, Lundberg, M et al., 2003, Mol.
Therapy
30 8:143-150 and Snyder, E.L. and Dowdy, S.F., 2004, Pharm. Res. 21:389-
393).
Steps (ii) and (iii) of the first screening method of the invention is carried
out basically
as described above with respect to the second method of the invention.
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Steps (ii), (iii) and (iv) of the second screening method of the invention is
carried out
basically as described above with respect to the third method of the
invention.
In the first screening method of the invention, the fluorescence values of
each cell
within the representative sample obtained in step (iii) are then compared with
a similar
sample which has not been treated with the compound to be tested. Different
approaches can be used for said purpose. In a preferred embodiment, the
fluorescence
values of the cells within the cell population are divided in four intervals
and each cell
is assigned to each interval depending on its fluorescence values so that the
percentage
of cells within each region of the tissue within each fluorescence interval
can be
determined. If the tested compound has promoted the mobilization of the stem
cells
from the stem cell niche, a reduction in the percentage of cells within the
upper quartile
within the area of the tissue known to contain the stem cells and/or an
increase in the
number of cells within the lower quartile in the areas of the tissue
containing the transit
amplifying cells or the differentiated cells in comparison with a sample which
has not
been treated with said compound will be observed.
In the second screening method of the invention, the telomere length values of
each cell
obtained in step (iv) are then compared with the telomere lengths of the cells
of a
similar sample which has not been treated with the compound to be tested.
Different
approaches can be used for said purpose. In a preferred embodiment, the
telomere
lengths values of the cells within the cell population are divided in four
intervals and
each cell is assigned to each interval depending on its telomere length so
that the
percentage of cells within each region of the tissue within each telomere
length interval
can be determined. If the tested compound has promoted the mobilization of the
stem
cells from the stem cell niche, this will be observed as a reduction in the
percentage of
cells within the upper quartile within the area of the tissue known to contain
the stem
cells and/or an increase in the number of cells within the lower quartile in
the areas of
the tissue containing the transit amplifying cells or the differentiated cells
in comparison
with a sample which has not been treated with said compound.
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As described above, image collection of the tissue samples can be carried out
using a
confocal microscope. In this case, several confocal images are collected
spanning the
whole tissue sample depth and a final image is obtained by merging the
different
confocal images using the maximun projection. Moreover, in a preferred
embodiment,
the determination of the fluorescence values is carried out on those regions
of the image
corresponding to the cell nuclei. For said purpose, the sections are
simultaneously
stained with a DNA dye, which allows the localization of the cell nuclei. The
pattern of
cell nuclei is then used to construct a mask that is used to select those
regions of the
image wherein the telomere-associated fluorescence is captured.
Tissue arrays
The authors of the present invention have also observed that the sensitivity
in the
determination of the telomere length of a cell within a test cell population
can be
improved by processing in parallel a plurality of samples, each containing a
cell
population of known and stable telomere length. The co-processing of the
tissue under
study and of the control cell population is most adequately carried out by
using a tissue
microarray comprising all the different control cell populations. This
microarray can
then sectioned by conventional means and processed in parallel to the tissue
sample.
Thus, in another aspect, the invention relates to an array comprising at least
two
immobilised tridimensional cell populations being each cell population
physically
separated from the other(s) and wherein each cell population has a stable and
known
telomere length which is different to the average telomere length of the other
cell
population(s) of the array.
The tissue arrays according to the invention contain a plurality of different
cell
population samples in a single receiver block. The block material can be any
material
that is known in the art and that allows for the preparation of tissue sample
blocks that
will function in the methods and compositions of the invention. Those
materials include
agarose, gelatin, paraffin and others that will be understood by those of
skill upon
reading this specification. In a preferred embodiment, the block is made of
gelatine,
more preferably 5% gelatine. The receiver block is sectioned in the usual
manner with a
microtome, and the section is applied onto a specimen slide. The specimen
slide then
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contains a plurality of different tissue samples. Because of the large number
of tissue
samples on a single specimen slide, it is possible to stain or process all the
samples
under the same conditions. Multiple tissue samples may be taken from multiple
such
tissue specimens, and the multiple samples from a particular specimen are
similarly
placed at corresponding positions in the multiple recipient substrates. Each
of the
resulting substrates contains an array of tissue samples from multiple
specimens, in
which corresponding positions in each of the arrays represent tissue samples
from the
same tissue specimen. In particular examples, each substrate is then sectioned
into
multiple similar sections with samples from the same tissue specimen at
corresponding
positions of the sequential sections. The different sections may then be
subjected to
different reactions, such as exposure to different histological stains or
molecular
markers, so that the multiple "copies" of the tissue microarrays can be
compared for the
presence of reactants of interest. The large number of tissue samples, which
are repeated
in each of a potentially large number of sections of multiple substrates, can
be exposed
to as many different reactions as there are sections. For example, about
100.000 array
sections may be obtained from a set of 1000 tissue specimens measuring 15x15x3
mm.
This approach provides a high-throughput technique for rapid parallel analysis
of many
different tissue specimens. In a particular embodiment of the method, the
specimens are
embedded in embedding medium to form tissue donor blocks, which are stored at
identifiable locations in a donor array. The donor blocks are retrieved from
the donor
array, coordinates of particular areas in each of the tissue specimens in the
donor blocks
are determined, and tissue samples from the donor blocks (such as elongated
punches)
are retrieved and inserted into receptacles of corresponding size (such as
punched holes)
in different recipient tissue microarray blocks. After repeating this process
with multiple
donor blocks, to form a three-dimensional array of substantially parallel
elongated
samples from a variety of different specimens, the recipient tissue microarray
blocks are
then sectioned to make multiple similar tissue microarray sections that
include samples
of many different specimens. Each of these sections can then be subjected to
treatment
with multiple reagents, and subsequently analyzed for the presence of
biological
markers.
Preferably, the different samples of the array are formed by cells derived
from stable
cell lines. In a more preferred embodiment, the cell lines of the tissue
microarray are
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selected from the group of Hela 2, HeLa, MCF7, HeLa S3, 293T, L5178Y-S, MEFs
BL6 G3 Terc4-, MEFs BL6 wild type, HeLa 1211, MEFs 129Sv/BL6 wild-type and
L5178Y-R.
Methods for determining telomere length using image processing
In yet another aspect, the inventors have developed a method which allows the
determination of the telomere length in individual cells within images of
three-
dimensional cell populations labelled with a telomere-specific fluorescent
probe. Thus,
in another aspect, the invention relates to a method for determining the
telomere length
of a cell within a tridimensional cell population from a collection of at
least two
fluorescence microscopy images obtained using a fluorescently-labeled telomere-
specific probe and corresponding to different focal planes of said cell
population
comprising the steps of:
(i) converting the at least two fluorescence microscopy images
corresponding to different focal planes into a single image by adding
up the fluorescence intensities at each position within the image,
(ii) determining the average fluorescence intensity of said cell within the
image of the cell population obtained in step (ii) and
(iii) assigning to said cell an average telomere length value, wherein said
value is obtained by interpolation of the average intensity of the cell
within a data set of telomere length values and corresponding
fluorescence intensity values obtained from different cell populations
of known and stable telomere length processed by fluorescence
microscopy in parallel to the cell of the test cell population.
Step (i) comprises the merging or flatteling of the different confocal images
of the cell
polulation so as to obtain a single image wherein each pixel contains the
addition of the
intensities of the corresponding pixels at the same positions from each
confocal image.
The image is usually obtained by fluoresence microscopy analysis of said cell
population. For this purpose, a cell population has been previously contacted
with a
fluorescently-labelled telomere-specific probe. Using appropriate optical
filters to allow
excitation of the cells with the appropriate wave-length and capturing only
the emission
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wave-length, it is possible to capture an image wherein the optical density of
the stained
cells will be proportional to the amount of the probe bound to the telomeric
regions and,
indirectly, to the length of the telomere in the cell. When using a confocal
microscope,
the microscope can be programmed so as to obtain the different focal images at
given
5 focal lengths (preferably 1 gm).
The microscope is attached to an adequate image capture device having
digitalisation
means so that a digital image is obtained. A digital image, as used herein, is
a two-
dimensional array of pixels. Each pixel value relates to the amount of light
received by
10 the imaging capture device corresponding to the physical region of
pixel. Preferably,
image collection is carried out using a microscope, preferably a confocal
microscope,
attached to appropiate detectors (for instance, a CCD camera). For colour
imaging
applications, a digital image will often consist of red, green, and blue
digital image
channels. Those skilled in the art will recognize that the present invention
can be
15 applied to, but is not limited to, a digital image channel for any of
the herein-mentioned
applications. Although a digital image channel is described as a two
dimensional array
of pixel values arranged by rows and columns, those skilled in the art will
recognize
that the present invention can be applied to non-rectilinear arrays with equal
effect. The
image file format can be any format used for digital images, including for
example, a
20 JPG format, a JPG2 format, a RAW format, a TIFF format, a PNG format, a GIF
format, or a BMP format. Depending on the size and the resolution of the
image, the
digital image can be stored in storage media readable in a computer, such as,
a CD, a
DVD, a web-hard disk, a memory card, etc., and then provided to users.
25 Step (ii) of the method of the invention comprises the determination of
the intensity of
the telomere-specific fluorescent emission within a given target cell of the
cell
population in the flattened image obtained in step (i). This step comprises
determining,
in first instance, the region of interest (ROI) within the wherein fluorescent
emission is
to be determined. The ROI may be a region corresponding to the whole cell
under study
30 which can be determined, by way of example, by overlying a bright filed
image of the
same cell population. Preferably, the ROI may be the region corresponding to
the
nucleus of the cell under study, which is identified by overlying a
fluorescent image
captured of the same cell population labelled with a DNA-specific fluorescent
probe
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with the image under study. Once the ROI is identified, the determination of
the
intensity level of each pixel within the ROI is usually carried out using a
personal
computer, a workstation, a network computer or a personal digital assistant
which can
determine the intensity level of each pixel in the cell under study. The
intensity of the
telomere-associated fluorescence can be measured as "average grey value" and
is
usually calculated by dividing the summation of the intensities of all pixels
of the ROI
under study by the number of pixels. These values are thus the average
fluorescence
intensity over the whole ROI and not the average of the intensities in those
pixels
corresponding to the individual telomeres.
It will be appreciated that background noise may interfere with the
determination of the
of pixel intensity. In order to avoid this problem, digital thresholding is
usually applied
to distinguish desired intracellular fluorescence from unwanted background
fluorescence. Because background fluorescence (including cell autofluorescence
as well
as non-specific fluorescence due to non-specific binding of the telomere-
specific probe
to non-telomeric structures) is more diffuse and is less intense than telomere-
specific
fluorescence, contribution to the total fluorescence measurement from
background
fluorescence is reduced by ignoring light intensities which are below a
specified value.
In a preferred embodiment, the correction of probe accessibility and cell
ploidy is
carried out by normalising the fluorescence values of the cell within the
image obtained
in step (i) using the fluorescence values of the same cell within a
corresponding image
obtained using a fluorescently-labeled centromeric.
The skilled person will appreciate that other corrections algorithms may be
applied to
the image to remove any artifacts introduced by the image capture system. For
example,
"quality control algorithms" may be employed to discard image data based on,
for
example, poor exposure, focus failures, foreign objects, and other imaging
failures.
Generally, problem images can be identified by abnormal intensities and/or
spatial
statistics.
Step (iii) of the method of the invention is carried out once the average gray
values for
each cell are determined. Step (iii) comprises assigning to said cell an
average telomere
length value, wherein said value is obtained by interpolation of the average
intensity of
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the cell within a data set of telomere length values and corresponding
fluorescence
intensity values obtained from different cell populations processed by
fluorescence
microscopy in parallel to the cell of the test cell population.
The skilled person will appreciate that the data set used for determining the
telomere
length values of selected cells within the test image can be obtained from
images of
reference cell lines wherein said images have been captured immediately before
obtaining the images of the test cell population, in which case, the average
fluorescence
intensities and the telomere lengths in each cell type are stored as a dynamic
data base
until the fluorescence values of the cell or cells under study are available
for
interpolation. In a preferred embodiment, the images of the test cell
population and the
images of the different control cell populations are collected sequentially in
an
automated fashion by using a microscopy with a motorised stage.
In a preferred embodiment, the determination of the image density of the cells
under
study is carried out not on the whole cell surface but on those areas of the
image which
correspond to the cell nuclei. In order to identify the cell nuclei, the same
cell
population which is under study is also stained with a fluorescent DNA dye and
an
image is captured using adequate filters to detect fluorescence emission by
said DNA
dye. The resulting image comprises spatial information of the location the
cell nuclei
within the cell image and is then used as mask to select those areas of the
image derived
from the telomere-specific fluorescence which correspond to cell nuclei. Thus,
in a
preferred embodiment, the method of the invention comprises, previous to step
(i), the
selection of those regions of the cells within the cell image corresponding to
cell nuclei
using a mask obtained by fluorescence microscopy analysis of the cell
population using
a DNA-specific fluorescent dye and the determination of the average
fluorescence
intensity in step (i) is carried out on the regions of the image which have
not been
masked.
In yet another embodiment, the invention relates to a computer program
including
encoded means to carry out the steps of the methods according to the
invention. The
computer program is provided on a computer-readable media. Thus, in another
aspect,
the invention relates to a computer-readable support comprising encoded means
adapted
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to carry out the steps of the methods of the invention. Any method or
technology
suitable for storing information can be used for storing the program of the
invention. By
way of example, the invention comprises any readable medium such as RAM, ROM,
EEPORM, flash memories or other types of memory, CD-ROM, DVD or other types of
optic storage media as well as magnetic tapes, hard drives and other types of
devices for
magnetic storage. Alternatively, the program may be hosted in a remote storage
device.
In this case, the instructions encoded in the program are delivered by a
telematic
communication system such as wireless network, internet, local area networks,
wide
band networks, direct connections via a USB serial port or via model, (ISDN)
or digital
subscriber lines (DSL); satellite links as well as any other communication
types known
to the skilled person.
The following methods and examples illustrate the invention.
EXAMPLES
EXAMPLE 1
Experimental procedures
Mice, treatment regimens, and mouse sample collection
Unless otherwise specified, all mice used in this study were males of
approximately 2
months of age and from a C57BL6 genetic background. Mice of a FVB genetic
background (2 months-old) were also used in Supplementary Figure Sl.
Generation and
characteristics of the Terc¨/¨ and the K15-EGFP mice were previously described
(Morris et al., 2004; Blasco et al., 1997).
To induce epidermal stem cell "mobilization" (activation, migration and
proliferation),
tail skin in the telogen (resting) phase of the hair cycle was topically
treated every 48 h
with TPA (20 nmol in acetone) for a total of four doses. Control mice of each
genotype
were treated with acetone alone. 24-hours after the last TPA treatment, mice
were
sacrificed and the tail skin analysed.
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For telomere length analyses, samples from mouse tail and back skin, small
intestine,
cornea, testis and brain were harvested and fixed o/n in neutralbuffered
formalin at 4 C,
dehydrated through graded alcohols and xylene, and embedded in paraffin. Prior
to
embedding, dissected skin was cut parallel to the spine in order to obtain
longitudinal
hair follicle sections. The intestinal tract was flushed with PBS and rolled
up in a
compact circle using longitudinally oriented jejunal sections for analysis.
For cornea
and testis analyses, whole eyes and testis were cut in half prior to
dehydration. Finally,
brain was coronal-dissected to harvest the rostral hippocampus. In all cases,
5 "M
sections were used for QFISH and immunostaining analyses.
Telomerase assay
Telomerase activity was measured with a modified telomere repeat amplification
protocol (TRAP) as described (Blasco et al., 1997). An internal control for
PCR
efficiency was included (TRAPeze kit Oncor, Gaithersburg, MD). Hela cells were
included as a positive control for telomerase activity.
Confocal quantitative telomere fluorescence in situ hybridization (confocal Q-
FISH) on histological sections
For Q-FISH, paraffin-embedded tissue sections were hybridized with a PNA-tel
Cy3-
labelled probe and telomere length was determined as described (Gonzalez-
Suarez et
al., 2000; Munoz et al., 2005; Zijlmans et al., 1997). Slides were
deparaffinized in three
xylene washes (3 m each), then treated for 3 m with a 100, 95 and 70% ethanol
series,
followed by telomere Q-FISH protocol performed as described (Samper et al.,
2000).
DAPI, Cy3 signals were acquired simultaneously into separate channels using a
confocal ultraspectral microscope (Leica TCS-5P2-A-OBS-UV) using a PL APO
20x/0.70 PH2 as lens with Leica LCS software and maximum projections from
image
stacks (10 sections at steps 1.0 [tm) were generated for image quantification.
The DPSS-
561 laser (Cy3 laser) was hold at a constant intensity to capture all the
mouse tissues
images.
Generation of topographic telomere length maps on histological sections or
"telomapping"
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High throughput quantitative image analysis was performed on confocal images
using
the Metamorph platform (version 6.3r6; Molecular Devices, Union City, CA). The
DAPI image was used to define the nuclear area and the Cy3 image to quantify
of
telomere fluorescence. In all cases, background noise was subtracted from each
image
5 prior to making qualitative measurements. The DAPI images were signal-
intensity
thresholded, segmented and converted to 1-bit binary image. The binary DAPI
mask
was applied to the matching Cy3 to obtain a combined image with telomere
fluorescence information for each
nucleus. Cy3 fluorescence intensity (telomere fluorescence) was measured as
"average
10 gray values" (total gray value/nuclei area) units (arbitrary units of
fluorescence). These
"average telomere fluorescence" values always represent the average Cy3 pixel
intensity
for the total nuclear area, and not the average value of individual telomere
spot
intensities, therefore ruling out that differences in nuclear size may
influence telomere
length measurements. A code of four colours was used to classify the nuclei
according
15 to their average telomere fluorescence. Telomere fluorescence ranges
were initially set
up to allocate in each range roughly 1/4 of the total cells of a given tissue
in wild type
mice of 2
months of age. Subsequently, telomere fluorescence ranges were fine adjusted
to better
delineate the location of stem cell compartments in different tissues.
Telomere
20 fluorescence ranges of a given tissue obtained in this manner were then
maintained
constant between genotypes, treatment and ages to facilitate comparisons.
Finally,
telomere fluorescence values for each histological region (i.e. skin sections
were
subdivided in hair follicle bulge, hair follicle bulb, hair follicle
infundibulum, and
interfollicular epidermis) were exported to Excel and the frequency histograms
were
25 generated. A macro created using the Metamorph platform allowed the
automated and
user-controlled processing of the DAPI and Cy3 images to obtain the telomap
images
(available upon request).
To avoid differences due to variation in section thickness, we used paraffin
slices of the
30 same thickness (5 [tM) in all the tissues analyzed. Additionally, to avoid
possible
variations due to different roughness of the paraffin on the slices, confocal
capture
conditions were set to cover the entire fluorescence signal (maximum
projections of 10
sections at steps of 1.0 [tM). To avoid differences in day-to-day staining
efficiency
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variation, Q-FISH and immuno-Q-FISH staining, as well as image capture were
performed for each given tissue in the same day.
Finally, to control for differences in ploidy as well as probe accessibility,
we used a
Cy3-labelled PNA probe directed against mouse major satellite repeats (5"-TCG-
CCA-
TAT-TCC-AGG-TC-3'). As shown in Supplementary Figs. S2ac, no significantly
differences in centromeric fluorescence were detected between different skin
and testis
compartments, again ruling out that differences in "probe accessibility" or
ploidy
account for the observed
differences in telomere length in the skin or the testis. Furthermore, we
ruled out that
differences in nuclear size between different compartments (ie, skin) could
account for
the observed differences in telomere length (Supplementary Fig. 2b).
Similarly, as
shown in Figs.2d,e, no significant differences in centromeric fluorescence
were detected
between GFP+ and GFP- sorted cells from the K15-EGFP mouse model, again ruling
out that differences in "probe accessibility" or ploidy account for the
observed
differences in telomere length between between GFP+ and GFP- cells. To better
assess
differences in centromeric signal, maximum projections of 16-bits confocal
images
were obtained form paraffin sections. Finally, to verify whether image
analyses by
conventional Q-FISH and telomapping give similar results, the same confocal
image of
tail skin was quantified using the TFL-TELO program (gift from Dr. Lansdorp,
Vancouver) and the Metamorph platform ( version 6.3r6; Molecular Devices,
Union
City, CA) (see Supplementary Fig. S3).
Immunohistochemistry
Skin (5 "M) sections were used for immunohistochemistry (IHC). Prior to IHC,
slides
were de-paraffinized, re-hydrated, immersed in 10 mM citrate solution and
epitopes
retrieved by three high-power, 5 min microwave pulses. Slides were washed in
water,
blocked in 1:10 dilution of normal goat serum (Vector Labs) and incubated with
primary antibodies: CD34 at 1:200 (RAM34, BD Biosciences), and keratin 15 at
1:500
(LHK15, NeoMarkers). Slides were then incubated with secondary biotinylated
antibodies from Vector labs (goat anti-rabbit at 1:200 or goat anti-mouse at
1:200),
followed by signal development with an immunoperoxidase reagent (ABC-HRP,
Vector
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Labs) and DAB (Sigma) as the substrate. Sections were lightly counterstained
with
hematoxylin and analyzed by light microscopy.
Cell sorting and telomere length measurements in K15-EGFP mice
Eight week-old K15-EGFP mice were sacrificed and back skin and tail skin were
harvested, fixed o/n in neutral-buffered formalin at 4 C, dehydrated through
graded
alcohols and xylene, and embedded in paraffin. Simultaneously, part of the
tail skin was
collected and soaked in Betadine for 5 m, in a PBS antibiotics solution for 5
m, in 70%
ethanol for 5 m, and in PBS-antibiotics solution for 5 m. Tail skin was peeled
off using
forceps and floated on the surface of lx trypsin (Sigma) solution (4m1 on 60
mm cell
culture plate) for 3 h at 37 C. Tail skin was then transferred to a sterile
surface and the
epidermis separated from the dermis using forceps, and minced and stirred at
RT for 30
m in serum-free Cnt-02 medium (CELLnTEC Advanced Cell Systems AG, Bern,
Switzerland). The cell suspension was filtered through a sterile teflon mesh
(Cell
Strainer 0.7 m, Falcon) to remove cornified sheets. Keratinocytes were then
collected by
centrifugation (160 g) and counted. Freshly isolated keratinocyte suspensions
from
K15-EGFP mice were then sorted with a fluorescence-activated cell sorter
(FACS)
using a MoFlo (DakoCytomation, Glostrup, Denmark). Cells were excited with a
488-
nm laser and GFP signals collected via the FL1 channel (510 to 550 nm),
sorting them
in an "enrichment" mode into GFP-positive and GFP-negative cells. The sorted
cell
suspensions were centrifuged onto microscope slides using a cytospin (Cytospin
3;
Thermo Shandon, Pittsburgh, PA). After air-drying, cells on slides were fixed
in
methanol/acetic acid (3:1) during 1 h and dried o/n. Telomere FISH was
performed as
described calculating the telomere fluorescence of the whole nuclei (Munoz et
al., 2005;
Samper et al., 2000). For telomere length quantification on interphase nuclei,
Cy3 and
DAPI images were captured at 100x magnification using a COHU CCD camera on a
Leica Leitz DMRA (Leica, Heidelberg, Germany) microscope, and the telomere
fluorescence was integrated and quantified using spot IOD analysis in the TFL-
TELO
program (Zijlmans et al., 1997) (gift from Dr P.Lansdorp, Vancouver). The
telomere
fluorescence of individual nuclei was represented by frequency histograms. In
parallel,
combined GFP-immunostaning/telomere-QFISH was performed in paraffin-embedded
sections of back and tail skin from K15-EGFP mice to quantify telomere length
in GFP
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positive and negative cells. Slides were deparaffinized, re-hydrated, immersed
in 10
mM citrate solution and epitope retrieved
by three high-power, 5 m microwave pulses. Slides were then rinsed in water,
permeabilized in 0.25% TritonT" X-100/PBS, blocked in BSA 10%/PBS for 30 at RT
and incubated with BD Living Colours AV Monoclonal Antibody JL-8 (Becton
Dickinson, San Jose, CA) at a 1:250 dilution for 30 m at R.T. After three
rinses with
.TweenTN120-PBS, slides were blocked in BSA 10%/PBS for 30 m at RT and
incubated
with goat antibody to mouse conjugated with AlexaTM 488 (1:500; Molecular
Probes,
Invitrogen) 30 m at RT. After three rinses with Fweenrm20-PBS slides were
fixed in
methanol/acetic acid (3:1) for 1 h and dried o/n in the dark. Telomere Q-FISH
was
performed as described (Gonzalez- Suarez et al, 2000; Munoz et al., 2005) with
minor
modifications to preserve GFP- immunostaining. DAP1, Cy3 and A1exa488 signals
were
acquired simultaneously into separate channels using a confocal ultraspectral
microscope (1....eica TCS-SP5-A-OBS- UV) using a PL APO 20x/0.70 PH2 as lens
with
Leica LAS AF software and maximum projection from image stacks (10 sections at
steps 1.0 p.m) were generated for image quantification.
Flow-FISH telomere length measurements in K15-EGFP mice
Freshly isolated keratinocyte suspensions (EGFP+ and EGFP-) from KA 5-EGFP
mice
were fixed in methanol/acetic acid (3:1), permeabilized with methanol 100% and
washed in PBS. Cells were blocked in BSA 10% PBS for 15 m at RT and incubated
with 13D Living Colours AV Monoclonal Antibody 11.-8 (Becton Dickinson, San
Jose,
CA) at 1:250 dilution for 30 min at RT. After two washes in Tween20-PBS cells
were
blocked in BSA 10% PBS for 15 m at RT and incubated with goat antibody to
mouse
conjugated with Alexa 647 at 1:500 dilution (Molecular Probes, Invitrogen) 30
m at RT.
After two washes in Tween20-PBS cells were fixed in Ibrmaldehyde 0.5% PBS for
5 m
and washed twice in PBS. Then telomere flow-FISH was performed as described
(Rufer
et al_ (998) using a F1TC labeled PNA-tel probe and Propidium Iodide (PI,
Sigma) to
counterstain DNA, and analyzed in a FACScantOrm eytometer (BD Biosciences).
Cells
with adequate size and complexity as determined by forward scatter and side
scatter
channels, were gated for GO/GI phase using the PI signal acquired in FP_
channel.
Their labeling for Alexa 467 was acquired in Fl.:4 channel and was used to
identify GFP
positive and negative cell populations. The telomere fluorescence as F1TC
signal was
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acquired in FL1 for both cell populations. To compensate for the contribution
of cellular
autofluorescence, fluorescence values of negative control cells (i.e. cells
hybridized in
the absence of the FITC PNA-tel probe) were subtracted from every sample.
L5178Y-R
and L5178Y-S cell lines with known telomere length of 10.2 and 79.7 kb
(McIlracth et
al., 2001), respectively, were processed in parallel and used to convert
fluorescence
values into kb. Negative controls for each fluorochrome and acquisition
settings were
established with unstained or single stained cell populations.
Telomapping of a paraffin-embedded array of human and mouse cell lines
The cell line array was developed using a range of different cell lines with
known
telomere length (Canela et al., 2007). Cell lines were cultured and 4x106
cells from
each one were used, washed in PBS, fixed in formaldehyde 4% PBS during 5 m at
RT,
washed twice in PBS and mixed with melted gelatine (Sigma) 5% PBS to generate
gelatine blocks after polymerization at 4 C overnight. Gelatine blocks were
embedded
in paraffin blocks as a classical fixed tissue, previously, every gelatin
block was stained
with blue metilene to allow its identification in the paraffin block. A small
cylindrical
cell-containing core of lmm diameter was performed in every paraffin block
using a
Manual Tissue Microarrayer MTA (Beecher, Sun Prairie, WI, USA) and inserted in
a
receptor paraffin block separated from each other 1.5 mm. A 4 "m section of
one of
these paraffin block containing every cell line was placed together with skin
sections on
the same slide and confocal Q-FISH was performed. Telomapping analysis were
carried
on images from skin follicles and interphase nuclei of every cell line, and
telomere
fluorescence values of the skin follicle compartments were converted in kb
using these
cell lines as calibration standard with stable and known telomere length
(Canela et al.,
2007).
TRF-based telomere length analysis of K15-EGFP adult keratinocytes
Isolated GFP+ and GFP- adult epidermal keratinocytes from K15-EGFP mice were
sorted as indicated above. A fraction of enriched GFP-negative cells and GFP-
positive
cells were included in agarose plugs following instructions provided by the
manufacturer (Bio-Rad), and TRF analysis was performed as previously described
(Blasco et al., 1997).
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Quantitative telomere length analyses on K15-EGFP skin sections
Quantitative image analysis was performed on confocal images using Metamorph
(version 6.3r6; Molecular Devices, Union City, CA). The DAPI image was used to
define the nuclear area, the Cy3 image for telomere fluorescence
determinations, and
5 the A1exa488 image to identify the GFP-expressing cells. In all cases,
background noise
was subtracted from each image prior to qualitative measurements. The DAPI
images
were signalintensity
thresholded, segmented and converted to 1-bit binary image. The binary DAPI
mask
was applied to both Cy3 and A1exa488 images obtaining topographic maps showing
10 telomere fluorescence and GFP staining for each nucleus or object.
Specific nuclear
masks for GFP-positive and GFP-negative cells were generated to allow
quantification
of telomere fluorescence in the two populations. The nuclear mask of GFP-
positive cells
was created by converting the combined image from DAPI mask and A1exa488 to 1-
bit
image, showing
15 only those nuclei that had an A1exa488-fluorescence above a minimum
threshold. The
nuclear mask for GFP-negative cells was created subtracting the GFP-positive
mask
from the DAPI mask. The three masks generated, DAPI, GFP-positive and GFP-
negative, were applied to the Cy3 image obtaining the combined images with
telomere
fluorescence information for all nuclei, GFP-positive or negative nuclei,
respectively.
20 The combined images were then analyzed as indicated above.
Isolation of newborn keratinocytes
Two days old mice were sacrificed, soaked in Betadine (5 min), in a PBS
antibiotics
solution (5 min), in 70% ethanol (5 min), and in a PBS antibiotics solution (5
min).
25 Limbs and tail were amputated, and the skin peeled off using forceps.
Skins were then
soaked in PBS (2 min), PBS antibiotics solution (2 min), 70% ethanol (1 min)
and in
PBS antibiotics solution (2 min). Using forceps, each skin was floated on the
surface of
lx trypsin (Sigma) solution (4m1 on 60mm cell culture plate) for 16 h at 4 C.
Skins
were transferred to a sterile surface, and the epidermis separated from the
dermis using
30 forceps, minced and stirred at 37oC for 30 min in serum-free Cnt-02 medium
(CELLnTEC Advanced Cell Systems AG, Bern, Switzerland). The cell suspension
was
filtered through a sterile teflon mesh (Cell Strainer 0.7 m, Falcon) to remove
cornified
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sheets. Keratinocytes were then collected by centrifugation (160 g) for 10 min
and
counted.
Isolation of adult keratinocytes
2-months old and 27-31 months old mice were sacrificed and tail skin was
collected and
soaked in Betadine for 5 m, in a PBS antibiotics solution for 5 m, in 70%
ethanol for 5
m, and in PBS-antibiotics solution for 5 m. Tail skin was peeled off using
forceps and
floated on the surface of lx trypsin (Sigma) solution (4m1 on 60 mm cell
culture plate)
for 3 h at 37 C. Tail skin was then transferred to a sterile surface and the
epidermis
separated from the dermis using forceps, and minced and stirred at RT for 30 m
in
serum-free Cnt-02 medium (CELLnTEC Advanced Cell Systems AG, Bern,
Switzerland). The cell suspension was filtered through a sterile teflon mesh
(Cell
Strainer 0.7 m, Falcon) to remove cornified sheets. Keratinocytes were then
collected by
centrifugation (160 g) and counted.
Clonogenic assays
103 mouse keratinocytes obtained from 2-days-old mice and 104 mouse
keratinocytes
from 2-months-old and 27-31-months-old mice were seeded onto mitomycin C (10
"g/ml, 2 hours) treated J2-3T3 fibroblast (105 per well, 6 well dishes) and
grown at
37 C/5% CO2 in Cnt-02 medium (CELLnTEC Advanced Cell Systems AG, Bern,
Switzerland). After ten days of cultivation, dishes were rinsed twice with
PBS, fixed in
10% formaldehyde and then stained with 1% Rhodamine B to visualizy colony
formation. Colony size and number were measured using three dishes per
experiment.
Statistical analysis
A Wilcoxon's ram sum test was used to calculate the statistical significance
of the
observed differences in the different assays. Microsoft Excel v.2001 and
Graphpad
Instat v3.05 were used for the calculations. In all cases, differences are
significant for
P<0.05; very significant for P<0.01; and highly significant for P<0.001.
EXAMPLE 2
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Cells with the longest telomeres are enriched at the hair follicle stem cell
compartment and show stem cell behaviour upon treatment with mitogenic
stimuli.
To evaluate whether telomere length could be used as a general marker to
anatomically
map stern cell compartments within adult tissues, confocal telomere
fluorescence in
hybridization (Confocal Q-FISH) was performed directly on tissue sections
coupled to a
single-cell highthroughput Metamorph image analysis platform (referred here as
"telomapping") (see Experimental Procedures for detailed description of the
technique
and of different controls performed). First, single-cell topographic telomere
length maps
were generated for skin sections from 2 month-old wild-type mice of a C57131.6
genetic
background (Experimental Procedures), which vere subdivided in four different
epidermal compartments: the hair follicle bulge where the hair follicle stem
cell niche is
located aumbar et al. 2004; Cotsarelis et al, 1990; Oshima et at, 2001; Morris
et al,
2004), the hair follicle bulb and the infundibulwn where the transit-
amplifying (TA)
cells reside. and the interfollicular epidermis (Figs. 1(0). In "resting"
untreated wild-
type mouse skin, it was observed that cells with the longest telomeres, 1800-
3000
arbitrary units of telomere fluorescence (Fig. 1 u; see Experimental
Procedures for
criteria to establish telomere length ranges within a given tissue) were
enriched at the
hair bulge, coinciding with the known stem cell niche (Tumbar et al., 2004;
Cotsarelis
et al., 1990: Oshima et al., 2001, Morris et at., 2004). Immunostaining with
the hair
follicle stem cell markers CD34 and keratin 15 (K15) 'further confirmed that
the longest
telomeres localized to the hair bulge (not shown). These results were
confirmed in age-
matched mice of a different genetic background (FVB background: Experimental
Procedures) (Fig. 2a,b). These findings indicate that telomeres are
progressively shorter
as cells move out from the stem cell compartment to the adjacent TA
compartments
(hair bulb and infundibulum), with the more differentiated layers of the
interfollieular
epidermis showing the shortest telomeres, in agreement with their longer
proliferative
and differentiation history. Of notice, these differences in telomere length
are unlikely
to be due to differences in "probe accessibility" or ploidy between different
skin
compartments as we did not find significant differences between compartments
when performing Q.-FISH with a centromerie major satellite probe
(Experimental Procedures; Fig. 3a). Furthermore, telomere length differences
are not
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likely to be due to differences in nuclei size between different skin
compartments as
telomere length is captured for the whole nucleus using the DAPI image
(Experimental
Procedures). Indeed, we did not find major differences in nuclear size between
the stem
cell compartment (hair bulge) and the interfollicular epidermis and
infundibulum
compartments that could account for the observed differences in telomere
length (Fig.
3b). Next, the skin telomapping results were validated using the conventional
quantitative telomere FISH technique (Q-FISH) on tissue sections (Experimental
Procedures). Q-FISH on tissue sections has been extensively used to obtain
quantitative
and accurate telomere length determinations both in mouse (Gonzalez-Suarez et
al.,
2000; Munoz et al., 2005) and human cells (Meker et al., 2002; 2004; Meeker
and De
Marzo, 2004). In particular, Fig. 4a shows a similar decrease in telomere
length when
comparing the hair bulge compartment to other skin compartments using
telomapping
or Q-FISH on tissue sections. Furthermore, there was a linear correlation in
telomere
length values obtained by these techniques (Fig. 4b). In order to calibrate
the
telomapping technique and to convert fluorescence values into kilobases,
telomapping
was performed on a paraffin-embedded array of human and mouse cell lines of
previously known telomere lengths (Canela et al., 2007) (Experimental
Procedures). As
shown in Fig. 5a, telomapping was able to detect differences of telomere
length of less
than 1 Kb (see comparisons between HeLa and Hela2 cell lines, and between
HeLaS3
and 293T cell lines; p<0.001 for both comparisons). Furthermore there was a
linear
correlation between telomapping results and Q-FISH telomere length results as
determined by conventional Q-FISH on metaphases Fig. 5b). Finally, using this
calibration, a decrease of telomere length was detected between the hair bulge
(stem cell
compartment) and the TA compartments of 1.3 Kb (hair bulb) and 3.5 Kb
(infundibulum) and of 9.8 Kb when comparing the hair bulge to the
interfollicular
epidermis (Fig. 5c).
To further test whether the longest telomeres map to stem cell compartments
and to
prevent the influence of possible telomerase activation on telomere length,
topographic
telomere length maps were generated from first generation telomerase-deficient
G1
Terc4- mice (Fig. lb) (Blasco et al., 1997; Ramirez et al., 1997). Similarly
to wild-type
skin, G1 Terc4- skin showed an enrichment of cells with the longest telomeres
(1800-
3000 a.u. of telomere fluorescence) in the bulge area of the hair follicle
with the shortest
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telomeres at the interfollicular epidermis (Fig. 1 b), thus confirming that
the longest
telomeres are enriched at the stem cell compartment. However, the percentage
of cells
with 1800-3000 arbitraty units of telomere fluorescence was lower in all skin
compartments compared to control wild-type mice, in agreement with the fact
that G1
Terc4- mice lack telomerase activity (Fig. lb). Similarly, average telomere
fluorescence
was lower in G1 Terc4- mice compared to wild-type mice in all skin
compartments
(Fig. 6). These results demonstrate that the cells with the longest telomeres
are enriched
at the hair follicle stem cell compartment, while the cells with the shortest
telomeres are
located in the outer skin layers, indicating that telomeres are shorter as
cells go from the
more primitive to the more differentiated skin compartments. Furthermore,
these results
indicate that telomerase activity is important to maintain the overall
telomere length of
different skin compartments in the mouse, as first generation telomerase-
deficient G1
Terc-/- showed a marked decrease in telomere length compared to age-matched
wild-
type controls in all skin compartments.
To address whether cells with the longest telomeres within the hair follicles
also show
characteristic stem cell behaviour, wild-type and G1 Terc¨/¨ mice were treated
with the
mitogenic stimulus TPA, which triggers migration and proliferation
("mobilization") of
stem cells out of the niches in the TA compartments. Wild-type TPA-treated
skin
showed a
decreased in the percentage of the cells with the longest telomeres at the
hair bulge with
an accumulation of these cells to the TA compartments (hair bulb and
infundibulum),
coinciding with an enlargement of these compartments and thickening of the
interfollicular epidermis (compare Fig. lc to Fig. la). As a consequence, the
absolute
number of cells containing the longest telomeres decreased at the hair bulge
and
concomitantly increased in the other epidermal compartments (Fig. ld;
significant in all
cases). Furthermore, the total number of epidermal cells showing 1800-3000
a.u. of
telomere fluorescence significantly increased in TPA-treated skin compared to
the
untreated wild-type skin (significant, P<0.05; Fig. le), suggesting net
telomere
elongation associated to TPA-induced proliferation in TA compartments (Fig.
le).
Telomere length histograms also showed decreased frequency of long telomeres
in hair
bulge cells upon TPA treatment, which was concomitant with increased telomere
length
in cells located at the TA compartment and the interfollicular epidermis (Fig.
6).
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To address whether these effects were dependent on telomerase activity, TPA-
treated
G1 Terc4- skin was studied. Similarly to TPA-treated wild-type skin, G1 Terc4-
skin
showed a reduction of the percentage of cells with the longest telomeres at
the hair
5 bulge (Fig. lc) coincidental with an enlargement of the TA compartments
(compare Fig.
lc to Fig. lb), suggesting that these cells mobilized and proliferated in
response to TPA.
However, in contrast to TPA-treated wild-type skin, G1 Terc4- TPA-treated skin
did not
show increased percentage of cells with the longest telomeres at the TA
compartments
and the interfollicular epidermis (Fig. lc,d). Indeed, the total number of
epidermal cells
10 showing the longest telomeres was decreased in G1 Terc4- TPA-treated
skin compared
to untreated skin (very significant P<0.01; Fig. le), suggesting telomere
shortening as
the result of TPA-induced proliferation in the absence of telomerase activity.
In
agreement with this, telomere length histograms of TPA-treated G1 Terc4- mice
showed a decreased frequency of long telomeres in all hair follicle
compartments (Fig.
15 6). These results indicate that telomerase actively participates in
telomere maintenance
upon migration and proliferation (mobilization) of stem cells in response to
TPA
treatment, in contrast, mobilization of stem cells in the absence of
telomerase results in
telomere shortening in all skin compartments in G1 Terc4- mice.
20 EXAMPLE 3
Isolated hair bulge cells from K15-EGFP mice show the longest telomeres
The results described above indicate that cells with the longest telomeres are
enriched at
stem cell compartments. To further address this issue, purified skin hair
bulge cells (hair
25 follicle stem cell compartment) was studied in order to determine
whether these cells
also showed the longest telomeres. For this, K15-EGFP transgenic mice were
used, in
which the K15-expressing hair bulge cells are identified by a positive GPF
expression
(Morris et al., 2004) (Experimental Procedures). GFP-positive cells from these
mice
have been previously shown to have stem cell properties and to contribute to
some
30 aspects of skin regeneration (i.e., wound healing) as well as to have a
higher in vitro
clonogenic potential than GFP-negative cells (Morris et al., 2004; Ito et al.,
2005). To
this end, we sorted K15- EGFP skin keratinocytes into GFP-negative (GFP-) and
GFP-
positive (GPF+) cell populations (Experimental Procedures). In agreement with
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previously published data, approximately 4% GFP+ cells were recovered from
total
K15-EGFP tail epidermis reflecting on the relatively low abundance of hair
bulge Cells
with stem cell properties (Morris et al., 2004; Ito et al., 2005). As control
for enrichment
in stem cells, in vitro clonogenic assays were performed, where individual
colonies are
proposed to derive from single stem cells (Flores et al., 2005). Purified GFP+
cells
formed 3 times more colonies than GFP- cells in clonogenic assays, in
agreement with
the notion that they are enriched in hair bulge stem cells (Morris et al.,
2004; Ito et al.,
2005) (Figure 7a). More over, GFP+ cells were more abundant in the big-size
colonies
when using total unsorted K15-EGFP keratinocytes, also supporting the notion
that
K15-GFP+ cells are enriched in stem cells (Fig. 7b). Importantly, using
conventional Q-
FISH on cytospin-plated interphasic cells (Experimental Procedures), it was
found that
the GFP+ keratinocytes showed longer telomeres than the GFP- keratinocytes
(highly
significant, P<0.001; Fig. 8a,b). To rule out possible differences in ploidy,
the number
of telomere signals per nuclei were quantified and found no significant
differences
between sorted GFP+ and GFP- cells (P=0.457; Fig. 8c). In addition,
differences in
telomere length due to differential "probe accessibility" could be ruled out
by
performing Q-FISH with a centromeric major satellite probe as control
(Experimental
Procedures; Figs, 8d,e). The decline in telomere length between GFP+ and GFP-
cells
was validated using an independent quantitative telomere FISH technique based
on flow
cytometry known as Flow-FISH (Experimental Procedures). Two mouse cell lines
of
known telomere length were also included in the Flow-FISH analysis in order to
convert
telomere fluorescence values into kilobases (Example 1). Again, purified K15-
EGFP+
cells showed significantly longer telomeres than K15-EGFP- cells both in young
(0.5
year-old) and old mice (1.5 year-old) (P<0.01; Fig. 8f). We estimated a
telomere
shortening of 6 Kb between K15-EGFP+ hair bulge keratinocytes (a population
enriched in stem cells) and K15-EGFP- keratinocytes (a population enriched in
differentiated cells) (Fig. 8f), which corresponds to an approximately 16%
decline in
telomere length. Finally, longer telomeres in K15-EGFP+ hair bulge cells were
also
confirmed when using a Southern blot-based technique known as "telomere
restriction
analysis" or TRF, which is not based on fluorescence (Example 1) (Fig. 8g). Of
interest,
concomitantly with the decreased telomere length in the differentiated skin
compartments, it was also observed a reduction in telomerase activity when
comparing
K15-EGFP+ hair bulge keratinocytes and K15-EGFP- keratinocytes (Fig. 2h),
which
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may contribute to the observed telomere attrition associated to
differentiation (see also
Figs. 1, 4 and 5).
Next, GFP fluorescence was combined with confocal telomere QFISH directly on
skin
sections from K15-EGFP mice to address whether the GFP+ cells within the hair
follicle co-localized with the longest telomeres on skin histological sections
(Experimental Procedures). As shown in Fig. 3a, GFP+ cells precisely localized
to the
hair follicle bulge, in agreement with the fact that K15 labels stem cell
niches (Morris et
al., 2004). Interestingly, these GFP+ cells also showed the longest telomeres
compared
to the GFP- cells (highly significant, P<0.001; Fig. 9a,b). Telomapping of
GFP+ and
GFP- skin cells also indicated a 17% decrease in telomere length between both
compartments, similarly to the decrease obtained by Flow-FISH (see above). In
addition, it was calculated that 59.3% of the cells with the longest telomeres
(red color
after telomapping) were GFP+, while this percentage dropped to 5.5% in cells
with the
shortest telomeres (green after telomapping) (Fig. 9c). All together, these
results suggest
that more than 50% of epidermal cells with the longest telomeres are K15-
expressing
hair bulge cells (GFP+ cells), which in turn have been shown to be enriched in
stem
cells (Morris et al., 2004; Ito et al., 2005) (see also Fig. 7).
EXAMPLE 4
The longest telomeres are a general feature of different mouse stem cell
compartments (small intestine, cornea, testis, brain)
All together, the above-described findings suggest that the most primitive
compartments
within the skin are characterized by having cells with the longest telomeres
compared to
the more differentiated compartments. To generalize and test this hypothesis
to other
tissues besides the skin, telomapping was performed in histological sections
from small
intestine, cornea, testis and brain, where the corresponding stem cell
compartments have
been well characterized in the mouse. In mice, the small intestine stem cell
niche is
localized to the bottom of the intestinal crypts at approximately the +4
position, right
above the Paneth cells (positions +1 to +3) and below the TA compartment
(position
>+5), whereas the most differentiated cells are located at the intestinal
villi (see scheme
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in Fig. 10b) (Gregorieff et al., 2005; Marshman et al., 2002). Topographic
telomere
length maps of small intestine histological sections localized the cells with
the longest
telomeres (1700-3000 a.u. of telomere fluorescence; see Experimental
Procedures for
criteria on telomere length ranges) above the Paneth cells and in the TA
compartment
(Fig. 10a,c), in agreement with the known location of stem cell niches in the
small
intestine (Gregorieff et al., 2005; Marshman et al., 2002). In particular,
between
positions +1 to +3 (Paneth cells) only 16% of the cells showed the highest
telomere
fluorescence, while this increased to 37% between +4 and +5 positions
(putative stem
cells) (Fig. 10a,c). This percentage slightly decreased to 30% in the TA
compartment
(cells above the +5 position), further dropping to 4% in the differentiated
villi area (Fig.
10a,c). The differences in telomere fluorescence between the stem cell
compartment
and the other compartments were significant for all comparisons (Wilcoxon's
sum test,
P>0.05; Fig. 4d). Again, this telomere length distribution supports the notion
that the
longest telomeres are enriched at the most primitive compartments in the small
intestine.
Next, histological telomere length maps were generated for mouse cornea and
testis,
two other epithelial tissues where the SC compartment has been spatially
defined.
Corneal stem cells reside at the limbus, the peripheral zone of the cornea
lying above
the ciliary body (Fig. 10e) (Lavker et al., 2004; Lehrer et al., 1998). From
this location,
corneal stem cells migrate towards the central corneal epithelium as their
differentiation
program proceeds (Fig. 4e) (Lehrer et al., 1998). Telomapping of eye sections
revealed
that an average of 50% of the limbal cells possess the longest telomeres (1400-
3000 a.u.
of telomere fluorescence; see Experimental Procedures for criteria on telomere
fluorescence ranges), a percentage that gradually diminishes as cells move
centripetally
towards the centre of the cornea (Fig. 10e). The percentage of cells with the
longest
telomeres further increased to 68% within the limbal basal layer (see insert
in Fig. 10e),
a compartment where corneal SC are particularly enriched (Lehrer et al.,
1998).
Comparison of average telomere fluorescence between the limbus and the central
cornea further indicated that the corneal stem cell compartment harbours the
cells with
the longest telomeres (Fig. 4e; highly significant P<0.001).
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In mouse testis, spermatogenesis starts at the periphery of the seminiferous
tubules,
where the germ stem cells reside (Guan et al., 2006). By a series of mitotic
divisions
followed by meiosis, male germ stem cells give rise sequentially to
spermatogonia,
spermatocyte, spermatid and spermatozoa as they move to a more luminal
position
(Brinster et al., 2002). Telomapping of testis sections showed that the
periphery (1st and
2nd layers) of the seminipherous tubules presented the highest percentage of
cells with
long telomeres (1800-3000 a.0 of telomere fluorescence; see Experimental
Procedures
for criteria on telomere fluorescence ranges) (Fig. 10J). In contrast, the
lumen zone is
highly enriched with cells showing the shortest telomeres. Comparison of
telomere
fluorescence frequency histograms of the periphery and the lumen areas also
indicate a
decreased telomere length in the lumen compared to the periphery (highly
significant
P<0.001; Fig. 10J), reflecting on their differentiation program. Again, these
differences
in telomere length are unlikely to be due to differences in "probe
accessibility" or
ploidy between different testis compartments as we did not find significant
differences
when performing Q-FISH with a centromeric major satellite probe (Example 1;
Fig. 3c).
Finally, coronal sections of the adult mouse brain containing areas of
neurogenesis were
analyzed by telomapping. To date two different spatial stem cell niches have
been
characterized in the adult mouse brain: the subventricular zone (SVZ) of the
lateral
ventricule and the subgranular zone (SGZ) at the hippocampus (Alvarez-Buylla &
Lim,
2004; Gage, 2000). At the hippocampus, neural stem cells lie in the SGZ, an
area
located between the granular cell layer (GCL) and the hilus (H) (Fig. 10g).
From their
basal position, neural stem cells proliferate, migrate and differentiate into
the more
apical GCL (Fig. 10g) (Gage, 2000). Topographic telomere length mapping
revealed
that cells with the longest telomeres (1400-3000 a.u. of telomere
fluorescence) are
enriched at the SGZ, showing progressively shorter telomeres as they enter the
abutting
GCL (Fig. 10g). Average telomere fluorescence and telomere length
distributions also
indicated longer telomeres at SGZ compared to GCL (highly significant P<0.001;
Fig.
10g), further reflecting that the hippocampus stem cell compartment (SGZ) is
enriched
in cells having the longest telomeres.
EXAMPLE 5
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Telomere shortening with age in mouse stem cell compartments
Next, telomapping was used to address whether telomeres shorten in different
mouse
stem cell compartments with increasing age, which in turn could contribute to
stem cell
dysfunction with age. A role for telomere shortening in mouse aging and stem
cell aging
5 was previously suggested by the reduction in both median and maximum life-
span
(Garcia-Cao et al., 2005) as well as in stem cell functionality (Flores et
al., 2005) found
in early generation Terc4- mice which is progressively aggravated with
increasing
mouse generations concomitant with gradual reduction in telomere length
(Garcia-Cao
et al., 2005; Flores et al., 2005). Telomapping revealed that both the
percentage of cells
10 with the longest telomeres (Fig. 11a), as well as the average telomere
length (Fig. 11c),
decreased in all skin compartments at 2 years of age in wild-type C57B16 mice.
Similarly, average telomere length at the hair bulge cells decreased
significantly from
1780 268 a.u. to 1485 219 a.u. (p<0.001; Fig. 11c), demonstrating telomere
shortening
in hair follicle stem cells at old ages. In parallel, we performed telomapping
in the skin
15 of 6-month-old third generation (G3) Terc-deficient C57B16 mice. As
expected, G3
Terc-deficient mice showed a dramatic reduction of the percentage of cells
with the
longest telomeres, as well as of average telomere length in all skin
compartments (Fig.
11b), in agreement with their severe epidermal stem cell dysfunction (Flores
et al.,
2005). The decreased telomere length with aging in both the stem cell
compartment and
20 the more differentiated skin compartments was also confirmed by Flow-
FISH analysis
using the K15-EGFP reported mouse. In particular, Flow-FISH showed that both
sorted
GFP+ keratinocytes (enriched in stem cells) and GFP-keratinocytes (enriched in
differentiated cells) present a reduction of telomere length when comparing
0.5-year old
mice to 1.5 year-old mice (see Fig. 111). Similarly to skin, we detected
telomere
25 shortening in other mouse stem cell compartments including the small
intestine, cornea,
testis and brain when comparing 2 month-old mice to 2 year-old mice (Figs. 12-
15),
further supporting the notion that telomeres shorten at old ages in different
stem cell
compartments of the mouse, which in turn may result in age-related stem cell
disfunction.
Of notice, telomere shortening with age in Mus muscu/us male germ cells is in
agreement with previously reported telomere shortening with age in Mus spretus
testis
when comparing young (0-11 month-old) to old animals (>12 month-old) (Coviello-
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66
McLaughlin & Prowse, 1997). These findings suggest that, at least in the
mouse,
telomeres shorten with age in the male germ line. Finally, comparison of
telomere
shortening with age in all tissues studied here (Fig. 11d,e), indicates that
telomere
erosion rates vary at different ages, ranging from a slight reduction in
length from 2
month-old to 1 year-old animals to a rapid telomere loss when comparing 2
month-old
to 1 year-old mice, both in the stem cell and differentiated compartments.
These results
indicate that the mechanisms of telomere length maintenance decline more
rapidly at
advances ages, which in turn may contribute to stem cell aging and therefore
to aging
phenotypes.
EXAMPLE 6
Reduced clonogenic potential of mouse skin cells with age
The fact that telomeres shorten with age at different stem cell compartments
in the
mouse, may suggest that the mechanisms for telomere length maintenance decline
more
rapidly at advances ages, and that this telomere shortening may contribute to
stem cell
aging and therefore to aging phenotypes. To address this, the functionality of
mouse
epidermal stem cells at different ages was compared using clonogenic assays
(Example
1), which reflect on the proliferative potential of epidermal stem cells
(Flores et al.,
2005). In agreement with their shorter telomeres, keratinocytes directly
isolated from
27-31 month-old mice formed significantly fewer colonies than those derived
from
2month-old mice (P<0.001; Fig. 16), indicating a decreased clonogenic
potential of
epidermal cells with aging. These results are in agreement with previous
findings in
human skin keratinocytes that showed decreased clonogenic potential with
increasing
age (Barrandon & Green, 1987).
BIBLIOGRAPHY
Alvarez-Buylla, A., and Lim, D.A. (2004). For the long run: maintaining
germinal
niches in the adult brain. Neuron 41, 683-686.
Baird, D.M., Rowson, J., Wynford-Thomas, D., and Kipling, D. (2003). Extensive
allelic variation and ultrashort telomeres in senescent human cells. Nature
genetics 33,
203-207.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
67
Barrandon, Y., Green, H. (1987). Three clonal types of keratinocyte with
different
capacities for multiplication. Proc Natl Acad Sci U S A. 84, 2302-2306.
Blasco, M.A., Lee, H-W., Hande, P., Samper, E., Lansdorp, P., DePinho, R.,
Greider,
C.W. (1997). Telomere shortening and tumor formation by mouse cells lacking
telomerase RNA. Cell 91, 25-34
Blasco, M. A. (2005). Telomeres and human disease: cancer, ageing and beyond.
Nature Reviews Genetics 6, 611-622
Braun, K.M., Niemann, C., Jensen, U.B., Sundberg, J.P., Silva-Vargas, V.,
Watt, F.M.
(2003). Manipulation of stem cell proliferation and lineage commitment:
visualisation
of label-retaining cells in wholemounts of mouse epidermis. Development 130,
5241-
5255
Brinster, R.L. (2002). Germline stem cell transplantation and transgenesis.
Science 296,
2174-2176.
Bryan, T.M. et al., (1997) Evidence for an alternative mechanism for
maintaining
telomere length in human tumors and tumor-derived cell lines. Nature Medicine
3:1271-
1274.
Cawthon et al., (2003) Association between telomere length in blood and
mortality in
people aged 60 years or older, Lancet 361, 393-395.
Cawthon, R.M. (2002). Telomere measurement by quantitative PCR. Nucleic acids
Research 30, e47.
Chan, S.W., and Blackburn, E.H. (2002). New ways not to make ends meet:
telomerase,
DNA damage proteins and heterochromatin. Oncogene 21, 553-563.
Collins, K., Mitchell, J.R. (2002). Telomerase in the human organism.
Oncogene, 21,
564-579.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
68
Canela A, Vera E, Klatt P, Blasco MA. (2007). High-throughput telomere length
quantification by FISH and its application to human population studies. PNAS,
104,
5300-5305.
Cotsarelis, G., Sun, T.T., Lavker, R.M. (1990). Label-retaining cells reside
in the bulge
area of pilosebaceous unit: implications for follicular stem cells, hair
cycle, and skin
carcinogenesis. Cell 61, 1329-1337.
Coviello-McLaughlin, G.M., and Prowse, K.R. (1997). Telomere length regulation
during postnatal development and ageing in Mus spretus. Nucleic Acids Res 25,
3051-
3058.
de Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards
human
telomeres. Genes and Dev 19, 2100-2110.
Epel, E.S. et al., (2004) Accelerated telomere shortening in response to life
stress.
Proc.Natl.Acad.Sci USA, 101:17312-17315.
Flores, I., Benetti, R., and Blasco, M.A. (2006). Telomerase regulation and
stem cell
behaviour. Current Opinion Cell Biology 18, 254-260.
Flores, I., Cayuela, M.L., Blasco, M.A. (2005). Effects of telomerase and
telomere
length on epidermal stem cell behavior. Science 309, 1253-1256.
Freulet-Marriere, M.A., Potocki-Veronese, G., Deverre, J.R., and Sabatier, L.
(2004).
Rapid method for mean telomere length measurement directly from cell lysates.
Biochemical and biophysical research communications 314, 950-956.
Fuchs, E., Tumbar, T, and Guasch, G. (2004). Socializing with the neighbours:
stem
cells and their niche. Cell 116, 769-778.
Gage, F.H. (2000). Mammalian neural stem cells. Science 287, 1433-1438.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
69
Garcia-Cao., I., Garcia-Cao, M., Tomas-Loba, A., Martin-Caballero, J., Flores,
J.M.,
Klatt, P., Blasco, M.A, and Serrano, M. (2006). Increased p53 activity does
not
accelerate telomere-driven aging. EMBO Reports 7, 546-552.
Gonzalez-Suarez, E., Samper, E., Flores, J.M. & Blasco, M.A. (2000).
Telomerase-
deficient mice with short telomeres are resistant to skin tumorigenesis. Nat
Genet 26,
114-117.
Gregorieff, A., Pinto, D., Begthel, H., Destree, O., Kielman, M., Clevers, H.,
(2005).
Expression pattern of Wnt signaling components in the adult intestine.
Gastroenterology
129, 626-638.
Guan, K. et al. (2006) Pluripotency of spermatogonial stem cells from adult,
mouse
testis. Nature 440, 1199-1203.
Harley, C.B. et al., (1990) Telomeres shorten during ageing of human
fibroblasts.
Nature 345:458-460.
Harrington, L. (2004). Does the reservoir for self-renewal stem from the ends?
Oncogene 23, 7283-7289.
Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R.J., Cotsarelis,
G. (2005).
Stem cells in the hair follicle bulge contribute to wound repair but not to
homeostasis of
the epidermis. Nat Med 11, 1351-1354.
Kim, N.W. et al., (1994) Specific association of human telomerase activity
with
immortal cells and cancer, Science, 266:2011-2015.
Lansdorp, P.M. (2005). Role of telomerase in hematopoietic stem cells. Ann N Y
Acad
Sci 1044, 220-227.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
Lavker, R.M., Tseng, S.C., and Sun, T.T. (2004). Corneal epithelial stem cells
at the
limbus: looking at some old problems from a new angle. Exp Eye Res 78, 433-
446.
Lehrer, M.S., Sun, T.T., and Lavker, R.M. (1998). Strategies of epithelial
repair:
5 modulation of stem cell and transit amplifying cell proliferation. J Cell
Sci 111 (Pt 19),
2867-2875.
Marshman, E., Booth, C., and Potten, C.S. (2002). The intestinal epithelial
stem cell.
Bioessays 24, 91-98.
Mason, P.J., Wilson, D.B., Bessler, M. (2005). Dyskeratosis congenita ¨ a
disease of
dysfunctional telomere maintenance. Curr Mol Med 5, 159-170.
McIlrath et al. (2001) Telomere length abnormalities in mammalian
radiosensitive cells.
Cancer Res 61, 912-915.
Meeker et al. (2002). Telomere length assessment inhuman archival tissues:
combined
telomere fluorescence in situ hybridization and immunostaining. Am J Pathol
160,
1259-1268.
Meeker, A.K., Hicks, J.L., Gabrielson, E., Strauss, W.M., De Marzo, A.M., and
Argani,
P. (2004). Telomere shortening occurs in subsets of normal breast epithelium
as well as
in situ and invasive carcinoma. Am J Pathol 164, 925-935.
Meeker, A.K. and De Marzo, A.M. (2004). Recent advances in telomere biology:
implications for human cancer. Curr Opin Oncol 16, 32-38.
Moore, K.A. and Lemischka, I.R. (2006). Stem cells and their niches. Science
311,
1880-1885.
Morris, R.J., Liu, Y., Marles, L., Yang, Z., Trempus, C., Li, S., Lin, J.S.,
Sawicki, J.A.,
Cotsarelis, G. (2004). Capturing and profiling adult hair follicle stem cells.
Nat
Biotechnol 22, 411-417.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
71
Moyzis, R.K., Buckingham, J.M., Cram, L.S., Dani, M., Deaven, L.L., Jones,
M.D.,
Meyne, J., Ratliff, R.L., and Wu, J.R. (1988). A highly conserved repetitive
DNA
sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc.
Natl.
Acad. Sci. USA 85, 6622-6626.
Munoz, P., Blanco, R., Flores, J.M., and Blasco, M.A. (2005). XPF nuclease-
dependent
telomere loss and increased DNA damage in mice overexpressing TRF2 result in
premature aging and cancer. Nature Genetics 10, 1063-1071.
Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K., Barrandon, Y. (2001).
Morphogenesis and renewal of hair follicles from adult multipotent item cells.
Cell 104,
233-245.
Prowse, K.R., Greider, C.W. (1995). Developmental and tissue-specific
regulation of
mouse telomerase and telomere length. Proc Natl Acad Sci U S A. 92, 4818-4822.
Ramirez, R.D., Wright, W.E., Shay, J.W., and Taylor, R.S. (1997). Telomerase
activity
concentrates in the mitotically active segments of human hair follicles. J.
Invest
Dermatol 108, 113-117.
Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E., Lansdorp, P.M. (1998).
Telomere length dynamics in human lymphocyte subpopulations measured by flow
cytometry. Nat Biotechnol. 16, 743-747.
Samper, E., Goytisolo, F.A., Slijepcevic, P., van Buul, P.P., and Blasco, M.A.
(2000).
Mammalian Ku86 protein prevents telomeric fusions independently of the length
of
TTAGGG repeats and the G-strand overhang. EMBO rep 1, 244-252.
Sarin, K.Y., Cheung, P., Gilison, D., Lee, E., Tennen, R.I., Wang, E.,
Artandi, M.K.,
Oro, A.E., Artandi, S.E. (2005). Conditional telomerase induction causes
proliferation
of hair follicle stem cells. Nature 436,1048-1052.
CA 02723950 2010-11-09
WO 2009/138117 PCT/EP2008/055791
72
Therkelsen, A.J., Nielsen, A., Koch, J., Hindkjaer, J., and Kolvraa, S.
(1995). Staining
of
human telomeres with primed in situ labeling (PRINS). Cytogenetics and cell
genetics
68,
115-118.
Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W.E., Rendl, M.,
Fuchs, E.
(2004). Defining the epithelial stem cell niche in skin. Science 303, 359-363.
Valdes et al., (2005) Obesity, cigarette smoking, and telomere length in
women. Lancet,
366, 662-664.
Zijlmans, J.M., Martens, U.M., Poon, S.S., Raa,p A.K., Tanke, H.J., Ward,
R.K.,
Lansdorp, P.M. (1997). Telomeres in the mouse have large inter-chromosomal
variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. U S A. 94,
7423-
7428.
