WO2009138117A1 - Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées - Google Patents

Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées Download PDF

Info

Publication number
WO2009138117A1
WO2009138117A1 PCT/EP2008/055791 EP2008055791W WO2009138117A1 WO 2009138117 A1 WO2009138117 A1 WO 2009138117A1 EP 2008055791 W EP2008055791 W EP 2008055791W WO 2009138117 A1 WO2009138117 A1 WO 2009138117A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
telomere
fluorescence
cell population
Prior art date
Application number
PCT/EP2008/055791
Other languages
English (en)
Inventor
Ignacio FLORES HERNÁNDEZ
Andrés CANELA RODRÍGUEZ
María Antonia Blasco Marhuenda
Original Assignee
Fundación Centro Nacional De Investigaciones Oncológicas Carlos Iii
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fundación Centro Nacional De Investigaciones Oncológicas Carlos Iii filed Critical Fundación Centro Nacional De Investigaciones Oncológicas Carlos Iii
Priority to CA2723950A priority Critical patent/CA2723950C/fr
Priority to PCT/EP2008/055791 priority patent/WO2009138117A1/fr
Publication of WO2009138117A1 publication Critical patent/WO2009138117A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • 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 telo mere-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.
  • 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).
  • 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.
  • telomere restriction fragment assay 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.
  • TRF is a time-consuming technique which requires plenty of cells and only provides an average telomeric length of the cell population under study without giving an indication of telomere length in individual cells.
  • FISH quantitative fluorescent in situ hibridisation
  • 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-specif ⁇ c probe (Rufer et al., 1998; Baerlocher et al., 2006).
  • flow-FISH flow fluorescent in situ hybridisation
  • 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-specif ⁇ c probe and normalized to the signal obtained with an AIu repeat- specific probe.
  • this method requires a constant number of AIu repeats in the genome.
  • fluorescence length 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).
  • hybridization assay Reulet-Marriere et al, 2004
  • PRIMS primed in situ labeling
  • 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.
  • stem cell niches 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 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.
  • LRCs long-term retention cells
  • SP side population
  • WO07124125 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.
  • the invention relates to a method for the determination of the telomere length of a cell in an immobilised tridimensional test cell population comprising
  • the invention provides a method for the identification of stem cells in a cell population which comprises
  • the invention provides a method for the identification of stem cells in a test cell population which comprises
  • telomere length value 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.
  • 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
  • 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
  • the invention in a sixth aspect, 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 which is different to the average telomere length of the other cell population(s) of the array.
  • 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- specif ⁇ c probe and corresponding to different focal planes of said cell population comprising the steps of:
  • 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.
  • FIG. 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.
  • telomere-def ⁇ cient mice 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-def ⁇ cient mice.
  • FIG. 1 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.
  • telomere 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.
  • FIG. 3 Controls for the telomapping technique, a. Quantification of centromere 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
  • 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.
  • FIG. 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.
  • FIG. Telomere distribution pattern of wild type and Gl Ter ⁇ r f ⁇ 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-def ⁇ cient Gl T ere " 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 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.
  • 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 Gl Terc ⁇ ⁇ mice.
  • FIG. 7 Clonogenic potential of GFP+ and GFP- K15-EGFP sorted 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.
  • FIG. 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
  • telomere spots per nuclei in sorted GFP+ and GFP- cells indicate that there are no differences in ploidy between these two populations.
  • 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. 2b. Two independent mice were used for the analysis. n total number of nuclei used for the analysis, f.
  • FIG. 9 Telomapping maps the longest telomeres to the EGFP + 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 Alexa488 fluorescence (GFP immunostaining) and the combined DAPI+GFP image. Note that GFP-expressing cells localize to the bulge area of the hair follicle, the known putative stem cell niche.
  • telomere length maps generated according to GFP 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 ⁇ m.
  • n number of nuclei analyzed.
  • FIG. 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 histological section generated from confocal telomere Q-FISH 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 ⁇ m. Nuclei are coloured according to their average telomere fluorescence in arbitrary units (a.u.).
  • telomeres 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).
  • TA transient amplifying
  • b Scheme representing the small intestine compartments of villi and Lieberk ⁇ hn crypts. The crypts are further divided in (i) the Paneth cells at the bottom of the crypt between positions +1 and +3, (ii) the stem 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 (red color), while the villi are enriched in cells with the shortest telomeres (green color). Average and standard deviation for the percentages are indicated.
  • n number of nuclei per compartment analyzed for telomere FISH. Number in parenthesis indicates the cell position in the crypt. Vertical coloured 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, e, f, g. Right panels show representative telomere length pseudo-colour images of histological sections from cornea (e), testis (f) 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 ⁇ M.
  • telomere fluorescence comparisons between each of the stem cell compartments and the corresponding differentiated compartment are highly significant (PO.001).
  • 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, including the hair bulge where the stem cells are located.
  • a third generation G3 Terc- def ⁇ cient mouse is shown for comparison.
  • n number of nuclei per compartment analyzed for telomere FISH.
  • telomere fluorescence in the indicated stem cell compartments at the indicated age Note a faster rate of telomere loss at > 1 year or age.
  • 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.
  • 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 Zerc-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 ⁇ m. Nuclei are coloured according to their average telomere fluorescence in arbitrary units (a.u.).
  • 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 fluorescence within each epidermal compartment. Right panels show telomere fluorescence histograms of nuclei in each compartment.
  • 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.
  • FIG. 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 ⁇ m. 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.
  • 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.
  • FIG. 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 ⁇ m. 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.
  • 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.
  • telomere length of every single cell in an immobilized tridimensional cell population using fluorescence microscopy on said cell population followed by a semiautomatic image analyses.
  • 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.
  • 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
  • tridimensional cell population 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).
  • a tridimensional cell population 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.
  • the test cell population is a tissue sample selected from the group of skin, small intestine, testis, 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 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 chromosome, including centromeric regions. Accordingly, telomere-specif ⁇ c oligonucleotides may be designed using telomeric sequences that are well known in the art.
  • 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- 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.
  • Probes suitable for use in the present invention include probes specifically directed to the simple tandem repeat (TTAGGG) n .
  • 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).
  • MGB minor groove binder
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • a "minor groove binder" (MGB) moiety binds to the minor groove of DNA with high affinity.
  • 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” 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 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 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.
  • PNA peptide nucleic acid
  • the probe that specifically binds to the telomere is a probe that comprises the sequence (CCCT AA) 3 .
  • the telomeric probe is a PNA.
  • the telomeric probe is a PNA that comprises the sequence (CCCT AA) 3 .
  • 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.
  • 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.
  • 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.
  • the cells may be treated with pepsine at pH 2 at 37 0 C.
  • 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.
  • the amount of hybridization probe used is between 0.1-10 ⁇ g/ml, preferably 0.3 ⁇ g/ml.
  • the hybridization medium and hybridization conditions are selected so as to favour hybridization of the 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.
  • a hybridization medium is selected which has a low ionic strength and typically contains a buffer, denaturing agent and blocking reagent. Suitable buffers include Tris and Hepes.
  • the hybridization medium containing the hybridization probe may be applied to the morphologically preserved biological materials. Generally, 5 to 50 ⁇ l, preferably 30 ⁇ l 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 0 C, most preferably 3 minutes at 80 0 C.
  • Hybridization is carried out for about 0.1 to 24 hours, most preferably 2 hours, at 4 to 40 0 C, preferably 25 0 C. After hybridization, the slides are washed with buffer (e.g. formamide/TBS/Tween).
  • buffer e.g. formamide/TBS/Tween
  • the methods of the invention for detecting and/or quantitating multiple copies of a repeat sequence in cell suspensions about 10 to 1000 ⁇ l, preferably 200 ⁇ l 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.
  • 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).
  • buffer e.g. formamide/BSA/Tween; Tris/NaCl/Tween/BSA
  • 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.
  • 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.
  • Table 1 provides a list of possible fluorescence dyes that can be coupled to telomeric-specific probes.
  • 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 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
  • the average telomere length is know can be used as control cell population in the second method of the invention.
  • the cell lines HeIa 2, HeLa, MCF7, HeLa S3, 293T, L5178Y-S, MEFs BL6 G3 Terc "7" , MEFs BL6 wild type, HeLa 1211, MEFs 129Sv/BL6 wild-type and L5178 Y-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).
  • 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.
  • 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.
  • 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).
  • TRF telomere restriction fragment assay
  • 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.
  • 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.
  • 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.
  • fluorescence intensity in the cells associated to the telomere-specif ⁇ c probe can envisaged to determine fluorescence intensity in the cells associated to the telomere-specif ⁇ c probe.
  • the determination of the fluorescence signal is preferably carried out on an image of the cells obtained by fluorescence microscopy on a section of said tissue sample or said preparation of immobilized cells.
  • the tissue In case the sample to be analysed is embedded in paraffin, the tissue must be first deparaff ⁇ nized prior to the analysis using the methods of the invention. Typically, deparaff ⁇ nisation is carried out by applying sequential washes with an organic solvent (e.g. xylene) and rehydrated using ethano I/water mixtures of decreasing ethanol concentrations (100, 95 and 70%).
  • an organic solvent e.g. xylene
  • 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, dextran, pectine, carboxymethylcellulose.
  • the cells are embedded in gelatine.
  • 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.
  • the images 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 emisi ⁇ n: Fura/FITC or 535/50 for FITC detection.
  • the images are collected using a confocal microscope.
  • 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.
  • confocal microscopy allows collection of ten X/10 ⁇ m 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).
  • the method 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-specif ⁇ c 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.
  • 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 DNA dye must emit at a wavelength which allows the capturing of the telomere fluorescence without interference from the DNA fluorescence.
  • the telomere-specif ⁇ c probe is labeled with Cy3 and the DNA is labeled with DAPI.
  • 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.
  • 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.
  • 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.
  • 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, 111.), or as “Human Chromosome-Specific Centromeric Probes", from Open Biosystems (Huntsville, Ala.). Alternatively chromosome specific centromeric oligonucleotides may be designed using known sequences.
  • chromosome 1 Waye et al., (Genomics (1987) 1 :43-51); Hardas et al., (Genomics (1994) 21:359-63); Solus et al., (Somat. Cell. MoI. Genet. (1988) 14:381- 91); chromosome 2: Ostroverkhova et al., (Am J Med Genet. (1999) 87:217-20; Matera et al., (Hum MoI Genet.
  • 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.
  • chromosome 7 Mark et al., (Exp MoI 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 8: Zhao et al, (Ann. Clin. Lab. Sci. (1998) 28:51-6); Macoska et al, (Urology (2000) 55:776-82); Mark et al., (Exp. MoI. Pathol.
  • 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 MoI. 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.
  • 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.
  • 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.
  • 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.
  • the centromeric probe comprises the sequence TCGCCATATTCCAGGTC (SEQ ID NO:3).
  • 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).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • MGB minor groove binder
  • 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 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-Alexa647, Cy3 and PE-Alexa647, 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) requires converting the average fluorescence intensity obtained from the cells to an average telomere length value.
  • This step is preferably carried out by interpolation.
  • 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.
  • 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.
  • 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.
  • 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. Identification of stem cells using telomere-dependent fluorescence intensity values
  • telomere length can be used to identify those compartments within tissues that comprise stem cells.
  • stem cells contain certain telomerase activity but rarely divide and are found in protected microenvironments or niches.
  • telomere shortening the presence of telomerase activity results in that the telomere length decreases more slowly that the rest of somatic cells.
  • telomere length decreases more slowly that the rest of somatic cells.
  • the assay is advantageous over the assays known to date because it is tissue- 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
  • telomere length has been used to identify stem cell compartments in the hair follicle, confirming the known location of the stem cell niche in the hair follicle bulge.
  • the assay has been validated studying other tissues wherein the location of the stem cell niche is known.
  • 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 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).
  • the invention relates to a method for the identification of stem cells in a cell population which comprises (i) 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,
  • “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.
  • 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, Mammary stem cells Cardiac stem cells Pituitary stem cells Cancer stem cells
  • cell population 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).
  • 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” 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%,
  • the representative sample may be formed by the totality of cells of the cell population.
  • the determination of 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.
  • 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-specif ⁇ c probe.
  • Each flow-FISH experiment begins with the acquisition of the premixed calibration (MESF) beads.
  • MEF premixed calibration
  • CV mean fluorescence and coefficient of variation
  • 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 FIl (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 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.
  • 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.
  • the fluorescence signal may be influenced by changes in nuclear size and differences in ploidy. Therefore, the signal obtained using telomeric-specif ⁇ c 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.
  • 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. Identification of stem cells based on telomere length
  • the authors of the present invention have also improved the method for the 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.
  • 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.
  • longer 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).
  • 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
  • telomere length value 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.
  • 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.
  • the distribution of telomere lengths within the cell population is divided in quartiles and the cells whose telomere length is found in the upper quartile are then considered as candidate stem cells.
  • 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.
  • 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.
  • 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
  • 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
  • tissues wherein the stem cell niche is known.
  • any tissue which is known to contain a population of stem cells and whose spatial distribution is known may be used in the screening methods of the invention.
  • tissues that may be used in the present invention include skin, intestine, cornea, testis and central nervous system.
  • 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.
  • 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).
  • 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).
  • the stem cells are located in the peripheral region of the seminiferous tubes (Guan et al., 2006, Nature 440, 1199-1203).
  • 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).
  • the method involves in step (i) contacting a tissue with a compound whose activity as promoter of stem cell mobilization wants to be studied.
  • the contacting step can be carried out in several different ways.
  • 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 is possible within the present invention as long as the compound is able to reach the stem cell niche of the target tissue.
  • the compound may be administered orally, intradermically, parenterally and the like.
  • the contacting step may be carried out on the isolated tissue maintained under perfusion by providing the compound to be tested to the perfusion media.
  • 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.
  • the invention contemplates no limitation as to the type of compound that can be tested.
  • the candidate compound is a molecule with low molecular weight, it is enough to add said molecule to the culture medium.
  • the candidate compound is a molecule with a high molecular weight (for example, biological polymers such as a nucleic acid or a protein)
  • 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.
  • 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.
  • any of the aforementioned methods can be used to allow its entrance in the cell interior.
  • a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV-I 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, MoI. Therapy 8:143-150 and Snyder, EX. and Dowdy, S.F., 2004, Pharm. Res. 21 :389-393).
  • a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the determination of the fluorescence values is carried out on those regions of the image corresponding to the cell nuclei.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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 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.
  • tissue samples from the donor blocks such as elongated punches
  • 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.
  • the different samples of the array are formed by cells derived from stable cell lines.
  • the cell lines of the tissue microarray are selected from the group of HeIa 2, HeLa, MCF7, HeLa S3, 293T, L5178Y-S, MEFs BL6 G3 Terc "7” , MEFs BL6 wild type, HeLa 1211, MEFs 129SWBL6 wild-type and L5178 Y-R.
  • 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-specif ⁇ c fluorescent probe.
  • 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- specif ⁇ c 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
  • Step (i) comprises the merging or flattering 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.
  • a cell population has been previously contacted with a fluorescently-labelled telomere-specif ⁇ c probe.
  • appropriate optical filters to allow excitation of the cells with the appropriate wave-length and capturing only the emission 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.
  • the microscope can be programmed so as to obtain the different focal images at given focal lengths (preferably 1 ⁇ m).
  • a digital image is a two- dimensional array of pixels. Each pixel value relates to the amount of light received by the imaging capture device corresponding to the physical region of pixel.
  • image collection is carried out using a microscope, preferably a confocal microscope, attached to appropiate detectors (for instance, a CCD camera).
  • a digital image will often consist of red, green, and blue digital image channels.
  • the image file format can be any format used for digital images, including for example, a JPG format, a JPG2 format, a RAW format, a TIFF format, a PNG format, a GIF format, or a BMP format.
  • 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.
  • Step (ii) of the method of the invention comprises the determination of the intensity of the telo mere-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 which can be determined, by way of example, by overlying a bright filed image of the same cell population.
  • 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-specif ⁇ c fluorescent probe with the image under study.
  • 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.
  • background noise may interfere with the determination of the of pixel intensity.
  • 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-specif ⁇ c probe to non-telomeric structures) is more diffuse and is less intense than telomere-specif ⁇ c fluorescence, contribution to the total fluorescence measurement from background fluorescence is reduced by ignoring light intensities which are below a specified value.
  • 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.
  • corrections algorithms may be applied to the image to remove any artifacts introduced by the image capture system.
  • quality control algorithms may be employed to discard image data based on, for example, poor exposure, focus failures, foreign objects, and other imaging failures.
  • 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 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 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.
  • 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.
  • 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.
  • 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-specif ⁇ c fluorescence which correspond to cell nuclei.
  • 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.
  • 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.
  • the invention relates to a computer-readable support comprising encoded means adapted 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.
  • 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.
  • the program may be hosted in a remote storage device.
  • 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.
  • 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.
  • mice Treatment regimens, and mouse sample collection
  • 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).
  • 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.
  • TPA 20 nmol in acetone
  • 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.
  • 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 0 C, dehydrated through graded alcohols and xylene, and embedded in paraffin.
  • dissected skin 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.
  • whole eyes and testis were cut in half prior to dehydration.
  • brain was coronal-dissected to harvest the rostral hippocampus. In all cases, 5 "M sections were used for QFISH and immunostaining analyses.
  • telomere 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). HeIa cells were included as a positive control for telomerase activity.
  • TRAP modified telomere repeat amplification protocol
  • telomere length was determined as described (Gonzalez- Suarez et al., 2000; Munoz et al., 2005; Zijlmans et al., 1997). Slides were deparaffmized 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).
  • Cy3 signals were acquired simultaneously into separate channels using a confocal ultraspectral microscope (Leica TCS-SP2-A-OBS-UV) using a PL APO
  • 561 laser (Cy3 laser) was hold at a constant intensity to capture all the mouse tissues images.
  • telomere length maps were generated on histological sections or "telomapping"
  • 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 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 was measured as "average 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 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.
  • telomere fluorescence ranges were fine adjusted to better delineate the location of stem cell compartments in different tissues. Telomere 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 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).
  • paraffin slices of the same thickness 5 ⁇ M
  • confocal capture conditions were set to cover the entire fluorescence signal (maximum projections of 10 sections at steps of 1.0 ⁇ M).
  • Q-FISH and immuno-Q-FISH staining, as well as image capture were performed for each given tissue in the same day.
  • Immunohistochemistry Skin 5 "M) sections were used for immunohistochemistry (IHC). Prior to IHC, slides were de-paraff ⁇ nized, 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).
  • 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 Ix trypsin (Sigma) solution (4ml on 60 mm cell culture plate) for 3 h at 37 0 C.
  • Ix trypsin Sigma
  • 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 MoFIo (DakoCytomation, Glostrup, Denmark).
  • FACS fluorescence-activated cell sorter
  • telomere FISH was performed as described calculating the telomere fluorescence of the whole nuclei (Munoz et al., 2005; Samper et al., 2000).
  • telomere length quantification on interphase nuclei Cy3 and DAPI images were captured at 10Ox 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.
  • Telomere Q-FISH was performed as described (Gonzalez- Suarez et al, 2000; Munoz et al., 2005) with minor modifications to preserve GFP- immunostaining.
  • DAPI, Cy3 and Alexa488 signals were acquired simultaneously into separate channels using a confocal ultraspectral microscope (Leica 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 ⁇ m) were generated for image quantification.
  • 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.
  • Tween20-PBS cells were fixed in formaldehyde 0.5% PBS for 5 m and washed twice in PBS.
  • telomere flow-FISH was performed as described (Rufer et al., 1998) using a FITC labeled PNA-tel probe and Propidium Iodide (PI, Sigma) to counterstain DNA, and analyzed in a FACScanto cytometer (BD Biosciences).
  • telomere fluorescence as FITC signal was acquired in FLl 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.
  • telomere length was known.
  • 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 0 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).
  • 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). 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 the Alexa488 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 Alexa488 images obtaining topographic maps showing telomere fluorescence and GFP staining for each nucleus or object.
  • 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 Alexa488 to 1-bit image, showing only those nuclei that had an Alexa488-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.
  • the combined images were then analyzed as indicated above.
  • mice 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). 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 Ix trypsin (Sigma) solution (4ml on 60mm cell culture plate) for 16 h at 4 0 C.
  • Ix trypsin Sigma
  • Skins were transferred to a sterile surface, and the epidermis separated from the dermis using 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 sheets. Keratinocytes were then collected by centrifugation (160 g) for 10 min and counted.
  • mice 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 Ix trypsin (Sigma) solution (4ml on 60 mm cell culture plate) for 3 h at 37 0 C.
  • Ix trypsin Sigma
  • 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.
  • mice 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.
  • EXAMPLE 2 Cells with the longest telomeres are enriched at the hair follicle stem cell compartment and show stem cell behaviour upon treatment with mitogenic stimuli.
  • telomere fluorescence in situ hybridization 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).
  • telomere length maps were generated for skin sections from 2 month-old wild-type mice of a C57BL6 genetic background (Experimental Procedures), which were subdivided in four different epidermal compartments: the hair follicle bulge where the hair follicle stem cell niche is located (Tumbar et al, 2004; Cotsarelis et al, 1990; Oshima et al, 2001; Morris et al, 2004), the hair follicle bulb and the infundibulum where the transit-amplifying (TA) cells reside, and the interfollicular epidermis (Figs. ⁇ a,b).
  • telomeres In "resting" untreated wild- type mouse skin, it was observed that cells with the longest telomeres, 1800-3000 arbitrary units of telomere fluorescence (red color in Fig. Ia; 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 al., 2004). Immunostaining with the hair follicle stem cell markers CD34 and keratin 15 (Kl 5) further confirmed that the longest telomeres localized to the hair bulge (not shown).
  • 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 interfollicular epidermis showing the shortest telomeres, in agreement with their longer proliferative and differentiation history.
  • 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 centromeric major satellite probe (Experimental Procedures; Fig. 3a).
  • telomere length differences are not 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 telo mapping results were validated using the conventional quantitative telomere FISH technique (Q-FISH) on tissue sections (Experimental Procedures).
  • telomere length determinations 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).
  • 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.
  • Fig. Ab there was a linear correlation in telomere length values obtained by these techniques
  • 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.
  • 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).
  • telomere length maps were generated from first generation telomerase-def ⁇ cient Gl Terc ⁇ ⁇ mice (Fig. Ib) (Blasco et al., 1997; Ramirez et al., 1997).
  • Gl Terc ⁇ ⁇ 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 telomeres at the interfollicular epidermis (Fig.
  • telomere fluorescence was lower in all skin compartments compared to control wild-type mice, in agreement with the fact that Gl Terc ⁇ mice lack telomerase activity (Fig. Ib).
  • average telomere fluorescence was lower in Gl T ere " mice compared to wild-type mice in all skin compartments (Fig. 6).
  • telomere activity is important to maintain the overall telomere length of different skin compartments in the mouse, as first generation telomerase-def ⁇ cient Gl T ere-/- showed a marked decrease in telomere length compared to age-matched wild- type controls in all skin compartments.
  • telomeres are shortened telomeres within the hair follicles.
  • wild-type and Gl T ere-/- 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. Ic to Fig. ⁇ a).
  • telomere fluorescence significantly increased in TPA-treated skin compared to the untreated wild-type skin (significant, P ⁇ 0.05; Fig. Ie), suggesting net telomere elongation associated to TPA- induced proliferation in TA compartments (Fig. Ie).
  • 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).
  • TPA-treated Gl Terc ⁇ skin was studied.
  • Gl Terc ⁇ skin showed a reduction of the percentage of cells with the longest telomeres at the hair bulge (Fig. Ic) coincidental with an enlargement of the TA compartments (compare Fig. Ic to Fig. Ib), suggesting that these cells mobilized and proliferated in response to TPA.
  • TPA-treated skin did not show increased percentage of cells with the longest telomeres at the TA compartments and the interfollicular epidermis (Fig. ⁇ c,d). Indeed, the total number of epidermal cells showing the longest telomeres was decreased in Gl Terc ⁇ TPA-treated skin compared to untreated skin (very significant P ⁇ 0.01; Fig. Ie), suggesting telomere shortening as the result of TPA- induced proliferation in the absence of telomerase activity.
  • telomere length histograms of TPA-treated Gl Terc ⁇ mice showed a decreased frequency of long telomeres in all hair follicle compartments (Fig. 6).
  • telomeres are enriched at stem cell compartments.
  • purified skin hair bulge cells hair follicle stem cell compartment
  • 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 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).
  • GFP- GFP-negative
  • GFP+ GFP-positive
  • 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, Sd,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).
  • telomere shortening 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. Sf), which corresponds to an approximately 16% decline in telomere length.
  • telomere restriction analysis or TRF, which is not based on fluorescence (Example 1) (Fig. Sg).
  • TRF telomere restriction analysis
  • GFP fluorescence was combined with confocal telomere QFISH directly on skin sections from Kl 5 -EGFP mice to address whether the GFP+ cells within the hair follicle co-localized with the longest telomeres on skin histological sections (Experimental Procedures).
  • Fig. 3a GFP+ cells precisely localized to the hair follicle bulge, in agreement with the fact that Kl 5 labels stem cell niches (Morris et al, 2004).
  • 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).
  • telomere length between both compartments, similarly to the decrease obtained by Flow-FISH (see above).
  • 59.3% of the cells with the longest telomeres red color after telomapping
  • this percentage dropped to 5.5% in cells with the shortest telomeres (green after telomapping) (Fig. 9c).
  • telomeres 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).
  • telomeres are a general feature of different mouse stem cell compartments (small intestine, cornea, testis, brain)
  • telomeres compared to the more differentiated compartments.
  • 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.
  • 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 in Fig.
  • 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. 10 ⁇ ,c), in agreement with the known location of stem cell niches in the small intestine (Gregorieff et al., 2005; Marshman et al., 2002).
  • telomere fluorescence 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. 10 ⁇ ,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. 10 ⁇ ,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. Ad). Again, this telomere length distribution supports the notion that the longest telomeres are enriched at the most primitive compartments in the small intestine.
  • 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. 1Oe) (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.
  • telomere fluorescence a percentage that gradually diminishes as cells move centripetally towards the centre of the cornea (Fig. 1Oe).
  • the percentage of cells with the longest telomeres further increased to 68% within the limbal basal layer (see insert in Fig. 1Oe), 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. Ae; highly significant P ⁇ 0.001).
  • spermatogenesis starts at the periphery of the seminiferous tubules, where the germ stem cells reside (Guan et al., 2006).
  • meiosis 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).
  • 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. 10/), reflecting on their differentiation program.
  • telomere length is 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).
  • 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. 1Og). Average telomere fluorescence and telomere length distributions also indicated longer telomeres at SGZ compared to GCL (highly significant P ⁇ 0.001; Fig. 1Og), further reflecting that the hippocampus stem cell compartment (SGZ) is enriched in cells having the longest telomeres.
  • telomere shortening 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 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 Terc ⁇ ⁇ 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).
  • telomere shortening was shortened in hair follicle stem cells at old ages.
  • G3 Zerc-deficient C57B16 mice we performed telomapping in the skin of 6-month-old third generation (G3) Zerc-deficient C57B16 mice.
  • telomere length was also confirmed by Flow-FISH analysis using the K15-EGFP reported mouse.
  • 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. 11/).
  • telomere 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.
  • telomere shortening with age in Mus musculus 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- 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • Telomere shortening occurs in subsets of normal breast epithelium as well as in situ and invasive carcinoma. Am J Pathol 164, 925-935.
  • Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang.
  • 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.

Abstract

L'invention porte sur des procédés et des réactifs pour la détermination de la longueur de télomères dans des sections de tissu par la technique de cartographie télomérique des cellules individuelles basée sur une étape d'hybridation in situ fluorescente utilisant une sonde spécifique de télomères et sur une étape d'interpolation utilisant une courbe d'étalonnage corrélant l'intensité de fluorescence et la longueur des télomères obtenue à partir d'une collection de lignées cellulaires de longueur de télomères connue. L'invention porte en outre sur des procédés pour l'identification de niches de cellules souches à l'intérieur de tissus et pour l'identification de composés aptes à déclencher la mobilisation de cellules souches à l'aide de la longueur des télomères comme critère pour l'identification de cellules souches et qui reposent sur la technique de cartographie télomérique de cellules individuelles de l'invention.
PCT/EP2008/055791 2008-05-12 2008-05-12 Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées WO2009138117A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA2723950A CA2723950C (fr) 2008-05-12 2008-05-12 Procedes et reactifs pour la determination de la longueur de telomeres d'une maniere semi-automatique de chaque cellule individuelle dans une population de cellules immobilisees
PCT/EP2008/055791 WO2009138117A1 (fr) 2008-05-12 2008-05-12 Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2008/055791 WO2009138117A1 (fr) 2008-05-12 2008-05-12 Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées

Publications (1)

Publication Number Publication Date
WO2009138117A1 true WO2009138117A1 (fr) 2009-11-19

Family

ID=40386117

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/055791 WO2009138117A1 (fr) 2008-05-12 2008-05-12 Procédés et réactifs pour la détermination de la longueur de télomères d'une manière semi-automatique de chaque cellule individuelle dans une population de cellules immobilisées

Country Status (2)

Country Link
CA (1) CA2723950C (fr)
WO (1) WO2009138117A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018534564A (ja) * 2015-10-23 2018-11-22 ノバルティス・エイジーＮｏｖａｒｔｉｓ ＡＧ 視野に存在する選択された細胞のバイオマーカー陽性率の値を導き出す方法
EP3954269A1 (fr) * 2014-05-02 2022-02-16 Massachusetts Eye & Ear Infirmary Classement de coloration cornéenne à la fluorescéine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041016A1 (fr) * 1995-06-07 1996-12-19 Geron Corporation Procedes de mesure de la longueur d'un telomere
WO2004035597A2 (fr) * 2002-10-16 2004-04-29 Women And Infants Hospital Of Rhode Island, Inc. Procede permettant d'evaluer le risque d'echec de la reproduction par mesure de la longueur d'un telomere
WO2008081451A2 (fr) * 2007-01-03 2008-07-10 Monaliza Medical Ltd. Méthode et trousse d'analyse du matériel génétique d'un foetus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041016A1 (fr) * 1995-06-07 1996-12-19 Geron Corporation Procedes de mesure de la longueur d'un telomere
WO2004035597A2 (fr) * 2002-10-16 2004-04-29 Women And Infants Hospital Of Rhode Island, Inc. Procede permettant d'evaluer le risque d'echec de la reproduction par mesure de la longueur d'un telomere
WO2008081451A2 (fr) * 2007-01-03 2008-07-10 Monaliza Medical Ltd. Méthode et trousse d'analyse du matériel génétique d'un foetus

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FLORES IGNACIO ET AL: "Telomerase regulation and stem cell behaviour", CURRENT OPINION IN CELL BIOLOGY, vol. 18, no. 3, June 2006 (2006-06-01), pages 254 - 260, XP002518438, ISSN: 0955-0674 *
FLORES IGNACIO ET AL: "The longest telomeres: a general signature of adult stem cell compartments", GENES & DEVELOPMENT, vol. 22, no. 5, March 2008 (2008-03-01), pages 654 - 667, XP002518436, ISSN: 0890-9369 *
POON S S S ET AL: "TELOMERE LENGTH MEASUREMENTS USING DIGITAL FLUORESCENCE MICROSCOPY", CYTOMETRY, ALAN LISS, NEW YORK, US, vol. 36, no. 4, 1 August 1999 (1999-08-01), pages 267 - 278, XP009023307, ISSN: 0196-4763 *
SALDANHA SABITA N ET AL: "Assessment of telomere length and factors that contribute to its stability.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 270, no. 3, February 2003 (2003-02-01), pages 389 - 403, XP008103386, ISSN: 0014-2956 *
SIEGL-CACHEDENIER IRENE ET AL: "Telomerase reverses epidermal hair follicle stem cell defects and loss of long-term survival associated with critically short telomeres", JOURNAL OF CELL BIOLOGY, vol. 179, no. 2, October 2007 (2007-10-01), pages 277 - 290, XP002518437, ISSN: 0021-9525 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3954269A1 (fr) * 2014-05-02 2022-02-16 Massachusetts Eye & Ear Infirmary Classement de coloration cornéenne à la fluorescéine
US11350820B2 (en) 2014-05-02 2022-06-07 Massachusetts Eye And Ear Infirmary Grading corneal fluorescein staining
US11844571B2 (en) 2014-05-02 2023-12-19 Massachusetts Eye And Ear Infirmary Grading corneal fluorescein staining
JP2018534564A (ja) * 2015-10-23 2018-11-22 ノバルティス・エイジーＮｏｖａｒｔｉｓ ＡＧ 視野に存在する選択された細胞のバイオマーカー陽性率の値を導き出す方法
EP3365000A4 (fr) * 2015-10-23 2019-03-20 Novartis AG Procédé de dérivation d'une valeur de positivité de biomarqueur en pourcentage pour des cellules sélectionnées présentes dans un champ de vision
EP3719143A1 (fr) * 2015-10-23 2020-10-07 Novartis AG Procédé de dérivation d'une valeur de positivité de biomarqueur en pourcentage pour des cellules sélectionnées présentes dans un champ de vision
US11480570B2 (en) 2015-10-23 2022-10-25 Novartis Ag Method of deriving a value for percent biomarker positivity for selected cells present in a field of view

Also Published As

Publication number Publication date
CA2723950C (fr) 2016-07-12
CA2723950A1 (fr) 2009-11-19

Similar Documents

Publication Publication Date Title
Flores et al. The longest telomeres: a general signature of adult stem cell compartments
Sereti et al. Analysis of cardiomyocyte clonal expansion during mouse heart development and injury
Jansz et al. Smchd1 regulates long-range chromatin interactions on the inactive X chromosome and at Hox clusters
US8084203B2 (en) Methods for the determination of telomere length in a semi-automatic manner of every single cell in a immobilized cell population
Prieto‐Granada et al. A genetic dichotomy between pure sclerosing epithelioid fibrosarcoma (SEF) and hybrid SEF/low‐grade fibromyxoid sarcoma: a pathologic and molecular study of 18 cases
Prater et al. Mammary stem cells have myoepithelial cell properties
Pruszak et al. CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells
Teixeira et al. Cyclin E deregulation promotes loss of specific genomic regions
Quade et al. Intravenous leiomyomatosis: molecular and cytogenetic analysis of a case
Tichy et al. Single stem cell imaging and analysis reveals telomere length differences in diseased human and mouse skeletal muscles
Potapova et al. Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes
Didiot et al. Nuclear localization of huntingtin mRNA is specific to cells of neuronal origin
Achcar et al. Mammalian mastermind like 2 11q21 gene rearrangement in bronchopulmonary mucoepidermoid carcinoma
Wu et al. Subtype-specific tumor-associated fibroblasts contribute to the pathogenesis of uterine leiomyoma
Verheggen et al. Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis
CN108004299B (zh) 一种使用基因组dna测定端粒长度的荧光定量原位杂交(q-fish)方法
Broughton et al. Cardiac interstitial tetraploid cells can escape replicative senescence in rodents but not large mammals
Delalande et al. The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells
Basta et al. A Sall1-NuRD interaction regulates multipotent nephron progenitors and is required for loop of Henle formation
Choi et al. Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH) to detect RNA in tissue: simple and fast tissue RNA diagnostics
Wang et al. Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function
US20180142295A1 (en) Regulator targeting fatty acid synthase and method of using the same for improving meat quality
Hayes et al. Discovering novel oncogenic pathways and new therapies using zebrafish models of sarcoma
Aida et al. Determination of telomere length by the quantitative fluorescence in situ hybridization (Q-FISH) method
CA2723950C (fr) Procedes et reactifs pour la determination de la longueur de telomeres d'une maniere semi-automatique de chaque cellule individuelle dans une population de cellules immobilisees

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08750248

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2723950

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08750248

Country of ref document: EP

Kind code of ref document: A1